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Immobilization of growth factors using cellulose binding domain-cytokine fusion proteins Jervis, Eric J. 1998

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Immobilization of Growth Factors Using Cellulose Binding Domain-Cytokine Fusion Proteins Eric J. Jervis B.A.Sc, The University of British Columbia, 1986 M.A.Sc, The University of British Columbia, 1992 A thesis prepared in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Faculty of Graduate Studies Department of Chemical Engineering We accept this thesis as conforming to the reauired standard The University of British Columbia October, 1998 © Eric Jervis, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date Q ^ o W 2D DE-6 (2/88) 11 \ • Abstract This study describes the application and characterization of cellulose binding domain ( C B D ) -cytokine fusion proteins for the cultivation of cytokine dependent cells. The affinity, binding reversibility and surface diffusivity of type-U C B D s are analyzed. Binding of C B D s to crystalline cellulose is irreversible with respect to protein dilution in the solution phase. The surface diffusion rates of an exo-p-l-4-glycanase (Cex), an endo-P-l-4-glucanase (CenA), and their respective isolated C B D s , are quantified using fluorescence recovery after photobleaching analysis. Greater than 70% of bound molecules are mobile on the cellulose surface ( D s u r f ~ 3 x 11 2 10" cm /sec). Surface diffusion rates are dependent on surface coverage density and temperature ( E a ~ 45 kJ/mol.K). These attributes of the C B D suggest that C B D fusion proteins are not simply immobilized on the cellulose surface, but rather localized at it, so as to retain 2-dimensional mobility. Localization of cytokines to cellulose via a C B D affinity tag, provides a convenient method for adsorbing growth factors to a solid phase (cellulose) in a surface-active form. Bioactivity and long term stability of CBD-cytokine adsorption to microcrystalline cellulose under cell culture conditions is demonstrated. Cellulose-bound fusion proteins of murine stem cell factor with CBDcex ( C B D - S C F ) , murine interleukin-3 with C B D C e n A (CBD-IL3) and murine interleukin-2 with CBDcenA (CBD-IL2) , stimulate the proliferation of their respective factor dependent cells. Cellulose localization of C B D - S C F results in a significant increase in the persistence of tyrosine activation of the S C F receptor. Furthermore, when cells are incubated with cellulose-bound C B D - S C F , activated receptors and C B D - S C F co-localize at the cellulose surface. In contrast to the surface diffusion of C B D C e x , C B D - S C F does not diffuse on crystalline cellulose. This is likely a result of the formation of C B D - S C F dimers (through S C F domain dimerization) at the cellulose interface. iii Table of Contents ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xi ACKNOWLEDGEMENTS xiv CHAPTER 1 INTRODUCTION 1 THESIS OBJECTIVES 1 PROJECT JUSTIFICATION 3 i) Growth factor-mediated regulation of cellular function 3 ii) Culturing of factor-dependent cells 8 iii) Mass transport of growth factors 16 RESEARCH APPROACH 31 i) Molecular engineering of growth factors 31 ii) Biomaterials for cell culture applications 32 iii) Cellulose binding domain technology 35 iv) System configuration 40 v) Confocal imaging 42 vi) Fluorescence recovery after photobleaching analysis 45 vii) The biology of growth factors used in this study 47 CHAPTER 2 MATERIALS AND METHODS 56 PROTEIN PRODUCTION AND PURIFICATION 56 i) Preparation of CBDs 56 ii) Preparation of CBD-IL3 56 Hi) Preparation of CBD-IL2 57 iv) Preparation of CBD-SCF 57 v) Protein labeling with fluorescein 58 CHARACTERISTICS AND PREPARATION OF CELLULOSE MATERIALS 59 i) Valonia ventricosa microcrystalline cellulose 59 ii) Bacterial microcrystalline cellulose 59 Hi) Commercial-grade Avicel cellulose 60 ISOTHERM ANALYSIS 60 i) Fluorometric CBD assay 60 ii) Adsorption of CBDs to V. ventricosa cellulose 61 Hi) Adsorption of CBDs to BMCC 62 F R A P ANALYSIS : 63 i) FRAP with the BioRad MRC600 CLSM 63 ii) FRAP data analysis 64 C E L L CULTURE 64 i) Cell lines 64 ii) Bioassays 65 C L S M IMAGING 67 i) CLSM of fixed cells 67 ii) Live cell confocal imaging 68 CHAPTER 3 CHARACTERIZATION OF CBD BINDING TO CRYSTALLINE CELLULOSE 70 INTRODUCTION 70 RESULTS 72 i) Langmuir adsorption model 72 ii) RSA-modified Langmuir model 75 V iii) Low Surface concentration isotherms 79 iv) Multiple classes of binding sites model 81 v) Adsorption of FITC-labeled CBD to cellulose 86 vi) CBD adsorption to V. ventricosa cellulose 91 vii) Reversibility of CBDCexadsorption to cellulose 94 DISCUSSION 95 CHAPTER 4 CBDS DIFFUSE ACROSS THE SURFACE OF CRYSTALLINE CELLULOSE 98 INTRODUCTION 98 RESULTS 100 i) Photobleaching analysis of FITC-CBD on crystalline cellulose 100 ii) Surface diffusion rates for different cellulose binding proteins 108 iii) Surface diffusion rate as afunction of CBD surface coverage density 110 iv) Surface diffusion rate as afunction of temperature 113 DISCUSSION 116 CHAPTER 5 PRODUCTION AND BIOACTP7ITY OF CBD-CYTOKINE FUSION PROTEINS 120 INTRODUCTION 120 RESULTS 122 i) Vector Design for Expression of CBD-IL3 in E. Coli 722 ii) Expression of CBD-IL3 in E. Coli 124 iii) CBD-IL3 is active when bound to cellulose 125 iv) Strain selection for expression of CBD-IL3 in E. Coli 128 v) Production of CBD-IL3 infedbatch fermentation ofE. coliJMWl 130 DISCUSSION 135 (II) A C B D FUSION PROTEIN FOR THE CELLULOSE-LOCALIZATION OF MIL-2 137 i) CBD-IL2 is active when bound to cellulose 137 ii) CBD-IL2 produced in animal cells binds reversibly to cellulose 139 DISCUSSION 142 EVALUATION OF C B D - M E D I A T E D CYTOKINE IMMOBILIZATION 143 CHAPTER 6 CBD-SCF ADSORPTION TO CELLULOSE 144 INTRODUCTION 144 RESULTS ]47 i) Western blot analysis of CBD-SCF binding to BMCC 147 ii) CBD-SCF binds to BMCC with high affinity 149 Hi) CBD-SCF binding to crystalline cellulose is stable and irreversible 153 iv) CBD-SCF does not diffuse across the surface of crystalline cellulose 156 DISCUSSION 160 CHAPTER 7 ANALYSIS OF THE BIOACTIVITY OF CBD-SCF 163 INTRODUCTION: 163 RESULTS: 166 i) Cellulose bound CBD-SCF has increased molar specific activity 166 ii) B6SutA cells are motile 170 Hi) SCF-responsive cells adhere to CBD-SCF on cellulose 173 iv)C-kit activation is prolonged with CBD-SCF bound to cellulose 176 DISCUSSION: 178 CHAPTER 8 CYTOSTRUCTURAL CHANGES INDUCED BY CBD-SCF STIMULATION 183 INTRODUCTION: 183 RESULTS: 185 i) Exposure to soluble SCF causes rapid internalization of c-kit 185 ii) Receptor patching occurs within 60 seconds of SCF addition 187 Hi) C-kit co-localizes with CBD-SCF presented on BMCC 190 iv) BMCC fibers presenting CBD-SCF are internalized by B6SutA cells.: 194 v) C-kit co-localizes with CBD-SCF presented on V. ventricosa cellulose sheets 196 vi) lmaging c-kit co-localization with tyrosine-P following CBD-SCF stimulation 200 vii DISCUSSION 203 CHAPTER 9 CONCLUSIONS AND RECOMMENDATIONS 209 CHAPTER 10 REFERENCES 213 viii List of Tables T A B L E 3-1: BINDING CONSTANTS FOR C E N A , C B D C E N A AND C E X ON B M C C 80 T A B L E 7-1: E D 5 0 FOR C B D - S C F WITH OR WITHOUT ADDED B M C C 170 List of Figures FIGURE 1-1: T H E 3-D STRUCTURE OF C B D C E X 38 FIGURE 1-2: POTENTIAL RATE-LIMITING PROCESSES IN CELL STIMULATION BY CELLULOSE-ADSORBED C B D -CYTOKINES 41 FIGURE 3-1: ISOTHERM FOR THE ADSORPTION OF C B D C E N A TO B M C C 74 FIGURE 3-2: SCATCHARD PLOT OF ISOTHERM DATA FOR ADSORPTION OF C E N A AND ITS ISOLATED BINDING DOMAIN TO B M C C AT 3 0 ° C AND P H 7.0 75 FIGURE 3-3: SCHEMATIC REPRESENTATION OF THE C B D BINDING TO THE 110 AND 111 CRYSTAL FACES 77 FIGURE 3-4: DOUBLE RECIPROCAL PLOT OF THE ADSORPTION OF C E N A TO B M C C 81 FIGURE 3-5: B M C C LABELED WITH CBD C E x TAGGED WITH F I T C 83 FIGURE 3-6: ISOTHERM FOR THE ESTIMATION OF BINDING AFFINITIES AND CAPACITIES OF B M C C FOR C B D C E X 85 FIGURE 3-7: DEMONSTRATION OF EQUIVALENCE OF C B D C E X AND F I T C - C B D C E X 87 FIGURE 3-8: AVICEL LABELED WITH FITC-TAGGED C B D C E X 90 FIGURE 3-9: V. VENTRICOSA CELLULOSE LABELED WITH C B D C E X TAGGED WITH F I T C 92 FIGURE 3-10: ISOTHERM ANALYSIS OF C B D BINDING TO V. VENTRICOSA 93 FIGURE 4-1: IMAGE SEQUENCE COLLECTED DURING F R A P ANALYSIS 101 FIGURE 4-2: PHOTOBLEACHING ANALYSIS OF F I T C - C B D ON V. VENTRICOSA 103 FIGURE 4-3: PHOTOBLEACHING ANALYSIS OF F I T C - C B D ON V. VENTRICOSA 105 FIGURE 4-4: DEPENDENCE OF RECOVERY TIME ON BLEACH SPOT DIAMETER 109 FIGURE 4-5: SURFACE DIFFUSION RATE AS A FUNCTION OF BOUND C B D CONCENTRATION 112 FIGURE 4-6: DIFFUSION COEFFICIENT AS A FUNCTION OF TEMPERATURE 114 FIGURE 4-7: ARRHENIUS PLOT FOR C B D C E X BINDING TO V. VENTRICOSA CELLULOSE 115 FIGURE 5-1: VECTOR DESIGN FOR EXPRESSION OF C B D - I L 3 IN£. COLI 123 FIGURE 5-2: EXPRESSION OF C B D - I L 3 INE. COLI 125 FIGURE 5-3: C E L L PROLIFERATION ASSAY OF C B D - I L 3 ACTIVITY 127 FIGURE 5-4: STRAIN SELECTION FOR EXPRESSION OF C B D - I L 3 IN E. COLI 129 X FIGURE 5-5: ELECTROPHORESIS ANALYSIS OF C B D - I L 3 DEGRADATION PRODUCED IN A VARIETY OF E. COU STRAINS 130 FIGURE 5-6: RECOMBINANT JM101 E. Cou GROWTH DURING EXPRESSION OF C B D - I L 3 IN FEDBATCH FERMENTATION 132 FIGURE5-7: EXPRESSION OF C B D - I L 3 IN E. COU DURING FEDBATCH FERMENTATION 134 FIGURE 5-8: CELLULOSE-BOUND C B D - I L 2 STIMULATION OF C T L L CELL PROLIFERATION 138 FIGURE 5-9: IL-2 BIOACTIVITY IN CULTURES STIMULATED WITH SOLUBLE C B D - I L 2 140 FIGURE 5-10: IL-2 BIOACTIVITY IN THE SUPERNATE OF CULTURES STIMULATED WITH C B D - I L 2 BOUND TO AVICEL 141 FIGURE 6-1: C B D - S C F DIMERIZES WITH M S C F ON THE CELLULOSE SURFACE 149 FIGURE 6-2: BINDING ISOTHERM FOR FITC-LABELED C B D - S C F ON B M C C 152 FIGURE 6-3: C B D - S C F BINDS EFFECTrVELY IRREVERSIBLY TO CELLULOSE UNDER CELL CULTURE CONDITIONS 155 FIGURE 6-4: C B D - S C F DOES NOT DIFFUSE ACROSS THE SURFACE OF CRYSTALLINE CELLULOSE 159 F I G U R E 7-1: POTENTIAL INTERACTIONS IN CELLULOSE-BOUND C B D - S C F CELL STIMULATION 164 FIGURE 7-2: AVICEL-BOUND C B D - S C F HAS INCREASED MOLAR SPECIFIC ACTIVITY 168 FIGURE 7-3: B 6 S U T A CELLS ARE MOTILE 172 FIGURE 7-4: SCF-RESPONSIVE CELLS ADHERE TO C B D - S C F ON CELLULOSE 175 FIGURE 7-5: C-KIT ACTIVATION IS PROLONGED WITH C B D - S C F BOUND TO CELLULOSE 178 FIGURE 8-1: C L S M IMAGING OF M 0 7 E CELLS STIMULATED WITH SOLUBLE R H S C F 187 FIGURE 8-2: LIVE-CELL C L S M OF B6SUTA CELLS STIMULATED WITH SOLUBLE F I T C - C B D - S C F 190 FIGURE 8-3: C L S M OF B 6 S U T A CELLS GROWN WITH C B D - S C F BOUND TO B M C C 192 FIGURE 8-4: B M C C FIBERS PRESENTING C B D - S C F ARE INTERNALIZED BY B 6 S U T A CELLS 195 FIGURE 8-5: B M C C FIBERS STAINED WITH F I T C - C B D C E x AND C B D - S C F ARE INTERNALIZED BY B 6 S U T A CELLS.. 196 FIGURE 8-6: C L S M OF CELLS CULTURED WITH C B D - S C F BOUND TO A V. VENTRICOSA CELLULOSE SURFACE 200 FIGURE 8-7: ANTI-PHOSPHOTYROSINE C L S M OF CELLS CULTURED WITH C B D - S C F BOUND TO A V. VENTRICOSA CELLULOSE SURFACE 202 xi List of Abbreviations BD Brownian dynamics BM bone marrow BMCC bacterial microcrystalline cellulose BMP bone morphogenic protein BMT bone marrow transplant BSA bovine serum albumin CBD cellulose binding domain CCD charge coupled device CFU colony forming units c-kit S C F receptor CLSM confocal laser scanning microscopy CRU competitive repopulating unit CTLL cytotoxic T-lymphocyte Da Damkoehler number ECM extracellular matrix E D 5 0 effective dose yielding 50% maximum effect EGF epidermal growth factor FACS fluorescence activated cell sorting FCS fetal calf serum FGF fibroblast growth factor FITC fluorescein isothiocyanate xu FRAP fluorescence recovery after photobleaching G-CSF granulocyte colony stimulating factor GM-CSF granulocyte macrophage colony stimulating factor hGH human growth hormone HME hematopoietic microenvironment H-SFM hybridoma serum-free medium IL-2 interleukin 2 IL-3 interleukin 3 IL-4 interleukin 4 IL-6 interleukin 6 IMDM Iscove's modified Dubelcco's medium IPTG isophenyl-thio-(3-D-galactopyranoside LTBMC long-term bone marrow cultures LTC-IC long-term culture initiating cells MCAC metal chelate affinity chromatography M-CSF macrophage colony stimulating factor MNC mononuclear cells NK natural killer cells PBS phosphate buffered saline PDGF platelet-derived growth factor PMT photomultiplier tube S/N signal-to-noise XU1 stem cell factor tumor necrosis factor fractional surface saturation xiv Acknowledgements I wish to dedicate this thesis to my wife Cara Dubeski. Without her infinite energy, love and understanding, this thesis would not have been possible. I also wish to dedicate this thesis to my parents, Eric and Violet Jervis. Their love and guidance has always given me the confidence to follow my own path. Finally, I wish to dedicate this thesis to my mentor and colleague Doug Kilburn. Doug always maintained a stimulating and open research environment for my development as a student, scientist and a teacher. I wish to acknowledge the input and guidance of my cosupervisor, Chip Haynes, and my thesis committee members, J. Piret, D . Brooks and P. Lansdorp. Their insights and discussions have been a constant source of ideas over the course of this work. I gratefully acknowledge the expert technical assistance, discussion and collaboration of the following individuals: J. Alimonti , G . Doheny, M . Guarna, R. Graham, P. Carew, N . Gilkes, E . Kwan, E . Amandoron-Akow, D . Haddow, D . Hasenwinkle, T. Warren, R. Deane, G . Lesniki , A . Ozin, R. Turner, M . Weiss, E . Humpheries, J. Greenwood, H . Ziltner, P. Johston, S. Teh, R. Mil le r . Thus his path had been a circle, or an ellipse or a spiral or whatever, but certainly not straight; straight lines evidently belonged only to geometry, not to nature and life. Yet he had faithfully obeyed the exhortation and self-encouragement of his poem, even after he had long forgotten the poem and the awakening he had then experienced. Herman Hesse, Magister Ludi - The Glass Bead Game (1943) 1 Chapter 1 Introduction Thesis Objectives Recent advances in bone marrow transplantation and other cell-based graft therapies have intensified the need for technological improvements in primary cell cultivation systems (Asonuma and Vacanti, 1992; Crane et al, 1995). Examples of potential applications include the expansion of donor cells for bone marrow transplantation (Eaves et al, 1991) and wound healing (Marks et al, 1991), or the purging of malignant cells from a patient's explanted cells for autografting in cancer therapy (Coulombel et al, 1985). The development of cell culture techniques for the growth of cells for medical purposes has rapidly developed into a new area of intense research, often referred to as 'tissue engineering' (Bell , 1991; C ima et al, 1991; Edgington, 1992; Edgington, 1994; Putnam and Mooney, 1996; Langer, 1997; Vunjak-Novakovic et al, 1998). Tissue engineering applies principles of engineering and life sciences to the development of biological substitutes that restore, maintain, or improve tissue function (Langer and Vacanti, 1993). However, improved bioreactor systems which provide the appropriate conditions for cell growth and development are still required. A major objective in tissue engineering is the development of cell culture methods which provide the environmental conditions necessary for the growth and maintenance of specific cell phenotypes. Insight into the conditions required for primary cell culture has been obtained by the examination of normal tissues. A common feature in many tissues is the binding of growth regulatory signals by specific extracellular matrix ( E C M ) molecules. The E C M provides not only structural components, which support the development of tissue morphology, but also growth factor signaling context, which modulates the cellular response to growth factors. The 2 central role of growth factor presentation within the E C M suggests that the localization of factors to tissue culture surfaces might be used to mimic the in vivo environment. The engineering of cell culture supports which present surface-immobilized growth factors offers several advantages. These advantages include significantly reduced requirements for cytokines due to increased cell-proximal factor concentrations and the potential increased efficacy of ECM-presented factors. This thesis w i l l examine the interactions between growth factor-dependent cells and growth factors presented on tissue culture surfaces, to provide necessary data for the eventual development of materials for the cultivation of factor-dependent cells. Methods of growth factor surface-localization, which exploit specific noncovalent association of factors with the cell culture surface, w i l l be studied. Specifically, cell stimulation by growth factors adsorbed to cellulose matrices through cellulose binding domain (CBD) fusion protein technology w i l l be investigated. The use of hematopoietic-derived cell lines as a model in this study, addresses an immediate biomedical requirement for improved systems for the expansion of hematopoietic cells for adoptive immunotherapy or transplantation following chemotherapy. Thesis Statement The use of CBD-cytokine fusion proteins and cellulose-based tissue culture matrices facilitates the spatial regulation of growth factor stimulation in cell cultures providing a means to: (a) establish a reservoir of growth factors within a tissue culture vessel; (b) produce a matrix-bound signal to localize the response to a factor; (c) present a localized signal to stimulate cell polarization; and, (d) modulate receptor activation through control of ligand availability. 3 Project Justification In vivo, the activation and growth of progenitor cells involves complex interactions with stroma which provide an important source of soluble and surface-presented growth factors (Kittler et al, 1992; Long et al, 1992; Sensebe et al, 1997). Cytokines and growth factors are frequently found in a surface-bound form located on the membrane of stromal cells (Sasaki et al, 1995) or bound to extracellular matrix ( E C M ) components (Alon et al, 1994; Somasundaram and Schuppan, 1996). The mode of cytokine presentation to cells influences the intensity and persistence of growth factor signaling (Slanicka-Krieger et al, 1998). Therefore, the ability to manipulate the presentation of factors in culture systems should provide an additional degree of control in regulating the stimulation of factor-dependent cells. i) Growth factor-mediated regulation of cellular function Hematopoiesis Hematopoiesis is the process by which blood cells are generated from a small population of pluripotent hematopoietic stem cells (Dexter et al, 1984; Sutherland et al, 1990). Because of the limited life span of mature blood cells (Cronkite, 1988), the human hematopoietic system must produce up to 400 bil l ion blood cells per day (Koller et al, 1993). Hematopoiesis is the result of the combined actions of accessory cells and microenvironmental factors, which stimulate and regulate progenitor cell proliferation and differentiation. The existence of hematopoietic stem cells was first shown by transplantation of limiting numbers of bone marrow cells in mice. T i l l and McCul loch (1980) observed that following bone marrow injection into lethally irradiated mice, discrete colonies formed in the spleen. They developed the spleen colony forming assay as a means of quantifying presumptive bone marrow stem cells. 4 The blood cell hierarchy can be divided into two major classes: myeloid and lymphoid. A t each stage of the hematopoietic cell maturation sequence, cells undergo significant proliferation as well as differentiation, so that each successive step in the maturation pathway contains more cells. The major site of hematopoiesis in human adults is the bone marrow (Koller and Palsson, 1993). Only a small number of progenitor cells are found in the circulation of healthy adults (Lowry and Tabbara, 1992). Stem cells are defined by their ability to self-renew and give rise to all of the cells of the various lineages. The existence of stem cells has subsequently been verified in the long-term competitive repopulation assay (Fraser et al, 1992). In this assay lethally irradiated mice (i.e. hematopoietic ablated) are injected with limiting dilutions of candidate stem cells. Under these conditions, the ability to regenerate a complete hematopoietic system from a single cell has been demonstrated (Osawa et al, 1996). Hematopoietic growth factors and cytokines Polypeptide growth factors and cytokines are a general class of small proteins which regulate the growth, death and differentiation of many target cell types including hematopoietic cells (Morstyn and Burgess, 1988). These factors elicit a cellular response by binding to specific receptors presented on the membrane of responsive cells. Much in vitro work has been performed with hematopoietic cells to identify the critical factors affecting their proliferation and differentiation (Eaves et al, 1991; Koller et al, 1995; Petzer et al, 1996). These studies have established a relatively well-defined set of growth stimulatory factors required for extended cell maintenance ex vivo. Hematopoietic growth factors are secreted by stromal and accessory cells within the bone marrow microenvironment (Dexter et al, 1984; Dexter et al, 1990; Temeles et al, 1993). Hematopoietic factors are also released systemically by effector cells, whereby they influence cell 5 activation and inflammation (Cavallo et al, 1994). Many growth factors induce a range of responses (functional pleiotropy) dependent upon cell type and state (Nicola, 1994). Furthermore, different growth factors may induce similar responses in target cells (functional redundancy) (Metcalf, 1992; Miyajima et al, 1992). Thus, in growth factor-mediated signaling, there is not a simple one-to-one correspondence between factor and response. Rather, growth factor signaling is a complex regulatory network and cascade in which the eventual signaling outcome results from the integration of many extracellular and intracellular signals. Hematopoietic factors exert their biological functions by interacting with specific transmembrane receptors (Nicola, 1989; Park and Gi l l i s , 1990; McKinst ry et al, 1997). These receptors are composed of three major domains: the extracellular domain is responsible for ligand binding; a transmembrane domain anchors the receptor in the membrane, and 'transmits' the ligand binding status of the extracellular domain to the third major domain, the intracellular domain which transduces the ligand binding event to the intracellular compartment. Receptors are generally displayed at low levels (i.e. 50 - 5000 receptors per cell) (Brizzi et al, 1991) although some factor-dependent cell lines may display up to 100,000 receptors per cell (Murthy etal, 1990). Hematopoietic receptors fall into three basic classes: the hematopoietic receptor superfamily, the receptor tyrosine kinases, and functionally distinct soluble forms of either. The hematopoietic receptor superfamily includes receptors for interleukin 2 (IL-2) (Nelson et al, 1994), IL-3 (Clark-Lewis et al, 1986), IL-6 (Hibi et al, 1996), and granulocyte macrophage colony stimulating factor ( G M - C S F ) (Goodall et al, 1993). This class of receptors is defined by its lack of tyrosine kinase activity and a conserved motif in the extracellular domain (Miyajima, 1992). Upon ligand binding, this class of receptors form either homo or hetero (e.g. IL-2, IL-3) 6 oligimers, which then cause conformational changes in the intracellular domains to form binding sites for secondary molecules in the signaling cascade. The second class of hematopoietic growth factors are defined by their intrinsic tyrosine kinase activity and conserved extracellular domain motifs. This class includes receptors for fibroblast growth factor (FGF) (Friesel and Maciag, 1995), stem cell factor (SCF) (Reith et al, 1990) and platelet-derived growth factor (PDGF) (Heldin et al, 1985). Upon binding of the ligand, these receptors form dimers which stimulate receptor trans-autophosphorylation and subsequent activation of secondary messenger molecules (Williams, 1989). Three different dimerized-ligand motifs have been identified: P D G F is a homodimer, human growth hormone (hGH) has two equivalent binding faces, and S C F forms a noncovalent homodimer. In each case, the ligand bridges two receptor molecules to bring the intracellular domains into contact for trans-activation. Soluble forms of many growth factor receptors have also been identified. Soluble receptors are created either by protease cleavage of the extracellular domain of intact receptors (e.g. tumor necrosis factor (TNF) (Olsson et al, 1989), S C F (Reith et al, 1990) and macrophage colony stimulating factor (M-CSF) (Downing et al, 1989)), or by alternative splicing during R N A transcription (e.g. IL-4 (Mosley et al, 1989), granulocyte colony stimulating factor (G-CSF) (Fukunaga et al, 1990) and IL-6 (Hibi et al, 1990)). Olsson et. al. (1992) suggest that soluble receptors may (a) function as receptor competitors, thereby attenuating available soluble cytokine; (b) function to stabilize the growth factor by protecting it against proteolysis, thus increasing its effective half-life; or (c) possibly act to cause dissociation of growth factors from the E C M and thereby increase the local availability of soluble factor. 7 Clinical applications of growth factors Cytokines have been used in a number of clinical applications (Broxmeyer and Vadhan-Raj, 1989). G M - C S F has been tested in several clinical trials, for applications ranging from A I D S therapy (Manfredi et al, 1997) to anemia treatment (Gibson et al, 1994) and progenitor mobilization for transplantation (Redei et al, 1997). In human marrow failure syndromes, the application of pharmacological doses of growth factors has been evaluated for supplementing stromal cell function or overcoming receptor abnormalities (Wright et al, 1989). Factors (e.g. IL-2 and IL-3) have been used to stimulate mature T-cells as an adjuvant in immunization schemes (Vondrys et al, 1997). Therapies for the treatment of myelodysplasia with recombinant G M - C S F (produced in yeast) infused continuously for two weeks, showed a marked increase in total leukocyte counts (Dunbar et al, 1991). Other factors including IL-3 and S C F have also been used in clinical trials for myelodysplasia. None of these studies has shown improvements over those found with G M - C S F . Two major applications of cytokines for bone marrow transplant ( B M T ) have been developed. The first has examined the application of G M - C S F and G - C S F to combat graft versus host disease and pancytopenia. Following B M T , mature blood cell numbers are greatly reduced for 2 to 8 weeks (Hogge et al, 1991). Administration of doses up to 16 mg/kg-day resulted in more rapid increases in cell numbers although the duration of the severe pancytopenia was not reduced (Nemunaitis, 1992; Nemunaitis, 1993). The other major application of cytokines in B M T is the mobilization of hematopoietic stem cells into the peripheral blood (Nemunaitis and Rosenfeld, 1993). In clinical trials, G M - C S F has been demonstrated to mobilize significant numbers of stem cells. G M - C S F and other factors, including IL-3 and S C F , have been evaluated in clinical trials for their ability to reduce the toxic effects of 8 chemotherapeutic agents on the hematopoietic system. In general, these studies have demonstrated that the administration of growth factors may reduce the duration and magnitude of pancytopenia. ii) Culturing of factor-dependent cells Tissue engineering In 1992, the total annual cost in the United States of treating patients who had suffered tissue loss or failure exceeded $400 bill ion dollars (Langer and Vacanti, 1993). This represents approximately 8 mil l ion surgical procedures annually. Generally, these procedures involve either organ transplant, reconstructive surgery or the application of mechanical devices. However, each of these approaches has important limitations (Langer and Vacanti, 1993). Perhaps the most significant problem is the critical shortage of suitable donor tissues. For example, only 10% of those patients requiring liver transplant receive a compatible donor tissue. The engineering of new tissues by ex vivo culture of primary cells addresses this critical lack of donors for tissue and organ transplant therapies. Current examples of engineered tissues include skin (Yannas et al, 1982), bone marrow (Eaves and Eaves, 1988) and liver (Davis and Vacanti, 1996). However, outstanding challenges remain, including a) developing approaches to block unwanted host reactions, b) improving organ storage methods prior to transplantation, c) controlling the regeneration and formation of complex multicellular structures, and d) understanding how the organ directs and synchronizes cell function. A major challenge in tissue reconstruction is the development of scaffolding which specifically directs tissue formation: the scaffold must be porous enough to promote close cell association, must be biocompatible, biodegradable and mechanically strong, and has to provide adhesive surfaces to cells (Shinoka et al, 1995). Natural biopolymers such as collagen and 9 laminin have been used to line the wall of tubes used as nerve guidance conduits (Valentini et al., 1992). Artif icial scaffolds have been synthesized using polymers of lactic acid and glycolic acid. Addition of polylysine to the lactic acid polymers has permitted the incorporation of R G D sequences into the scaffolding to promote cell attachment. Extensive work has been performed in modeling the interactions between cell surface receptors and the microenvironment, including research on how distribution of ligand affects cell migration, and how receptor clustering affects shear resistance of cells on culture supports. Current problems in the reproducible manufacture of highly porous polymer structures limit the application of artificial cell matrix development for transplantation (Mikos etal., 1993; Wald etal, 1993). Microenvironment control is critical in establishing and maintaining cell function. Various groups have used collagen sandwiches to culture functional liver tissues (e.g. Rinkes et al, 1994). Promoting the eventual invasion of blood vessels (angiogenesis) to supply blood to the engineered tissue can be accomplished by providing sites of local slow-release of growth factor activity. Essential growth factors may be incorporated into porous polymer matrices and controlled or slow-release accomplished by hindered diffusion of the factor through the polymer matrix (Murray et al, 1983; Langer and Moses, 1991). To accelerate bone healing, bone morphogenic protein ( B M P ) has been incorporated into porous polymer particles (Watrous and Andrews, 1989). Release occurred as the factor-entrapping particles degraded. In this way, a steady local supply of B M P was supplied to enhance bone formation. Another approach to tissue regeneration uses polymer matrices to deliver growth factors to wound sites to stimulate normal tissue repair. Sheardown et al. (1993) developed slow-release polymer matrices for the slow and continual addition of E G F to the site of corneal wounds. This strategy enhanced wound closure and corneal regeneration. 10 Several companies are developing skin prosthesis through the culture of a patient's epidermal cells. However, for significant epidermal loss (e.g. burn victims), up to a 10,000-fold increase in cell number is required for body surface coverage. Skin cell cultures are generally performed using a feeder-layer of irradiated 3T3 fibroblasts. This stromal layer supplies E C M components and undefined factors to support rapid growth of epidermal layers. The current limitation of this therapeutic approach remains the significant cell growth period required to produce large epidermal sheets from small biopsies. Several groups currently use fibroblasts and keratinocytes isolated from human foreskins. A second approach grows artificial skin on mesh-like sheets of lactic acid-glycolic acid copolymers (Hansbrough et al, 1992). The epidermal layer is given mechanical strength and shape by the polymeric support, but the eventual biodegradation of the support matrix permits incorporation of the epidermal graft. Hematopoietic cell culture Applications of hematopoietic cell culture include the generation of cells for transplantation following radiation and chemotherapy (Sutherland et al, 1994), the manipulation of culture conditions for tumor purging (Eaves et al, 1993), and the transfer of genes into hematopoietic progenitor cells for gene transfer therapies (Conneally et al, 1998; Hughes et al, 1992). Other therapies include the transplantation of mature cells, including platelets, dendritic cells and T-lymphocytes (Korbling and Champlin, 1996). The goal of each of these cell-based therapies is to supply the correct cell phenotype and cell number to improve patient health. There are three basic sources of hematopoietic cells: bone marrow, peripheral blood and umbilical cord. Bone marrow is harvested directly from the pelvis or sternum by multiple needle aspiration (Lenarsky, 1995). Progenitor cells can be mobilized from the bone marrow into the blood by the administration of growth factors (e.g. G M - C S F and G-CSF) (Korbling and Champlin, 1996). 11 With this approach, progenitor cells can be harvested without the donor undergoing the rather painful bone marrow harvesting operation. Finally, progenitor cells can be isolated from the umbilical cord of newborns (Broxmeyer et al, 1992). The most significant limitation for the use of cells derived from umbilical cord blood for transplantation, is the low number of progenitors obtained from cord blood samples. Assay Methods In vitro, hematopoietic progenitor cells are defined by functional assays. Various levels of lineage commitment (i.e. the development of specific cell phenotype) have been measured in cell culture systems. One of the most common assays measures the ability of cells to initiate the formation of a variety of mature hematopoietic cell colonies following extended periods of culture (long-term culture initiating cells (LTC-IC) (Petzer et al, 1996). A primitive cell population, the competitive repopulating unit (CRU) , has been identified in mice (Fraser et al, 1992). C R U cells are distinguished by their ability to completely restore hematopoietic function in lethally irradiated mice at limiting dilutions of transplanted cells; the limiting dilution assay has confirmed that complete repopulation is possible following transplantation of a single cell. Dexter-type Cultures Dexter (Dexter et al, 1977) developed an in vitro method for the culture of bone marrow cells using irradiated stromal-cell feeder layers. This technique maintained cell populations which could regenerate hematopoiesis in irradiated mice. The feeder layer cells produced the cytokines, growth factors and E C M components necessary to support L T C - I C maintenance. The maintenance of human L T C - I C (Sutherland et al, 1993) and expansion of the primitive hematopoietic cell population in these cultures demonstrates the feasibility of cultivating primitive hematopoietic progenitor cells (Koller and Palsson, 1993). 12 Emerson (1991) suggested that there are five main areas in which liquid cultures of hematopoietic cells fail to mimic the necessary in vivo environment for efficient cell expansion: (1) failure of quiescent cultured stromal cells to secrete hematopoietic growth factors, (2) lack of a 3-D geometry with stromal cells and E C M at high density, (3) accumulation of toxic metabolites in traditional batch cultures, (4) degradation of the stromal and hematopoietic progenitor cells by mature cells and their products which accumulate in liquid cultures, and (5) depletion of stem cells resulting from frequent sampling of nonadherent cells. Various non-stromal-supported culture systems have been developed in recent years for the culture of hematopoietic cells. Batch and fedbatch cultures have been run in several configurations, including culture in gas-permeable Teflon™ bags (Moore and Hoskins, 1994), T-flasks and stirred suspension cultures (Zandstra et al., 1994). Perfusion Cultures Caldwell et al. (1991) suggested that the clearest difference between the hematopoietic microenvironment ( H M E ) and Dexter culture is that the rate of delivery of plasma proteins and nutrients to the bone marrow and the rate of removal of cellular metabolites are far higher in vivo. Caldwell developed a perfusion schedule for wellplates and flasks to better approximate the in vivo environment. The in vivo perfusion rate is estimated as 0.1 ml/cc-minute (Dexter et al., 1990). Wi th Caldwell's feeding schedule, cell proliferation was directly proportional to the total serum flux through the growth chamber. Also, the growth factor production was dependent on the medium perfusion rate. A t low perfusion rates no G M - C S F was detectable in the media (limit of detection 0.01 ng/ml), but when feeding schedules were changed from 3.5 media changes per week to 7 per week, G M - C S F was detected. 13 Koller et al. (1993) used a novel perfusion bioreactor and the addition of combinations of cytokines to expand human hematopoietic cells from umbilical cord blood. Perfusion cultures stimulated with IL-3, S C F , and IL-6 resulted in significant expansion of primitive progenitor cells. Static cultures gave only G M - C F U (colony forming units). In an earlier report, Koller et al. (1992) showed that reduced oxygen tension increased hematopoiesis in long term culture of human stem cells. Nearly twice as many C F U were detected at eight weeks in cultures maintained at 5% 0 2 (atm) compared to those maintained at 20% 0 2 (atm). The degradation of the stromal layer was also slower at reduced oxygen tensions. Oxygen gradients across the bioreactor were not measured. At the low oxygen concentrations used in that perfusion bioreactor, anaerobic conditions may have existed near the exit. Schwartz et al. (1991) found that medium exchange rates significantly affected total cell production, the cell types produced, and the physical appearance of the cell culture over time. However, after 10-12 weeks, cell production ceased in all cultures regardless of conditions. In their experiment, the media hydraulic retention times were 1, 3.5 and 7 days. The 3.5 day hydraulic retention time was reported to promote maximum cell production. It is difficult to draw conclusions from direct comparisons of their data. The culture medium was changed using a batch exchange step which resulted in the 1 day time sample being completely replaced with fresh medium (i.e. no preconditioning). However, the 3.5 and 7 day hydraulic retention time cultures were exchanged by replacing 50% of the medium with fresh media, thereby maintaining the cells in preconditioned media. A bioreactor system has been reported that uses porous collagen microspheres in a packed bed perfusion reactor (Wang and W u , 1992). The collagen spheres were 500-600 u,m in diameter with a 20-40 | i m pore size. The authors suggest that the use of highly porous collagen 14 microspheres provided a natural extracellular matrix ( E C M ) and large surface area for cell attachment and growth. Murine cells were cultured for up to 120 days in their perfusion reactor. Growth factors in hematopoietic cell culture Engineered Stromal Cells Culturing hematopoietic cells would be simplified i f the stromal feeder layer required in Dexter cultures could be replaced or eliminated. Sutherland et al. (1991) used a genetically engineered fibroblast cell line to replace the stromal layer. Fibroblasts were transfected to produce G - C S F , G M - C S F and IL-3 alone or in combination. Cotransfection of G - C S F and IL-3 into the feeder cells enhanced the maintenance of L T C - I C cultures significantly. Factor concentrations measured in the culture supernatant were below the level of detection of their assay (0.1 ng/ml). Non-transformed cells were also able to support L T C - I C maintenance. It was determined that this effect was due to the secretion of S C F by the fibroblast cell line. Another study which used genetically engineered cells to supply IL-3, showed that IL-3 can enhance multilineage hematopoiesis in the long-term culture of human cells (Otsuka et al, 1991). Furthermore, secreted factors triggered responses that were not obtainable by simple addition of cytokine to the culture supernatant. A single addition of up to 100 ng/ml did not support cell proliferation, whereas 3 doses of 10 ng/ml over 3 consecutive days did. The duration of exposure of the cells to IL-3 may be the critical parameter which determines the magnitude of the growth response. It was not determined whether the increased growth response obtained with the genetically engineered stromal cells was due solely to the high local concentration of IL-3 secreted by the support cells, or to synergy with other factors also produced by the stromal layer. 15 Stroma Free Cultures More recently, Sutherland et al. (1993) have used combinations of cytokines to replace the stromal layer in Dexter cultures. S C F or G - C S F plus IL-3 substituted for feeders in the maintenance of L T C - I C . There was no enhancement of L T C - I C numbers with these factors over cultures with a stromal feeder layer. L u et al. (1993) examined the replating capacity of single sorted C D 3 4 + + + cells. Wi th appropriate cytokine additions (SCF 50 ng/ml, G M - C S F 200 U/ml , G - C S F 200 U / m l , IL-3 200 U /ml , and E P O 2 U/ml) a subset of cells demonstrating a high proliferative potential could be serially replated at least 5 times, demonstrating self-renewal capacity. However, this effect was dependent upon serum addition, indicating that some other essential factor was missing from their cytokine mixture. Lansdorp and Dragowska (1993) cultured adult human bone marrow in L T C using serum-free media supplemented with IL-6, IL-3, S C F and E P O . Cultures were initiated with cell samples highly enriched for the CD34+, CD45RA 1 0 , and CD71 1 0 phenotype. Concentrations of cytokines used were IL-6: 10 ng/ml, IL-3: 20 ng/ml, S C F : 50 ng/ml, and E P O : 2 U / m l . In these cultures they observed that the majority of CD34+ cells did not actively proliferate during the culture, in contrast to the CD34" cells which did. Their results show that in these serum-free cultures, hematopoiesis was maintained from a pool of slowly proliferating or completely non-proliferating progenitors, and that upon activation these cells showed a variable self-renewal capacity as well as a limited and variable ability to generate daughter cells. They concluded that it may therefore be difficult to expand adult bone marrow cells for clinical purposes in bone marrow transplants. 16 Multi-factor Cytokine Analysis Factorial experiment designs have recently been reported in which cytokine combinations and concentrations were varied and L T C - I C and C F U - G M were monitored as response variables (Petzer et al, 1996; Petzer et al, 1997; Zandstra et al, 1997). The/Ztf-ligand, IL-3 and S C F were the only factors required for stimulation of the generation of L T C - I C after 10-day liquid suspension cultures. In addition, only T P O or /Ztf-ligand stimulated L T C - I C expansion when added alone. Dose response analysis further revealed that L T C - I C were maximally expanded at cytokine concentrations 30-fold higher than that required for colony forming cell (CFC) generation. In optimally stimulated cultures, up to 50-fold expansion of L T C - I C was reported. For ex vivo bone marrow stem-cell expansion to be clinically useful, a robust system must be developed in which process outputs (e.g. cell type and numbers) can be predicted and controlled. Koller et al. (1997) examined the variability of C F C output as a function of the type of l iquid cell culture. They demonstrated that C F C output variability was significantly reduced when stromal cells were used to support CD3 4+Lin cells. Interestingly, process variability did not correlate with variability of the expression of a variety of phenotype markers, including C D 3 8 , Thy-1 and c-kit. Ce l l number output variability (± S.E.) was 2.9 logs for soluble cytokine supplemented cultures versus 0.8 logs for stromal-supported cultures. The authors suggest that this difference is due to differences in the mode of factor presentation: stromal-presented versus soluble. iii) Mass transport of growth factors Growth factors and the cellular microenvironment Tissues are composed of cells, E C M and void space. The E C M is generally formed by the secretion of E C M components by fibroblast and endothelial cells (Siczkowski et al, 1992; 17 Mayani et al, 1992). The E C M gives the tissue its structure and provides the microenvironment in which cells develop. Furthermore, the E C M constitutes a barrier to diffusion and convection in tissues, which slows dispersion of factors thereby localizing their effects. The spatial localization of factors presented bound to an insoluble matrix, is thought to be a key feature in tissue morphogenesis (Ingber, 1992; Drubin and Nelson, 1996; Chen etal, 1997). The in vivo hematopoietic microenvironment is made up of a network of cells (fibroblasts, endothelial cells, macrophages and adipocytes) and E C M (collagen, laminin, fibronectin and proteoglycans), which physically support the hematopoietic cells and influence their proliferation and differentiation (Mayani et al, 1992; Verfaillie et al, 1994; Kle in , 1995). The stromal cells and E C M constituents direct the development and differentiation of hematopoietic cells in vivo (Fukushima and Ohkawa, 1995) and in vitro (Spooncer and Dexter, 1984). Stromal cells are the major source of cytokines in the bone marrow (Dexter et al, 1990). Proteoglycans are a major component of the extracellular matrix. There are four major forms: heparin sulfate, chondritin sulfate, keratin sulfate and hyaluronic acid (Ruoslahti and Yamaguchi, 1991). The binding properties of these E C M proteins are primarily determined by the glycosaminoglycan carried on the proteoglycan. Proteoglycans are an abundant and ubiquitous tissue component and are likely to capture a majority of intercellular growth factors for which they have affinity (Ruoslahti and Yamaguchi, 1991). It has been suggested that the combined action of diffusible factors and nondiffusible matrix signals may be an important mechanism for localizing responses of cells to a cytokine that is widely distributed within the organism (Nathan and Sporn, 1991). In their review, Ruoslahti and Yamaguchi (1991) suggest that "growth factors and cytokines were meant to act over a short range only and their immobilization at the cell surface and in the intercellular spaces accomplishes this". 18 The role of E C M in localizing growth factors was first suggested by the absence of measurable levels of G M - C S F in hematopoietic cell cultures supported on stromal cell layers (Gordon et al, 1987). The critical role of G M - C S F in supporting granulopoiesis was well established, yet it was not found in the supernatants of cell cultures. Gordon et al. (1987) demonstrated that compartmentalization to the E C M was caused by G M - C S F association with glycosaminoglycans. This has since been observed with other growth factors, including F G F (Klagsbrun, 1990) and IL-3 (Roberts et al, 1988). These factors associate in the E C M with proteoglycans in an active form. Indeed, basic F G F has a higher affinity for its receptor when F G F is bound to glycosaminoglycans (Yayon and Klagsbrun, 1990). Binding to the E C M protects growth factors from proteolytic degradation, and serves as an important reservoir of factors (Brunner et al, 1991). This reservoir can then function as a rapid signaling element when factors are released by E C M degrading enzymes (Miyazawa et al, 1996). Association of growth factors with previously laid down E C M can form gradients to direct cell migration (Hecht, 1992), or serve as tissue-type cues to effector cells. E G F and F G F are potent inducers of metalloproteinases, which alter matrix structure. The central role of the E C M is clear when the range of interactions between E C M and cells are considered: (1) Adherence of cells to E C M induces cells to secrete cytokines. For example, adherence to fibronectin induces secretion of G M - C S F (Thorens et al, 1987) and CSF-1 (Eierman et al, 1989), and adherence to collagen induces synthesis of IL-1 (Dayer et al, 1986) and T N F - a (Eierman et al, 1989). (2) Cytokines affect the expression of cell adhesion receptors. For example, TGF-p and IL-1 increase the expression of integrin receptors in a variety of cells (Enenstein et al, 1992; Hertle et al, 1995). Indeed, some cells w i l l respond to E C M binding in a manner indistinguishable from cytokine-induced signaling (Ding et al, 1990). Adhesion of hematopoietic progenitor cells to stroma inhibits proliferation (Verfaillie and Catanzaro, 1996). Furthermore, spatial regulation of growth factor availability enables the development of cell polarity, when receptors are captured at factor-presenting interfaces ( M c K a y et al, 1991). (3) Adhesion of cells to E C M influences the response of cells to factors. For example, adherence of cells to fibronectin and collagen determines whether cells proliferate or differentiate in response to F G F (Ingber and Folkman, 1989). Also , neutrophils do not respond to TGF-oc when cells are not adherent to E C M through a P2 receptor (Nathan and Sanchez, 1990). The direct role of E C M in receptor activation has been demonstrated with F G F (Miao et al, 1996). Stimulation of acidic F G F (aFGF) responsive cells requires the presence of heparin sulfate. A s discussed above, many growth factor receptors are activated upon aggregation following ligand binding. Wi th aFGF, dimerization of the receptor is driven by aFGF which is bound to the E C M . Thus, the E C M acts as a bridge to bring the receptor domains into closer proximity for trans-activation. One important corollary is the potential for aFGF agonist activity of monomeric E C M components. A n aFGF molecule binding to a monomeric E C M analog would be ineffective at stimulating receptor oligerimization. Thus, metalloproteases which degrade E C M may also function to down-regulate aFGF activity by sequestering F G F into an inactive form. 20 Reaction and diffusion of growth factors In vitro studies have shown that E C M proteins play a dynamic role in the regulation of hematopoiesis, by indirectly regulating and localizing growth factors secreted into the microenvironment (Zuckerman, 1989). To date, no comprehensive analysis of the complex interactions between the E C M , growth factors, and growth factor cell-surface receptors has been published. Recently however, it was suggested that the combined actions of reaction and diffusion in bone marrow (BM) gives rise to a fractal structure of the B M . Naeim et al. (1996) examined 148 microscope fields from bone marrow biopsies. The mean percent cellularity was 56 ± 10%. The rest of the microscopic field was either E C M or empty space. The authors estimated the fractal dimension of the observed cell distribution to be 1.67 ± 0.09. They point out that this fractal dimension is consistent with diffusion-limited aggregation processes. In such processes, one starts with a seed particle (cell) at the center of some defined region. Diffusing species (e.g. cytokines) are released in the vicinity of the open region and diffuse until they contact the seed. If this process is repeated over and over a fractal structure wi l l develop with a fractal dimension between 1.6 and 1.7. The apparent diffusion-limited aggregation morphology of B M suggests that the development of B M may indeed be dependent upon cytokine diffusion. It is interesting to note that only normal B M had a fractal structure. B M samples derived from leukemic B M s did not have a fractal structure, suggesting that diffusion processes had become insignificant due to the development of cytokine-free cell division. When considering the extent to which diffusive processes govern cell behavior, one can define a dimensionless group as a measure of the significance of diffusion rates relative to reaction rates. The Damkoehler number (Da) is the ratio of the reaction and diffusion rate constants. If Da is very much greater than unity (Da » 1), the system is diffusion rate limited. 21 Under these conditions, the reaction terms in the governing differential equations may be neglected and the diffusion rate constant determined. On the other hand, i f Da is much less than unity (Da « 1), the system is reaction rate limited and the reaction rate parameters and ligand site density may be determined from the recovery data. The estimation of the Da in cellular systems is complicated by the nonhomogeneous distribution of cytokines and receptors. The true Da is only estimable for soluble ligand and receptors in a well-mixed finite volume. In physiologically relevant systems, the receptor is typically displayed on the surface of a target cell. Thus, the reaction rate estimated by observing the depletion of cytokines from solution, is only an apparent rate by which the cytokine must not only diffuse to the surface of the cell but also diffuse at the surface of the cell, in order to interact with a cell membrane-presented factor. Consumption rates for S C F have recently been reported for hematopoietic cells. Zandstra (1997) observed a biphasic consumption profile following addition of rhSCF to unstimulated M 0 7 e cells. The first phase was governed by an initial rapid association of S C F with cell surface receptors. This consumption rate was a function of initial S C F concentration, and ranged from 25 fg/cell-day with an initial dose of 1 ng/ml SCF, to 280 fg/cell-day at 100 ng/ml S C F . The initial phase was followed by a second slower consumption phase, in which depletion of S C F was assumed to be governed by receptor turnover rates and cell growth. Consumption rates were also dose dependent in the second phase, and ranged from 2 to .7 fg/cell-day over approximately the same concentration range as above (i.e. little S C F was depleted from the media during the initial phase). For this simple model we can write the apparent Da as 22 where qscF is the cell specific S C F consumption rate, D is the factor diffusion rate and C b is the bulk concentration of the factor. Cel l stimulation wi l l be diffusion rate limited when D a » 1. Based on the consumption rates estimated by Zandstra (1997), cell stimulation w i l l not be diffusion rate limited unless the bulk S C F concentration is significantly less than 1.0 ng/ml. Thus, in the simple case in which the cell is assumed to be an adsorbing sphere, the uptake of S C F is far from diffusion rate limited at soluble factor concentrations typically employed in cell cultures. Clearly, this analysis does not consider the details of encounter-limited receptor binding at the cell membrane. A general theory describing mass transfer of proteins diffusing and subsequently binding to discrete cell surface receptors, was developed by Berg and Purcell (1978). Their approach was to model the cell surface as a disk covered with discrete binding sites. The binding process was separated into three distinct stages: (1) diffusion of factors from the bulk media to the disk proximal region, (2) lateral diffusion in the plane of the disk between cell-surface receptors, and (3) binding of diffusing species to unoccupied receptor sites. The Berg-Purcell formulation predicts a 1.5-fold higher effective factor concentration (i.e. with respect to the number of factor-receptor interactions at a given factor concentration) for a flat plate over a sphere. This is because a flat plate appears smaller than a sphere to a diffusing ligand, due to the curvature of the sphere. The Berg-Purcell theory states that, in the vicinity of the receptor, diffusion is 2-dimensional (i.e. between receptors) rather than 3-dimensional (i.e. from the bulk solution to the surface). Brownian motion is the trajectory described by a particle (e.g. protein) as a result of collisions between itself and surrounding solvent molecules. Einstein (1905) and Smoluchowski (1906) solved the equation of motion of a particle undergoing Brownian motion. In their formulation, 23 the deterministic Newton's equation for motion of a particle in a viscous solution was replaced by a stochastic equation, in which the forcing and viscous terms are the result of ensemble-averaged random forcing terms arising from collisions with the solvent molecules. The trajectory is assumed to be a Markovian process (infinitesimally short memory, uncorrelated with direction or velocity) in which the forcing terms have zero mean and variance and are proportional to the absolute temperature of the system. Brownian dynamics (BD) models have been used to simulate the ligand cell-receptor system, in which the cell was modeled as a sphere covered with discrete disks representing receptors (Northrop 1988). That analysis agreed with the theoretical predictions obtained in the Berg-Purcell analysis. The binding rate estimate for a single receptor decreases rapidly as the number of receptors on the cell surface increase, due to competition between receptors for ligand. Significantly, the binding rates are half maximal when only 5% of the cell surface is covered with receptor disks. Northrup (1991) suggested that B D simulation methods may be employed in systems which, although perhaps conceptually simple, are analytically intractable because of (multiple) reactive boundary conditions. For example, the effects of coulombic interaction on binding rates were studied by Northrop and co-workers (1984). In such cases, the apparent reaction rates are significantly increased when attractive forces exist between the ligand and receptor. The effect of geometry on the binding process may also be studied in greater detail with the B D approach than is possible using analytical solutions to the governing reaction/diffusion equations. If, for example, binding interactions depend upon the angle of approach between the ligand and receptor, this effect could be included in the simulation. 24 The regulation of cell growth by autocrine factors and the effects of innoculum cell density were examined using a B D model (Charnick, 1990). Lauffenburger's analysis assumed a dynamic equilibrium between the production of growth factor, its binding to cell surface receptors, and the diffusion of the factor from the cell surface into the bulk medium. Because of the low concentrations of factors produced by cells, mass transport limitations were assumed to prevent cells from being stimulated by factors once they exit the cell proximal microenvironment. For the diffusion values investigated by the researchers, only a small fraction of the growth factor produced by a cell was captured by its own receptors (autocrine stimulation), while a significant fraction of the ligand bound to neighboring cells (paracrine stimulation) before being lost into the bulk medium. The authors suggest that this is one of the major reasons for minimum innoculum cell density requirements for the initiation of tissue cultures. Their analysis also indicates that the effects of bulk mixing on factor transport to the cell surface is negligible (< 10%) under the mild mixing conditions typical of mammalian bioreactor systems. A more realistic model of the cell considers a cell in suspension as a reflecting sphere covered with a number of discrete, ligand-adsorbing receptors (Berg, 1993). The adsorbing sites are assumed to be very small relative to the size of the cell (clearly the case for the c-kit receptor). The cytokine flux through the sphere (i.e. S C F consumption) depends upon the number of adsorbing patches on its surface. With a low number of sites, the overall flux ( / ) approaches that of the sum of fluxes for individual adsorbing patches, given by: I=4DsCb (1-2) where s is the radius of the adsorbing patch, D the diffusion coefficient and Cb the bulk factor concentration. A s the number of patches becomes large, the adsorptive flux approaches that of 25 an adsorbing sphere (Equation 1-2). The diffusive flux to a cell covered with an arbitrary number of receptors (N) is also given by Berg (1993): / = - (1-3) where I s is the flux to a completely adsorbing sphere of radius a. For example, a cell with 2.5 x 10 4 randomly distributed receptors, a radius of 8 um, and an effective binding surface radius of 1 nm, w i l l have 50% of the flux to a fully adsorbing sphere. Zandstra (1997) reported a S C F dose-dependent depletion of c-kit following stimulation: unstimulated cells presented approximately 4 x 10 5 receptors per cell, while maximally stimulated cells presented about 5 x 10 4 receptors per cell following receptor internalization after the addition of S C F . Thus, it is likely that at the receptor densities reported for the M 0 7 e (Turner et al, 1995) or B6SutA (Liu et al., 1994) cell lines, the uptake of soluble S C F is reaction rate limited. iv) Growth factor immobilization for cell culture The application of immobilized factors in tissue culture has been examined only recently in the literature. Applications of immobilized factors range from the development of controlled drug-release technologies (Langer, 1996; Sanchez et al, 1996) to the construction of tissue regeneration systems (Hubbell et al, 1991; Edgington, 1994). Processes which use immobilized factors have the advantage of maximizing cell-proximal concentrations of exogenous factors to provide cell stimulation, while maintaining relatively low concentrations of these factors in the bulk media. This partitioning of factors to cell-proximal regions of the artificial E C M , mimics the in vivo situation by creating an artificial juxtacrine stimulation configuration (Mayani et al, 1992). 26 Roberts et al. (1988) used Matrigel™ (Becton Dickinson, M A ) , a commercially available artificial E C M , to bind IL-3 and G M - C S F . Factor-dependent cell lines for IL-3 and G M - C S F were then used to track the activity of bound cytokine. Binding studies revealed that all IL-3 and G M - C S F remained within the Matrigel™. Most of the binding was to heparin sulfate, since all of the cytokine activity could be removed from the Matrigel by the addition of heparinase. The importance of heparin was confirmed by the complete functional replacement of Matrigel with heparin sulfate immobilized on cell culture dishes. Both native and recombinant (nonglycosylated) growth factors bound in an active form on the heparin sulfate matrix. The immobilization of F G F on various E C M components has been studied in vitro for application in wound healing therapy (Roy et al, 1993). The authors reported a significant increase in cell growth when the growth factor was incorporated within fibrin. The fibrin substrate, which contained fibronectin, was shown to be a good support for fibroblast spreading and presentation of F G F to cells. The incorporation of hyaluronic acid into the f ibrin/FGF matrix further increased cell proliferation. It was presumed that the hyaluronidase secreted by the fibroblasts was able to release immobilized growth factor through local biodegradation of the hyaluronic acid component of the E C M . Edelman et al. (1991) bound b F G F to heparin sulfate covalently coupled to a sepharose carrier. Heparin was shown to stabilize the b F G F and bind strongly ( K d ~10" 9 M" 1 ) when covalently coupled to sepharose beads. The sepharose bead was embedded within a calcium alginate microsphere to slow loss of factor from the bead. The resulting release kinetics were biphasic: release of b F G F from the heparin, followed by subsequent diffusion of the factor through the gel matrix. 27 Previous efforts to immobilize cytokines or growth factors include covalent attachment either directly to surfaces or through linking tethers (Cuatrecasas, 1969; Horwitz et al, 1993; Ito et al, 1996; K u h l and Griffith-Cima, 1996). Ito et al. (1997) investigated the use of immobilized transferrin and insulin in anchorage-dependent and independent cell cultures. These researchers showed that immobilized factors stimulated the proliferation of anchorage-dependent cell lines. They suggested that enhanced growth was due to a cross-linking of receptors by the immobilized factors, and an inhibition of down-regulation of stimulatory signals by inhibiting the endocytosis of bound receptors. N o effect was seen for anchorage-independent cells, suggesting that adhesion of cells to the immobilized-factor surface was required for signal transduction. Covalent immobilization of the insulin and transferrin to a poly-(methyl methacrylate) f i lm was performed with glutaraldehyde (Liu et al, 1992). The authors did not distinguish between total immobilized protein and actual units of immobilized active factor. In their discussion they suggest (but do not prove) that lower concentrations of insulin or transferrin may be used i f they are maintained in cell-proximal regions of the bioreactor. In another study which investigated the advantages of immobilized insulin, it was shown that the co-immobilization of insulin with cell adhesion proteins (i.e. fibronectin or collagen) accelerated the growth of anchorage-dependent cells cultured in serum-free medium (Ito et al, 1991). In the Ito group's most recent report on immobilized insulin (Ito et al, 1997), insulin bound to thermo-responsive polymers yielded an order of magnitude decrease in the observed ED50 (effective dose yielding 50% maximum effect). Covalently immobilized epidermal growth factor (EGF) generated a higher mitogenic response than soluble E G F (Chen et al, 1997). E G F was derivatised with N-p-4-azidobenzoyloxy) succinimide and was then photo-immobilized onto polystyrene tissue culture 28 plates with U V irradiation. Wi th immobilized E G F , up to a two-fold increase in the apparent growth rate was reported compared to non-immobilized derivatized E G F . However, soluble E G F induced a higher growth rate then either immobilized or soluble derivitized E G F . Interestingly, the apparent E D 5 0 was unchanged by immobilization in either comparison. This indicates that immobilization increased the cell's capacity to respond to the E G F , while not increasing the cell 's E G F sensitivity (or conversely the E G F potency). Immunofluorescent imaging with anti-phosphorylated-tyrosine antibody was used to demonstrate activation of cells on EGF-immobi l ized surfaces. Chen et al. suggest that the increased cell response to the immobilized factor may have been due to an inhibition of receptor migration in the cell membrane following complexation with the E G F ligand. They suggest that this may reduce the rate of receptor deactivation by phosphatases. Another approach to immobilizing E G F was developed by Kuhl and Griffith (1996). Their approach employed extended poly-(ethylene oxide) (PEO) spacers between amino-silane modified glass and the N-terminus of E G F . This method was developed with the intention of presenting factors with enhanced lateral mobility at the surface. Tethered E G F had a similar mitogenic effect on primary rat hepatocytes as soluble E G F , as measured by D N A synthesis rates. Physically adsorbed E G F did not stimulate cells when loaded at comparable densities onto the tissue culture plate. Lopina et al. (1996) used a similar technique to immobilize galactose to mimic high affinity branched oligosaccharides at the interface. Cellular interaction with the surface was only stimulated by galactose spaced from the surface with the P E O tether. No stimulation was observed with directly coupled sugars. The chemical immobilization of IL-2 to cell culture supports has been studied by several groups (Crum and Kaplan, 1991; Kaplan, 1991; Horwitz et al, 1993). The motivation for these 29 studies was to uncouple receptor ligand binding from subsequent receptor aggregation and internalization events, to better understand IL-2 mediated cell activation. Horowitz used plasma-activated polystyrene membranes to immobilize IL-2 through covalent binding with primary amino groups on the DL-2. IL-2 has several lysine residues distributed throughout the molecule. Thus, it is l ikely that the technique employed by Horowitz produced a surface with IL-2 bound in several conformations depending upon the lysine involved in the linkage. The covalent immobilization procedure of Horowitz released only 0.25% of the bound IL-2 protein per day. This amount of soluble IL-2 was shown to be insufficient to stimulate cell proliferation. Significantly, when cells were cultured on membrane-bound IL-2 they did not proliferate. However, approximately 15% of the cells were able to proliferate in response to added soluble IL-2 after 36 hours culture on the prepared IL-2 membranes. Although the maintenance of cell viability in their system was quite low, the authors concluded that the immobilization of the IL-2 decoupled the proliferative and cell viability maintenance responses. Furthermore, they concluded that immobilized IL-2 was not internalized by cells, although cell-associated IL-2 was not measured. Perhaps the most significant problem with their covalent immobilization approach was the inability to monitor the loss of IL-2 bioactivity during the immobilization and hence to estimate the actual surface density of bioactive IL-2. It has been observed that receptor tyrosine kinases may activate similar pathways yet elicit different responses in the cell. Although N G F and E G F activate similar pathways, E G F signals proliferation while N G F stimulates differentiation (Traverse et al, 1992). Apparently, the response of PC-12 cells is determined by the duration of a specific signal transduction intermediate ( E R K ) activation in the ras signaling pathway. Wi th E G F , E R K is only transiently activated, while with N G F , activation is prolonged. The researchers suggested that the prolonged 30 activation of E R K permitted time for its translocation to the nuclear compartment, where it interacts with nuclear transcription factors. Growth factor presentation methods which cause a change in the duration of receptor activation, might therefore induce alternative differentiation decisions by modulating the intensity or duration of the activation of the signaling cascade. 31 Research Approach i) Molecular engineering of growth factors Molecular engineering of growth factors and growth factor mimics has produced several novel approaches to enhancing growth factor activity and availability in vivo. The observation of synergy between IL-3 and G M - C S F , and the identification of a common receptor subunit, suggested that a fusion of IL-3 and G M - C S F (PLXY-321) might have increased efficacy (Vadhan-Raj, 1994). This hybrid provided a significantly enhanced biologic effect (10-fold greater proliferation) via multiple cross-linking of G M - C S F and IL-3 receptors in cell culture. However, Immunex (Seattle, U S A ) has stopped development of PLXY-321 after it failed to show clinical benefits in Phase-HI clinical trials. A second approach to growth factor induced signaling enhancement, has been to re-engineer growth factors to modulate the affinity of the ligand receptor interaction. Reddy et al. (1994 and 1996) demonstrated that by reducing the affinity of E G F for its receptor, an increase in the molar specific activity of the factor can be achieved. The enhancement was attributed to a decrease in the rate of factor clearance, due to destabilization of E G F binding to the receptor. Mutagenesis of IL-3 has produced a variant which was selected for its stem cell stimulating capacity and decreased inflammatory response (G.D. Searle Company: Skokie, U S A ) . These mutant deletion derivatives of human IL-3 were produced as a fusion to the filamentous phage surface protein pIU for cloning and screening. Phage display and site-directed mutagenesis have been used to generate human EL-6 variants with strongly enhanced receptor binding activity and bioactivity (Toniatti et al, 1996). Homology modeling and site-directed mutagenesis of hIL-6, suggested that the binding interface for the receptor consisted of the C-terminal portion of the D-helix and residues contained in the 32 A B loop. For screening and isolation of mutation libraries, mutants were displayed on filamentous phage surfaces and sorted separately for binding to immobilized receptor. Mutants were found which, when expressed as soluble proteins, showed a 10- to 40-fold improvement in receptor binding; a further increase (of up to 70-fold) was achieved by combining variants isolated from different libraries. Phage display is also being used to identify small-peptide mimics of growth factors (Wrighton, 1996). One of the most recent examples of molecular engineering of growth factors, involves the fusion of IL-6 with its soluble receptor (Fischer, 1997). The soluble form of the DL-6 receptor functions to allow cells which do not express the IL-6 receptor to respond to IL-6 stimulation (i.e. the soluble form of the receptor complexes with the cytokine which is then able to bind to a shared receptor signalling subunit (gpl30) located in the target cell membrane) (Gabay et al, 1995). Fischer used a flexible linker domain to genetically fuse the IL-6 receptor to IL-6. The fusion protein was one to two orders of magnitude more active than IL-6 in stimulating cell proliferation. Significantly, the fusion protein preferentially stimulated the proliferation of more primitive progenitor cells in S C F and IL-3 supplemented cell cultures. ii) Biomaterials for cell culture applications There is considerable interest in the development of novel biomaterials for cell growth, differentiation and phenotype maintenance in in vitro (Ertel et al, 1994) and in vivo (Sundaram et al, 1991) systems. Work has focussed on the development of novel polymers and the characterization of protein adsorption (Haynes and Norde, 1995) and cell adhesion (Healy et al, 1994) as a function of surface properties. Generally, investigations consider the charge, hydrophobicity and surface reactive groups presented by the surface. It has been suggested by several authors that characterization studies should consider the three main interactions occurring 33 at the biomaterial surface: the role of the substrate and surface chemistry, the role of adsorbed macromolecules, and the role of cellular response to both. Ideally, engineered surfaces should present an interface which enhances the attachment and spreading of certain cells, while minimizing (passivating) the surface to other cell types and proteins (Elam and Elam, 1993). Thus biocompatibility may be considered as a measure of the balance between enhancing desired reactions while minimizing undesired reactions. Whi le cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human hematopoietic cells in culture has only recently been addressed (Laluppa et al, 1997). Laluppa screened a variety of commercially available polymers, metals and glass substrates, for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood C D 3 4 + cells and mononuclear cells were evaluated for expansion of total cells and C F U of granulocytes and monocytes. Significantly, human hematopoietic cultures were found to be sensitive to the substrate material. Materials that supported expansion at or near levels achieved with tissue culture polystyrene, included Teflon™ perfluoroalkoxy, Teflon™ fluorinated ethylene propylene, cellulose acetate, titanium, polycarbonate and polymethylpentene. The detrimental effects of a number of the materials tested on hematopoietic cultures, were caused by either protein adsorption or leaching of toxins. Factors such as cleaning, sterilization and re-use, also significantly affected the performance of some materials as culture substrates. The results demonstrated that many materials approved for blood contact or considered biocompatible, are not suitable for use with hematopoietic cells cultured in serum-free medium. 34 Cellulose as a cell culture material In general, desirable characteristics of biomaterials for cell culture include stability, so that the matrix does not degrade during culture; inertness, so that the matrix does not present or release cytotoxic compounds; and hydrophilicity, so that cells are able to make suitable contacts with the surface. Cellulose is a biocompatible material (Knospe et al, 1989; Mandolfo et al, 1997), and is already used in several types of bioreactors (e.g. hollow fiber reactors) (Liu et al, 1991; Nordon et al, 1996; Qiang et al, 1997). Regenerated cellulose is currently utilized in several extracorporeal devices used in kidney dialysis (Basile and Drueke, 1989; Mujais et al, 1995). The low nonspecific protein adsorption of cellulose and the ability to specifically localize growth factors through C B D fusion protein technology, afford the significant advantage of cell-type adhesion selectivity (i.e. adhesion ligands for a specific cell type may be bound to the surface, thus allowing cell retention selectivity). Cellulose is a naturally occurring polymer of linked 1,4 [3-D-glucose residues. It is the major structural component of higher plants and is therefore the most abundant natural polymer. The structure of crystalline cellulose derived from the cell wall of Valonia ventricosa, has been solved by x-ray diffraction (Gardner and Blackwell , 1971; Blackwell and Kolpak, 1975). The unit cell is a cellobiose unit. Glucose chains are oriented in a parallel fashion with extensive hydrogen bonding between sheets (separated by ~ 0.78 nm) and between parallel chains (separated by ~ 0.82 nm). In V. ventricosa cellulose, individual cellulose chains are packaged into crystals having nearly square cross-sections, with an average side length of 18 nm (Revol, 1986). Crystalline cellulose is generally referred to as type-I cellulose. This form can be converted to type-II cellulose by treatment in concentrated hydroxide (mercerization). Type-II cellulose is thought to exist in anti-parallel sheets with a much lower degree of crystallinity. It is 35 believed that the extensive hydrogen bonding network of crystalline cellulose is disrupted during mercerization, which allows the cellulose chains to re-form into the less organized type-U isoform. Cellulose is a hydrophilic polymer which wi l l associate with (near) monolayers of water, which are resistant to evaporation and freezing (Fielden et al, 1988). The cellulose used for cell culture should be highly crystalline, with a relatively uniform particle size and minimum intraparticulate pores and interparticulate voids. B M C C produced by the bacterium Acetobacter xylinum has these desired properties (Gilkes et al, 1992). The cellulose vesicle produced by the alga V. ventricosa is also highly crystalline; it can be attached to glass surfaces to give a thin, uniform cellulose surface to which C B D s can be adsorbed. Avice l , while only ~ 50% crystalline, can be used when cellulose particles ca. 100 microns in diameter are required. This cellulose wi l l be useful when spatial control of C B D - S C F availability is required for cell attraction and adhesion experiments. Each of these cellulose forms provides a unique geometry for the presentation of C B D - S C F : microfibril, planar and particulate respectively. iii) Cellulose binding domain technology Origin, structure and function of cellulose binding domains Cellulases, amylases, chitinases and other enzymes involved in the hydrolysis of insoluble polysaccharides, are typically modular enzymes with a distinct catalytic domain joined to one or more accessory domains (Tomme et al, 1995). Cellulases are classified as either exo, acting at the ends of the glucose polymer (exoglucanases) or endo, acting on intermediate regions within the glucose polymer (endoglucanases). Recently, glycosyl hydrolases have been classified into a set of 45 distinct families, based on sequence homology and hydrophobic core sequence analysis (Henrissat et al, 1989). Enzymes either cause inversion (single displacement reaction) or 36 retention (double displacement reaction) upon hydrolysis of the glycositic linkage. The endo versus exo activity of these enzymes has recently been attributed to the configuration of the catalytic site. Structural analysis of endo-acting enzymes generally shows that this class of enzyme has an open catalytic site, characterized by a groove which acts to orient the glycosidic linkage for hydrolysis (Meinke et al, 1995). In contrast, exo-acting enzymes generally have a tunnel-shaped active site which is thought to restrict the access of polymer chains to the active site. The catalytic domain of many glycosyl hydrolases is generally linked to substrate binding domains through one or more linker domains. Most linker domains are rich in proline, hydroxyamino acids and alanine or glycine residues (Gilkes et al, 1991). Repeated proline-hydroxyamino acid motifs are thought to impart structural stability, while maintaining the flexibility necessary for efficient access of catalytic domains to glycosidic bonds. Linker domains are often glycosylated, which likely enhances stability by preventing proteolysis (Langsford et al, 1987). Structure prediction of linking domains indicates that these sequences adopt an extended alpha-helical morphology, which suggests that these domains provide spacing between catalytic and substrate binding domains. Cellulose-binding domains (CBDs) are found in most fungal and bacterial cellulases. They can be classified into 15 families on the basis of amino acid sequence similarities (Tomme et al, 1995). Family II is the largest. The C B D s of seven cellulases and xylanases including CenA (an endo-(3-l-4-glucanase) and Cex (a mixed exo-p-l-4-glucanase/xylanase) from the bacterium Cellulomonas fimi, belong to this family. The substrate-binding domains appear to assist in the hydrolysis of insoluble substrates, because lower activities are generally observed following their removal by proteolysis or genetic manipulation. Proteolytic removal of C B D s from catalytic 37 domains generally results in a significantly lower enzyme activity on crystalline and semi-crystalline substrates (Gilkes et al, 1988). C B D s disrupt the structure of ramie fibers (Din et al, 1991) and release small particles of cellulose from ramie and cotton fibers. These effects appear to be restricted to the disruption of relatively weak interactions between cellulose microfibrils, and do not involve the disruption of the microfibrils themselves. Indeed, there is no evidence that C B D s disrupt the crystalline structure of cellulose. C B D s range in size from 33-36 amino acids (type I) to about 100 residues (type II) and to 130-170 residues (type IH). Most C B D s have conserved aromatic residues which are thought to participate in the CBD-substrate binding interaction (Din et al, 1994; Tomme et al, 1995). Furthermore, type II C B D s have two highly conserved cystine residues which form disulfide bridges to stabilize the 3-D structure of the C B D . The role of C B D s in directing substrate specificity has been investigated (Gilkes et al, 1984; Tomme et al, 1996). In general, C B D s w i l l bind to most forms of cellulose. However, the availability of highly crystalline cellulose (e.g. bacterial microcrystalline cellulose ( B M C C ) or cellulose derived from the cell walls of V. ventricosa or purely amorphous cellulose, e.g. phosphoric acid swollen cellulose), has revealed that some C B D s have binding specificity which may play a role in targeting specific cellulases to their preferred substrate ( U B C Cellulase Group, personal communication). Biophysical characteristics of Type-II CBDs The 3-D structure of C B D C e x has recently been solved by N M R (Xu et al, 1995). The molecule has a compact p-barrel motif with no oc-helix content (Figure 1-1). These findings and others (Bray et al, 1996) implicate three tryptophan residues (W54, W72 and W17) exposed on a planar face of the molecule, in binding interactions with cellulose. This putative binding face presents to the cellulose surface a cluster of hydrophobic residues flanked by hydrogen bond 38 donors and acceptors. This motif is preserved across the C B D type-II family. Using titration microcalorimetry, we recently demonstrated that dehydration effects dominate the driving force for binding of C B D C e x to crystalline cellulose (Creagh et al, 1996). We proposed that a C B D -cellulose complex is formed when a number of the hydrophobic residues along the C B D binding face make sufficient contact to dehydrate both the binding face and the underlying sorbent. Dehydration of the interface facilitates the formation of hydrogen-bonding pairs between the protein and the cellulose surface. Each hydrogen bond interaction has a modest affinity and dissociation rate, but the sum of interactions results in irreversible association. Figure 1-1: The 3-D structure of CBD C e x The 3-D structure has been solved by N M R . The three tryptophan residues shown exposed on the bottom planar face have been implicated in C B D binding to cellulose. 39 The mechanism of C B D adsorption to cellulose is still under investigation. The conservation of aromatic residues in most C B D families, has led to the suggestion that these residues are critical in directing C B D association with substrate. Indeed, mutagenesis studies, in which aromatic residues are replaced with alanines, reveal that significantly lower association affinities result as aromatics are replaced (Linder et al, 1995; U B C Cellulase Group, personal communication). The conditions required to desorb C B D s from cellulose vary considerably. In some cases, C B D s must be denatured with agents such as guanidinium hydrochloride, urea or triethylamine while other C B D s , or C B D s bound to less crystalline substrates, can be desorbed with double-distilled water (Greenwood et al, 1992; Ong et al, 1995). CBDs for localization of fusion proteins to cellulose C B D s from cellulases make effective affinity tags for the purification and immobilization of proteins (Greenwood et al, 1992) and the immobilization of cell attachment factors (Wierzba et al, 1995) to cellulose. In general, cellulose has low protein binding affinity (Ong et al, 1991), while C B D s bind to cellulose with a high apparent affinity (Gilkes et al, 1992). A C B D can be linked to the N or C terminus of a fusion partner, and these chimera proteins retain the properties of both fusion domains (Greenwood et al, 1992; Tomme et al, 1996). These properties suggest that a genetic fusion of a C B D and a growth factor might produce a chimera protein to facilitate the immobilization of a factor to a cellulose extracellular matrix. The application of C B D s for protein immobilization was first demonstrated by constructing fusions between enzymes: C -terminal fusion between C B D c e x and [3-glucosidase from Agrobacteriwn (Ong et al, 1991) and an N-terminal fusion between CBDcenA and alkaline phosphatase (Greenwood et al, 1989). In both cases, the fusion protein bound tightly to various forms of cellulose, while retaining enzyme 40 activity. Furthermore, the enzymes bound effectively irreversibly, as demonstrated by the long-term application of these fusion proteins in immobilized enzyme columns (Ong et al, 1991). iv) System configuration There are several potential rate-controlling steps in cell stimulation by cellulose-adsorbed CBD-cytokine fusion proteins. The pathways for cell stimulation by cellulose-adsorbed fusion proteins are illustrated in Figure 1-2. If we consider an immobile target cell, CBD-cytokine fusions can interact with cytokine receptors only by diffusion of molecules to the target cell. In general, diffusion can occur either through protein desorption followed by solution diffusion of the CBD-cytokine to target-cell receptors, or potentially through direct surface diffusion of the cellulose-adsorbed CBD-cytokine fusion. Indeed, i f the CBD-cytokine fusion is immobile while adsorbed to the cellulose, cell stimulation may be limited by the desorption rate (koff) of the C B D . In addition, the analysis of Northrop (1988) suggests that desorbed fusion proteins wi l l eventually be lost to the bulk solution. 41 Bu lk Solution surface FIGURE 1-2: Potential rate-limiting processes in cell stimulation by cellulose-adsorbed CBD-cytokines If we consider an immobile target cell, CBD-cytokine fusion proteins can interact with cytokine receptors only by diffusion of molecules to the target cell. In general, diffusion can occur either through protein desorption followed by diffusion in solution ( D s o i u t i o n ) of the C B D -cytokine to target-cell receptors, or potentially through direct surface diffusion (D s u r f a c e ) of the cellulose-adsorbed CBD-cytokine fusion. If the CBD-cytokine fusion is immobile when adsorbed to the cellulose ( D s u r f a c e = 0), cell stimulation may be limited by the desorption rate (kof f )of theCBD. In this thesis, we w i l l characterize the interaction of CBD-cytokine fusion proteins with cellulose and target cells. Initially, we wi l l consider the interaction of isolated C B D s with crystalline cellulose. Measurements w i l l be performed of the CBD-cellulose affinity constant, the kinetics of cellulose binding, and the cellulose-adsorbed C B D surface-diffusion coefficient. These measurements wi l l permit the identification of potential rate-controlling processes in the contacting of cellulose-adsorbed fusion proteins and target cells distributed across the cellulose 42 matrix. In addition, confocal imaging of fluorescently-tagged C B D s bound to crystalline cellulose w i l l be used to determine the distribution of C B D s on cellulose and image various celluloses which w i l l be employed in cell culture experiments. Following characterization of isolated C B D s , CBD-cytokine fusion proteins w i l l be examined. Three different fusion proteins wi l l be tested to evaluate the feasibility of C B D -mediated growth factor immobilization: CBD C e n A - inter leukin-3 (CBD-IL3) , C B D C e n A -interleukin-2 (CBD-IL2) , and in particular C B D C e x - s t e m cell factor ( C B D - S C F ) . Experiments w i l l be performed to identify potential rate-controlling steps for cell stimulation and the cellulose binding properties unique to each CBD-cytokine fusion protein. It is anticipated that the motility of target cells may also significantly impact on the rate of cell stimulation. Therefore, live cell imaging w i l l be performed to evaluate potential chemo-sensory responses of factor-dependent cells to cellulose-localized factors. Finally, confocal fluorescence microscopy w i l l be used to image the interaction of cells with cellulose-presented factors and evaluate the cytophysical effects of cellulose-matrix factor presentation. v) Confocal imaging In normal "wide-field" optical imaging the image resolution is limited by optical aberrations within individual components (e.g. spherical and chromatic aberration), and the lack of contrast between closely spaced subjects (for a review see White et al, 1987). Digital imaging techniques which use intensified video or cooled charge coupled device (CCD) detectors can increase contrast by digitally amplifying the acquired image signal and then expanding pixel intensities to f i l l the dynamic range of the image display (e.g. video-enhanced Nomarski imaging). Another approach to increasing image contrast has been the development of fluorescent imaging techniques. In fluorescence microscopy, indicator dyes which fluoresce at 43 specific wavelengths are used to stain objects of interest. This results in specific illumination of labeled structures and hence a significant increase in image contrast. However, with fluorescence microscopy, material that is out of the focal plane adds to the intensity of the image. This can greatly decrease image contrast, resulting in an image blurred by out-of-focus light.' Marv in M i n s k i patented the confocal microscope in 1961. His invention included most of the essential elements common to modern confocal microscopes: illumination is focused by the objective lens to maximize excitation intensity in the plane of focus, a pinhole aperture is placed at the secondary image plane, and the specimen is scanned to produce a rasterized image of the specimen. The pinhole aperture is one of three basic techniques by which out-of-focus light can be rejected in fluorescence imaging. A second method of removing out-of-focus light, uses a technique known as deconvolution imaging. Wi th this approach, information about the optical transfer function of the microscope, usually generated from reference images of fluorescent microspheres, is used to remove out-of-focus light from images by determining the most-likely image plane from which each photon arose, and then reassigning photons to the correct image slices (Shaw, 1994). This technique is quite computationally intensive, but is finding application under a variety of circumstances. A third technique, which was first demonstrated by Webb et al. (1990), uses the "two-photon" effect. Wi th two-photon excitation, two photons arriving simultaneously at the same fluorophore, each with half of the energy required for fluorescence excitation, cause the fluorophore to be excited. Because the photon flux density is highest at the in-focus plane of the specimen, the laser intensity can be adjusted such that only the focal plane receives sufficient photon flux density for fluorophore excitation. In this way, the confocal effect is achieved by confocal excitation rather than confocal collection of emitted fluorescence. 44 The primary advantages of a confocal microscope include the rejection of out-of-focus light and the ability to 'optically section' relatively thick samples (~ 100 |im). Optical sectioning is achieved by scanning the sample through the focal plane of the microscope and collecting successive images to create a 3-D composite image of the subject. Confocal laser scanning microscopes ( C L S M s ) are limited by two fundamental characteristics of fluorophore dynamics: fluorescence photobleaching, which limits the number of available photons per fluorescent molecule (~ 10 5), and excited-state saturation, which limits the rate at which photons are emitted from fluorophores (primarily because of finite fluorescence-state lifetimes) (Sandison, 1995). These limitations are in addition to inefficiencies inherent in the collection and detection of emitted photons. Because the saturation and bleaching properties of fluorophores introduce more severe limitations than optical inefficiencies in modern C S L M s , it is generally more effective to integrate multiple scans at lower intensity, than to collect images at a high excitation power in a single scan (White, 1987). The confocal image signal-to-noise (S/N) ratio is optimum when the confocal pinhole aperture is slightly smaller than the first minimum of the Ai ry disk (i.e. the diameter of the first minimum of a diffraction limited spot). Confocal rejection of background fluorescence can increase the image S/N ratio by as much as 100-fold. Disadvantages of confocal imaging include increased photobleaching and increased photo-toxicity to live specimens because of the required high intensity fluorescence excitation. A s with all optical techniques, resolution is limited by the wavelength of probing light. For typical fluorophores (excited between 490 and 550 nm) lateral resolutions of about 250 nm is possible. The axial resolution of the confocal microscope is slightly lower (e.g. ~ 450 nm). Future instrumentation requirements include the development of objective lenses with longer working distance and high numerical aperture to allow the imaging of thicker specimens. 45 vi) Fluorescence recovery after photobleaching analysis One of the most powerful techniques available for the analysis of macromolecular motion is based on fluorescence recovery after photobleaching (FRAP) . F R A P allows the quantification of bound and unbound fluorescent species without prior separation. F R A P has been applied in the study of a wide range of processes: protein motion on cell membranes (Jacobson et al, 1987), motion of actin filaments in cell locomotion (Wang, 1985), macromolecular diffusion in solution (Kaufman and Jain, 1991), and diffusion in polymer mixtures (Hwang and Ediger, 1996; De Smedt era/., 1997). To perform a F R A P measurement on a surface to which a fluorescent ligand is bound, a spot is selected and the fluorescence in that region is bleached by a short pulse of high intensity light. Following bleaching, recovery of fluorescence in the bleached region is monitored using low intensity light. When only isotropic lateral diffusion occurs, the time required for 50% recovery of the fluorescent signal is inversely proportional to the lateral diffusion coefficient of the fluorescent molecule (Axelrod et al, 1976). One relevant extension of the spot technique is the bleaching of patterns over a large area of the sample. Tsay and Jacobson (1991), used this method to study nonisotropic diffusion and flow across a surface, using a video recorder to monitor fluorescence recovery and 2-D Fourier analysis of the recovery profiles. In systems in which ligand binding interactions occur, F R A P analysis is performed by fitting the measured fluorescence intensity recovery profile to theoretical fluorescence recovery equations derived from equilibrium binding reaction equations for the fluorescent species. Kaufman and Jain (1991) showed that the differential equations governing the fluorescence recovery process were comprised of 3 independent terms: diffusion, free binding association, and dissociation (a fourth term was included in their analysis to negate the association term as the 46 surface became saturated). Determination of binding or diffusion parameters is possible, by selecting length and recovery time scales such that one effect dominates the other. The antibody immobilized-antigen interaction studied by Kaufman and Jain (1990), was reaction rate limited for a bleach spot size of 40 \im monitored for 20 seconds during recovery. F R A P techniques have recently been applied with the confocal light microscope (Blonk et al. 1992). The confocal microscope (BioRad MRC600) was shown to be capable of performing the spot photobleaching experiments. Blonk derived equations which extended the 2-D analysis of Axelrod (1976) to the 3-D situation encountered with the confocal microscope. In the final analysis however, they demonstrated that the 2-D calculation method of Axelrod was a good approximation i f a low magnification (10X) and low numerical aperture (0.5) objective lens was used. Using their methods, the instrument yielded good estimates of diffusion coefficients up to a maximum of 2 x 1 0 6 cm 2/s. F R A P using 2-photon excitation has also recently been demonstrated (Kubitscheck et al, 1996). F R A P has been used to measure the diffusion of a-D-glucan maltohydrolase on insoluble starch (Katchalski-Katzir et al, 1985), the diffusion of bovine serum albumin on glass (Tilton, 1990), and of collagenase adsorbed to a peptide substrate covalently coupled to an insoluble support (Gaspers et al, 1996). In each of these systems, surface mobility of the adsorbed enzyme was thought to arise because the proteins made multiple binding contacts with the sorbent surface. Individual bonds at contact points were presumed to be weak enough to permit diffusion across the surface, but the ensemble of bonds maintained the protein at the surface. Indeed, it was suggested that the combined effect of multiple weak interaction sites on a single molecule, and the dynamics of protein structure and folding oscillations, could result in a molecular motion similar to that observed for a centipede (Henis et al, 1988). 47 vii) The biology of growth factors used in this study Interleukin-3 IL-3 is a glycosylated protein of 140 amino acids in mouse (23-28 kDa) and 133 amino acids in human. Human and murine JJL-3 are species specific. Glycosylation does not affect the activity of IL-3 since natural and recombinant sources, including nonglycosylated material derived from genetically engineered E. coli cells, have similar specific activity (Ziltener et al, 1988). Glycosylated and nonglycosylated forms of IL-3 interact equally well with extracellular matrix ( E C M ) material in vitro (Roberts et al, 1988). The major physiological sources of IL-3 are from stimulated T-cells (Schrader et al, 1981) and activated mast cells (Grabbe et al, 1994). IL-3 is not detectable in the blood of normal animals, and has a half life of only 40 minutes when injected intravenously (Crapper et al, 1984). Schrader suggested that IL-3 derived from activated T-cells is unlikely to be involved in steady state hematopoiesis because the release of IL-3 is localized to the site of inflammation (Schrader, 1991). Because IL-3 concentrations are below detectable limits in the bone marrow, cell stimulation by IL-3 presumably results from direct cell contact with the bone marrow stromal cells which secrete EL-3, or indirectly by association with DL-3 bound within the E C M (Morris etal, 1991). Target cells of IL-3 include progenitor cells of every lineage derived from pluripotent stem cells (Hile et al, 1983), as well as some endothelial cells (Colotta et al, 1993). The function of IL-3 signaling in endothelial cells remains unclear, but IL-3 induces a chemotactic response at physiological concentrations (10-100 ng/ml) (Colotta et al, 1993) and stimulates proliferation and expression of endothelial-leukocyte adhesion molecule-1 (Brizzi et al, 1993). In vitro, IL-3 prevents cell death and promotes the survival and proliferation of several hematopoietic cell types. IL-3 dependent cells undergo apoptosis i f they are deprived of IL-3 in cell culture. It has 48 been suggested that this mechanism may be effective in rapidly terminating responses to IL-3 following termination of IL-3 release (Schrader, 1991). The cellular consequences of stimulation by DL-3 include activation of protein kinase C (Farrar et al, 1985), and increased levels of intracellular A T P and glucose (Whetton et al, 1986). The high affinity receptor ( K d 300 pmol/litre) for murine and human IL-3 consists of two subunits (Miyajima et al, 1993). The two receptor subunits associate at the cell membrane to form a high affinity receptor capable of transducing a signal from the IL-3 binding event into the cytoplasm. These subunits belong to a family of growth factor receptors which lack intrinsic tyrosine kinase activity (Miyajima et al, 1993). Rather, these receptors form associations with kinases at the membrane. The IL-3 receptor (IL-3r) associates with the proto-oncogene p53/56 lyn kinase (Torigoe et al, 1992). Phosphorylation of tyrosine and serine residues of the 140 kDa (3-chain of the IL-3r is an early consequence of IL-3 binding to target cells. Maximal phosphorylation occurs within 15 minutes of addition of IL-3 (Sorensen et al, 1989). The number of IL-3r expressed on cells ranges from 115,000 on an IL-3 dependent cell line (B6SutA) (Murthy et al, 1989) to less than 100 on umbilical cord endothelial cells (Brizzi et al, 1993). A study using genetically engineered fibroblast cells to supply EL-3, showed that IL-3 can enhance multilineage hematopoiesis in the long term culture of human cells (Otsuka et al, 1991). Furthermore, secreted factors triggered growth responses that were not obtainable by simple addition of cytokines to the culture supernatant. The duration of exposure of the cells to IL-3 may be a critical parameter which determines the magnitude of the growth response. Whether the increased growth response obtained with the genetically engineered stromal cells was due solely to the high local concentration of IL-3 secreted by the support cells, or to synergy with other factors also produced by the stromal layer, was not determined. 49 Analysis of the binding of IL-3 to the factor-dependent cell line B6SutA cells gave an apparent K d of 1 nmol/litre (Murthy et al, 1989). The IL-3 E D 5 0 (effective dose yielding 50% maximum effect) for this cell line was in the pmol/litre range, as measured by tritiated thymidine incorporation during D N A synthesis. This indicates that very few receptors need to be occupied to elicit a biological response. The large number of receptors expressed on these cells may function to quickly dampen the IL-3 signal by internalizing and degrading excess IL-3 following initial cell stimulation. In this way, a single local release of IL-3 might bring about a single wave of IL-3 induced signaling. Interleukin-2 IL-2 is a pleiotropic hematopoietic cell growth factor, primarily active on lymphoid cell progenitors and mature T-cells (Smith, 1980). IL-2 is heavily glycosylated with an unglycosylated molecular weight of 15 kDa. IL-2 has been tested in a number of clinical applications including ex vivo cultivation of bone marrow aspirates for the treatment of leukemia (Klingerman et al, 1993), use as an adjuvant for vaccinations (Nunberg et al, 1989), and facilitation of immune response enhancement when treating intracellular infectious diseases (Kaplan et al, 1991). In hematopoietic cell culture, IL-2 induces a range of effects, including the activation of natural killer (NK) cells and the increased secretion of other growth factors (Vujanovic et al, 1993). The DL-2 receptor is a heterotrimer, composed of a high affinity (P-subunit) protein and a low affinity (a-subunit) protein required for high affinity binding of EL-2, and the y-subunit protein, which is required for signal transduction (for a review see Taniguchi and Minami , 1993). These three chains interact in various combinations to produce functional receptors with distinct characteristics. The interaction of the (3 and y chains is required for signal 50 transduction. However, the high affinity ( K d - 1 0 " M" 1 ) form of the receptor requires the association of all three of the receptor chains. Upon ligand binding, IL-2 receptors aggregate in the cell membrane and are then internalized in clatherin-coated pits (Lowenthal et al, 1986). The interaction of the cell surface IL-2 receptor with IL-2, obeys simple binding kinetics and produces a linear Scatchard plot (Lowenthal et al., 1985). The response of resting IL-2 dependent cells to the addition of IL-2 in vitro has been studied by a number of researchers. The application of the logistic function to the modeling of the stimulation response of an IL-2 dependent cytotoxic T-lymphocyte cell-line ( C T L L ) has been described (Hooton et al, 1985). Hooton et al. argue that the combined actions of an exponential increase in cell growth and a finite and diminishing supply of growth factor, naturally gives rise to a sigmoid growth dose response function. In their model for cellular response to IL-2 stimulation, Hooton assumed that cells exist in either of two states: quiescent or active (in which the cell is committed to moving through the cell cycle). The probability of any particular cell entering the active state, was defined by the concentration of growth factor to which the cell was exposed. Cit ing earlier studies in which inhibitors of lysosome formation blocked the degradation of DL-2 (Robb et al., 1981), the model was designed to include the effect of signal attenuation by ligand-receptor internalization. It was noted that this feature predicts the down-regulation of receptors on the cell surface at increased IL-2 concentrations (i.e. a larger fraction of receptors are not on the cell surface because they are being cycled through the lysosomes). A dialysis perfusion bioreactor for the expansion of lymphokine-activated killer cells for adoptive immunotherapy has been described (Murata et al, 1991). This reactor supported 2.7 x 10 7 cells/ml with greater than 90% viability. The medium was continuously perfused. Batch additions of IL-2 were made to the reactor at "appropriate" intervals. Compared to standard T-51 flasks, basal media, IL-2 and serum consumption, were reduced by 20%, 84% and 96% respectively. Doubling times, surface molecule phenotype and cytolytic activities were found to be similar for cells produced in the perfusion bioreactor or in standard tissue culture plates. Stem Cell Factor The calculated molecular weight of murine S C F (mSCF) is 18.5 kDa based on the amino acid sequence. S C F is normally heavily glycosylated, and ranges from 28 to 40 kDa when expressed in C H O cells. S C F contains 2 intramolecular disulfide bonds which are essential for full bioactivity (Jones et al, 1996). Murine and rat SCFs can stimulate human cells with nearly full bioactivity, but human S C F has an 800-fold lower specific activity on murine cells (Langley et al, 1994). The N-terminus of S C F is sensitive to truncation: deletion of the first 3 amino acids impairs cell proliferation and receptor binding, and deletion of amino acid 4 or beyond completely abolishes bioactivity (Langley et al, 1994). The 14 C-termini residues can be deleted from S C F with retention of full bioactivity. The bioactive form of S C F is a noncovalently linked homodimer (for a recent review see Broudy et al, 1996). The homodimer forms a symmetric pair of binding faces to which a pair of c-kit receptors bind and subsequently activate through cross-phosphorylation (Philo et al, 1996). The dimerization equilibrium association constant (Keq) is estimated to be ~ 3 x 10 8 M " 1 (Hsu et al, 1997). Using site specific mutagenesis, Hsu et al, (1996) demonstrated that substitution of residues on the putative dimerization interface significantly reduced molar specific activity. Engineering a cystine residue at the interface, designed to form covalently linked dimers, increased the specific activity 10-fold, consistent with the idea that receptor activation is driven by dimerized ligands. 52 Only the first 3 Ig-like extracellular domains of c-kit are required for formation of c-kit dimers on S C F dimers (Lemmon et al, 1997). Lemmon et al. were unable to detect single c-kit molecules associated with a S C F dimer, suggesting that two c-kit receptors bind simultaneously to a S C F dimer. Using isothermal titration calorimetry, an association constant (K a ) of 1.8 x 10 7 M " 1 was determined for c-kit binding to S C F . The reaction is enthalpically driven with a A H of -8.7 kcal/mol at 25°C. The K a is somewhat lower than that measured with intact cells (Lev et al, 1992), but the authors suggest that this is due to the increased degrees of freedom of the receptors in solution. Additional evidence against the interaction of single c-kit monomers with S C F dimers arises from the shape of the S C F dose response curve. At high ligand concentrations, single c-kit receptors should be bound-up by the high concentrations of S C F dimer, resulting in decreased cell proliferation. There are few reports in the literature of this phenomena, which argues against a two step mechanism, whereby a single c-kit binds to a S C F dimer which then binds to a second c-kit receptor to form the active complex. S C F exists in both membrane-bound and soluble forms (Pandiella et al, 1992). The differential effects of stromal versus soluble presentation of S C F , include the enhanced ability to support hematopoiesis (Toksoz et al, 1992) and primordial germ cell survival (Matsui et al, 1991). Transgenic experiments with obligate soluble or membrane-bound forms of S C F , differentially induced the proliferation of erythroid or myeloid progenitor cells, respectively (Majumdar et al, 1996). Wi th soluble S C F , myelopoiesis but not erythropoiesis was recovered when compared to S C F knockout mice. In contrast, membrane-bound S C F had the reverse effect. This result indicates that the mode of S C F presentation is important in determining the biological outcome of S C F signaling. In vitro, S C F presented on the membrane of stromal cells is more potent than soluble S C F , stimulating more persistent tyrosine kinase activity of its 53 receptor (Miyazawa et al, 1995) and supporting longer lived bone marrow cultures (Toksoz et al, 1992). The importance of S C F in progenitor cell development and colony formation is well established (Broudy et al, 1997). However, the concentrations of factors typically used in colony formation assays are significantly higher than those measured in vivo (Lowry, 1992). This paradox has stimulated researchers to examine modes in which low levels of growth factors, such as those found in the supernatants of Dexter-type liquid cultures, give rise to normal colony formation numbers in stroma-free liquid cultures. Lowry demonstrated that colony formation in cultures stimulated with a cytokine mixture containing 100-fold less cytokine than optimal levels, could be recovered through the addition of 100 ng/ml S C F . They suggested that S C F acts as an "anchor" factor, reducing the ED50 of other growth factors required for colony formation. The significance of S C F in chemotaxis has been studied in Boyden chamber assays (Meininger et al, 1992; Kinashi and Springer, 1994; Okumura et al, 1996). In this apparatus, a membrane is used to separate two chambers: the bottom chamber containing the chemo-attractant and the top chamber containing the cells. After 3 hours incubation with cells under normal cell culture conditions, the membrane is recovered and the cells which have migrated into the membrane, up the chemo-attractant gradient, are enumerated. Mast cells migrate into the partitioning membrane in response to S C F placed in the lower chamber (Meininger et al, 1992). When equal amounts of S C F were added above and below the membrane, cells did not migrate into the membrane. Thus, S C F acted to stimulate chemotaxis but did not stimulate chemokinesis. Cells with point mutations in the kinase domain of c-kit did not respond to gradients of S C F , indicating that receptor tyrosine kinase activity was required for the chemotactic response. Antibody to S C F blocked cell migration in a dose-dependent manner. 54 A common feature of S C F stimulation is the rapid ligand-induced internalization and degradation of the ligand-receptor complex (Yee et al, 1993). Receptor is cleared from the cell membrane by internalization in a ligand dose-dependent manner with a half-life of 30 to 40 minutes. Ligand-receptor complexes are typically endocytosed in clatherin coated pits. A recent study with fetal-derived mast cells, used fluorescence-activated cell sorting ( F A C S ) and immunocytochemical analysis to demonstrate that c-kit was rapidly internalized with S C F , and that the reappearance of c-kit on the cell membrane required R N A synthesis (Shimizu et al, 1996). Mast cell adhesion to fibroblasts expressing the transmembrane form of S C F has been used to demonstrate the role of S C F in mast cell adhesion (Adachi et al, 1992). In co-culture experiments, mast cells rapidly bound to preformed fibroblast monolayers. In contrast to the requirement of an active kinase domain for chemotaxis, the signaling deficient mutant receptor was fully active in promoting mast cell attachment to fibroblasts. Specificity was demonstrated by the ability of monoclonal antibody directed against c-kit to block mast cell attachment. In addition, bone marrow-derived hematopoietic progenitor cells also interact directly with stroma membrane-presented S C F through the c-kit receptor (Kodama et al, 1994). Adhesion is completely blocked by the addition of anti-c-kit monoclonal antibody, demonstrating that other stromal cell-presented factors were not involved. The addition of 200 ng/ml S C F was able to block about 50% of cells from binding to stroma. The significance of the mode of S C F presentation in c-kit dephosphorylation kinetics has been demonstrated in two recent reports. Miyazawa et al. (1995), used fibroblasts stably transformed with the gene for the membrane-anchored form of S C F . They reported that the membrane-bound form extended receptor phosphorylation by at least 90 minutes, compared to 55 soluble S C F . The prolonged stimulation was correlated with a decrease in the rate of c-kit turnover in stimulated cells. In a second paper by the same group (Kurosawa et al, 1996) anti-receptor antibodies were used to activate c-kit. To achieve receptor activation it was necessary to bind the monoclonal antibody to surfaces coated with anti-mouse antibody. Soluble or directly surface-bound anti-c-kit antibody (YB5.B8) inhibited cell growth. Receptor dephosphorylation kinetics were slowed by at least 2 hours, and correlated with a decrease in c-kit internalization. Similarly, the strong binding of C B D - S C F on crystalline cellulose may impede the rapid patching and internalization of c-kit and thereby prolong receptor signaling. 56 Chapter 2 Materials and Methods Protein Production and Purification i) Preparation of CBDs Cellulases and their isolated binding domains were supplied by the U B C Cellulase Group. The genes encoding the exoglycanase Cex or the isolated C B D C e x were subcloned into the p T Z E 0 7 vector and expressed in E. coli JM101. The gene fragments encoding the catalytically inactive mutant of the endoglucanase CenA (Asp252Ala) and the isolated C B D C e n A were subcloned into the vector pUC18 and expressed in E. coli JM101. Fermentations were carried out in a 20-litre Chemap fermenter at 37°C. Cellulases and their isolated C B D s were purified by affinity chromatography on Avice l PH101 ( F M C ; County Cork, Ireland), a microcrystalline form of cellulose. Contaminating oligosaccharides from the Avice l affinity column were removed by size exclusion chromatography on a Superose-12 column (Pharmacia; Uppsula, Sweden ). ii) Preparation of CBD-IL3 The C B D - I L 3 expression vector was constructed by R. Graham (unpublished results). The plasmid (PTUglO) was a derivative of pTrc99A. This plasmid carries the tac promoter and kanamycin resistance markers. A plasmid containing the Cex leader peptide, the P/T linker domain, the gene fragment encoding C B D c e n A and the gene fragment encoding alkaline phosphatase (pTUglO-CBD-phoA) was used as the starting point (Greenwood et al, 1989). The gene fragment encoding phoA was replaced with the Nhe-HindUI digest of the gene encoding mIL-3 (pTUglO-CBD-IL3) . The plasmid was transformed into E. coli JM101 by standard methods. Cultures were grown in 2-litre shake flasks to an OD600 of 1.0 at 37°C. Isopropyl-(3-D-thiogalactoside (IPTG) (0.5 m M ) was then added, and the cultures shifted to 30°C for a further 8 57 hours. Cells were harvested by centrifugation and lysed in a French Press™. The resulting cell lysate was clarified by centrifugation at 14,000 rpm for 20 minutes. Clarified supernatants were either frozen at - 7 0 ° C until use, or bound immediately to Avice l and washed extensively to remove E.Coli proteases. iii) Preparation of CBD-IL2 The construction of the C B D - J L 2 expression vector was described by Ong (Ong et al, 1995). Briefly, a plasmid containing the g D l herpes simplex leader peptide, the P/T linker domain, and the gene fragment encoding C B D C e n A were ligated into the p S V L plasmid (Pharmacia; Uppsula, Sweden) to give the plasmid p S V L - g D l - C B D C e n A - P T - I L 2 . C O S cells were transfected with the vector and grown in D M E M supplemented with 10% F C S (fetal calf serum) (Gibco B R L ) , 100 units penicillin ml" 1 and 100 tig streptomycin ml" 1 . Cells were cultured to late exponential phase and then the supernatant harvested by centrifugation at 1500 g. The supernatant was stored at -20°C until use. Cells produced between 150-600 mg CBD-1L2 per litre of culture supernatant. iv) Preparation of CBD-SCF The construction of the C B D - S C F expression vector and production of the hybrid are described by Doheny (1997). The gene segment encoding the extracellular domain of murine S C F was fused to the gene segment encoding the cellulose binding domain of the Cellulomonas find cellulase Cex. The mammalian signal sequence was replaced by the Cex signal sequence, and the hybrid gene ligated into the expression vector pTugAS (Graham et al, 1995) which had been modified to encode kanamycin resistance (Dr. R. Graham, unpublished construct). A polyhistidine affinity tag was inserted after the Cex signal peptide processing site to facilitate subsequent purification by metal chelate affinity chromatography ( M C A C ) . The resulting 58 plasmid ( p S C F / C B D 1.0) was transformed into E. coli JM101 by standard methods. Cultures were grown in 2-litre shake flasks to an OD6oo of 1.0 at 37°C at 250 rpm. Isopropyl-p-D-thiogalactoside (0.5 m M ) was then added, and the cultures shifted to 30°C at 150 rpm for a further 8 hours. Cells were harvested by centrifugation and the periplasmic fraction recovered (22). C B D - S C F was purified from the periplasmic fraction by M C A C . Protein concentrations were determined by absorbance at 280 nm. v) Protein labeling with fluorescein CBDcex has two amino groups which react with fluorescein isothiocyanate (FITC): the N -terminus and a single surface-exposed lysine residue. Neither is on or near the putative binding face of the C B D (Xu et al, 1995). C B D c e n A also has 2 potential reaction sites, which are sufficiently removed from the binding face so that FLTC-labeling does not influence adsorption characteristics. Proteins were labeled by standard procedures. To summarize briefly, 0.15 mg FITC was added per mg of protein at 1 mg protein per ml . The p H was adjusted to approximately 9 to initiate the reaction and the solution was gently mixed in the dark at 4°C for 5 hours. The labeled protein was passed twice through a 5 ml Sephadex G50 column (Pharmacia) in a 50 m M phosphate buffer mobile phase to separate unbound FITC from labeled protein. Fractions containing significant amounts of protein were pooled, and the absorbence at 280 nm and 495 nm was used to determine the number of F ITC molecules bound per protein. On average, 1.5 moles F I T C bound per mole of C B D protein. A slightly higher conjugate ratio of ca. 2.2 was found for whole enzymes. Protein solutions were stored in the dark at 4°C until use. 59 Characteristics and Preparation of Cellulose Materials i) Valonia ventricosa microcrystalline cellulose V. ventricosa is a marine algae which grows in many temperate marine environments. Its cell wall has a multilamellar structure organized with each lamella positioned orthogonal to its neighbor. Each lamella contains several parallel layers of cellulose microfibrils (Revol, 1992). The individual microfibrils are highly crystalline, with a square cross-section of approximately 18 nm corresponding to the 110 and the IK) crystallographic planes. Electron diffraction measurements show that the U 0 face of the microfibrils is preferentially oriented parallel to the cell wall . Both orientations of the microfibril longitudinal axis occur within each lamella. Preparation and cleaning of V. ventricosa cell walls were based on the method of Gardner and Blackwell (1971). For F R A P analysis, cell wall layers were carefully peeled apart under a dissecting microscope, typically into about six distinct sheets. The outermost and innermost sheets were discarded. Each of the remaining sheets was then floated and spread evenly onto a normal No . 1 glass coverslip (Baxter Canlab; Montreal, Canada). After drying, the sheet was trimmed to a 3.5 mm square and permanently fixed to the coverslip using a narrow border of Quickmount™ mountant (Fisher Scientific; Vancouver, Canada). Microscopic examination showed that the mountant did not permeate past the perimeter of the cellulose sheet. Mounted cellulose samples were stored at room temperature. ii) Bacterial microcrystalline cellulose B M C C was prepared from cultures of Acetobacter xylinum ( A T C C 23769) grown on peptone/yeast extract/glucose medium. One liter cultures in 25 cm by 30 cm covered trays were incubated at 30°C for 7 days, without shaking; the cellulose pellicles produced on the surface of 60 the medium were then harvested. The pellicles were cut into 1 cm squares and then washed extensively with double distilled water and extracted with 2 liters of 4% N a O H at 4°C for 24 h. Extraction was repeated 5 times with fresh N a O H . After washing in ddHiO, the cellulose was heated in 2.5 N HC1 under reflux for 1 h. The mixture was cooled and homogenized in a blender operated at full speed for 5 min. The B M C C was washed extensively with d d H 2 0 and then resuspended in 50 m M phosphate buffer containing 0.01% NaN3 to a concentration of 2 mg/ml and stored at 4°C. Preparation of B M C C fibrils for cell culture was similar to that above except no NaN3 was added prior to storage at 4°C. iii) Commercial-grade Avicel cellulose Avicel™ PH-101 ( F M C International; County Cork, Ireland) was size sieved in a set of sizing mesh trays. The 50 - 70 urn fraction was collected and washed twice in d d H 2 0 and then twice in 5 0 m M phosphate buffer and finally resuspended in 50 m M phosphate buffer (pH 7.0) at ~5 g/litre. The Avice l suspension was then autoclaved (121°C for 25 minutes) and then stored at 4°C until use. The crystallinity index of Avice l varies from 64% - 81%. Isotherm Analysis i) Fluorometric CBD assay Samples containing known amounts of FITC-labeled C B D were adsorbed to Av ice l as standards. Tubes were then placed on a rotating mixer for 24 hours at 25°C in the dark. Following binding, tubes were centrifuged at 10,000 rpm for 10 minutes. The Av ice l pellets were resuspended in 100 u l of 50-mM phosphate buffer and transferred to wells in a 96-well plate. The fluorescence of the F L T C - C B D adsorbed to the Avice l was determined using a 96-61 well plate fluorimeter ( J D E X X ; Portland, M A ) at an excitation wavelength of 488 nm and emission wavelength of 535 nm. Concentrations of F I T C - C B D were determined by reference to the standards. ii) Adsorption of CBDs to V. ventricosa cellulose To facilitate binding of proteins to V. ventricosa cellulose sheets, a short length of tubing was fixed over the dried cellulose sheet on the coverslip to form a well . Prior to binding C B D s , the well was filled with 50-mM phosphate buffer, incubated for 10 minutes, and then inverted and gently shaken to remove excess liquid. 400 ui aliquots of labeled protein diluted in 50 -mM phosphate buffer were then added to the wells. Equilibrium between the bound and free protein fractions was reached within 3 hours (data not shown), after which the supernatant was removed and set aside for subsequent determination of the unbound protein concentration. The sheet was rinsed thoroughly with 50 -mM phosphate buffer and then soaked for 30 minutes, with a buffer change after 15 minutes. The tube which formed the well over the cellulose sheet was removed, and the cellulose sheet was mounted over a small well drilled into a normal microscope slide (Baxter) (approximate volume 8 mm 3 ) containing 50 m M phosphate buffer. The coverslip was sealed around the well with silicon grease to prevent evaporation during imaging. For comparison with previous results, C B D c e x binding isotherms were determined using V. ventricosa cellulose from sheets disrupted by sonication. A new approach to C B D binding analysis was developed to permit analysis of protein concentrations at picomole levels. Disrupted cellulose and FJTC-labeled C B D s were added to an Eppendorf tube pre-blocked with bovine serum albumin to minimize nonspecific adsorption to the container walls. The filled tubes were placed on rotating mixers for 3 hours at 25°C in the dark to allow binding to come to equilibrium. Following binding, the tubes were centrifuged at 10,000 rpm for 10 minutes to 62 pellet the cellulose adsorbent. The supernate was collected and added to preblocked Eppendorf tubes containing a 25-fold excess of Avice l (based on saturation capacity) to concentrate the unbound F I T C - C B D . iii) Adsorption of CBDs to BMCC A l l unlabeled-CBD adsorption isotherm measurements were carried out at 30°C in 1.5 ml Eppendorf tubes containing 1 to 300 uJVI C B D mixed with 1 mg B M C C in 50 m M phosphate buffer (pH 7.0) at a final volume of 1.0 ml . Control tubes contained no B M C C . Each solution was vortexed for ca. 5 sec, and then rotated end-over-end overnight to allow the adsorption system to equilibrate. The samples were then centrifuged at 30°C and 10,000 rpm for 10 minutes to pellet the B M C C ; the clarified supernatant was passed through a 0.2 urn Acrodisc filter (Gelman Sciences). No protein adsorption to these filters could be detected. The depletion method, based on A280 readings of the supernatant, was used to calculate the amount of C B D adsorbed to the B M C C . Desorption isotherm data was taken by serial dilution of an equilibrated adsorption solution prepared in the manner described above. Each measurement was performed in triplicate. Binding isotherms for F L T C - C B D - S C F were determined using pre-blocked polypropylene Eppendorf tubes. B M C C (40 ug) was mixed with varying concentrations of C B D - S C F labeled with F ITC in 1 ml of phosphate-buffered saline (PBS). The tubes were placed on rotating mixers for 3 hours at 25°C in the dark to allow binding. Tubes were then centrifuged at 10,000 rpm for 10 minutes to pellet the B M C C . The supernatants were transferred to tubes pre-treated with B S A and containing 3.2 mg Avice l cellulose to adsorb the unbound F L T C - C B D in the supernatant. 63 For partition coefficient estimation, C B D - S C F standards were prepared in a similar fashion and used to calculate the fraction of initial protein which bound to the celulose. FRAP analysis i) FRAP with the BioRad MRC600 CLSM The BioRad M R C 6 0 0 confocal microscope (BioRad; Richmond C A ) used for imaging and F R A P experiments, consists of laser scanning mirrors, filters for excitation and emission, and photomultiplier tube(s) (PMT) mounted onto a conventional Nikon Optiphot-U microscope. 10X (N.A. 0.8) and 60X ( N . A 1.4) objective lenses were used for imaging. A 100 m W K r / A r laser was used for excitation at 488 nm. Excited fluorescence intensity was measured using a 535 nm bandpass filter and P M T . The P M T gain was adjusted to maximize the dynamic range in all images. The P M T black level was set at 4.7 for all imaging. The confocal aperture is noted for each image in the figure legends. For F R A P analysis, a 0.06% transmission filter was placed in the laser path in front of the instrument's standard filter set, to attenuate the laser for recovery monitoring. The neutral density filter wheel on the BioRad instrument was set to 3 (3% transmission) during all imaging scans. A n image collected prior to bleaching was used to normalize fluorescence intensities to pre-bleach levels. The C L S M was then electronically zoomed (zoom = 8) so that only a small region of the surface was illuminated during laser scanning. One scan was performed at this high zoom to produce a large bleached reference region. The C L S M zoom was then returned to its normal setting (zoom = 2), and the neutral density filter wheel on the BioRad instrument set to 0 (100% transmission). Using six successive laser parking and shutter opening (-100 msec each) sequences, six bleached spots were produced for F R A P analysis. The neutral density filter was 64 then returned to the 3 position and recovery monitoring initiated. Fluorescence intensity was monitored until greater than 95% of fluorescence recovery had occurred. ii) FRAP data analysis The center of each bleach spot was determined by averaging several successive bleach spot images and selecting the pixel with the minimum intensity as the bleach spot center. The bleach spot intensity profile was then radially averaged to obtain an intensity cross-section with spatial heterogeneity of the crystalline cellulose microfibrils averaged out. A Gaussian curve was fit by nonlinear least squares regression to the averaged radial profile of fluorescence intensity within the bleach spot. In all cases, the averaged spot profile was well fit by a Gaussian function. The parameters from the Gaussian fit (width, depth and offset) were used to calculate the fluorescence intensity at the spot center. The offset value (fluorescence intensity at three spot diameters from the spot center) was used to estimate the "background" fluorescence bleaching occurring during recovery monitoring. Laser intensity was attenuated to minimize bleaching during recovery monitoring (less than 5%). Smoothed recovery time-profiles were normalized against the initial pre-bleach fluorescence. The surface diffusion coefficient and mobile fraction were determined from the normalized recovery curves by nonlinear regression of the parameters in the series solution for spot F R A P diffusion analysis developed by Axelrod (1976). Cell culture i) Cell lines B6SutA cells (Murthy et al, 1989) were cultured in Iscove's modified Dubelcco's medium (EVIDM) containing 10% fetal calf serum (FCS, Stemcell Technology; Vancouver, B C and G I B C O B R L ; Grand Island, N Y ) and supplemented with 5% murine spleen cell conditioned 65 media (HemoSt im™ M2100, StemCell Technologies; Vancouver, B C ) . Cells were grown to late exponential phase (8 x 10 5 cells/ml) before use in Bioassays. M 0 7 e cells (Avanzi et al, 1990) were maintained in 10% F C S plus 10"5 M 2-mercaptoethanol (2 -ME, Sigma Chemicals; St. Louis, M O ) and 5 ng/ml G M - C S F (Novartis; Basel, Switzerland). TF-1 cells (Ala i et al, 1992) were cultured in EVIDM containing 10% F C S supplemented with 2 ng/ml r h G M - C S F (StemCell Technologies; Vancouver, B C ) . Cells were grown to late exponential phase (8 x 10 5 cells/ml) before use in bioassays. C T L L - 2 cells (Gillis and Watson, 1981) were cultured in EVIDM containing 10% fetal calf serum and supplemented with 10 U / m l murine IL-2 (kindly provided by the laboratory of H.S. Teh). ii) Bioassays Purified C B D - S C F diluted in 20 ul of hybridoma serum-free medium ( H - S F M , Gibco B R L ; Grand Island, N Y ) supplemented with 200 ug/ml human transferrin, 10 ug/ml bovine insulin and 10 mg/ml bovine serum albumin (BSA) was mixed with 50 u l of a B M C C suspension in P B S (3.5 ug B M C C / m l ) in each well of 96 well tissue culture plates (Costar Corp.; Cambridge, M A ) . After 12 hours incubation at 37°C in an atmosphere of 5% CO2, 100 u l of s H - S F M containing 2 x l 0 4 B 6 S u t A cells were added to the mixture. After incubation of the cultures for 48 hours, cell stimulation was estimated by measuring cell metabolic activity with standard MTT-based assays (Indrova et al, 1997). Recombinant murine S C F produced in E. coli ( R & D Systems; Minneapolis, M N ) was used as control. Factor-dependent cell lines were grown to late exponential phase (three days in the case of M 0 7 e and TF-1 cells, and two days in the case of B6SutA cells), in Iscove's modified Dubelcco's 66 medium (EVIDM) containing 10% F C S and supplemented as follows: B6SutA medium was supplemented with 5% murine spleen cell conditioned media (HemoSt im™ M2100, StemCell Technologies; Vancouver, B C ) , M 0 7 e medium was supplemented with 1 ng/ml rhSCF and 5 ng/ml rhIL-3 (StemCell Technologies; Vancouver, B C ) , and TF-1 medium was supplemented with 2 ng/ml r h G M - C S F (StemCell Technologies; Vancouver, B C ) . The cells were washed three times in supplemented-H-SFM (sH-SFM), diluted 1:4 and added to liquid cultures. Cells were then allowed to proliferate in s H - S F M in the presence of S C F or C B D - S C F , with or without 1 ug B M C C / m l for 48 hours at 37°C, 5% C 0 2 . Viable cells were determined by trypan blue exclusion in hemocytometric counts. Dose-response curves were fitted using Origin V.4.0 (Microcal Software Inc.; Northampton, M A ) , based on the 24 individual data points from each set. E D 5 0 values were calculated using the same software. Non-viable cells constituted less than 5% of the total cell number in all cases and were not included in the statistical analysis. C T L L - 2 cells were used for the assay of IL-2 bioactivity according to standard methods. To describe briefly, serial 2-fold dilutions of sample (0.2 jam filter sterilized) were added in duplicate to aliquots of 1.5 x 10 4 cells in a 96-well flat bottom microtitre plate. After 24 hours culture, the tetrazolium salt M T T (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma, St. Louis M O ) was added at 500 mg/ml. During the subsequent 4 hour incubation, metabolically active cells convert the M T T to a blue formazan product which was quantified after dissolution in 1 M HC1 by measurement of the absorbance at 600 nm in a microtitre plate reader. The IL-2 activity of the sample was calculated relative to a murine IL-2 standard. The dilution of the IL-2 standard for which a 50% maximal response was measured was defined as having 1 unit/ml activity. 67 CLSM Imaging i) CLSM of fixed cells Cells were cultured in R P M I supplemented with 10% F C S at 37°C in a 5% C 0 2 atmosphere until late exponential phase. Cells were then washed two times in S F M , resuspended in S F M and then cultured for 6 hours in the absence of growth factors. After 6 hours, hSCF was added to replicate wells containing 1 x 10 6 cells/ml at a range of concentrations covering the h S C F dose response curve for these cells: 100, 25, 6.25, 1.54 and 0 ng/ml. After 20 minutes, under normal cell culture conditions, cells were fixed with 3.5% paraformaldehyde, rinsed 3 times in P B S and then permeabilized with 0.5% Triton X-100 for 10 minutes. The fixed cells were blocked for 20 minutes in 5% bovine serum albumin (BSA) and then labeled with antibody diluted in 5% B S A for 1 hour. Primary antibody (monoclonal murine anti-human c-kit [YB5.B8]) was used at 1 ug/ml. Secondary antibody was fluorescein isothiocyanate conjugated goat anti-mouse IgG (human serum preadsorbed) (Sigma; St. Louis M O ) . Actin was stained with phalloidin conjugated to Texas Red at 1 unit/ml (Molecular Probes, Eugene OR). Cells were then washed and resuspended in 95% glycerol, 5% P B S . SlowFade™ (Molecular Probes, Eugene OR) was added at 2.5% as antifade. Immunofluorescence was imaged using the BioRad M R C 6 0 0 laser scanning system mounted on a Nikon Axiophot microscope fitted with a 6 0 X 1.4 N . A . objective lens. Fluorescein and Texas Red were imaged independently using standard filter sets. For the analysis of cells stimulated with B M C C - b o u n d C B D - S C F , B6SutA cells were grown as above to late exponential phase. Cells were then washed twice in s H - S F M , resuspended in s H - S F M , and then cultured in the absence of growth factors. After 6 hours, soluble C B D - S C F or C B D - S C F adsorbed to B M C C was added to replicate wells containing 1 x 10 6 cells/ml. After 20 minutes under normal cell culture conditions, cells were fixed with 4.0% paraformaldehyde, 68 rinsed 3 times in P B S and then permeabilized with 0.5% Triton X-100 for 10 minutes. The fixed cells were blocked for 20 minutes in 5% B S A and then labeled for 1 hour with antibody diluted in 5% B S A . Primary antibodies were as follows: polyclonal rabbit anti-SCF and monoclonal biotin-conjugated rat anti-mouse c-kit receptor (Pharmigen; San Diego, C A ) . Secondary labels were: FITC-conjugated goat anti-rabbit IgG (Sigma; St. Louis M O ) , Streptavidin-Cy5 conjugate (Evergreen Labs; Edmonton, A B ) ; and phalloidin-Texas Red conjugate (Molecular Probes; Eugene, OR) . For analysis of receptor activation, sheets of cellulose were prepared from the cell walls of the marine algae Valonia ventricosa. Sheets dried onto glass coverslips were incubated with either C B D c e x , S C F or C B D - S C F . After washing, B6SutA cells were cultured for 20 hours on the cellulose surfaces. For antibody labeling, cells were fixed, permeabilized and blocked as described above. Primary antibodies were as follows: polyclonal rabbit an t i -CBDc e x ; monoclonal biotin-conjugated rat anti-mouse c-kit receptor (Pharmigen; San Diego, C A ) ; monoclonal, mouse anti-phosphotyrosine (UBI; Lake Placid, N Y ) . Secondary labels were: F ITC-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG (Sigma; St. Louis M O ) , and Streptavidin-Texas Red conjugate (Gibco; Grand Island, N Y ) . Immunofluorescence was imaged using the BioRad M R C 6 0 0 laser scanning system mounted on a Nikon Axiophot microscope fitted with a 60X - 1.4 N . A . objective lens. ii) Live cell confocal imaging For live-cell imaging, 8 x 10 5 M 0 7 e cells were cultured in 1 ml of Hepes-buffered H - S F M in a covered 2.5 cm petri dish fitted to a C L S M stage heater. Cells were added to the stage dish and then incubated for 10 minutes to allow the cells to settle to the bottom of the culture chamber (adjacent to the objective lens). P M T gain and black levels were set so that the image field was 69 completely dark prior to the addition of fluorescenated ligand. Following collection of an initial image stack, FlTC-tagged C B D - S C F was added by gently pipetting a 5[il aliquot of protein into the culture chamber. 70 Chapter 3 Characterization of CBD Binding to Crystalline Cellulose Introduction The widespread occurrence of C B D s in cellulase enzymes implies that enzyme adsorption plays an important role in cellulose degradation (Gilkes et al, 1984; Gilkes et al, 1988). Furthermore, the construction of genetic fusion proteins between C B D s and other proteins, for the immobilization of the fusion-partner protein onto cellulose, has been demonstrated to have several practical applications (Ong et al, 1991; Greenwood et al, 1992; Tomme et al, 1996). Thus, a detailed characterization of the adsorption of C B D s to cellulose w i l l have important implications, both for understanding the action of cellulases on insoluble substrates and for the development of fusion protein strategies for protein immobilization and purification on cellulose. In this chapter we perform binding isotherm analysis of a cellulase (CenA) and two isolated binding domains ( C B D C e n A and C B D C e x ) - Adsorption isotherms were constructed using the depletion method, wherein protein remaining in solution following mixing with the solid phase adsorbent w i l l be measured to construct bound versus free protein equilibrium isotherm plots. Analysis of the characteristic shape of the isotherm was used to determine appropriate binding models. Fluorescent imaging using FITC-tagged C B D s was used to investigate the extent of binding heterogeneity on the surface of the sorbent. The studies performed in this chapter build on previous studies by characterizing binding to highly crystalline cellulose substrates. The basic repeating unit of cellulose is cellobiose. The projected surface area of a family LI C B D , such as C B D c e x , covers approximately 30 cellobiose units in a crystalline lattice. This large differential in protein to ligand size, creates the possibility of substantial blockage of 71 potential binding sites at the cellulose surface. A s a result, analysis of C B D binding isotherms by classic Langmuir theory oversimplifies the system, and may lead to incorrect (at least on an absolute thermodynamic scale) estimates of binding constants and capacities. Indeed, it is likely that surface packing of the adsorbed protein and protein interactions of adsorbed molecules, may play a significant role in determining the characteristics of the adsorption isotherm. These effects w i l l be considered in this chapter. Three types of cellulose materials were used in this work: Avice l , B M C C and V. ventricosa cellulose. These three celluloses provide distinct morphologies suitable for different experimental techniques: Av ice l is a 40-70 urn particulate, B M C C is a submicron microfibril, and V. ventricosa cellulose is used as micron-thick sheet made up of parallel arrays of 1-2 urn fibers. To examine C B D binding heterogeneity and cellulose surface accessibility and porosity, fluorescenated C B D c e x was used to perform C L S M on these cellulosics. The equivalence of FJTC-tagged C B D s to non-tagged C B D s for binding studies was also investigated. 72 Results i) Langmuir adsorption model In its simplest form, protein adsorption at a solid interface can be characterized by the Langmuir adsorption isotherm (Haynes and Norde, 1993). Wi th the Langmuir model, the adsorption of a C B D onto crystalline cellulose is assumed to be an equilibrium reaction in which a single C B D reacts with one or more of the repeating cellobiose units on the cellulose surface. At equilibrium, the adsorption reaction is described by the following: K - M , , A~[N]*[F] where [B] is the concentration of bound ligand (umol/gram cellulose), [F] is the concentration of free ligand (uM), [N] is the concentration of available binding sites (u.mol/g cellulose), and K a is the equilibrium association constant ( u M 1 ) . In deriving Equation 3-1, we have assumed that each binding site on the cellulose surface acts independently of all other sites. If a single ligand interacts with only one lattice unit and there are no positive or negative cooperativity effects: [N]=[N0]-[B] 3-2 where [N 0 ] is the concentration of binding sites in the absence of ligand (umol lattice sites/g B M C C ) . Substitution of Equation 3-2 into 3-1 yields the Langmuir adsorption isotherm equation. 73 [ B = L _ ^ J — ^ L J 3 _ 3 l + ^ a * [ F ] Equil ibrium adsorption isotherms for CenA and its isolated binding domain are shown in Figure 3-1. The molar saturation capacity of the B M C C for the C B D is about 10% higher than that for the intact enzyme, as might be expected from differences in their respective molecular sizes. A common means of analyzing adsorption isotherm results is to plot the data using various transformations to linearise the adsorption equation. One popular transformation is the Scatchard plot where the ratio [B]/[F] is plotted against the concentration of bound protein [B]. If the adsorption isotherm is well fit by the Langmuir model, the plotted data should form a straight line. However, as shown in Figure 3-2 the transformed data yield a concave-up plot. Potential causes for failure of Langmuir theory are many and include (a) the existence of multiple classes of binding sites, (b) negative-cooperativity between adsorbed proteins, and (c) the presence of overlapping potential binding sites. Thus, simple fitting of the Langmuir isotherm to the adsorption of C B D onto B M C C is not appropriate. In the following sections alternative strategies for the analysis of isotherm data w i l l be investigated. 74 9.0 0.0 + 1 1 1 1 1 i 0 5 10 15 20 25 30 Free CenA (JIM) Figure 3-1: Isotherm for the adsorption of CBD C e n A to BMCC Binding isotherm at 30°C for C B D C e n A ( • ) and CenA. ( • ) on B M C C in 50 m M phosphate buffer at p H 7.0 measured by the conventional solution depletion method (n = 6 replicates) 75 [B] (mmol ligand /g cellulose) Figure 3-2: Scatchard plot of isotherm data for adsorption of CenA and its isolated binding domain to BMCC at 30°C and pH 7.0 CBDcenA ( • ) and CenA. ( • ) (n = 6 replicates). ii) RSA-modified Langmuir model A concave-up Scatchard plot w i l l be generated for binding systems in which the adsorption of ligands blocks binding sites due to the packing configuration of adsorbed species. Figure 3-3 shows a schematic representation of C B D C e n A molecules binding to the 110 and the 111 crystal 76 faces of a cellulose microfibril. The cellobiose unit cell has dimensions 1.6 x 1.0 nm (Gardner and Blackwel l , 1971). When projected normal to a cellulose crystal face, C B D c e n A occupies an area covering approximately 29 repeating cellobiose units. The dimensions of the C B D therefore greatly exceed the dimensions of the elemental cellobiose repeating unit. A s a result, the ligand blocks several lattice units and the surface of the crystal may be considered as an array of overlapping potential binding sites. Under these conditions, the number of available binding sites is described by a probability function which depends not only on [B] but also on the spatial arrangement of the bound ligands on the cellulose surface (Stoclet et al, 1996). If the C B D is modeled as a large ligand and the cellulose surface as a lattice of overlapping potential binding sites, Equation 3-2 must be modified to account for the accelerated decrease in the population of free ligand "finding" vacant sites of the appropriate lattice dimensions as the lattice becomes saturated. The exclusion of potential binding sites by steric hindrance w i l l occur when any two bound C B D s are separated by a distance less than that required for a third C B D to adsorb. A probability distribution P([B]), a function of [B], is therefore introduced and serves to reduce the apparent number of free sites as the lattice becomes saturated: r,P([B])N0*Ka*[F] 3 4 1 + B Figure 3-3: Schematic representation of the CBD binding to the 110 and 111 crystal faces. The packing of the crystal units are taken from Gardner and Blackwell 's X-ray diffraction estimates of the unit cell (Gardner and Blackwell , 1958). The 3-D structure of C B D c e x (Xu et al., 1996) (~ 3.1 nm x 3.1 nm x 4.7 nm (Xu et al, 1996)) is projected onto the V. ventricosa cellulose fibril crystal (~ 18 nm x 18 nm x 1 mm (Revol, 1988)): Image A is an end-on view of the cellulose fiber, while image B is a side view of the fiber (the backbone structure of the C B D is shown projected onto the cellulose fiber in B showing the size of the C B D "footprint" on the cellulose lattice). 78 Although an analytical solution to determine P([B]) as a function of lattice and ligand size exists for one dimensional systems (e.g. protein adsorption to D N A ) , no such solution exist for higher dimensionality systems. Monte Carlo simulations were therefore performed to examine the characteristic form of P([B]) (results not shown). Simulations used a square lattice (with periodic boundary conditions) and a rectangular adsorbing ligand. It was assumed that a C B D was irreversibly adsorbed to the solid phase and that no molecular rearrangements occurred due to surface diffusion of adsorbed molecules. Potential adsorbing sites on the homogeneous lattice were selected at random, and then the site was evaluated for its capacity to fully accommodate an adsorbing C B D . For these simulations, 1000 trials were performed at a given surface coverage of adsorbed C B D s to determine the probability of a free ligand finding an unoccupied site of sufficient size to permit adsorption. Following each set of trials, a C B D was added to the surface (at the first free site found in the previous set of trials) and the simulation repeated. The sequence of adsorption trials and ligand addition was repeated until no successful binding trials occurred (10,000 trials were performed in the final run to ensure that the surface was saturated). Trials performed over the range of adsorption capacities were then used to determine the site exclusion probability density function as a function of fractional surface saturation (i.e. r/rmax). The applicability of Monte Carlo simulations of this type is sensitive to assumptions made in formulating the trials. For example, because little is known about the dynamics of C B D adsorption, the actual size of the lattice area occupied by an adsorbed C B D , or processes occurring following adsorption (e.g. dangling ligands, lattice disruption and surface rearrangement of adsorbed C B D s ) , care must be taken in drawing specific conclusions from these simulations. However, in general it was found that surface packing effects only become significant above 30% saturation. Furthermore, below 20% saturation, adsorbed-ligand packing 79 configuration effects are insignificant. This suggested that adsorption analysis can be carried out at low surface coverage to estimate the intrinsic affinity constant of C B D for B M C C . Provided No can be determined independently, this greatly simplifies the analysis and removes the necessity of making specific assumptions regarding the packing of adsorbed C B D s . iii) Low Surface concentration isotherms To avoid the complications inherent in R S A analysis, one may consider adsorption at only very low values of [F],which correspond to low surface coverages, where it can be assumed that ligands are packed in such a way that any two nearest C B D s do not exclude the binding of a third ligand. Under these conditions, Equation 3-2 may be rewritten as [N]=[N0]-a*[B] 3-5 where a is the number of lattice units occupied by a single ligand molecule. Substitution of Equation 3-5 into Equation 3-3 and rearranging using the double reciprocal transformation yields 1 1 1 | a 3 6 [B]~ Ka*[N0)*[F]+[Nti) The double reciprocal plot is appropriate for this analysis because it emphasizes data at lower concentrations (Figure 3-4). The slope ( l / K a [ N 0 ] ) and the intercept (a/N 0) of transformed adsorption data were calculated for CenA, C B D c e n A and Cex (see Table 1). 80 Unique estimates for K a , N 0 and a cannot be derived solely from this isotherm analysis. To obtain an estimate of the number of potential binding sites presented by the cellulose crystal, we must consider the structure of the cellulose microfibrils used for the adsorption isotherms. B M C C is synthesized as a long ribbon composed of 50 to 80 aggregated microfibrils (White and Brown, 1981). During preparation, these ribbons are partially disrupted into bundles of microfibrils with cross-sections on the order of 40 nm (111 face) by 15 nm (110 face). Given a density of 1.5 g/cm for crystalline cellulose (Meyer and Misch , 1937), and assuming that the smallest surface area unit C B D s have access to is the 40 x 15 nm fibrils, B M C C cellulose presents approximately 101 umol of lattice units per gram. Using this approximation, absolute K a and a values were estimated from the adsorption isotherms (Table 3-1). Table 3-1: Binding Constants for CenA, CBD C e n A and Cex on BMCC Binding parameters were calculated from adsorption data plotted in double reciprocal form. The values for the absolute affinity constant (K a ) and the C B D lattice occupancy number (a) were calculated assuming 101 | imol of cellulose lattice units per gram of B M C C . Ligand K r (1-g)"1 K a ujvr 1 a/No g ujnol"1 a mol / mol C e n A 40.4 ± 3.3 0.40 ± 0.03 0.33 ± 0 . 0 1 32.9 ± 1.4 CBDcenA 45.3 ± 2 . 1 0.45 ± 0.02 0.36 ± 0.02 39.2 ± 0 . 2 Cex 33.2 + 5.5 0.33 ± 0.05 0.28 ± 0 . 1 1 27.0 ± 11.4 81 0 H 1 1 1 1 1 — I 0 5 10 15 20 25 30 1/[Free CenA] (tiM 1 ) Figure 3-4: Double reciprocal plot of the adsorption of CenA to BMCC Binding parameters were calculated from adsorption data plotted in double reciprocal form (n = 6 replicates). Binding parameters were estimated by fitting a straight line to the limiting slope of the transformed data (see Table 3-1). iv) Multiple classes of binding sites model Although its simplicity is appealing, Equation 3-3 and the dilute surface R S A equivalent (Eq. 3-6) assume that all free binding sites on the cellulose surface are equivalent. For a sorbent surface offering a heterogeneous array of binding sites, application of Equation 3-6 yields an 82 average binding constant which describes the relative strength of binding, but cannot be used to calculate binding energetics (i.e. AG) associated with each class of sites. The validity of the one-site model for the analysis of C B D c e x binding, was checked by imaging B M C C after adsorption at either high or low concentrations of FJTC-tagged C B D c e x -Figure 3-5 shows typical images obtained for (A) subsaturating adsorption (T = 0.3 rmax) or (B) saturating levels (T = rmax). The images, collected with a 60X (N.A. 1.4) lens, are the average of three successive scans at a zoom of 2 X . The scale bar represents 5 (im. The confocal aperture on the BioRad 600 was set to 3. A t low surface coverage, the adsorbed C B D appears to be evenly distributed on the sorbent B M C C fibers. In contrast, at monolayer coverage there are a small number of relatively bright regions on many B M C C fibers. The absence of brighter regions in the low surface coverage images, suggests that the labeling heterogeneity is the result of isolated regions with lower binding affinity but significantly higher available specific surface area. These low affinity regions could be due to several effects, including physical damage to fibrils during disruption of the B M C C pellicles or imperfections during biosynthesis leading to noncrystalline regions on the fiber. 83 25 urn Figure 3-5: BMCC labeled with CBD C e x tagged with FITC Images for (A) subsaturating adsorption (r/rmax ~ 0.3) or (B) saturating levels (T ~ rmax) are shown. A t low surface coverage, the adsorbed C B D appears to be evenly distributed on the sorbent B M C C fibers. In contrast, at near monolayer coverage there are a small number of relatively bright regions on some B M C C fibers. Therefore, an alternative method for modeling C B D association with cellulose was used which assumes two distinct classes of binding sites: a high affinity class, and a low affinity class. Wi th this approach, the second class of binding sites are included in the standard Langmuir-type model for adsorption isotherm analysis: 84 l+KaH*[F] l + KaL*[F] where N H and N L are the total concentrations of the high and low affinity sites respectively (umol/g), and K a j j and KaL are their respective binding affinities (uM" 1). K a , the Langmuir affinity constant, is then equal to Kan divided by 29 sites/molecule (i.e. the number of cellobiose residues occupied by an adsorbed molecule of C B D c e x (Xu et al, 1995) and N 0 , the Langmuir site concentration, is multiplied by 29 sites/molecule to give [ N H ] . A typical isotherm prepared with C B D bound to B M C C is presented in Figure 3-6. Results for this analysis yield the following binding parameters: N H = 3.43 ± 0.04 |imol/g B M C C , K a H = 6.3 ± 14 x 10 7 M " 1 , N L = 0.9 ± 0.05 ujnol/g B M C C , and K a L = 1.1 ± 0.06 x 10 6 M " 1 . Although the two-site model is likely not an exact representation of the equilibrium binding reaction, it does acknowledge the heterogeneous nature of the binding reaction. 85 4.5 1 -0.5 -0 -| 1 1 . 1 1 0 1 2 3 4 (CBDcex) (uM) Figure 3-6: Isotherm for the estimation of binding affinities and capacities of BMCC for CBDcex Binding isotherm at 30°C for C B D C e x on B M C C in 50 m M phosphate buffer at p H 7.0, measured by the conventional solution depletion method. The ascending isotherm ( • ) and the descending isotherms (A) from two jump-off points are shown. 86 v) Adsorption of FITC-Iabeled CBD to cellulose Several experimental techniques employed in this work required the use of FITC-tagged CBDcex for binding studies. Thus, it was essential that the equivalence of C B D - F I T C to non-tagged C B D be established. The amino acid sequence of C B D c e x reveals that there are only two potential sites for isothiocyanate-mediated fluorophore conjugation to this C B D : Lys-28 and the N-terminus both present amino-bonds susceptible to this linkage chemistry. The 3-D structure of CBDcex indicates that both the N-terminus and lysine are surface-exposed and removed from the putative binding face (Xu et al, 1995). Therefore, it is reasonable to assume that attachment of fluorescein to either of these residues wi l l not significantly affect the binding properties of this C B D . To confirm this assumption, mixtures were prepared in which the ratio of labeled to unlabeled protein was varied. Isotherms were measured for mixtures containing 5%, 10% and 30% labeled CBDcex at 30°C on B M C C in 50 raM phosphate buffer at p H 7.0 (Figure 3-7). To verify the equivalence of these two species (tagged and nontagged), binding capacities and initial slopes were calculated for each mixture. N o significant change in the capacity or the affinity of the cellulose for the C B D molecule was seen. The affinity constants and saturation capacities of the C B D on cellulose for the mixtures tested were in quantitative agreement with isotherms prepared with unlabeled C B D , indicating that F ITC labeling of the C B D did not affect its binding properties. 87 6 H 1 1 1 1 1 2 3 4 5 (CBDcexXuM) Figure 3-7: Demonstration of equivalence of CBD C e x and FITC-CBDC e x Mixtures were prepared in which the ratio of labeled to unlabelled protein was varied: 5% ( • ) , 10% (A) and 30% ( • ) labeled C B D c e x - No significant difference in the capacity or the affinity of the cellulose for the two species (tagged and nontagged) was found. Quantitative fluorescence microscopy and isotherm analysis showed that F I T C - C B D fluorescence intensity per mole of bound protein was independent of surface concentration, indicating that F ITC self-quenching is not significant at surface concentrations up to saturation. Unbound fractions from the isotherms -were also analyzed to ensure that the ratio of F ITC to the 88 total concentration of protein in the mixtures had remained constant following adsorption. No significant change in the ratio of F ITC (A495) to C B D C e x (A280) was seen. It was thus verified that FJTC-CBDcex could be used as an effective tracer for the adsorption of C B D C e x to crystalline cellulose. To prepare adsorption isotherms at very low concentrations of protein, it was necessary to develop an assay protocol capable of measuring nanogram quantities of protein. Therefore, an assay was developed which measured the amount of FITC-labeled C B D remaining in supernatants of binding reactions following adsorption equilibrium. Supernatant was collected and equilibrated with a large excess of particulate cellulose. The cellulose was washed and then the cellulose-bound fluorescence was measured in the Pandex fluorescence concentration analyzer. Av ice l is a particulate semi-crystalline cellulose and is ideally suited for application in microtiter plate-based fluorescenated protein assays, because it is readily separated from supernatants. Prior to use, all Avice l samples were sieved to provide a uniform preparation of 40-70 \im particles. Normal phase-contrast microscopy revealed an assortment of rod shaped particles and irregular aggregates. Figure 3-8 shows images of Avice l particles stained with FITC-tagged C B D C e x . Sufficient protein was used for r/rmax to be about 75%. Clearly, labeling of the Avice l particles is not as uniform as that for B M C C . Bright regions may represent areas of increased surface coverage and could be the result of higher affinity or higher specific surface area. Relatively dim regions were also noted on some particles, even though other areas on the same particles were of typical bright intensity. Optical cross-sections were produced across the axis indicated in Figure 3-8a. The axial resolution in this image (Figure 3-8b) is approximately 0.25 (im. Two things must be noted in these images. First, the 89 laser on the confocal microscope can only penetrate about 20 um into this material. Thus, the lack of fluorescence noted on the bottom of the cross-section is a result of the opacity of the cellulose. Second, the ring of fluorescence noted around the top of the cross-section indicates that the C B D does not penetrate into the Avice l particle. In some particles, fissures were found which permitted the penetration of the C B D several microns into the particle. In these cases, fluorescence could be detected within the fissure. 90 Figure 3-8: Avicel labeled with FITC-tagged CBD C e x Confocal images of Avice l particles stained with FITC-CBDcex (Image A ) . Sufficient protein was used for T/Tmax to be about 75% for imaging. A n optical cross-section was produced across the axis indicated (Image B) . The ring of fluorescence noted around the top of the cross-section indicates that the C B D does not penetrate into the Avice l particle. The images were collected with a 60 X (N.A. 1.4) lens, and the scale bar represents 5 Jim. The images are the average of three successive scans at a zoom of 2. The confocal aperture on the BioRad 600 was set to 3. 91 vi) CBD adsorption to V. ventricosa cellulose Figure 3-9a shows images of a mounted V. ventricosa cellulose sheet stained with F1TC-CBDcex- The parallel-array microfibril structure and uniformity of the surface is evident. V. ventricosa cellulose has a very high degree of crystallinity (> 95% (Gardner and Blackwell , 1971)), and a high binding capacity for family II C B D s . Figure 3-9b shows a cross-section perpendicular to the surface at the axis indicated in Figure 3-9a. The sheets of V. ventricosa cell walls prepared for our studies are approximately 1 |im thick. The axial resolution of the confocal microscope under our imaging conditions is on the order of 0.25 Lim. From these images, it is clear that the C B D has access to all cellulose structures larger than the optical resolution of the microscope (~ 0.25 urn). In particular, it is likely that the C B D s have access to at least some elemental fibrils. Figure 3-10 shows the binding isotherm for FITC-labeled C B D c e x on disrupted V. ventricosa cellulose fibers. The use of fluorescently labeled protein permitted data to be collected at much lower protein concentrations than have been reported previously. The adsorption isotherm data was analyzed by nonlinear regression, using the model which includes two classes of binding sites (Equation 3-7). The saturation capacity of V. ventricosa cellulose was determined independently using a mixture of labeled and unlabeled protein. Three parameters were regressed from the adsorption isotherm: high and low affinity constants and the fraction of high affinity sites: [NJ = 5.0 umol/g B M C C , KaR = 5.0 x 10 7 M " 1 , [Nn] = 1.2 umol/g B M C C , and KaL = 1-0 x 10 6 M " 1 . These binding parameters agree well with the values presented previously, determined using unlabeled C B D and B M C C . 92 Figure 3-9: V. ventricosa cellulose labeled with CBD C e x tagged with FITC. Images are of the mounted cellulose sheet stained with the fluorescein-labeled binding domain from the exoglucanase Cex (FITC-CBDcex) (Image A ) . The ca. 0.5 | i m fibers of packed microfibrils are evident. These fibers are stacked into lamella oriented at right angles. Several orthogonal layers make up each sheet. Image B shows a cross-section of the surface at the axis indicated in the figure. The sheet is approximately 1.0 fim thick. The image was collected with a 60X (N.A. 1.4) lens, and the scale bar represents 5 u.m. The images are the average of three successive scans at a zoom of 2. The confocal aperture on the BioRad 600 was set to 3. 93 Figure 3-10: Isotherm analysis of CBD binding to V ventricosa Binding isotherm at 30°C for C B D c e x on V. ventricosa cellulose in 50 m M phosphate buffer at p H 7.0, measured by the FJTC-CBDcex solution depletion method. The ascending isotherm is shown. Binding parameters derived from a two site model for binding are in good agreement with our earlier studies using unlabeled C B D and bacterial microcrystalline cellulose. About 85% of the binding has a high apparent affinity of about 5.0 x 10 7 M " 1 . The remaining 15% of the sites have a lower affinity of about 1 x 10 6 M " 1 . V. ventricosa cellulose binds about 6.2 umol CBDcex per gram of cellulose. Points are means of triplicate binding reactions. 94 vii) Reversibility of CBDcex adsorption to cellulose In order to use a C B D for protein immobilization, the desorption rate of the C B D must be very slow. To address this point, desorption (descending) isotherms were attempted. To desorb C B D from B M C C , the free protein in solution following adsorption is diluted by buffer exchange and re-equilibrated. Figure 3-6 shows the desorption results obtained where two points were selected with which decreasing concentrations of free protein were incubated overnight. If the C B D was bound to the cellulose in a reversible manner, each of these " jump-off points would have returned to the ascending isotherm trace following re-equilibration. Clearly, this is not the case. There is considerable hysteresis in the binding isotherm for C B D c e x to crystalline cellulose: the adsorption phase is characterized by a standard ascending hyperboloid, while in the desorption phase only slight amounts of protein are released from saturated B M C C , while no protein is released from lower surface-concentration jump-off points. A second, more sensitive technique for the detection of binding reversibility using FITC-tagged CBDcex was also used. With this approach, several tubes were prepared with constant amounts of B M C C and increasing concentrations of F I T C - C B D . After equilibration, the cellulose was washed three times in P B S and then re-equilibrated overnight. No fluorescence was found in the supernatant following re-equilibration. Thus, under these conditions, no CBDcex desorbed from the B M C C after binding within the limits of our fluorescent C B D detection method (i.e. limit of detection ca. 10 picogram). 95 Discussion In this chapter, we have examined the adsorption of CenA, its isolated binding domain (CBDcenA), and the binding domain isolated from Cex (CBD C ex)- In each case, initial slopes of adsorption isotherms were steep, indicative of a high affinity. Crystalline cellulose has a high capacity and high affinity for family II C B D s . Scatchard analysis of adsorption isotherms yielded plots that were nonlinear (concave upward). This deviation from linearity is usually attributed to multiple classes of binding sites or interaction between adsorbing species (negative cooperativity); graphical analysis alone cannot distinguish or resolve these possibilities (Klotz, 1989). However, confocal imaging of CBD-stained B M C C suggests the existence of at least two classes of binding sites. Thus, this effect must be included in a realistic adsorption model. However, effects from binding site overlap cannot be discounted. For adsorption of C B D s to crystalline cellulose, the substrate should be considered as an array of overlapping potential binding sites. The dimensions of the C B D s greatly exceed those of the repeating cellobiose lattice units exposed on the surface of the cellulose microfibrils. Thus, it is reasonable to assume that adsorption of C B D s involves interaction with more than one lattice unit. A s noted for the one-dimensional system studied by McGhee and von Hippie (1974), the concentration of free sites is a function not only of the number of ligands bound but also their distribution on the lattice. Unlike the one-dimensional case however, it is not possible to derive an analytical isotherm model based on conditional probability analysis. In the absence of definitive information as to the configuration of bound molecules, one can perform isotherm analysis at low ligand concentrations where the effects of site exclusion due to bound-ligand configuration can be ignored. Under these conditions, a relative association constant can be determined from the limiting slope of the adsorption data plotted in the form 1/[B] vs. 1/[F]. 96 Monte Carlo simulations revealed that this is appropriate for surface coverage below 30% saturation (data not shown). It is important to note that we have demonstrated that C B D s are essentially irreversibly adsorbed to microcrystalline cellulose. This is significant for at least two reasons. First in analyzing binding isotherms we have applied the Langmuir-type models to fit adsorption data. However, implicit in this model is the assumption of binding reversibility. Thus our analysis is only valid on the ascending isotherm: only an apparent association constant can be calculated. Secondly, implications of binding irreversibility to enzyme action on insoluble substrate must be considered. If enzymes adsorb irreversibly to cellulose, then apparent enzyme activity w i l l depend upon the ability of cellulases to sufficiently degrade proximal cellulose to facilitate their release, or cellulases must be free to diffuse at the surface of the cellulose substrate. Otherwise, once an enzyme had degraded susceptible proximal cellulosic linkages, its activity would be lost: surely an inefficient use of enzyme activity. In comparing isotherm results prepared with different batches of B M C C , we noted significant differences in the total capacity of the cellulose for C B D s . This is likely due to differences in the preparation (disruption) of the B M C C pellicles. Therefore, it is necessary to prepare a standard batch of B M C C in order to compare different C B D s . It would likely be beneficial (for groups studying cellulase adsorption) to prepare a large batch of "standard B M C C " , to be made available to all laboratories, in order to facilitate absolute comparison of binding affinities and protein capacities. It w i l l be necessary to investigate the adsorption of C B D s using other techniques to unequivocally select one binding model over another. In particular, the rearrangement of C B D s following binding to the cellulose substrate through surface diffusion (a phenomenon observed 97 for amylases on insoluble starch (Henis et al, 1988; Katchalski-Katzir et al, 1985), w i l l rule out models based on packing configurations and site exclusion. However, the application of C B D s for protein immobilization onto crystalline cellulose does not depend upon such a detailed understanding of C B D adsorption. We have shown in this chapter that C B D s have a high affinity for crystalline cellulose, that cellulose has a high specific capacity for C B D s , and that C B D C e x binds essentially irreversibly to crystalline cellulose. These characteristics make the application of C B D fusion proteins for immobilization of the fusion partner a very attractive strategy. In the following chapters, we w i l l investigate the potential for molecular rearrangement of adsorbed C B D s , and examine the application of C B D fusion protein technology for immobilization of growth factors for cell culture. 98 Chapter 4 CBDs Diffuse Across the Surface of Crystalline Cellulose Introduction Little is known about substrate accessibility on the surface of crystalline cellulose. Presumably the marked differences in the activities of endoglucanases on crystalline substrates relates in large part to their ability to disengage microfibrils from the crystalline array and thus access new sites. However, the processivity of the enzyme may also be important. For example, the Cellulomona fimi endoglucanases CenA, CenB and CenD display almost equal activity on Av ice l but their activities on highly crystalline B M C C vary by two orders of magnitude (Meinke etal., 1993). CBDcex and C B D C e n A are family U cellulose binding domains (Tomme et al, 1995). In the previous chapter, we demonstrated that C B D c e x and C B D C e n A do not dissociate from cellulose after binding; dilution of the free C B D , at otherwise constant conditions, does not result in desorption over time (Creagh et al., 1996). This suggests that the enzymes must freely diffuse across the substrate surface in order to gain access to susceptible bonds, since enzyme activity is enhanced by the presence of a C B D (Gilkes et al, 1988). To test this hypothesis, we w i l l use F R A P to directly measure the rate of C B D mobility on a cellulose surface. Enzymes or C B D s , labeled with a fluorescent tag, w i l l be adsorbed to sheets of crystalline cellulose microfibrils prepared from the cell walls of V. ventricosa. Following adsorption and extensive washing to remove unbound proteins, a small well-defined region of the surface-bound fluorescent molecules w i l l be irreversibly photobleached using a high intensity laser pulse. Recovery of 99 fluorescence in the bleached region wi l l then be monitored by C L S M to determine the diffusive mobility of the bound molecules. Furthermore, we w i l l use F R A P analysis to test the validity of an adsorption model based on a two-dimensional extension of the steric-exclusion theory of McGhee and von Hippel (McGhee and von Hippel, 1974) for C B D c e x adsorption to crystalline cellulose. Steric-exclusion theory assumes that adsorbed protein molecules are static, and thus molecular rearrangement following adsorption does not affect molecular packing at the interface. If this analysis shows that adsorbed C B D s are indeed mobile following binding, we may conclude that steric-exclusion effects do not need to be incorporated into the two-site isotherm model proposed in Chapter 3 for CBD-cellulose adsorption. 100 Results i) Photobleaching analysis of FITC-CBD on crystalline cellulose Figure 4-1 shows typical images recorded from F R A P analysis of FITC-labeled C B D c e x on V. ventricosa cellulose. Fluorescence intensity measurements of the surface just prior to bleaching (Figure 4-la) were used to normalize subsequent measurements to prebleach intensities. Gaussian-profile spots were bleached with a series of high intensity laser pulses in the pattern shown (Figure 4- lb) , and the fluorescence intensity recorded over time by successive imaging of the bleached spots and surrounding area. Approximately seven minutes after bleaching, substantial fluorescence recovery is evident in the bleached spots and at the interface between the bleached reference region and the surrounding unbleached area (Figure 4- lc) . Figure 4-2a shows the time profiles for fluorescence intensity at the center of the bleach spot, the unbleached region of the cellulose sheet, and the center of the bleached reference region. Under the monitoring conditions selected, background bleaching was less than 5% during recovery. The bleach spot recovery profiles were therefore analyzed directly without compensating for bleaching as a result of monitoring. Fluorescence recovery in the large bleached reference region was less than 2%, indicating that no highly diffusive species were present on the surface or in solution, and that significant chemical recovery of the bleached FITC was not occurring (Stout and Axelrod, 1995). 101 1 Figure 4-1: Image sequence collected during FRAP analysis Prior to bleaching, an image is collected of the region to be bleached (zoom = 2) (a). This image is analyzed to obtain the initial fluorescence intensities. The C L S M is then electronically zoomed (zoom = 8) so that only a small region of the surface is illuminated during laser scanning. One scan is performed at this high zoom to produce a large bleached reference region (b). The C L S M zoom is then returned to 2, and six bleached spots are rapidly produced with six successive 100 msec laser exposures (b). Fluorescence intensity is monitored for several minutes until recovery is greater than 95% complete (c). Surface diffusion coefficients and mobile fractions were determined by nonlinear regression of F R A P data with the analytical solution developed by Axelrod for the 1-D radial diffusion equation applied to a bleach spot with a Gaussian intensity profile (Axelrod et al, 1976). 102 OO He) n 1 fit) = I 4-1 n=0 l + n l + ( — ) L T* J where f(t) is the normalized fluorescence intensity at the bleach spot center, t is time, x d is the characteristic diffusion time for the molecular species, and K, the bleach rate constant, is related to the sensitivity of the system to bleaching. The measured fluorescence intensities were normalized by the fluorescence intensity recorded just prior to bleaching. The first twenty terms in the series given in Equation 4-1 were used to regress Td and K to normalized F R A P recovery curves, such as that shown in Figure 4-2. Several initial guesses were used for each fitting to ensure unique convergence of parameters during fitting. 103 200 40 + 20 - I E E E E Z Z Z ^ ^ 0 100 200 300 400 T ime Post Bleaching (sec) Figure 4-2: Photobleaching analysis of FITC-CBD on V. ventricosa Time profiles of the fluorescence signal for the bleach spot center (x), the unbleached region of the cellulose surface (o) and the center of the bleached reference region (•). Less that 5% background bleaching occurred during recovery monitoring. Less than 2% of the bleached fluorescence recovered in the reference region during monitoring. 104 The diffusion coefficient is related to the estimated characteristic diffusion time x d by D = 4-2 where co, the half width of the Gaussian profile (at e2 times the spot profile depth), was obtained by regression of the initial bleach spot profile. The mobile fraction, R, was determined from the long-time recovery intensity by where F(» ) is the effective infinite-time recovery, F(0) the fluorescence just after bleaching, and F k ( i ) the fluorescence intensity prior to bleaching. Figure 4-3 shows normalized F R A P results for C B D C e x at 60% maximal surface coverage. Under these conditions, the 2-D diffusion coefficient for C B D c e x on crystalline cellulose is 6.0 ± 0.9 x 10"'1 cm 2/sec. This diffusion coefficient is more than 4 orders of magnitude slower than the free solution diffusion rate of 10"6 cm 2/sec estimated from the Einstein equation for a globular protein with a mean diameter of 3 nm. If diffusion is strictly stochastic (i.e. no preferred direction or orientation) the measured or calculated diffusion rate corresponds to a cellobiose unit-cell transit time of approximately 0.18 msec. R = 4-3 105 1.2 0.2 + 0 H 1 1 1 1 1 0 100 200 300 400 Time Post Bleaching (sec) Figure 4-3: Photobleaching analysis of FITC-CBD on V. ventricosa This is a typical recovery profile used for the estimation of the diffusion coefficient and the mobile fraction of CBDc e x at a surface coverage density of ca. 60%. Fluorescence intensity is normalized by the pre-bleach fluorescence signal. Under these conditions, the diffusion rate for CBDcex on crystalline cellulose is 6.0 ± 0.9 x 10" cm /sec. The mobile fraction of C B D C e x is 70% ± 5%. 106 Based on the maximum recovery of fluorescence, the mobile fraction of C B D c e x on the crystalline cellulose surface is 70% ± 5%. Therefore, on the time scale of these experiments, the majority of the C B D adsorbed to the lattice surface is mobile. F R A P analysis repeated on cellulose sheets stored in buffer at 4°C for 48 hours, yielded similar diffusion parameter estimates. There were no obvious changes in the morphology of the cellulose surface or microfibril packing with incubation time, indicating that the C B D had not altered the cellulose structure. Control experiments were performed to examine the effect of void spaces, and the resulting potential for hindered diffusion due to molecular sieving within the cellulose fibril network of the V. ventricosa cellulose sheets. FITC-labeled myoglobin was prepared as previously described. Myoglobin was selected because it is similar to C B D c e x in size (17.5 kDa) and has no measurable affinity for crystalline cellulose. Mounted cellulose sheets were incubated for 4 hours with FITC-labeled myoglobin at a molar concentration 5 times the saturation level for C B D C e x , and then washed in 50 m M phosphate buffer. No increase in cellulose surface fluorescence was observed, indicating that no FITC-myoglobin was bound to, or trapped within, the microcrystalline cellulose fibril network. Identical results were obtained when the cellulose sheet was preincubated with unlabelled C B D c e x , indicating that the C B D s did not modify the cellulose microfibril structure. F R A P experiments performed with unwashed cellulose sheets containing free FITC-myoglobin, gave fluorescence recovery rates at least 3 orders of magnitude faster than those observed for C B D s bound to the cellulose surface. This shows that the fibril network of the V. ventricosa cellulose sheet imposes little hindrance to solution diffusion of the labeled protein molecule, presumably because the void space is made up of pores much larger than individual protein molecules, and thus that the cellulose fiber matrix entraps very little protein. 107 A series of F R A P experiments were performed with three different objective lenses (60, 40 and 20X) to create a range of initial bleach spot diameters, to determine i f the observed fluorescence recovery for bound C B D C e x in F R A P experiments was due to surface diffusion or exchange between bound and unbound C B D s . For a diffusion-limited process, the characteristic fluorescence recovery time scales with the square of the half-width of the initial bleach spot diameter (see Equation 4-2). For a recovery process dominated by exchange from solution, two possible rate-limiting cases must be considered. If the desorption step is rate limiting, x d w i l l be independent of CO. If the desorption step is not rate limiting, Td w i l l scale linearly with co2 and the slope w i l l yield a diffusion coefficient on the order of 10"6 to 10"7 cm 2/sec (i.e. the solution diffusion rate of the protein). As expected for a diffusion-limited process, Td scales linearly with (02 (Figure 4-4). From Equation 4-2, the slope of the line in Figure 4-4 yields a diffusion coefficient of 3.0 x 10" cm /sec. This value is in good agreement with the diffusion coefficients calculated from the observed recovery curves, and is at least four orders of magnitude slower than the estimated diffusion rate of a small globular protein in solution. The dependence of the mobile fraction estimate on the initial bleach spot radius was also examined. Over a range of characteristic recovery times, the contribution to the fluorescence recovery of a relatively slow process would become more significant at larger spot sizes. A s a consequence, the estimated mobile fraction of molecules would increase as co decreases. However, in our experiments the mobile fraction of adsorbed C B D s was independent of the initial bleach spot size (data not shown). Therefore, a single characteristic time constant adequately characterizes the observed fluorescence recovery. The second-order dependence of recovery time on bleach spot size and the independence of the mobile fraction on the 108 characteristic recovery time, strongly support our contention that fluorescence recovery results from surface diffusion of C B D s adsorbed onto microcrystalline cellulose. ii) Surface diffusion rates for different cellulose binding proteins Table 4-1 presents recovery results for two different Cellulomonas fimi cellulases and their respective C B D s at 60% surface coverage. The exoglucanase Cex has little activity on crystalline cellulose (Gilkes et al, 1992); hence, enzymatic modification of the cellulose surface during the course of diffusion experiments was not a concern. The endoglucanase C e n A is moderately active on crystalline cellulose. A catalytically-inactive mutant of CenA was therefore used to prevent surface degradation (Damude et al, 1995). This mutant binds substrate with wild-type affinity, but is unable to cleave substrate because the acid catalyst Asp252 is mutated to alanine. Diffusion coefficients and mobile fractions are presented for equivalent molar concentrations of protein. In both cases, the whole enzyme has a higher diffusion rate than the isolated binding domain, with Cex having a somewhat higher diffusion rate than CenA. The mobile fraction of C e n A is about 85%, compared to 70% for Cex. The mobile fractions appear to be a function of the C B D domain and do not depend upon whether the domain is isolated or part of the enzyme. 109 600 (Bleach Spot Half-Width)2 (mm2) Figure 4-4: Dependence of recovery time on bleach spot diameter A series of F R A P experiments performed with three different objective lens magnifications (60 [•] , 40 [ • ] and 20 [•] X ) were used to create a range of initial bleach spot diameters. The estimated characteristic recovery time is plotted against the initial bleach spot radius squared. A linear relationship is observed, as expected for a diffusive process. The slope of the fitted line 11 2 yields a diffusion coefficient estimate of ~ 3.0 x 10" cm /sec. This value is in good agreement with the diffusion coefficient for CBDcex on crystalline cellulose, determined using nonlinear regression fitting of Axelrod's series solution to the measured recovery curve. 110 Molecule Diffusion Coefficient (cm2/sec) Mobile Fraction CenA 2.9 ±0.5 (10"n) 0.85 ± .07 CBDcenA 1.9±0.4(10" n ) 0.89 ± .07 Cex 4.1±0.5(10" n ) 0.65 ± .05 CBDcex 3.1 ±0.4(10" H ) 0.65 ± .06 Table 4-1: Diffusion coefficients for CenA, Cex and their isolated C B D s F R A P results for two different Cellulomonas fimi cellulases and their respective C B D s at 60% surface coverage. Diffusion coefficients and mobile fractions are presented for equivalent molar concentrations of protein. In both cases, the whole enzyme has a significantly higher diffusion rate than the isolated binding domain. The mobile fractions appear to be a function of the C B D domain and do not depend upon whether the domain is isolated or part of the intact enzyme. iii) Surface diffusion rate as a function of CBD surface coverage density Figure 4-5 shows diffusion coefficients and mobile fractions regressed from F R A P measurements of C B D c e x on V. ventricosa cellulose at various fractions of the maximal surface coverage (r/rmax). Measurements were carried out at 25°C in 50 m M phosphate buffer (pH 7.0). Measured binding isotherms for C B D s on the prepared sheets were used to estimate fractional surface coverage densities. The maximal protein loading was 0.4 nmol C B D c e x per c m 2 cellulose sheet. The average sheet thickness was approximately 1.0 urn, as determined by imaging several cross-sections using a confocal microscope. Each sheet therefore represents a total cellulose I l l volume of approximately 1.2 x 10"5 c m 3 of cellulose, or approximately 147 jLLg of cellulose per c m 2 of cellulose sheet, based on a crystalline cellulose density of 1.5 g/cm 3 . These results give a binding capacity of 5.5 | imol C B D per gram of V. ventricosa cellulose. This capacity agrees well with values from isotherms prepared using disrupted cellulose sheets, indicating that most of the surface of the undisrupted sheet is available for binding. The diffusion rate of C B D c e x increases with surface coverage up to a r / r m a x of ~ 0.9, after which the estimated diffusion rate decreases as the surface becomes saturated (Figure 4-5). A t low surface coverage the diffusion rate is about 3.0 x 10"" cm 2/sec, increasing to a maximum of about 1.2 x 10"10 cm 2/sec at r / r m a x of ~ 0.9. The mobile fraction of C B D C e x also increases slightly as a function of 1 7 r m a x (Figure 4-5 inset). A t low surface coverage the mobile fraction is approximately 60%. A t high surface coverage density (i.e. 80% r m a x ) , the mobile fraction reaches a maximum of about 85%. 112 C M 1.6E-10 1.4E-10 °> 1.2E-10 <± 1.0E-10 •e " 8.0E-11 g CO 5 6.0E-11 4.0E-11 2.0E-11 0.0E+00 1 .2 0.9 B £ 0.8 5 0.7 0.6 0.5 0 < > - 1 • I + 0.2 0.4 0.6 r / T m a x 0.8 Figure 4-5: Surface diffusion rate as a function of bound CBD concentration Diffusion coefficients as a function of the maximal surface coverage r/rmax. Points are means of 12 individual-spot F R A P analyses, error bars show ± 1 standard error from the mean. The inset shows the estimated mobile fraction of C B D C e x as a function of r/rmax. 113 iv) Surface diffusion rate as a function of temperature To date, no systematic examination of protein surface diffusion as a function of temperature had been performed. To address this, we added an objective lens heater to the C L S M so that temperature studies could be performed for C B D C e x diffusion on V. ventricosa cellulose. F R A P analysis was performed at increasing temperatures from 19°C to 41 °C in 2 degree increments (Figure 4-6) (r/rm a x ~ 90%) Hysteresis was investigated by performing F R A P measurements at 37, 29 and 27°C as the cellulose sheet cooled. Diffusion rates generally increased with increasing temperature. There was no apparent hysteresis in the C B D diffusion rate dependence on temperature. The inset in Figure 4-6 shows the dependence of the mobile fraction on temperature. The mobile fraction is independent of temperature, suggesting that the surface immobile fraction is dependent on the cellulose surface properties and not the energetics of diffusive jumps. A n Arrhenius law equation for surface diffusion on metals has been suggested by Gomer (1990): D = D0 e x p ( - £ „ / kT) 4-4 where Ea is the activation energy for diffusive motion, k the Boltzman constant, and D 0 is the thermodynamic prefactor. The significance of the prefactor is unclear in the CBD-cellulose system, because the temperature dependence of protein folding and solvent phase limit the extrapolation of diffusivity estimates from Equation 4-4. In general, the activation energy term and D0 are functions of temperature and surface coverage (Gomer, 1990). Thus, linear plots of ln(D) versus 1/kT can be expected over only limited temperature ranges. A n Arrhenius plot for the diffusion rate dependence on temperature is shown in Figure 4-7. 114 20 25 30 35 T emperature (C) 40 Figure 4-6: Diffusion coefficient as a function of temperature F R A P analysis was performed at increasing temperatures from 19°C to 41°C in 2 degree increments. The inset shows the effect of temperature on the mobile fraction of molecules. The mobile fraction is insensitive to the temperature. 115 Figure 4-7: Arrhenius plot for CBD C e x binding to V. ventricosa cellulose A linear plot of ln(D) versus l / k b T was obtained over the temperature range studied. Linear regression of the Arrhenius model yielded E a = 45 kJ/mol 'K, D 0 = 0.008 cm 2/sec. 116 Discussion Both CBDcex and C B D c e n A are family n cellulose binding domains (Tomme et al, 1995). CBDcex binds irreversibly to crystalline cellulose; dilution of the free C B D , at otherwise constant conditions, does not result in desorption over time (Creagh et al, 1996). How then does the bound enzyme find available substrate when it is distributed across the cellulose surface? Our F R A P results indicate that the irreversibly adsorbed enzyme finds reactive (3-1,4-glucopyranoside linkages by diffusing in two dimensions across the cellulose surface. Surface diffusivities of Cex and the inactive mutant of C e n A are similar; both diffuse approximately 30% faster than their respective isolated binding domain. The observed surface mobility of adsorbed C B D brings into question the validity of assuming that a portion of the nonlinearity in the Scatchard plot for C B D c e x adsorption to B M C C is based on steric exclusion of potential binding sites, as suggested by the two-dimensional extension of the steric-exclusion theory of McGhee and von Hippel (von Hippel and McGhee, 1972). Steric-exclusion theory assumes that adsorbed protein molecules are static, which is not supported by our F R A P results. A large fraction of bound C B D molecules are mobile, and can thus redistribute on the surface so that binding-site exclusion does not occur and close packing of adsorbed C B D s is possible. Therefore, we conclude that a two-site adsorption model is more appropriate to explain CBD-cellulose adsorption data. Several groups have noted a concentration dependence of diffusion coefficients for proteins in bi l ipid membranes (e.g. Abney et al, 1989; Minton, 1989) or bound nonspecifically at surfaces (Tilton et al, 1990). In each of these studies, the rate of surface diffusion decreased with increasing concentration of protein. For single sorbate diffusion on a homogeneous surface containing a single class of adsorption sites, these results are supported by theory which predicts 117 near zero order dependence at low surface coverage, with a strong decrease in surface diffusivity as the sorbate surface concentration approaches the jamming limit (i.e. surface saturation) (Scalettar et al, 1988). In accordance with this simple theory, measured surface diffusivities for CBDcex on crystalline cellulose (Figure 4-5) are insensitive to surface concentration at r / r m a x < 0.4. A marked drop in the diffusion coefficient is also observed with increasing surface coverage near surface saturation. However, in contrast to theory, we observe an increase in the diffusion coefficient with increasing surface coverage over the range of 0.4 < r / r m a x < 0.9 . Clearly, this system is not well described by the simple model of a single self-diffusing species on a homogeneous crystalline lattice. The failure to capture the complex C B D diffusion rate dependence on T with existing simple models based on a single diffusing species on a homogeneous binding surface, suggests that the crystalline cellulose surface presents a heterogeneous array of binding sites. Adsorption isotherms for C B D c e x on crystalline cellulose are well fit by a model which recognizes two distinct classes of binding sites on the cellulose surface (Creagh et al, 1996). F R A P analysis measures the mean self-diffusion rate. The increase in C B D diffusion rate observed with increasing I 7 r m a x could therefore be the result of averaging between a self-diffusion rate of C B D s on high affinity sites, diffusing at ca. 3 x 10"" cm 2/sec, and an increasing fraction of C B D s bound at lower affinity sites, diffusing at a much higher rate. This interpretation is consistent with the two-site model for adsorption. The observed results can be reproduced in simulations in which 20% of binding interactions are of a lower-affinity type, with a second diffusion coefficient two orders of magnitude greater than that for proteins bound to the high affinity sites. This higher rate is in the same range as that reported for B S A adsorbed nonspecifically to a polymethylmethacrylate fi lm (Tilton et al, 1990). 118 Geometric considerations of the lamella structure of the V. ventricosa cell wall , indicate that some fraction of bound C B D s may move axially with respect to the scanning laser probe. Electron microscopy of C B D s absorbed at low surface coverage suggests that C B D s have a preference for crystal edges or for one of the crystalline faces of the cellulose microfibril (Gilkes et al., 1993). Thus, i f the C B D has a preference for the 220 crystal plane, preferentially oriented parallel to the laser scanning axis, the increase in diffusion coefficient with r /T m a x may be a result of the unequal partitioning of C B D s between the two crystal faces at lower r/rmax. Molecules which diffuse on surfaces parallel to the laser scanning axis w i l l appear to diffuse slower, because translation along the laser axis is not observable in our experiments (although fluorescence intensity is). Our experiments do not allow the determination of whether there is diffusion direction anisotropy resulting, for instance, from the migration of C B D s along the cellulose fiber axis. Relatively little can be said about the nature of the immobile fraction of C B D molecules. The mobile fraction of bound C B D molecules was ca. 70%, and increased only slightly with increasing r/rmax. The immobile species may be due to the existence of sites which promote a very slow diffusion rate, or to the trapping of adsorbed C B D s on chain ends or discontinuities in the cellulose crystal. Since the spacing of accessible cleavage sites on the cellulose surface is not known, we cannot determine unequivocally whether or not the rate of surface diffusion limits cellulase activity. However, based on our measurements this seems unlikely. A t the diffusion rates reported here, the C B D w i l l traverse several hundred lattice units on the cellulose crystal in one minute. C e n A has only moderate activity on crystalline cellulose, with 0.23 moles of reducing sugar being released per mole of enzyme per minute for B M C C degradation (Meinke et al., 119 1993). Other C. fimi cellulases are more active on crystalline cellulose substrate, with turnover rates up to ca. 10.0 moles of reducing sugar per mole of enzyme per minute. These low rates suggest that surface diffusion of C B D s does not limit substrate catalysis. The binding energies for the high and low affinity binding of C B D C e x on B M C C are -44.5 and -34.5 kJ/mol respectively (Creagh et al, 1996). These values are of the same order as the diffusion activation energy (E a) 45 kJ/mol. Surface mobility of the protein is believed to occur because at any instant a portion of CBD-cellulose contacts are broken. The sequential breaking and reassociation of interactions over the entire binding face results in the 2-D mobility of the protein. Thus the jump activation energy may only represent a small fraction of the true molecular desorption energy. Indeed, the effective irreversibility of the C B D adsorption onto cellulose, suggests that the C B D is making several contacts which must be sequentially broken to facilitate surface diffusion, and that the true Gibbs free energy of desorption is some large multiple of the jump activation energy. 120 Chapter 5 Production and Bioactivity of CBD-cytokine Fusion Proteins Introduction In previous chapters we investigated some of the properties of C B D s which may impact the utility of C B D fusion protein technology for cytokine immobilization. We have demonstrated that C B D s bind with high affinity to crystalline cellulose, and that adsorption is irreversible with respect to protein dilution in the solution phase. Furthermore, F R A P analysis has revealed that cellulose-bound C B D s diffuse in 2-D on the cellulose surface. These properties of C B D s strongly suggest that a CBD-cytokine fusion protein strategy can be used to localize cytokines to cellulose extracellular matrices. To be effective, a CBD-cytokine must bind to cellulose with the high affinity of the C B D , while retaining the bioactivity of the cytokine fusion partner. Preliminary investigations demonstrated the potential for CBD-mediated growth factor immobilization (Greenwood, 1993). However, that study identified several problems with the approach. First and foremost was the difficulty expressing intact C B D - I L 2 in E. Coli. We address this issue in two ways. First, we use an alternative fusion partner: murine IL-3. The gene fragment was integrated into a new expression vector developed in the laboratory of the Cellulase Group at U B C (Roger Graham, personal communication). Second, the C B D - I L 2 gene fragment was transferred into an animal cell host (COS) (Judy Alimont i , unpublished results). The bioactivity and expression of these fusions is investigated to evaluate the feasibility and efficacy of C B D fusion protein mediated growth factor immobilization. Specific questions such as vector design for purification, expression yield optimization and fusion protein bioactivity are addressed. 121 The characterization of a genetic fusion of the C B D from the endoglucanase CenA (CBDcenA) with murine interleukin-3 (IL-3) is used to demonstrate the feasibility of C B D -mediated growth factor localization to cellulose. IL-3 is important in a number of cellular responses. It has a high affinity for a number of extracellular matrix components (Roberts et al, 1988), suggesting that cells normally interact with ECM-presented IL-3. The C B D - I L 3 fusion is expressed in E. Coli. This system wi l l serve as a model to investigate the expression of C B D -cytokine fusion proteins in bacterial hosts. The C B D - I L 2 construct is expressed transiently in the mammalian cell line C O S (J. Al imont i , unpublished results). In a collaboration with J. Alimonti , the bioactivity of Avicel-bound C B D -IL2 is tested with the IL-2-dependent cell line C T L L . Furthermore, the CBD-DL2 fusion is used to investigate the properties of CBD-cytokine fusion proteins expressed in a mammalian cell host, and how this expression system affects the cellulose binding properties of the fusion protein. 122 Results (I) A CBD Fusion Protein for the Cellulose-Localization of mIL-3 i) Vector Design for Expression of CBD-IL3 in E. Coli Deletion analysis and anti-peptide antibody mapping of mIL-3 shows that the removal of 16 N-terminal or 20 C-terminal amino acids results in very little loss of IL-3 bioactivity (Ziltener et al, 1988). Thus, there was no obvious rational for selecting between an N or C-terminal fusion of a C B D to IL-3. A convenient vector (pTug9080) for the insertion of the IL-3 gene at the C -terminal of C B D c e n A was available and therefore used for these studies. Previous experience with the expression of C B D - I L 2 in E. coli highlighted two potential difficulties (Greenwood, 1993): proteolysis in the linker region between the domains of C B D fusion proteins, and the need to denature the C B D fusion to release it from cellulose during purification. Figure 5-1 shows the expression vector for C B D - I L 3 with its constituent components: C. fimi leader sequence (LP) for export of the protein product, CBDcenA gene fragment, a linker peptide composed of the P/T region from CenA, a Factor X a cleavage site for separation of the C B D domain from the IL-3 domain, the gene coding for mIL-3, and finally a 6 histidine affinity tail (H6) on the C-terminus to facilitate purification (R. Graham, unpublished results). Wi th C B D at the N-terminal of the IL-3 and the H6 tail at the C-terminal, the IL-3 is sandwiched between two affinity tags. This is intended to permit the purification of the CBD-EL3 fusion protein to homogeneity, free of contaminating degradation products. The fusion protein is first isolated by binding to a Ni-Agarose column (via the His-tag). After elution from the N i column, this step should yield two species: full length C B D - I L 3 and IL-3 degradation products derived from proteolysis of the fusion protein. For use in immobilized cytokine experiments, the eluate from the N i columns is bound to cellulose and then washed extensively with P B S . This approach 123 should produce a homogeneous cellulose-bound sample consisting of only CBD-TL3. A n y free IL-3 resulting from cleavage in the linker region of the hybrid protein, would contain only the His tag and so would not bind to the cellulose. (B) F X a site Ip CBD PT mlL3 H6 c Figure 5-1: Vector Design for Expression of CBD-IL3 in E. Coli (A) The vector used the plasmid pTug9080, which contained the gene for kanamycin resistance (kan). (B) The gene was made up of a C. fimi leader sequence (lp) for export of the protein product to the periplasm of E. coli., the C B D C e n A gene fragment (CBD) , a linker peptide composed of the PT region from CenA, a Factor X a cleavage site ( F X a ) for separation of the C B D domain from the IL-3 domain, the c D N A coding for mIL-3, and finally a 6 histidine C -terminus tag for the purification of the fusion protein on Ni-agarose (H6). 124 ii) Expression of CBD-IL3 in E. Coli E. coli (JM101) was transfected with pTug9080 using standard methods, and positive transformants were selected on kanamycin-supplemented agar plates. Colonies were selected at random from these plates and used directly to seed 100 ml shake flask cultures. These cultures were grown overnight at 37°C on shaker tables. Cells and supernatant were then separated by centrifugation and the supernate set aside for later bioassay. The cell pellet was resuspended in lysis buffer and then lysed by two passes through a French Press™ minicell. Analysis by S D S - P A G E indicates that the majority of product protein was cell associated (data not shown). Figure 5-2 shows a Coomassie blue stained S D S - P A G E gel for lysate fractions derived from batch E. coli cultures: induction with I P T G was performed after 5 hours of culture. Samples were bound to Avice l , which was then washed to remove nonbinding proteins and boiled for five minutes in S D S - P A G E loading buffer to solubilize the bound proteins. The gel demonstrates that the recombinant product protein (at ca. 31 kDa) accumulated during the culture following induction with IPTG. Note however, that a significant band (at ca. 21 kDa) also accumulated in the lysate. Samples from the growth curve (Figure 5-2A) were run on a separate gel, transferred to a nitrocellulose membrane and then probed with either sheep anti-IL3 or rabbit ant i -CBD. Several bands reacted with the anti-IL-3 antibody. The topmost band {ca. 33 kDa) is likely the unprocessed fusion protein (i.e. leader sequence intact). The major band (ca. 31 kDa) is the product band of interest (i.e. reacts with ant i-CBD and (Blot B) and anti-IL-3 (Blot C) . Note that this band accumulated during the culture. The anti-CBD blot demonstrates that degradation occurred in all expression trials. The major band on this blot (ca. 21 kDa) corresponds to the C B D / P T domain of the fusion protein, showing a significant degree of fusion protein 125 degradation. This result is consistent with those found for the expression of C B D - I L 2 (Greenwood, 1993). f a l Culture Time (hr) 2 4.3 5.2 6.1 6.4 7 7.3 7.6 8 — ? ~ = ^ S = » J * ^ Culture Time (hr) Culture Time (hr) 2 5.2 6.4 8 1 1 2 5.2 6.4 8 Figure 5-2: Expression of CBD-IL3 in E. coli S D S - P A G E analysis of protein from cell lysates. (A) Coomassie blue stained gel showing an Avicel-bound protein recovered from a batch culture time course. The gel demonstrates that product protein (ca. 31 kDa) accumulated during the culture following induction with IPTG. Note however, that a significant band (ca. 21 kDa) also accumulated. Western blots of every-other time point of the same batch culture timecourse as in A are also shown: rabbit anti-CBDcenA (Blot B) or sheep anti-IL-3 (Blot C). Several bands reacted with both antibodies. The topmost band (ca. 33 kDa) is likely the unprocessed fusion protein (i.e. leader sequence intact). The major band (ca. 31 kDa) is the product band of interest. The ant i-CBD probe blot demonstrates that degradation occurred, with the major band (ca. 21 kDa) (Blot A) corresponding to the C B D / P T domain of the fusion (i.e. this protein does not react with the anit-IL-3 probe). iii) CBD-IL3 is active when bound to cellulose The bioactivity of C B D - I L 3 present in E. coli lysate was tested using the IL-3-dependent cell line B6SutA (Figure 5-3). FL-3 bioactivity was determined in the crude lysate and the Avice l -126 bound C B D - I L 3 fraction. The bioactivity remaining in the lysate following binding to excess Avice l , was also assayed. The bioactivity in the crude lysate samples shows a nonmonotonic profile with maximum bioactivity at a dilution of about 1:200. The inhibitory effect is l ikely the result of cytotoxic components in the E. coli lysate. A t higher dilutions the bioactivity follows the expected dose-response profile. C B D - I L 3 bound to Avice l stimulated the proliferation of B6SutA cells in a dose-dependent fashion. There was no evidence of inhibition at high concentrations, indicating that E. Co/j'-derived cytotoxic products were not concentrated onto the cellulose. It is apparent that a significant amount of IL-3 bioactivity remained in the culture lysate following adsorption to Avice l . This finding is consistent with the results from the Western blot analysis showing multiple IL-3 bands. Thus, because of proteolysis and release of the C B D , only a fraction of the IL-3 bioactivity actually bound to the cellulose sorbent. Control samples of mIL-3 did not bind to Avice l cellulose, nor did Avice l affect the bioactivity of control mIL-3 (data not shown). 127 8.E+5 10 100 1000 10000 Sample Dilution Figure 5-3: Cell proliferation assay of CBD-IL3 activity Bioactivity was determined in the crude lysate and Avicel-bound C B D - I L 3 fractions using the IL-3 dependent cell line B6SutA. Wells were initially seeded with 2 x 105 cells/ml and then cells cultured for 48 hours. The bioactivity in the crude lysate (•), the bioactivity remaining in the culture lysate following binding to excess Avice l (x), the Avicel-bound bioactivity (•) and the dose-response profile for an IL-3 reference sample (A) are shown. 128 iv) Strain selection for expression of CBD-IL3 in E. Coli Because of the excessive degradation of the C B D - I L 3 noted in the E. coli strain JM101, a panel of other strains were tested as expression hosts (Figure 5-4). It was anticipated that protease deficient strains might express the C B D - I L 3 fusion with lower rates of degradation. Each of the selected strains was transformed with the vector, selected on kanamycin-supplemented agar plates, and cultured in shake flasks as before. After 8 hours culture, the cells were collected and lysed in the French Press™ in lysis buffer which contained pepstatin, P M S F and E D T A for protease inhibition. Two approaches were used for screening the strain transforms. First, samples were bound to Avice l and then probed with rabbit anti-mIL3 followed by goat anti-rabbit alkaline phosphatase. The Avice l was then washed and reacted with alkaline phosphatase substrate. The resulting enzyme product was then assayed colorimetrically at 405 nm. The highest levels of intact C B D - I L 3 were produced by TOPP5 and JM101. The second analysis used S D S - P A G E gels as before to determine the degree of product degradation (Figure 5-5). C B D - I L 3 from cell lysates was bound to Avice l and run as previously. Significant degradation was observed in all strains. A Western blot using ant i -CBD as a probe, confirmed that the major band on the Coomassie stained gels was indeed the C B D / P T degradation product (ca. 21 kDa). These results highlight the need for the dual affinity-tag approach for purifying this fusion protein. JM101 was selected for further work, because the expression levels and cell growth rates were among the highest of the strains tested. 129 < t— CO 0 0.5 1.5 OD405 Figure 5-4: Strain Selection for expression of CBD-IL3 in E. coli After 8 hours culture, cells were collected and lysed in the French Press™ minicell Samples of lysate were bound to Avice l , which was then probed with rabbit anti-mIL-3 followed by goat anti-rabbit alkaline phosphatase conjugate. The Avice l was then washed and reacted with alkaline phosphatase substrate. The resulting product was then quantified by absorbance at 405 nm. 130 Figure 5-5: Electrophoresis analysis of CBD-IL3 degradation produced in a variety of E. coli strains After 8 hours culture, cells were collected and lysed. C B D - I L 3 from the cell lysates was bound to Avice l and run on S D S - P A G E gels. Significant product degradation was observed in the samples from all strains. A Western blot using anti-CBD as a probe, confirmed that the major band on the Coomassie stained gels was the C B D / P T degradation product (ca. 21 kDa). v) Production of CBD-IL3 in fedbatch fermentation of E. coli JM101 A fedbatch process for the growth of E. coli expressing recombinant veloped in collaboration with D . Hasenwinkle (Hasenwinkle et al, 1997). The process achieved cell densities of 90 g dry cell weight ( D C W ) per liter of culture. Furthermore, the optimized fedbatch protocol yielded up to 8 g/litre of C B D c e x - A feedback control scheme was developed based on dissolved oxygen concentration measurements during the fermentation. The cellular oxygen uptake rate is a strong indicator of the availability of nutrient. Under starvation conditions, the respiration rate greatly decreases compared to nonsubstrate limiting growth conditions. 131 With a single limiting nutrient, a rapid decrease in respiration can be used to signal nutrient depletion. A supervisory control program was developed to recognize this oxygen concentration profile shift resulting from substrate exhaustion and to then trigger a glucose feed pump. Sufficient feedstock was added in each cycle to bring the glucose concentration in the bioreactor to about 0.5 g/litre. A critical modification to the feeding control routine was the addition of a 15 second delay following the feed request signal. The starvation condition causes cells to scavenge acetate from the medium back into the T C A cycle. This removes the major inhibitory metabolic byproduct from the culture media every feeding cycle. To exploit this fedbatch technology and examine proteolysis of CBD-JJL3 during fedbatch production, a culture run was performed with the recombinant JM101 E. coli strain. The exponential-phase cell growth rate (see Figure 5-6) was 0.22 h"1 . The glucose feed cycling closely followed the protocols developed previously (Hasenwinkle et al, 1997). However, the final cell density obtained was only 85 A6oo units, equivalent to approximately 40 g/litre D C W (less than half the expected value). Ce l l growth stalled shortly after the addition of I P T G (22.5 hours of culture). In contrast, induction did not significantly hinder growth in cultures expressing CBDcex alone. 132 90 5 10 15 20 25 CULTURE TIME (hr) Figure 5-6: Recombinant JM101 E. Coli growth during expression of CBD-IL3 in fedbatch fermentation C e l l growth rate (see inset figure) was 0.22 hr"1, comparable to that obtained in fedbatch cultures of TOPP5 expressing C B D C e x - Cel l growth stalled immediately following addition of I P T G at 22.5 hours of culture. S D S - P A G E analysis was performed on cellulose-bound protein from the culture lysate and supernatant samples (Figure 5-7). Time course samples were diluted to normalize them to an OD600 of TO. Final product protein levels were on the order of 10 mg per liter of final culture 133 volume for intact protein and 25 mg per liter degradation product. Strong induction is evident by the immediate appearance of C B D - I L 3 in the lysate and supernate following addition of I P T G (Figure 5-7: lane 4). Unfortunately, there is also an immediate appearance of lower molecular weight proteolysis degradation product. Following induction, there appears to be nearly equal amounts of intact protein and degradation product. The levels of both proteins increase in the lysate up to 1 hour after induction. A t that time the ratio of degradation product to intact protein increased, suggesting that the rate of recombinant protein synthesis had decreased while the rate of proteolysis remained constant. The appearance of C B D - I L 3 and degradation products in the supernatant also followed closely after the addition of IPTG to the fermentation. Again, almost equal levels of whole molecule and degradation product were found. 134 ( H ) Culture Time (hr) 12.5 18.7 21.4 22.2 23.3 23.5 24.1 24.5 25 45 31 21 14 ( g ) Culture Time (hr) 18.7 21.4 22.2 23.3 23.5 24.1 24.5 25 4 5 ' 31 21 141 Figure 5-7: Expression of CBD-IL3 in E. coli during fedbatch fermentation Coomassie stained S D S - P A G E analysis of Avicel-bound lysate (A) and supernatant samples (B). Samples were diluted to normalize them to an OD600 of 10. The major band at 21 kDa is the C B D - P / T released by proteolysis of the fusion protein. Lane 2 of Gel B shows an isolated CBDcenA standard 135 Discussion In this section we have presented results that demonstrate that CBD-EL3 bound to cellulose is bioactive indicating that immobilization of growth factors for cell cultures using C B D fusion protein technology is a viable approach. However, proteolysis of the C B D - I L 3 fusion protein was a serious problem for this expression system. The staining patterns on a n t i - C B D C e n A and anti-IL3 Western blots are similar to the results obtained for CBD-JJL2 (Greenwood, 1993). C B D fusion proteins appear to be susceptible to cleavage at the P/T l inker-CBD junction. Following this cleavage, the ladder pattern observed in the Western blots is likely the result of processive protease degradation of the SCF-P/T linker fragment. A wide range of conditions were tested in an attempt to reduce C B D - I L 3 degradation. These included: fermentor temperature, dissolved oxygen concentration during fermentation, protease inhibitor cocktails used during purification, cell lysis method, and I P T G induction protocol. None of the conditions tested significantly reduced proteolysis. The marked growth inhibition following induction with IPTG of the JM101 clone expressing C B D - I L 3 in fedbatch culture, is in direct contrast to our results reported for C B D C e x expression. In those experiments, cell growth was not inhibited following induction in a variety of induction protocols. This suggests that it was a property of the C B D - I L 3 fusion which caused cell death. Following expression of C B D - I L 2 in batch culture, Greenwood (1993) reported the localization of C B D - I L 2 to the membrane fraction of fractionated E. coli cells. This suggested that the protein was becoming trapped in the membrane during export. It is interesting to note the initial rapid appearance of C B D - I L 3 in the culture supernate following induction, with no subsequent accumulation. This may be the result of E. Coli inner membrane fouling as the cells exports the 136 overexpressed nonnative proteins. This aspect was not studied further here, but may make an interesting immunofluorescence microscopy study. The proteolysis of the C B D - I L 3 fusion protein during E. coli harvesting and purification, and the difficulty in renaturing fusion proteins eluted from Avice l , necessitated the inclusion of a second affinity tag on the C-terminus of the protein. Wi th this tag, the protein could first be bound to an alternate affinity matrix, eluted, and then bound to cellulose for use. The use of two tags would ensure that only intact fusion protein was bound to the cellulose matrix. The construct was modified to add a 6 histidine tag on the C-terminus of the C B D - I L 3 fusion. Under denaturing conditions designed to ensure exposure of the His6 tag, no C B D - I L 3 bound to a N i -sepharose matrix. Therefore, either the C-terminus of the fusion protein was truncated during synthesis and purification, or the expression vector 3' terminus had errors. It was determined that the expression vector did have errors, and would require a new round of genetic engineering and cloning to add the required His6 tag to the C B D - I L 3 fusion construct. Before this was initiated, other cytokine fusion proteins were investigated to determine the best fusion pair for further study. 137 (II) A CBD Fusion Protein for the Cellulose-Localization of mIL-2 i) CBD-IL2 is active when bound to cellulose IL-2 bioactivity was concentrated onto cellulose, directly from tissue culture supernatants from transient transfections of C O S cells with the C B D - I L 2 construct. The binding capacity of Av ice l was estimated to be at least 500 Units/mg (note: absolute estimates of bound protein mass are not possible because unpurified C O S culture supernatants were used for these studies). Only about half of the initial IL-2 bioactivity in the culture supernatant bound to an excess of cellulose, indicating some degradation or nonbinding variants of the C B D - I L 2 fusion. Native IL-2 does not bind to Av ice l cellulose (Greenwood, 1993). For cell cultures C B D - I L 2 was bound to Avice l to yield approximately 50% surface binding capacity saturation. A t a r/rmax of approximately 50%, approximately 65% of the soluble IL-2 bioactivity bound to Avice l . The Avice l was washed and allowed to equilibrate with P B S three times following binding. Only 10% of bound bioactivity was lost following the three washes. A three-fold serial dilution of CBD-IL2-loaded Avice l was then made into tissue culture wells and C T L L cells added. Every 24 hours a set of wells was sacrificed, the cells counted, and the IL-2 bioactivity released into solution analyzed. Figure 5-8 shows cell counts obtained over the duration of the culture. B y 24 hours culture, the controls without C B D - I L 2 (with or without Avicel) had stopped growing. In contrast, all other cultures grew vigorously over the first 24 hours, indicating the stimulatory effect of C B D - I L 2 . B y 48 hours of culture, the cell proliferation dependence on C B D - I L 2 dose is evident. A t each dose of C B D - I L 2 , the Avicel-bound and soluble C B D - I L 2 wells performed similarly with respect to cell proliferation, although soluble C B D - I L 2 wells had a slightly higher final cell counts. 138 3.0E+06 100 Culture Time (hrs) Figure 5-8: Cellulose-bound CBD-IL2 stimulation of CTLL cell proliferation IL-2 bioactivity was concentrated onto cellulose directly from tissue culture supernatants. A three-fold serial dilution series (100 U / m l • , 33 U / m l • , 11 U / m l A , 3.7 U / m l X, 1.2 U / m l •) of Avicel-bound CBD-DL2 (open symbols) or soluble IL-2 (solid symbols) was made into tissue culture wells. Every 24 hours a set of wells was sacrificed and the cells counted. B y 24 hours culture, the unstimulated control (*) and the blank Avice l control (*) stopped growing. B y 48 hours of culture, the cell proliferation dependence on C B D - I L 2 dose is evident. 139 ii) CBD-IL2 produced in animal cells binds reversibly to cellulose Figure 5-9 shows the measured bioactivity of soluble IL-2 during culture. The rate of IL-2 consumption is dependent upon initial IL-2 concentration. The consumption of available IL-2 continued for the entire course of the culture. Comparison of Figures 5-8 and 5-9 shows that cell growth ceased immediately following depletion of IL-2. IL-2 bioactivity in Avicel-bound C B D -IL2 wells is shown in Figure 5-10. The initial concentration of soluble IL-2 in these cultures was assumed to be 0.0 U / m l following binding and extensive washing of the Avice l . C B D - I L 2 bioactivity was released slowly from the cellulose into the culture supernate during the culture either by proteolysis of the bfusion or desorption of the C B D . As with the cultures with soluble C B D - I L 2 (without Avicel) , cell growth ceased upon the complete depletion of measurable soluble IL-2 bioactivity. 140 120 Culture Time (hrs) Figure 5-9: IL-2 bioactivity in cultures stimulated with soluble CBD-IL2 Soluble IL-2 bioactivity remaining in the culture supernatant of C T L L cultures (initial doses -100 U / m l • , 33 U / m l • , 11 U / m l • , 3.7 U / m l X, 1.2 U / m l •, 0.0 U / m l * ) was measured by recovering supernates at various times during the culture. The initial rate of IL-2 consumption depends upon IL-2 concentration, even though cell growth rates are comparable (Figure 5-8). The consumption of available IL-2 continued throughout the duration of the culture, and cell growth ceased immediately following depletion of IL2. 141 18 Culture Time (hrs) Figure 5-10: IL-2 bioactivity in the supernate of cultures stimulated with CBD-IL2 bound to Avicel C B D - I L 2 bioactivity was released from the cellulose (initial Avicel-loaded bioactivity - 100 U / m l • , 33 U / m l • , 11 U / m l • , 3.7 U / m l X, 1.2 U / m l •, 0.0 U / m l #) at a slow rate during the culture. The initial concentration of soluble C B D - I L 2 was assumed to be 0 U / m l following the final Av ice l wash. The soluble IL-2 bioactivity measured in Avicel-immobilized C B D - I L 2 cultures was never more than 30% of that found in the supernate of cultures stimulated with soluble C B D - I L 2 . 142 Discussion C B D - I L 2 initially bound to Avice l stimulates proliferation of the IL-2 dependent C T L L cell line. However, it is not clear from these studies i f the immobilized form of IL-2 is active, since C B D - I L 2 is released at a slow rate from Avice l cellulose into the culture supernate under cell culture conditions. This release is consistent with the lower binding affinity noted for C B D s produced in mammalian cells (Cellulase Group U B C , personal communication). It is now recognized that C B D s produced in animal cells are often glycosylated and that this glycosylation lowers the affinity of C B D s for cellulose. It is not clear whether C T L L cells are stimulated by cellulose-bound CBD-TL2. In both the soluble and Avicel-bound treatments, cell growth ceased as soon as soluble bioactivity concentrations fell below detectable levels. This suggests that, in this system, the cells are mainly responding to soluble C B D - I L 2 . The increase in IL-2 bioactivity during the initial phases of Av ice l cultures, suggests that the rate of C B D - I L 2 release from cellulose was higher than its consumption rate. The reversible binding of mammalian cell-produced C B D - I L 2 may therefore be of value in providing slow-release cytokine feeding kinetics. Such a tool could be used to stimulate immune responses through the sustained site-specific release of IL-2. Because IL-2 is a powerful homing and activation signal for T cells, the ability to provide a local supply of IL-2 offers several potential benefits to fight diseases (Hora et al., 1990). This approach should avoid the toxic effects observed with systemic treatments of high concentrations of soluble IL-2. 143 Evaluation of CBD-Mediated Cytokine Immobilization In this chapter, we have demonstrated that C B D s provide an effective means to immobilize cytokines for cell culture. However, there remain significant barriers to their practical application. First, the expression of intact fusion protein in E.Coli proved difficult. The problem stems from sensitivity of C B D fusion proteins to proteolytic degradation at the P/T linker. This is currently being addressed by mutagenesis studies and fragment sequencing to identify and remove sites for protease attack (Cellulase Group U B C , personal communications). It must be noted that these CBD-cytokine fusion proteins produced in E. Coli are stable following binding to cellulose. This suggests that the molecules themselves are stable, but are susceptible to as yet unidentified E. coli proteases. Second, C B D s expressed in animal cells have reduced long-term stability of the C B D -cellulose interaction. The identification and removal of these sites is an ongoing Ph.D. research project (Cellulase Group U . B . C . , personal communications). If glycosylation sites can successfully be removed from the C B D , expression of fusion proteins in animal cell hosts w i l l provide a viable means of producing factors which require specific post-translational processing. Difficulty encountered when eluting cytokine-CBD fusion proteins suggests that the C B D is not well suited as an affinity tag for purification of all proteins. Rather, the high affinity and essentially irreversible binding of E.Coli derived C B D fusions make C B D s an ideal approach for protein immobilization applications. Purification and proteolysis problems have motivated the design of a new expression strategy which employs a dual affinity tag. Although this scheme was not successfully demonstrated in this chapter, the design has been used for the expression of murine stem cell factor in E. coli (Doheny, 1996). 144 Chapter 6 CBD-SCF Adsorption to Cellulose Introduction Doheny (1996) demonstrated that a genetic fusion of C B D C e x and murine S C F ( C B D - S C F ) retained the functionality of both fusion partners, binding tightly to crystalline cellulose and stimulating SCF-dependent cell proliferation. The specific activity of soluble C B D - S C F was equivalent to that of S C F on a molar basis. Doheny's results, which are based on B6SutA cell proliferation bioassays, also showed that the bioactivity of C B D - S C F bound to B M C C is characterized by an ED50 of 65 p M , approximately one order of magnitude lower than that for soluble C B D - S C F at otherwise identical culturing conditions. The bioactive form of S C F is a dimer, (for a recent review see Broudy et al, 1997). The dimer forms a symmetric pair of binding faces, to which a pair of c-kit receptors bind and subsequently activate through cross-phosphorylation. S C F dimerization has an association 8 1 constant of about 3x10 M " . (Lu et al, 1995; Hsu et al, 1997). Using site specific mutagenesis, Hsu et al. (1997) demonstrated that substitution of residues on the putative dimerization interface significantly reduced molar specific activity. In contrast, engineering a cystine residue at the dimerization interface, designed to covalently link dimers, increased the specific activity 10-fold. This observation is consistent with the current belief that c-kit activation is driven by dimerized ligands. The enhanced bioactivity of the cellulose-adsorbed C B D - S C F , suggests that a large fraction of adsorbed molecules exist as a dimer, either with an adjacent bound C B D - S C F molecule or with an unbound factor at the fluid-surface interface. A t solution concentrations above the K<i (ca. 10"8 M ) for dimerization, C B D - S C F wi l l adsorb in its dimeric form to cellulose, which 145 provides a simple explanation for the observed bioactivity. However, Doheny also observed bioactivity when C B D - S C F was bound to the B M C C surface at concehntrations well below the S C F dimerization association constant. If dimerization is required for bioactivity, these data suggest that the K j for S C F dimerization of one or two cellulose-adsorbed C B D - S C F molecules is lower than that measured for S C F in solution. In solution, dimerization decreases the entropy of the molecular pair, by an amount proportional to the loss in translational and rotational degrees of freedom which results from the coupling of the two molecules. Thus, the entropy of mixing favors the monomer. Binding of C B D - S C F to the cellulose surface removes the entropic penalty of dimerization, since the molecule is confined to the surface. In general, this entropic gain would be offset by the added difficulty for two bound molecules to orient themselves favorably for dimerization. However, the structureless proline-threonine (P/T) linker permits considerable orientational freedom to the tethered S C F domain. Dimerization of a bound and a free C B D - S C F molecule should therefore be characterized by a K a higher than that in free solution by an amount equal to 1/2 the macromolecular entropy loss due to dimerization. The K a for dimerization of two bound C B D -S C F molecules should be greater still. A s shown in Chapter 3, adsorption of family II C B D s to crystalline cellulose is irreversible. Assuming that this is also true for C B D - S C F , dimerization of two surface-bound C B D - S C F monomers can therefore only occur through direct interaction by surface diffusion. This chapter focuses on characterization of the binding of C B D - S C F to B M C C , including analysis of binding reversibility. Biophysical properties of C B D - S C F which might affect its activity were examined. The composition of E. Co/i-derived C B D - S C F and its affinity for B M C C were characterized first. The susceptibility of some C B D fusion proteins to proteolytic degradation, made the 146 validation of sample composition crucial. Furthermore, the high affinity of S C F for dimerization may cause a portion of the cellulose-bound C B D - S C F to bind the degradation product S C F as a C B D - S C F : S C F heterodimer. This idea is consistent with results for CBD-alkaline phosphatase and CBD-|3-glucosidase fusion proteins, in which heterodimers bound to cellulose (Greenwood et al, 1992). L ike S C F , both alkaline phosphatase and p-glucosidase are dimers in their native state. Isotherm analysis was performed to estimate the binding affinity of C B D - S C F for B M C C cellulose. Also , the bioactivity and long term stability of C B D - S C F bound to B M C C under cell culture conditions was investigate. Finally, F R A P analysis was performed to measure the surface diffusivity of the C B D - S C F protein on B M C C . 147 Results i) Western blot analysis of CBD-SCF binding to BMCC The bifunctional structure of C B D - S C F complicates its biophysical characterization. Western blot analysis of purified C B D - S C F binding to B M C C is shown in Figure 6-1. Ge l A was labeled with rabbit anti-murine S C F . Gel B was labeled with rabbit a n t i - C B D C e x - The first three lanes are [1] proteins bound to cellulose; [2] soluble proteins after binding to B M C C ; [3] soluble proteins before binding. The "double tag" affinity purification scheme outlined previously was used for the purification of this C B D - S C F . In this method, only those molecules containing a His-6 domain are purified from lysates (i.e. C B D - S C F and the S C F domain from C B D - S C F fusion protein degradation). The degradation products from C B D - S C F inter-domain cleavage are evident in lane 3 of Ge l A . The sample contains two species: the purified C B D - S C F ( M W ~ 31 kDa), and a S C F degradation product ( M W - 2 1 kDa) resulting from proteolytic cleavage of the fusion protein at the C B D - P / T junction. Examination of blot B reveals that the sample contains only full length fusion protein and no free C B D , indicating that the double-tag purification system is effective and that degradation of the fusion protein occurred prior to purification on the Ni-sepharose column. Thus, the fusion is stable under the storage conditions used during these experiments: the C B D - S C F sample used for Figure 6-1 had been stored for 6 months at -70°C at 300 ug/ml. Lanes 2 and 3 of gel B demonstrate the binding of C B D - S C F to cellulose. Lane 2 reveals that nearly all CBD-containing molecules were concentrated onto the B M C C solid phase. Lane 1 of Ge l A shows that some free S C F domain also partitions to the cellulose. Control S C F does not bind to crystalline cellulose (data not shown). Therefore, it appears that the S C F domain of the intact C B D - S C F is interacting with a free S C F domain. A likely explanation for this is that 148 the 21 k D a S C F degradation product is interacting with bound C B D - S C F to form a heterodimer ( C B D - S C F : SCF) . Tests were conducted to determine wash conditions by which degradation product-free C B D -S C F could be prepared. Western blot analysis (gel A ) was performed on C B D - S C F bound to B M C C and washed under these conditions: [lane 4] high p H wash (pH 10.5, 50 m M Tris); [lane 5] low p H wash (pH 4.5, 100 m M acetate buffer); [lane 6] salt wash (pH 7.0, 50 m M phosphate buffer, 1 M NaCl ) ; [lane 7] double-distilled water wash; and [lane 8] m S C F control (pH 7.0, 50 m M phosphate buffer). The high and low p H washes removed most of the S C F from the C B D -S C F . However, these conditions also weakened the CBD-cellulose interaction (gel B) . The 1 M N a C l in P B S removed the majority of the free S C F (gel A - lane 6). Additionally, this wash preserved CBD-cellulose binding stability (gel B) . The double-distilled water wash did not disrupt S C F dimers or C B D - S C F binding to cellulose. This finding is inconsistent with expectations for the proposed hydrophobic-interaction-driven S C F dimerization reaction (Lu et al, 1995). In all future experiments, a 1 M N a C l in 50 m M phosphate buffer wash-step w i l l be used to remove non CBD-tagged S C F . This should ensure that the cellulose-adsorbed molecules are predominantly C B D - S C F (i.e. either the C B D - S C F monomer or the C B D - S C F : S C F - C B D homodimer). 149 CBDSCF Figure 6-1: CBD-SCF dimerizes with mSCF on the cellulose surface Western blot analysis was performed on samples bound to cellulose in 50 raM phosphate buffer and then washed under several different conditions. Blot A was probed with rabbit anti murine S C F . Blot B was probed with rabbit ant i -CBDc e x - The lanes were loaded as follows: [1] bound to cellulose; [2] soluble after binding; [3] before binding; [4] wash p H 10.5; [5] wash p H 4.5; [6] salt wash (1 M NaCl) ; [7] double-distilled water wash; and [8] m S C F (control). ii) CBD-SCF binds to BMCC with high affinity Figure 6-2 shows a binding isotherm for C B D - S C F on B M C C cellulose fibers. Isotherms were performed with FITC-tagged C B D - S C F . However, as was demonstrated in Figure 6-1, a significant amount of free S C F is present in the initial protein preparation due to proteolysis during purification. This makes it very difficult to estimate the initial concentration of C B D -S C F . Furthermore, the adsorption of non CBD-tagged S C F , present in C B D - S C F : S C F 150 heterodimers, makes it impossible to precisely quantitate an affinity constant for the C B D - S C F fusion protein. Thus, protein samples whose concentration is above the K a for S C F w i l l be comprised of C B D - S C F : S C F - C B D , C B D - S C F : S C F , and S C F : S C F . Therefore, an adsorption isotherm approach was developed in which only that FITC-tagged protein which remains bound to cellulose following a wash in 1 M N a C l (in 50 m M phosphate) is measured. In this way, a partition coefficient for C B D - S C F onto B M C C can be estimated, irregardless of the actual concentration of full length C B D - S C F in the protein sample. A series of dilutions of the initial protein stock solution (containing C B D - S C F and the non CBD-tagged S C F degradation product) was prepared. Each sample was then divided into two equal fractions: to one fraction was added B M C C in phosphate buffer, to the other was added an equal volume of buffer alone. The samples were equilibrated overnight at room temperature. A l l samples were then centrifuged (10,000 rpm for 25 minutes) and the supernatant collected. A large excess of Av ice l cellulose (~ 1000-fold excess adsorbent) was added to each supernatant sample and then the mixture equilibrated overnight at room temperature. The fluorescence of the protein bound to the Avice l particles was then measured in the Pandex 96-well plate fluorimeter. Washing steps with 1 N a C l in 50 m M phosphate (to remove any F I T C - S C F from the Avicel) were performed in the special vacuum plates of the Pandex assay system. Because only that protein which binds cellulose is measured with this assay approach, the partition coefficient of C B D - S C F to the B M C C adsorbent can be estimated without regard for the non CBD-tagged S C F contaminant in the initial sample. Binding data was analyzed linear regression. The estimated partition coefficient is 110 litre/gram B M C C adsorbent. This value is higher than the partition coefficient estimated from the data in Figure 3-1 for C B D c E N A (ca. 45 litre/gram B M C C ) , perhaps due to the potentially higher affinity of the double-CBD domain 151 C B D - S C F : S C F - C B D dimer, or to differences in the specific surface area of the B M C C preparation. 152 Figure 6-2: Binding isotherm for FITC-labeled CBD-SCF on BMCC Subsaturating concentrations of C B D - S C F were incubated with B M C C ( • ) . The inset shows a semilog plot of the isotherm data and fitted partition model. The solid line sows the partition coefficient fit of the initial slope. Points are means of triplicate binding reactions, C B D - S C F is reported in "arbitrary fluorescence units" of protein that remains bound to cellulose following a wash in 1 M N a C l in 50 m M phosphate buffer. 153 iii) CBD-SCF binding to crystalline cellulose is stable and irreversible The binding reversibility of the animal cell-produced C B D - I L 2 , although likely due to glycosylation of the C B D (Ong et al, 1994), and the dependence of C B D elution conditions on the fusion partner (Greenwood et al, 1992; Ong et al, 1991), make it important to verify the irreversibility of C B D - S C F association with cellulose. Furthermore, the degradation observed for the fusion protein during purification suggests that the molecule is relatively sensitive to proteolysis. Desorption of C B D - S C F or degradation of the fusion, thus releasing free S C F , would expose cells to a combination of soluble and matrix-bound signals. This would significantly impact the interpretation of cell culture experiments. The binding stability of C B D -S C F to cellulose was examined under storage conditions at 4°C in P B S , and during cell culture at 37°C in I M E M . C B D - S C F was bound to size-sieved Avice l cellulose (35 - 70 \xm mesh fraction) (r/rmax ~ 10%) and then washed in P B S containing 1 M N a C l , followed by two more washes in P B S . The washed cellulose was then resuspended in 100 u l of PBS and placed at 4°C in the dark. After 48 hours, the sample was centrifuged and the bioactivity in the supernate and Avicel-bound fractions estimated in cell proliferation assays (see Figure 7-1). The wash was found to contain less than l /10,000 t h of the Avicel-bound bioactivity. This is consistent with the high affinity and essentially irreversible binding of C B D c e x to cellulose, and indicates that the fusion protein is stable under these storage conditions. The stability of Avicel-bound C B D - S C F under cell culture conditions was confirmed using Transwell™ inserts in normal tissue culture plastic 24-well plates. These inserts contain a 0.44 | i m membrane which permits partitioning of the tissue culture well (see Figure 6-3). C B D - S C F was bound to Avicel-type cellulose (T/r m a x ~ 5%). The cellulose was then washed once in P B S 154 containing 1 M N a C l , followed by two more washes in P B S . The Avice l was placed in the Transwell™, thereby separating Avicel-bound C B D - S C F from B6SutA cells cultured in the bottom compartment, or added directly to the bottom compartment below the Transwel l™ membrane, so as to make contact with the test cells. After 48 hours in culture, cells were recovered and enumerated (Figure 6-3). Bioactivity was found only when the CBD-SCF-loaded Avice l and the cells were able to make direct contact. N o cell proliferation occurred in wells in which cells were separated from the Avice l . The sensitivity of B6SutA cells for S C F permitted the demonstration that less than 1/1000 t h of the Avicel-bound bioactivity could have been released over the 48 hour cell culture. The stability of C B D - S C F binding to cellulose in cell culture suggests that the fusion protein is not susceptible to proteolysis under cell cultures conditions. Thus, it can be assumed that cells interact solely with cellulose bound C B D - S C F during cell stimulation analysis. This simplifies interpretation of cell culture results and confirms that C B D - S C F does not stimulate cells through slow release from cellulose during cell culture, although active stripping of bound C B D - S C F by activated cells is not precluded by this analysis. Control wells with soluble mSCF, or soluble C B D - S C F placed above the membrane partition, demonstrated that these molecules could rapidly diffuse through the Transwell™ membrane insert. Control wells showed a response consistent with the dosage placed above the insert. Interestingly, control wells in which the cells were added into the Av ice l containing compartment above the membrane, grew to a very high density. These cells formed a packed mass around the Av ice l , which virtually filled the volume of the insert chamber. 155 S L F - C B D ( n g / m l ) Figure 6-3: CBD-SCF binds effectively irreversibly to cellulose under cell culture conditions Transwell™ inserts were used to partition normal tissue cell culture wells into two chambers separated by a 0.4 urn pore membrane. Avice l with bound C B D - S C F (following a high salt wash to remove S C F without the C B D tag) was placed in the upper chamber and B6SutA cells in the lower chamber (see inset figure). Following 48 hours culture, full bioactivity was found in the unseparated (•) wells while no bioactivity was measured in separated wells (•). 156 iv) CBD-SCF does not diffuse across the surface of crystalline cellulose We have hypothesized that growth factor localization through association with extracellular matrix components or presenting stromal cells, might be mimicked through the use of C B D technology. Furthermore, we have argued that covalent methods of growth factor immobilization cannot reproduce the lateral mobility noted for surface-presented growth factors in vivo (e.g. within the membrane of cells (Jacobson et al., 1976; Ljungquist et al, 1989) or through equilibrium association with extracellular matrix (Long et al, 1992; Schuppan and Ruhl , 1994). The surface mobility of C B D c e x on crystalline cellulose suggests that CBD-mediated cellulose association of fusion proteins might reproduce the lateral surface mobility of stromal cell-presented factors. Diffusion rates of receptors in the membranes of mammalian cells is on the same order as we have reported for C B D C e x (Ljungquist-Hoddelius et al, 1991). The diffusion rate for C B D - S C F on crystalline cellulose was measured using F R A P to better understand the surface dynamics of bound C B D - S C F . FITC-tagged C B D - S C F was incubated with prepared cellulose sheets for 30 minutes and then washed once in P B S containing 1 M N a C l and then rinsed twice in P B S (total washing time ~ 40 minutes). Immunofluorescence imaging confirmed that fluorescence was evenly distributed on the cellulose sheet. A t a surface loading of ~ 0.6 r/rmax, C B D - S C F does not diffuse under PBS-buffered conditions as measured using our F R A P technique (Figure 6-4). The diffusion rate is at least 2 orders of magnitude slower than that for the isolated C B D c e x domain. Furthermore, the mobile fraction is several-fold lower, indicating that C B D - S C F is essentially immobile over the time period (10 minutes) monitored here. F R A P analysis was carried out on C B D c e x to confirm that the observed immobility of C B D - S C F was not an artifact of cellulose preparation. 157 F R A P results have previously demonstrated that a dimeric form of C B D C e x (two C B D s joined by a proline-threonine linker region) is immobile when bound to V. ventricosa cellulose (data not shown). Since cellulose affinity is specific for the C B D , C B D - S C F dimers should be able to diffuse on the cellulose surface provided only one of the two C B D s associated with the dimer is specifically bound. However, i f both C B D s of the dimer are bound to cellulose, diffusion may be hindered by the inability of the two domains to move in the same trajectory. Our F R A P technique is not sufficiently sensitive to allow characterization of surface diffusion of bound C B D - S C F at total concentrations below the Kd for dimerization. However, the results from Western blot analysis suggest that the dimer, at least that between a bound and a free S C F molecule, can be disrupted by 1 M N a C l or by high pH. If either of these conditions also disrupt a C B D - S C F dimer in the bound state, F R A P analysis could then be used to determine the diffusivity of the C B D - S C F monomer. To investigate the effects of salt concentration on the surface-diffusion of C B D - S C F , the cellulose sheet was equilibrated with 1 M N a C l in P B S for 20 minutes following adsorption of C B D - S C F . The sheet was then mounted over the well in the microscope slide filled with P B S containing 1 M N a C l . F R A P measurements were performed for C B D c e x and C B D - S C F under these conditions (Figure 6-4). The diffusion rate and the mobile fraction were unchanged from that obtained in P B S alone for either molecule. Surface mobility as a function of p H was also tested. Solution p H below 7.0 could not be used because F ITC is not fluorescent under acidic conditions. The cellulose sheet was recovered from the microscope stage and re-equilibrated with 50 m M Tris buffer at p H 9.5 for 20 minutes. The sheet was remounted over the well in the microscope slide filled the Tris buffer. The diffusivities and mobile fractions changed for both C B D c e x and C B D - S C F (Figure 6-4). The 158 diffusion rate of C B D C e x decreased about 50% from that measured in P B S at p H 7.0. The mobile fraction increased slightly. Significantly, the C B D - S C F species was found to be mobile at this p H . The diffusion rate of the C B D - S C F was very similar to that of the isolated C B D C e x at p H 9.5 and the mobile fraction increased to about 50%. When combined with our results which show that the S C F interaction is disrupted at p H 9.5 (Figure 6-1), this indicates that adsorbed C B D -S C F exists predominantly as an immobile dimer, while the adsorbed monomer (prepared by disrupting S C F dimerization) is mobile. It is also possible that the observed effect isa result of changes in the conformation of the C B D - S C F molecule. Further experiments w i l l be required to conclusively identifiy the mechanism for C B D - S C F mobility at elevated p H . 159 3E-10 Si! C M E ^ 2E-10 i "o 8 | 1E-10 CO CO 0 CO CD 13 CO X 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Mobile Fraction CB Dcex + PBS High High Salt pH PBS High High Salt pH ft GO CO Q_ T3 CO 1 6 CBD-SCF Figure 6-4: CBD-SCF does not diffuse across the surface of crystalline cellulose CBDcex and C B D - S C F were adsorbed to V. ventricosa cellulose. F R A P analysis was performed under a variety of buffer conditions (PBS, PBS containing 1 M N a C l , 50 m M Tris buffer at p H 9.5) at a surface coverage density of ca. 30% at 25°C. The surface diffusivity and mobile fraction of C B D - S C F is significantly lower than that for C B D c e x -160 Discussion The partition coefficient of C B D - S C F for crystalline cellulose is consistent with that reported for other CBD C e x -con ta in ing fusion proteins (Gilkes et al, 1992; Ong et al, 1991). Furthermore, the formation of heterodimers of S C F and C B D - S C F is consistent with previous observations for CBD-alkal ine phosphatase and CBD-p-glucosidase fusion proteins (Greenwood et al. 1992). Clearly, the P/T linker affords sufficient conformational flexibility to permit macromolecular interactions at the cellulose interface. Cellulose washing conditions were determined for the elution of the S C F from the C B D - S C F , to facilitate the preparation of samples of cellulose-bound C B D - S C F free of the S C F degradation product. In general, elution conditions found for S C F from C B D - S C F on cellulose were consistent with literature data; L u et al. (1995) reported that low p H (4.5) or high p H (8.5) increased the apparent off-rate of the S C F dimer 100- and 10-fold respectively, while high salt ( 1 M NaCl) in P B S increased the apparent off-rate by ~ 25%. Each of these treatments removed S C F from C B D - S C F bound on cellulose. Binding of C B D - S C F to crystalline cellulose is irreversible with respect to protein dilution in the solution phase. A s a result, immobilization of S C F to cellulose via a CBDcex affinity tag provides a convenient method for retaining the factor in a perfusion culture environment in a surface-active form. Unlike more complex chemical conjugation methods for immobilizing cytokines, surface attachment using a C B D allows simple and precise control over the surface density of the factor, and therefore the potential to regulate cellular response. In addition, the attachment mechanism is highly specific, ensuring that all surface-localized S C F is presented to the solution phase in the same fully-active configuration. Irreversible cellulose-surface localization of C B D - S C F necessitates direct contact between factor-dependent cells and the cellulose surface for SCF-induced stimulation. Since the dimeric 161 form of surface-bound C B D - S C F is immobile, the magnitude and possibly the persistence of cellular response w i l l be dictated by the density of C B D - S C F dimers on the cellulose surface and the average cell surface area in direct contact with the cellulose. Regulation of accessible cellulose surface area in the culture environment is therefore important. In this work, B M C C microfibrils were used as a cellulose substrate. We have previously estimated the maximum surface area available for C B D binding on B M C C (Gilkes et al., 1992). For B M C C fibrils at maximum dispersion, approximately 1.3 x 10^ cm 2 /gram is be available for CBDcex (and presumably C B D - S C F ) binding. At 0.1 T m a x (i.e. -0.5 umole CBD-SCF/g ram cellulose), a u m 2 of accessible B M C C surface area wi l l contain approximately 2500 C B D - S C F molecules. A cell with a typical radius of 8 um has a minimum surface area of about 800 um^. Thus, for a cell to bind 50,000 receptors, less than 3% of the cell surface must be in direct contact with the cellulose microfibril (note: currently, there are no published estimates of the minimum number of c-kit receptors which must bind S C F in order for cell stimulation to occur). For the culture of factor dependent cells at 10 6 cells/ml, this would require about 0.2 c m 2 of cellulose surface area per ml , or 0.3 u.g B M C C / m l of culture which is easily achieved. Recall the three geometries of crystalline cellulose employed in this study: microfibril ( B M C C ) , planar (V. ventricosa) and particulate (Avicel). The effective irreversibility of C B D -S C F adsorption to cellulose implies that cells must make direct contact with the cellulose matrix for stimulation. Thus, the morphology of the cellulose may play a role in the rate of cell stimulation. For example, without cell motility, stimulation with B M C C microfibrils is limited by convective mixing, due to the very low diffusivity of the cellulose fibrils (i.e. the molecular weight of B M C C is » 10 6). 162 It is unclear how the application of the relatively large 50-u.m-sized Avice l particles w i l l affect cell stimulation. At low Avice l concentrations, cell encounters with S C F w i l l require cell motility. Because S C F is not released from the cellulose, cells cannot respond to a S C F gradient around the Avice l particle. Clearly, one must consider cell motility, cell adherence and bulk culture mixing when selecting the appropriate cellulose morphology and culture operation mode (e.g. mixed, static or perfusion). 163 Chapter 7 Analysis of the Bioactivity of CBD-SCF Introduction: This chapter compares the response of SCF-dependent cells to soluble versus cellulose-bound C B D - S C F stimulation. The responses considered included cell proliferation, cellulose adhesion, locomotion, chemotaxis and receptor activation. In general, the rate at which c-kit receptors encounter C B D - S C F on the cellulose surface might depend upon (a) the surface diffusion rate of the C B D - S C F , (b) the diffusion rate of c-kit receptors in the cell membrane, or (c) the speed of target cell migration across the cellulose surface (Figure 7-1). C B D - S C F depletion or concentration gradients w i l l form around a cell only i f the S C F consumption rate of the target cell differs from the supply rate. In Chapter 6 we demonstrated that the C B D - S C F surface diffusion rate on crystalline cellulose is less than 10" 1 2 cm 2/sec. Unassisted C B D - S C F surface diffusion can therefore be neglected during normal cell culture. In evaluating transport processes in the heterogeneous artificial-stroma culturing system, one must consider the large variation in length scales of the interactions which lead to receptor activation. A t the angstrom scale, short-range interactions of the immobilized factor with its receptor on the cell membrane are significant. Here, the details of the electrostatic interactions at the binding site, and orientation of the respective binding surfaces, become important. Determination of the kinetics of this process and whether it limits cell activation, requires knowledge of the encounter frequency between the growth factor and its receptor, and the fraction of encounters which lead to receptor binding. A s demonstrated by Northrop (1984), translational and rotational molecular diffusion processes at this scale may have significant 164 impact upon encounter frequencies, and may therefore be important in controlling reaction kinetics at the cell-cellulose interface. FIGURE 7-1: Potential interactions in cellulose-bound CBD-SCF cell stimulation The hypothesized sequence of events leading to the formation of activated patches of receptor/ligand complexes. Monomeric C B D - S C F forms stable homodimers on the cellulose surface (A). The C B D - S C F : S C F - C B D homodimer is immobile (B). In this state, ligand-receptor interaction is limited by the rate of membrane progression across the interface caused by cell migration (C), and the mobility of receptors in the cell membrane (D). Following initial c-kit interaction with ligand, a second receptor binds to the complex. The receptor then autophosphorylates (E). Following receptor activation, c-kit complexes aggregate by directing motion of bound C B D - S C F to form an activated patch of ligand-bound receptors (F), which activates signal transduction cascades (G). On the time-scale of our experiments, C B D - S C F binds effectively irreversibly to crystalline cellulose, and the adsorbed homodimer is relatively immobile on the cellulose surface under cell 165 culture conditions: once a dimer is formed on the cellulose, ligand/receptor encounter frequencies must be limited by cellular migration speeds and the diffusion rate of receptors in the cell membrane. However, because of large differences in length scale, a determining factor for gradient formation around B6SutA cells is likely the speed cell migration (i.e. consider the relative sizes, the C B D - S C F at ca. 4 nm versus the cell at ca. 15 | im). L ive cell microscopy w i l l therefore be used to observe cell behavior and estimate cell migration velocities, when cells are cultured with C B D - S C F bound to Avice l particles or V. ventricosa cellulose sheets. We w i l l perform growth curve analysis to determine the effects of cellulose-bound C B D - S C F on the ED50 and maximum cell growth rate of various cell types. Two different cellulose morphologies w i l l be used in cell proliferation experiments. Avice l w i l l be used because it provides a means of supplying C B D - S C F bioactivity on discrete 40-70 mm particles. Avice l w i l l also facilitate the investigation of cell adhesion and cell motility on stimulation. B M C C wi l l be used because it provides a disperse cellulose suspension which permits cell stimulation with cellulose-presented C B D - S C F in the absence of cell motility. In this thesis we wi l l not perform a rigorous investigation of the effects of cellulose morphology and source on cellulose-bound C B D - S C F cell stimulation. Rather, we have used that form of cellulose which possesses the best physical properties for each experiment. Dose response curves w i l l be prepared for B6SutA and the TF-1 and M 0 7 e human myeloid cell lines. These cell lines, each derived from a different cell lineage, w i l l be used to assess the generality of the cellular response noted with B6SutA (Doheny, 1996). Finally, we wi l l use Western blot analysis to measure the kinetics of c-kit activation and dephosphorylation when presented with cellulose-bound C B D - S C F . 166 Results: i) Cellulose bound CBD-SCF has increased molar specific activity Dose response curves for soluble C B D - S C F , Avicel-bound C B D - S C F , and a 24 hour re-equilibrated supernate from CBD-SCF-loaded Avice l , are shown in Figure 7-2. The concentration of the initial protein was determined by absorbance at 280 nm (from extinction coefficients calculated in Doheny, 1996). The concentration of Avicel-bound C B D - S C F bioactivity is plotted as the initial C B D - S C F concentration in the adsorption reaction prior to binding, minus the unbound fraction of S C F recovered from the adsorption reaction supernate. A l l cellulose samples were washed in 1 M N a C l after binding to remove non-CBD-tagged S C F bound to the cellulose in heterodimers. The shape of the dose response curves shown in Figure 7-2 is typical for growth factor-mediated cell stimulation. The E D 5 0 of the soluble C B D - S C F (301 pM) is in good agreement with published ED50 values ( R & D Systems: Minneapolis, M N ) . The maximum growth rate (tdoubie ~ 18 h"1) is also in close agreement with results obtained with commercial S C F and pokeweed mitogen stimulated spleen cell extract-conditioned media (Doheny, 1996). Cells grew to the same final density, indicating that the maximum cell growth rate over the 48 hour culture was unaffected by C B D - S C F immobilization. There was no apparent effect of Av ice l particles on the maximum cell specific death rate. The dose response curve for cells stimulated with Avicel-bound C B D - S C F was shifted to the left relative to the curve prepared with soluble C B D - S C F . This represents a decrease in the ED50 (i.e. increase in molar specific activity) of about 6-fold. The bound bioactivity in this experiment is comprised solely of homodimers of C B D - S C F because all non CBD-tagged S C F domains bound in heterodimers had been removed with a 1 M N a C l wash. The actual increase in the 167 molar specific activity of cellulose-bound C B D - S C F is somewhat higher than 6-fold i f one corrects for S C F eluted from the Avice l by the high salt wash. However, quantitative re-concentration of released S C F bioactivity following extensive washing of the Av ice l was not performed. The stability of the Avicel-bound C B D was tested by measuring the amount of bioactivity in the supernate of the Avicel-bound C B D - S C F following 24 hours re-equilibration in P B S . A s seen in Figure 7-2, less than l/20,000 t h of the bioactivity was released into the supernate. The re-equilibration was after the 1 M N a C l wash, which should have released all non-CBD containing S C F molecules. The stability of the immobilized form confirms the irreversibility of the C B D -cellulose interaction and the stability of C B D - S C F after purification. 168 2E+06 100000 Initial CBD-SCF (pM) Figure 7-2: Avicel-bound CBD-SCF has increased molar specific activity Dose response curves of B6SutA cells grown with ( • ) Ni-sepharose purified C B D - S C F , ( • ) Avicel-bound C B D - S C F , and the ( • ) 24 hr CBD-SCF-adsorbed Avice l re-equilibrated supernatant were prepared as outlined in the materials and methods chapter. 169 Previous experiments (Doheny, 1996) in which B M C C is added to culture media containing C B D - S C F (i.e. the cellulose is simply added to culture media containing C B D - S C F ) , indicate that the potency of C B D - S C F is increased when adsorbed to B M C C . In this work, Doheny's experiments were repeated with three different SCF-dependent cell lines (B6SutA, M 0 7 e and TF-1) to test the generality of the proliferative response resulting from C B D - S C F stimulation. Hemocytometer counts of viable cell numbers were used to directly measure factor-stimulated proliferation. The ED50 shift resulting from the adsorption of C B D - S C F onto B M C C was estimated using dose response curves for each treatment. Factor dependent cells were cultured to late log phase, washed in H - S F M , and then cultured in the presence or absence of 1 ug B M C C per milliliter of culture media, over a range of C B D -S C F or m S C F doses (5 p M - 5 nM) (the B M C C with adsorbed C B D - S C F was washed in 1 M NaCl-50 m M phosphate buffer prior to use). After 48 hours of culture, viable cell numbers were determined by trypan blue exclusion in hemocytometric counts. The four-parameter logistic dose-response function was fit to the cell counts to estimate the ED50 for the various treatments (Table 7-1). Consistent with the m S C F supplier's specifications, the ED50 for both m S C F and unbound C B D - S C F with M 0 7 e cells was ca. 150 p M , corresponding to ~3 ng S C F ml" 1 ( R & D Systems: Minneapolis, M O ) . Adsorption of C B D - S C F to B M C C , enhanced its potency 5-7 fold for all three cell lines (Table 7-1). Observation of the cultures prior to counting did not reveal any gross morphological differences. In contrast, the addition of B M C C to wells containing S C F slightly reduced the biological activity. Neither the cellulose nor the C B D alone possessed any intrinsic growth factor activity. In addition, the B M C C remained dispersed in the culture media after the 48 hours of culture. 170 Table 7-1: ED 5 0 for CBD-SCF with or without added BMCC Adsorption of C B D - S C F to cellulose enhances the proliferative effect on murine and human SCF-responsive cell lines. The half-maximal response (ED50) was calculated from dose-response curves of C B D - S C F and S C F with or without cellulose for B6SutA, M 0 7 e and TF-1 cell lines. Values are expressed as ED50 ± standard error. SCF-CBD (pM) SCF (pM) Cell line no with no with cellulose cellulose cellulose cellulose B6SutA 455 ± 58 65 ±9 629 * 65 807 ± 75 M07e 178 ± 42 37 ± 24 124 ± 30 308 ± 33 TF-1 160 ± 22 31 ± 16 349 ± 64 311 ± 53 ii) B6SutA cells are motile The irreversible adsorption of the C B D - S C F fusion protein, implies that direct cell contact with the cellulose surface is required for stimulation. To investigate cell locomotion, live cell imaging experiments were performed to observe the response of B6SutA cells to C B D - S C F bound to Av ice l cellulose. Cells were cultured in the presence of C B D - S C F adsorbed to 40-70 | i m Av ice l particles (5 micrograms C B D - S C F per milligram of Avice l : r/rmax -10%). A microscope and video camera were placed in a 37°C temperature-controlled room and allowed to 171 thermally equilibrate. Prewarmed cells and C B D - S C F loaded Avice l were then added to wells in 6-well tissue culture plates. Cells were initially thoroughly dispersed with Avice l particles. Individual B6SutA cells moved randomly during the first hour of culture. Ce l l motion was erratic. Cells would remain idle for several minutes and then move several hundred microns within a minute. Figure 7-3 shows a series of images taken from a time-lapse video of soluble C B D - S C F stimulated cells. The total elapsed time is 3 minutes, with 20 seconds per frame. The cell that enters the first frame at the top-center edge, is seen to traverse the entire width of the image frame (i.e. ~ 100 fim). Indeed, cells could be observed moving for short bursts at up to 100 | i m per minute. Note that the other two cells in the image sequence do not move as much as the third cell. 172 •i o 0 0 0 0 a Q 0 0 & d? 0 o o 53 o cP •7 •> *«* 6 < *° rc r> c O b Q Q 0 Figure 7-3: B6SutA cells are motile Cells were stimulated with soluble C B D - S C F and cultured in normal polystyrene tissue culture wells at 37°C on an inverted microscope equipped for time-lapse video microscopy. Cells were observed to move up to 100 |ira per minute. Cel l motion was erratic and apparently random when stimulated with soluble C B D - S C F . 173 iii) SCF-responsive cells adhere to CBD-SCF on cellulose Time-lapse microscopic observation of B6SutA cells cultured with Avicel-bound C B D - S C F shows that cells migrate to the Avice l particles and form large clumps around each particle (Figure 7-4a). These aggregates can be disrupted by 2 or 3 passes through a 100 (il pipette tip. It is interesting to note that not all cells within a cell aggregate are in contact with the Avice l particle. This suggests that either cells migrate through the cell mass to contact the surface for a short time, that intercellular trafficking of S C F is occurring, or that S C F is released from the cellulose. The dynamics of cell aggregate formation around Avice l particles w i l l be studied in the next section. Cells did not associate with cellulose-bound C B D c e x or with untreated Avice l particles (Figure 7-4b). B6SutA cells cultured in soluble S C F or C B D - S C F with or without serum also formed aggregates. The sequence of cell aggregate formation around the treated Avice l particles was noted. As cells move in apparent random motion they eventually contact other cells and adhere, slowly forming aggregates of 3 to 7 cells. Cel l aggregates continue to move in apparent random motion following association. As cells and aggregates move they eventually contact C B D - S C F adsorbed to an Av ice l particle. Following contact, cell aggregates would generally disperse around Avice l particles. Surprisingly, cells remained highly motile when in contact with the Avice l particle. Indeed, cells were able to move the Avice l particles. Particles were subsequently aggregated by cell bridging between Avice l particles. Considerable cell motion occurred within the cell aggregates. Although detailed cell tracking studies were not performed in this work, it appeared as though cell were able to migrate within the aggregate. 175 Figure 7-4: SCF-responsive cells adhere to CBD-SCF on cellulose B6SutA cells were cultured in normal polystyrene tissue culture wells at 37°C on an inverted microscope equipped for time-lapse video microscopy. Cells were stimulated with Av ice l -bound C B D - S C F (Panel A - three different image frames). Cells adhered to C B D - S C F adsorbed to Avice l surfaces. In contrast, untreated Avice l particles (Panel B - two different image frames), or those with C B D alone, did not stimulate cell attachment. Cells are approximately 18 (im in diameter. Qualitative tests for CBD-SCF-mediated cell adhesion to cellulose were also performed. Sheets of V. ventricosa cellulose are a convenient surface with which to access cell sticking. Cellulose sheets were prepared as in the F R A P analysis, except that the sheets were fixed to the glass coverslip using cell-compatible silicon-based glue (Silastic, Dow-Corning). The mounted cellulose sheet was incubated with sufficient C B D or C B D - S C F to reach approximately 10% surface saturation. B6SutA cells were then cultured on the loaded V. ventricosa cellulose sheets for 24 hours under normal conditions. Surface imaging with an inverted microscope at 4 0 X magnification revealed that cells remained firmly attached to C B D - S C F treated sheets, even when the cellulose sheet was washed by pipetting media across the surface. Only under the direct flow of rapid media ejection from a pipette could the cells be dislodged from the surface. B6SutA cells do not attach to glass cover slips or tissue culture plastic. Cells could be dislodged from untreated or CBDc e x -adsorbed cellulose sheets with slight agitation. These results confirm the direct role of the C B D - S C F - c-kit interaction in cell attachment. In chapter 8 confocal microscopy wi l l be used to interaction this interaction more closely. Cells grown on V. ventricosa cellulose sheets with bound C B D - S C F did not form the large cell aggregates noted with soluble or B M C C or Avicel-bound C B D - S C F . This suggests that the c-/c/?-mediated cell 176 interaction with cellulose-bound C B D - S C F is stronger than the cell-cell interaction noted with soluble growth factor. In all of the cell-attachment studies, the compact round shape of the cells was maintained and cells did not spread onto cellulose surfaces. Cells in culture on V. ventricosa cellulose surfaces were also motile. Ce l l motility was not dependent on the presence of C B D - S C F , indicating that cells could form stable, though perhaps weak, contacts with the cellulose surface of sufficient strength to permit cell locomotion. iv) C-kit activation is prolonged with CBD-SCF bound to cellulose A series of experiments was performed to investigate the effects of cellulose presentation of C B D - S C F on the kinetics of receptor phosphorylation. Phosphorylation kinetics of the c-kit receptor of the B6SutA cell line were measured for BMCC-presented and soluble C B D - S C F . B M C C was used to ensure the rapid and uniform contacting of cells with cellulose-presented C B D - S C F . Factor-deprived cells were cultured with various combinations of C B D - S C F , m S C F and B M C C for up to 2 hours. After 0, 10 , 30, 60 and 120 minutes of culture, a well from each treatment was sacrificed and prepared for immunoprecipitation. Ce l l lysates were, reacted with anti-murine c-kit antibody and then affinity precipitated with protein A beads. Western blots were performed with antibody specific for phosphorylated tyrosine residues (Antibody 4-G10 -Upstate Biotechnology; Lake Placid, N Y ) . C-kit was rapidly and transiently activated following the addition of soluble C B D - S C F (Figure 7-5a). This response profile is consistent with similar studies presented in the literature (Kurosawa et al., 1996; Yee et al, 1993). Receptor activation was maximal at the first time point (10 minutes) and then rapidly fell back to baseline levels by 60 minutes. S C F and soluble C B D - S C F have similar stimulation kinetics. In contrast to the transient activation seen with 177 soluble factor, C B D - S C F adsorbed to B M C C induced rapid and persistent activation of c-kit (Figure 7-5c). The exposure time for the gel in 7-5c is one tenth that of the gel in 7-5a so that variations in activation level may be visualized. A l l time point lanes for immobilized C B D - S C F have intensities similar to the 10 minute lane in the gel of 7-5a at the same blot exposure time. Consistent with the results of others (Kurosawa et al., 1996; Yee et al., 1993), activated c-kit was immunoprecipitated from the detergent-soluble fraction of cell lysates. Significantly, phosphorylated c-kit was not recovered from the soluble fraction of cell lysates in the B M C C -bound C B D - S C F stimulated cultures (Figure 7-5b). Rather, the activated receptor was associated with the insoluble fraction (Figure 7-5c). The insoluble fraction contained the B M C C and Triton X-100-insoluble cell debris. Reprobing of the Western blots with antibody against murine c-kit, revealed similar levels of receptor in the soluble fractions of both the B M C C - b o u n d and soluble C B D - S C F treatments. B M C C alone did not induce c-kit phosphorylation or inhibit phosphorylation by soluble mSCF. Controls without primary antibody, without cells or without S C F were all negative. 178 IMMUNOPRECIPITHTE PELLET SOLUBLE CBDSCF BMCC+CBDSCF BMCC+CBDSCF 10 30 68 120 10 30 60 120 10 30 60 120 ckit > Figure 7-5: c-kit activation is prolonged with CBD-SCF bound to cellulose The kinetics of c-kit activation in B6SutA cells stimulated with soluble C B D - S C F (soluble cell lysate fraction) (A), B M C C - b o u n d C B D - S C F (soluble cell lysate fraction) (B), and B M C C -bound C B D - S C F (insoluble cell lysate debris and B M C C fraction) (C) are shown. Western blots were prepared for cell lysates and then probed with anti-phosphorylated tyrosine. The exposure time of the B M C C - b o u n d C B D - S C F blot is l /10 t h that for the other two blots (similar exposure times resulted in all lanes in blot C being as dark as the 10' lane in the soluble C B D -S C F treated culture). No activated c-kit was detected in the soluble fraction of the B M C C -bound C B D - S C F sample. Discussion: C B D - S C F adsorbed to cellulose is an effective vehicle for the presentation of S C F to both human and murine target cells. The properties of each domain in the C B D - S C F fusion are retained, and correct orientation of S C F for receptor binding is achieved. Addition of cellulose to C B D - S C F decreased the E D 5 0 of the cellulose-presented S C F up to 7-fold over soluble material. Addition of B M C C to soluble S C F did not significantly affect its bioactivity. Although it has not 179 been proven here, the observed increase in specific activity, and the current belief that the S C F dimer drives receptor dimerization and therefore drives stimulation (Hsu, 1997), suggests that C B D - S C F is dimerized at the cellulose interface. S C F mutants with a low dimerization affinity have significantly reduced molar specific activity (Hsu, 1997), suggesting that a shift in dimerization equilibrium may be responsible in part for the enhancement of S C F bioactivity by immobilization to cellulose. Miyazawa (1995), using membrane-bound S C F presented by transformed fibroblasts, reported no enhanced bioactivity on M 0 7 e cell proliferation (measured using 3H-thymidine incorporation assays). However, the wide range of other factors presented and secreted by the transformed fibroblasts makes it impossible to draw definitive conclusions from their results. The binding capacity of cellulose for C B D - S C F (~ 4 umol/g B M C C ) means that less than microgram quantities of B M C C per milliliter of cell culture are required to stimulate cells. However, practical considerations such as cellulose handling, preparation and aliquoting require somewhat higher concentrations of cellulose to ensure uniform treatments of test cells. It was originally believed that micron-scale motions such as the diffusive flux of the C B D - S C F on the cellulose surface would influence cellular responses and associated kinetics. The diffusion of the growth factor on the cellulose surface between encounters with receptors, and the escape of factors into the bulk media would occur at this scale. It was also hypothesized that factor consumption by cells might create gradients on the cellulose surface which would cause cells to traverse the gradient through chemotaxis, or cause the C B D - S C F to diffuse down the induced factor gradient. It was shown in this chapter the B6SutA cells are highly motile and may move up to 100 (im/minute. On the other hand, C B D - S C F is essentially immobile on the V. ventricosa cellulose. Thus, our results suggest that growth factor gradients do not form around cells due to 180 the high motility of these cells relative to cellulose-adsorbed C B D - S C F . However, gradients may still form under a cell during accumulation of c-kit into patches at the cellulose interface. Receptor aggregation-driven redistribution of cellulose-adsorbed C B D - S C F w i l l be investigated with C S L M in the next chapter. The uptake of h S C F by M 0 7 e cells is biphasic: a rapid initial uptake is followed by a slower, prolonged consumption, presumably due to the reappearance of c-kit on the cell membrane (Zandstra, 1997). It is possible that the observed enhancement in molar specific activity of the cellulose bound C B D - S C F is due to a slowing of the initial rapid uptake phase, so that more S C F activity persists. B M C C - b o u n d C B D - S C F is only available when a cell makes direct contact with the cellulose. This can only occur through bulk mixing of the supernatant to bring the B M C C fibers into cell contact, or through cell motility. Both of these processes may result in receptor-ligand encounter frequencies much lower than that achieved with soluble factor. In results parallel to those of Miyazawa et al. (1995), cellulose localization of C B D - S C F resulted in a significant increase in the persistence of c-kit tyrosine activation. W e found no significant decrease in c-kit phosphorylation over a 2 hour period. In contrast, soluble C B D -SCF-induced phosphorylation was down-modulated within 30 minutes of stimulation. Significantly, we found that activated receptors are associated with the insoluble fraction following cell lysis. This, together with the prolonged stimulation of c-kit tyrosine kinase activity through ligand immobilization, implies that the receptor-ligand complex is trapped at the cell membrane-cellulose interface. Furthermore, the activated c-kit receptor even remained bound to C B D - S C F , which remained bound to cellulose, during cell lysis and immunoprecipitation. 181 With soluble S C F stimulation, activated receptors are found in the soluble fraction of the cell lysate, indicating release from the cell membrane during cell solubilization. Wi th the cellulose-bound C B D - S C F treatment, there was c-kit in the soluble fraction but these receptors were not phosphorylated. This suggests that each of the high affinity interactions in the complex, ( C B D to cellulose binding, S C F dimerization and c-kit binding to the S C F dimer), are stable under the mild detergent conditions required for cell lysis. In addition, it indicates that with the B M C C presentation of the C B D - S C F , not all of the c-kit was in contact with a C B D - S C F on a B M C C fibril. Ce l l motility may greatly affect the rate of cytokine stimulation when factors are localized to the cell 's substratum. A motile cell contacts new areas of the substratum as it moves, and thereby "grazes" on cytokine localized on the sorbent. B6SutA cell motility rates were up to 100 (im/minute. When the surface coverage density of C B D - S C F on the V. ventricosa cellulose sheet was about 0.2 pmol/u.m 2. This implies that the cell (15 urn diameter) could potentially contact 0.25 nmoles of S C F per minute, a number that is well above reported S C F consumption rates. The bioactivity of the soluble form of C B D - S C F , strongly suggests that C B D - S C F exists as either a homo or hetero-dimer in solution. Also, it is likely that any C B D - S C F adsorbed to cellulose as a monomer would rapidly form dimers on the cellulose, due to the surface diffusion observed for C B D s . Recall that the P/T linker between the domains of the fusion protein should permit considerable orientational freedom to the P/T-tethered S C F . Furthermore, because the C B D - S C F is bioactive when bound to cellulose, we hypothesize that C B D - S C F dimers are able to form, and that c-kit binds to cellulose-surface localized dimerized factor. Unwanted proteolytic cleavage of the C B D - S C F into constituent C B D and S C F domains results in co-purification of full length C B D - S C F and cleaved S C F on a Ni-Sepharose column 182 (recall that the His-6 tag is on the S C F domain). Thus, problems with C B D - S C F purity limit the range of feasible biophysical characterization experiments. Proteolytic degradation requires that both purification steps, His-6 tag on Ni-Sepharose followed by C B D adsorption to cellulose, be used to prepare a homogeneous population of full length molecules. The difficulty in eluting the C B D - S C F from cellulose, without denaturing the CBD-fusion protein, requires that most characterizations be performed on the cellulose solid phase. 183 Chapter 8 Cytostructural Changes Induced by CBD-SCF Stimulation Introduction: There are several potential mechanisms by which a stimulated cell can down-regulate a growth factor-induced response. These include receptor-ligand complex internalization, receptor degradation at the cell membrane, protease-mediated shedding of activated receptors, or induced receptor conformational changes which lower ligand affinity (for a review see Olsson et al, 1992). The rapid loss of c-kit from the cell membrane following stimulation with soluble S C F has been demonstrated by F A C S analysis (Zandstra, 1997). In Chapter 7, we showed that C B D -S C F bound on cellulose, causes c-kit positive cells to adhere to the cellulose matrix. As a result, the activation of c-kit was prolonged when C B D - S C F was presented on cellulose. Therefore, it is likely that the c-kit receptor and the cellulose-presented C B D - S C F form a stable and active complex. In this chapter, C L S M is used to image this interaction. The kinetics of c-kit internalization are biphasic (Yee et al, 1994). In pulse-chase studies with 1 2 5I-tagged S C F , the amount of bound labeled ligand, decreased by more than 50% 15 minutes after stimulation with S C F . The phosphorylation of c-kit is detectable seconds after the addition of S C F , with maximal activation by ten minutes and near complete dephosphorylation by thirty minutes. These kinetics suggest that i f aggregates are the active signaling state of the receptors, SCF-induced c-kit patching must occur very rapidly. Direct confocal imaging of the internalized c-kit-SCF complex has not been reported. In this study, cells stimulated with either soluble S C F or C B D - S C F presented on cellulose, were imaged to identify cytostructural responses induced by C B D - S C F presentation. 184 To investigate the kinetics of soluble SCF-induced receptor patching and internalization, live-cell C L S M imaging was performed with soluble, FTTC-labeled C B D - S C F . Fixed-cell imaging of C B D - S C F presented on fibers ( B M C C ) or sheets (V. ventricosa) were used to visualize the C B D -S C F ligand and the c-kit receptor. Immunolocalization with antibody specific for the C B D , S C F and c-kit w i l l be used. In fixed-cell experiments, phalloidin staining wi l l be used to differentiate the cell from its environment. Phalloidin is a fungal toxin with a high specific affinity for actin filaments (Knowles and McCul loch , 1992). Using phalloidin tagged with a fluorophore, is thus a convenient method to delineate the actin cell envelope and cytoplasm. C S L M imaging wi l l permit the 3-D spatial localization of the ligand-receptor complex at the membrane-cellulose interface. Staining with antibodies specific for phosphorylated tyrosine, w i l l be used to identify C B D -SCF-activated c-kit complexes at the cellulose interface. The findings of the previous chapter demonstrate that activated c-kit receptor is associated with the B M C C in cell lysates. Using C L S M imaging, one should therefore be able to resolve the polarization of activated c-kit to the loci of cell contacts with the C B D - S C F cellulose. Furthermore, cellulose presentation of C B D -S C F may serve to concentrate the signal, making it more amenable to direct immunofluorescence imaging. Immunofluorescent C L S M of cells cultured on planar V. ventricosa cellulose surfaces with bound C B D - S C F , permitted the investigation of c-kit polarization in response to surface-presented S C F . The diffusive mobility of receptors in the cell membrane suggests that the cellulose-presented C B D - S C F w i l l capture mobile c-kit when it encounters the S C F ligand. Confocal imaging should therefore reveal the polarization of c-kit to the cellulose interface. 185 Results: i) Exposure to soluble SCF causes rapid internalization of c-kit Cells stimulated with soluble S C F were imaged using C L S M to immuno-localize c-kit 20 minutes after stimulation. The dose dependence of cytokine internalization was also examined to determine whether c-kit trafficking was affected by cytokine concentration. Factor-starved M 0 7 e cells at several concentrations of S C F were incubated for 20 minutes and then quickly fixed in 4% P F M . Fixed cells were incubated with Y B 5 . B 8 antibody and then stained with FITC-labeled secondary antibody and Texas Red-labeled phalloidin. C L S M was used to image cells so that optical cross-sections could be collected through the cell body to spatially resolve c-kit. Figure 8-1 presents representative images obtained for M 0 7 e cells incubated with 100, 25, 6.25 1.55 or 0 ng/ml hSCF. The scale bar in the bottom left corner represents 15 um. Each row shows a series of optical sections (Sz - 1 . 0 um) through a typical cell. Red staining, from phalloidin-Texas Red binding to the actin cytoskeleton, shows the actin envelope of the cell membrane and the cytoplasmic compartment of the cell. The dark area within the cell envelope is the nucleus. Green fluorescence shows the distribution of anti-c-kit receptor antibody (YB5.B8) . Yel low patches within the cytoplasm are the result of actin (red) and c-kit (green) co-localization. Optical sectioning and image-stack volume or surface rendering, shows that the M 0 7 e cells are approximately 18 urn spheroids. Optical sectioning revealed that receptors are found in discrete bodies within the cytoplasm of stimulated cells. This was not observed in unstimulated cells. Furthermore, no significant Y B 5 . B 8 antibody staining was observed on the membranes of cells either with or without stimulation. Thus, the internalized clusters of c-kit l ikely represent a 186 considerable number of aggregated receptors. In general, the number of endosomes containing c-kit, and the absolute fluorescence signal observed for individual endosomes, was related to S C F dose. Although qualitative, these results are consistent with F A C S results reporting the S C F dose-dependent loss of c-kit on cell membranes and internalization of S C F on cultured mast cells (Yee et al, 1994). Absolute quantification is made difficult by the geometry of the samples and the potential for photobleaching during imaging. 187 H u y m l V v • /'J n g / m l 1 • 6.25 in) m l V * 1.55 n g / m l U I M J f i l l Figure 8-1: CLSM imaging of M07e cells stimulated with soluble rhSCF Factor-starved cells were cultured for 20 minutes at a range of S C F doses under normal cell culture conditions. Cells were then fixed and stained with mouse anti-human c-kit (green) and phalloidin (red). Each row shows a series of optical sections (8z - 1 . 0 um) through a single, representative cell, incubated with a particular S C F concentration. Yel low patches within the cytoplasm are the result of actin (red) and c-kit (green) co-localization in discrete bodies. The scale bar in the bottom left corner represents 15 urn. ii) Receptor patching occurs within 60 seconds of S C F addition B6SutA cells were cultured under physiological conditions for short periods on an inverted-C L S M with a heated stage. Hepes-buffered R P M I media was used for cell culture, and no 188 significant p H change was noted over the time course of the imaging experiments. B6SutA cells were cultured in 2.5 cm covered petri dishes fitted to the C L S M stage heater. Cells were added to the dish and cultured for 10 minutes to allow the cells to settle to the bottom of the culture chamber (adjacent to the objective lens). Cells were motile on the untreated bottom of the glass petri dish. Soluble, FITC-tagged C B D - S C F was added to cultures (100 ng/ml) and 3-D image sets were collected over time (Figure 8-2). To minimize photo-damage to cells caused by free radical release from excited fluorophores, confocal imaging was limited to 5 sections (at 3 | l m spacing). A n image stack was collected every 20 seconds for approximately 10 minutes, reported images are the projections of the middle 3 layers of each image stack. Considerable cell movement occurred during the imaging experiment, making it difficult to track individual cells. Typical images from the sequence are shown in Figure 8-2. P M T gain and black levels were set such that the image background field was completely dark prior to the addition of F I T C - C B D -S C F and cell auto-fluorescence was just visible (Figure 8-2 t 0 ) . Following addition of F ITC-C B D - S C F , the image brightness increased immediately in all areas of the image field not occupied by cells, indicating rapid mixing in the cell culture chamber. The exclusion of the F I T C - C B D - S C F from cells was consistent with the greater than 90% cell viability (as demonstrated by trypan blue exclusion). In the first few minutes following addition of F ITC-C B D - S C F a fluorescent halo develops around each cell, indicating a concentration of F ITC-tagged molecules on the cell membrane. Over the next few minutes, fluorescent patches developed on the cell membranes. These patches are likely the result of concentration of bound F I T C - C B D - S C F in receptor aggregates, which appeared to form prior to internalization. Each cell forms 3 or 4 patches on its membrane, 189 consistent with the fixed cell imaging results presented previously. Patches are discernible within the cell after 10 minutes. In control experiments, C B D c e x tagged with the fluorophore Texas Red (TxRd) (CBD-TxRd) was added with the F I T C - C B D - S C F . C B D - T x R d was excluded from the cells and did not concentrate on the cell envelopes over the course of the imaging experiments. 190 Figure 8-2: Live-cell CLSM of B6SutA cells stimulated with soluble FITC-CBD-SCF The time course of receptor patching in response to addition of soluble FITC-tagged S C F to the culture medium (-1.5 minutes between frame). In the first panel ( t 0 ) the slight autofluorescence of the cells is visible. Following addition of labeled C B D - S C F , image brightness increases immediately in all areas of the image field not occupied by cells. A fluorescent halo develops around each cell within seconds of the addition of labeled S C F , followed by the rapid formation of fluorescent aggregates on the cell membrane. Internalized aggregates were discernible within the cell volume within 3 minutes of SF addition. iii) C-kit co-localizes with CBD-SCF presented on BMCC In the previous chapter, it was noted that C B D - S C F does not diffuse when adsorbed to cellulose. This does not mean that the S C F is fixed (in a covalent sense) on the cellulose. For 191 example, receptor aggregation might involve receptor-driven translation of the C B D - S C F on the cellulose surface. It is also possible that the observed enhancement of specific activity of C B D -S C F when bound to cellulose, and the prolonged activation of c-kit, are the result of a stabilization of the receptor-ligand complex at the cellulose surface. In this section, C L S M imaging is used to determine the degree of c-kit patching and internalization in response to C B D -S C F adsorbed to B M C C . Three-color imaging is used to identify c-kit, C B D - S C F and actin. B M C C fibers provide micron-scale microcrystalline cellulose surfaces for the presentation of C B D - S C F to cells. The adsorption of C B D - S C F to these fibers facilitates the establishment of spatially discrete S C F signals. The diffusive mobility of receptors in the cell membrane suggests that the BMCC-presented C B D - S C F w i l l capture the mobile c-kit when it encounters the S C F ligand. Confocal imaging should therefore reveal the concentration of c-kit.on the B M C C fibrils, relative to the overall distribution of c-kit within the cell. B6SutA cells were cultured overnight in H - S F M without S C F . Prewarmed media containing B M C C fibers to which C B D - S C F had been adsorbed (r/rm a x ~ 25%) were then added to the cultures. After 20 minutes under normal cell culture conditions, the cells were harvested, washed, and then fixed and probed with various antibodies and fluorescent probes. Figure 8-3a shows a cell stimulated with soluble m S C F and probed with anti-SCF antibody. The formation of round patches on the cell membrane and in the cytoplasm is evident. These patches co-localized with c-kit staining (data not shown). In contrast, cells stimulated with C B D - S C F on B M C C show fibrous patches consistent with the shape of B M C C fibers when stained with anti-S C F antibody (Figure 8-3b). B M C C coated with C B D - S C F adhered to B6SutA cells expressing the S C F receptor. Fibers treated with C B D alone did not interact with cells (data not shown). Cells also bound tightly to B M C C fibers displaying a mixture of bound F I T C - C B D c e x and C B D -192 S C F . Apparently, cells interact directly with the fiber-presented C B D - S C F , causing the fiber to conform to the round shape of the cell envelope. Cells were counter-stained with anti-c-kit antibody (Figure 8-3c). The fibrous appearance of Figure 8-3b again suggests co-localization of the c-kit receptor with the BMCC-presented C B D -S C F . Co-localization is confirmed in Figure 8-3d. In this image, the cells have been stained with anti-SCF (green) and anti-c-kit (blue) antibodies, and with phalloidin-TxRd (red). Co-localization of S C F and the c-kit receptor is shown by the combination of green and blue colors, which result in aquamarine fibrous patches on the cell surface. The lack of "green-only" fibers on the cell surface, indicates that fibers are in contact with c-kit along most of their length. Some non-SCF associated c-kit staining was noted in some cells. It is unclear whether this represents newly synthesized c-kit or c-kit internalized without the C B D - S C F . A small aggregate of CBD-SCF-coated B M C C fibers is evident towards the right of panels B and D . The lack of anti-c-kit antibody staining of the non-cell associated fibers clearly demonstrates the specificity of the antibody. No staining was observed in control samples processed without primary antibodies. Furthermore, B M C C did not associate with cells treated with soluble S C F (data not shown). Figure 8-3: CLSM of B6SutA cells grown with CBD-SCF bound to BMCC Cells were stimulated for 20 minutes with soluble (A) or B M C C - b o u n d C B D - S C F (B). The fibrous morphology of the B M C C cellulose is evident in B . The distribution of the c-kit receptor on this cell is shown in C. Co-localization of receptor and ligand is demonstrated in D . The aquamarine stripes on the cell surface are a result of the co-localization of the S C F (green) and the c-kit receptor (blue). The cell cytoskeleton was counter-stained with phalloidin-Texas Red to delineate the cell envelope (red). The scale bar represents 5 |J,m. 194 iv) BMCC fibers presenting CBD-SCF are internalized by B6SutA cells We have demonstrated that soluble S C F is rapidly internalized following binding to c-kit. In contrast, C B D - S C F on B M C C forms a stable interaction with c-kit. Therefore, it is of interest to examine whether cellulose is internalized with the c-&zY-ligand complex during normal receptor endocytosis. B6SutA cells were incubated with B M C C with or without C B D - S C F for 20 minutes, fixed, and then stained with rabbit anti-SCF antibody (green), biotinylated rat anti-c-kit (blue) and phalloidin (red). Figure 8-6 shows a rotation series of a volume-rendered confocal image stack. The frames are at 30 degree intervals rotated about the z-axis. Co-localization in this image is indicated by the color combination of individual stains (e.g. aquamarine is green/blue and white is green/blue/red). From the rotation series it is clear that some B M C C fibers are fully contained within the cell envelope. The arrow in the center image points to a fiber near the center of the cell volume. Note that the fiber remains in the center of the cell body as the image is rotated about its axis. The fibrous morphology of the B M C C fibrils is retained, suggesting that the fiber was not compacted into an endosome at the cell membrane prior to internalization. There is no significant build-up of actin (red) along the length of the internalized fiber, although membrane ruffles do follow the fibrils along the exterior of the cell (Figure 8-3). Fibers stained with CBDcex-FITC did not interact with cells, and no internalization of fibers. was observed. Internalized B M C C fibers with bound F I T C - C B D - S C F were also observed in control experiments in which cells were not fixed or permeabilized prior to imaging. Figure 8-4: BMCC fibers presenting CBD-SCF are internalized by B6SutA cells B6SutA cells were incubated for 20 minutes with B M C C with bound C B D - S C F , fixed and then stained with rabbit anti-SCF antibody (green), biotinylated rat anii-c-kit (blue) and phalloidin (red). The figure shows a rotation series of a volume-rendered confocal image stack. Co-localization in this image is represented as the color combination of labels (e.g. aquamarine is green/blue and white is green/blue/red). The arrow (A) indicates the internalized fiber, while the other arrow (B) indicates a fiber associated with the cell membrane. 196 • • • • • •• • | f Figure 8-5: BMCC fibers stained with FITC-CBDCex and CBD-SCF are internalized by B6SutA cells B6SutA cells were incubated for 20 minutes with C B D - S C F and FITC-CBDcex bound to B M C C . Cells were not fixed or permeabilized prior to imaging. The figure shows a rotation series of a volume-rendered confocal image stack. The presence of FITC-CBDcex stained fibers confirms that B M C C fibrils were internalized. v) C-kit co-localizes with CBD-SCF presented on V. ventricosa cellulose sheets V. ventricosa cellulose sheets provide a centimeter-scale, planar, crystalline cellulose surface for the presentation of C B D - S C F to cells. The adsorption of C B D - S C F to these sheets thereby 197 facilitates the establishment of a spatially polarized S C F signal. B6SutA cells were cultured for up to 2 hours on V. ventricosa cellulose sheets mounted on normal glass coverslips. In one case, F I T C - C B D - S C F had been adsorbed to the cellulose (r/rmax < 5%), in the other, soluble S C F was added. The cells were fixed with 4% P F M and incubated with biotinylated rat anti-murine c-kit. Cells were then stained with phalloidin-TxRd and Cy5-streptavidin. Consistent with our observation of cell adhesion to C B D - S C F on cellulose, cells remained firmly attached to the cellulose sheet during antibody staining. In contrast, very few cells remained on the untreated V. ventricosa cellulose surface during antibody labeling. Three-color C L S M was performed with each channel collected independently. Optical sectioning had slice spacing of 0.15 (am. A 60 degree rotation was then projected independently for each channel using surface rendering algorithms in the public domain NUT Image program (developed at the U .S . National Institutes of Health). Final images were formed by recombining the independent R G B channels into a single 24-bit 3-color image. Figure 8-6 presents images of a cell cultured on the V. ventricosa-CBD-SCF surface for 20 minutes. The composite image is from four frames taken from an image stack projected through 60 degrees of rotation. The scale bar in the upper left-hand frame represents 10 um. Frames in Figure 8-6 are at 30, 45, 70 and 90 degrees to the surface normal (from top left). The images show the polarization of c-kit (green) at the cellulose interface coated with C B D - S C F (blue). The projections show that c-kit antibody bound only under cell bodies (red), and not elsewhere on the cell envelope, indicating a near-complete polarization of c-kit during the 20 minutes of cell exposure to the treated cellulose surface. In control samples stimulated with soluble S C F , no polarization of c-kit receptors was observed (data not shown). Samples not 198 treated with anti-c-kit primary antibody showed no binding of labeled secondary probes. Surfaces treated with C B D C e x only, did not bind cells or show regions of c-kit staining. The 30 and 45 degree projections show that anti-c-kit staining is completely polarized to the basal surface of the cell. The absence of internalized receptors is in direct contrast to images of c-kit internalization collected from soluble SCF-stimulated cells. Prior to imaging, a subset of prepared samples was shaken and rinsed vigorously to remove bound cells. Imaging of shaken surfaces revealed an apparent c-kit "footprint" from cells removed from the V. ventricosa cellulose surface, suggesting that receptors form stable interactions with cellulose-presented C B D - S C F . 200 Figure 8-6: CLSM of cells cultured with CBD-SCF bound to a V. ventricosa cellulose surface B6SutA cells were cultured for twenty minutes on a V. ventricosa cellulose sheet to which C B D - S C F was bound. A 60 degree rotation was then projected for each channel using surface rendering algorithms in N I H Image. Frames are at 30, 45, 70 and 90 degrees to the surface normal (from top left). Images show the polarization of c-kit (green) at the cellulose interface coated with C B D - S C F (blue). The slight green halo noted at the cellulose surface is due to a slight misallignment of images during 3-D projection. The scale bar in the upper left-hand frame represents 10 um. vi) Imaging c-kit co-localization with tyrosine-P following CBD-SCF stimulation The presentation of cellulose-localized C B D - S C F to factor-dependent cells, results in prolonged tyrosine phosphorylation and cellulose localization of c-kit. Furthermore, in the experiments to examine receptor phosphorylation kinetics, it was noted that the activated receptors are found in the insoluble fraction of the cell lysate, presumably associated with the insoluble cellulose phase. This shows that c-kit forms a stable complex with the cellulose-bound C B D - S C F . This phenomenon was imaged directly by culturing cells on V. ventricosa cellulose sheets with bound C B D - S C F . C L S M was then used to image the polarization of activated c-kit at the cellulose interface. Cells were cultured for 12 hours on V. ventricosa cellulose sheets under various treatments and then processed for imaging. The monoclonal antibody (4G-10) used in the immunoprecipitation Westerns was used for immunolabeling. Cells were counter-stained with biotinylated rat anti-c-kit antibody. Goat anti-mouse IgG-FITC (green) and Cy5-Streptavidin (red) were used as secondary stains. Two-color C L S M imaging was performed with a 60X lens and confocal aperture yielding a 8z of about 0.15 microns. Anti-phosphotyrosine antibodies co-201 localized with anti-c-kit antibodies in the optical slice taken at the cellulose surface (Figure 8-7a and b). There was very little staining except at the interface. These observations indicate that most of the anti-phosphotyrosine antibody labeling was due to activation of c-kit. However, other phosphorylated molecules associated with activated c-kit through SH2 docking sites on the activated receptor, such as activated P L C - y and G A P , may also have contributed to the observed signal. A n optical cross-section across the cell (at the slice indicated by the arrows in the figure) showed that receptors concentrated where the cell's membrane contacted the cellulose surface (Figure 8-7c and d). The association of activated c-kit with the cellulose is consistent with our results from immunoprecipitation Western blots. It should be noted that the image was collected after 12 hours of culture. This persistence of receptor activation is much longer than has been previously reported. It is unclear whether the imaged c-kit polarization is the result of receptor-ligand stabilization over the extended time course of this experiment, or i f it represents newly arrived receptor following normal receptor-ligand cycling. 202 H B > C D Figure 8-7: Anti-phosphotyrosine CLSM of cells cultured with CBD-SCF bound to a K ventricosa cellulose surface Antibody against phosphotyrosine bound to permeabilized cells (a) and co-localized in patches with anti-receptor antibody (b). A cross-section (Z-section) perpendicular to the cellulose surface passes through the cell at the plane indicated by the arrows in (a) and (b). Activation was primarily localized to the cellulose surface (c) and co-localized with the anti-receptor antibody (d). The scale bar in (c) represents 10 um; all panels have the same magnification. 203 Discussion The binding and localization of growth factors on the extracellular matrix is a key feature in tissue morphogenesis (Ingber, 1993; Drubin and Nelson, 1996). Indeed, the spatial regulation of growth factor availability establishes cell polarity when receptors are captured at a factor-presenting interface. In this work, C L S M imaging was performed to investigate the cytostructural changes induced in SCF-dependent cells by cellulose surface-presented C B D - S C F . The polarization of c-kit to the cell membrane-cellulose interface was demonstrated. Additionally, the formation of receptor aggregates in the cell membrane following soluble or cellulose surface-presented C B D - S C F cell stimulation was observed, consistent with results for IL-1 (Guo et al., 1995) and E G F (Chung et al., 1997; Gadella and Jovin, 1995). A common feature of S C F stimulation is the rapid ligand-induced internalization and degradation of the ligand-receptor complex (Yee et al, 1993). Receptor is cleared from the cell membrane by internalization in a ligand dose-dependent manner with a half life of 30 to 40 minutes. W e used immunofluorescent confocal imaging to determine the initial fate of the c-kit-S C F complex. Our results verify that c-kit and soluble S C F are internalized within 20 minutes of cell stimulation. Internalized c-kit localizes to endosomal structures in the cytoplasm and internalized receptor aggregates are partly actin-associated. The level of anti c-kit antibody staining correlated with the initial S C F concentration I which cells were incubated. This is consistent with results from F A C S analysis in which a dose-dependent decrease in cell surface c-kit receptor levels was observed (Zandstra, 1997). The dose dependence of c-kit internalization even at high concentrations of factor suggests that secondary processes such as receptor aggregation and coated-pit formation do not limit the rate of receptor cycling. Interestingly, the dose for maximal proliferative response for B6SutA cells (0.63 nM) 204 and M 0 7 e cells (0.12 nM) is much lower than the maximum concentration of 4.5 n M S C F used to stimulate cells in this imaging study. This supports the view that the proliferative response might be accompanied by a scavenging response in which ligand is rapidly cleared from the cell membrane by excess receptors. The very rapid formation of fluorescent halos observed on live cells stimulated with soluble F I T C - S C F implies that receptors are initially distributed evenly over the cell membrane. Following binding, receptors aggregate into clusters that are typically concentrated on one pole of the cell, consistent with the fixed-cell images, which show the cytoplasmic compartment pushed to one end of the cell body. The B6SutA cell line expresses up to 70,000 receptors on its cell membrane. Only background levels of antibody staining were observed on the membrane or cytoplasm of unstimulated cells, suggesting that the observed endocytic bodies in stimulated cells represent a significant concentration of receptors. The patches of internalized receptor imaged in Figure 8-1 may thus represent as many as 10,000 receptors. Patching of receptors in the cell membrane following ligand binding is known to result in their internalization in clatherin-coated pits. Again, the low staining of unstimulated cells indicates that c-kit receptor patches are not pre-formed in the cell membrane, but rather result from ligand binding. The disappearance of c-kit patches in the cell membrane 20 minutes after stimulation agrees with the kinetics of tyrosine-phosphorylated receptor down-regulation reported in Chapter 7. Baghestanian et al. (1996) saw no change in the cytoplasmic c-kit concentration in primary human mast cells or in a human mast cell line up to 3 hours after stimulation, but a substantially decreased level from 6 to 12 hours after stimulation. These results are in direct contrast to our findings, in which high levels of cytoplasmic c-kit staining are observed for M 0 7 e and B6SutA 205 cells for up to 2 hours after stimulation. The reason for this discrepancy is not clear. Baghestanian reported a 75% loss of cell surface c-kit 12 hours after c-kit stimulation, but was unable to determine the fate of these receptors. It is possible that lysosomal turnover of internalized receptors was much more rapid in their mast cells, or that receptor shedding was occurring. It has long been recognized that growth factor presentation in the extracellular microenvironment and on stromal cell membranes creates a cell proximal region of relatively high factor concentration (Taipale and Keski-Oja, 1997). Such surface-presentation should enhance cell stimulation rates by significantly increasing the probability of ligand-receptor encounters. Consider the cell microenvironment developed on a planar V. ventricosa cellulose sheet onto which C B D - S C F had been bound. In this system, each of the interactions, C B D binding to cellulose, S C F dimerization, and S C F dimer availability to c-kit (as determined by conformational and steric hindrance induced by the interface), affect the response of target cells to the surface-immobilized factor. We observed the polarization of c-kit in response to the planar geometry of C B D - S C F presentation on V. ventricosa cellulose sheets. The distinct receptor co-localization with C B D -S C F at the cellulose interface confirms that CBD-mediated growth factor presentation is an effective method for the spatial regulation of growth factor availability. The strength of cell association with treated V. ventricosa cellulose surfaces implies that a stable membrane-associated receptor-ligand-cellulose complex was formed. Soluble SCF-induced c-kit phosphorylation and subsequent de-phosphorylation is generally complete within 20 minutes, regardless of dose. These phosphorylation kinetics are on the same order as those observed for receptor internalization (Zandstra, 1997). Preliminary imaging 206 studies were performed to probe activated signal transduction complexes on the cellulose with antibodies specific for phosphorylated tyrosine. We found activated c-kit receptors polarized to the cellulose interface (Figure 8-7). Significantly, these images were prepared from cells cultured overnight on prepared cellulose sheets, indicating a marked extension of normal c-kit activation (Chapter 7). However, it remains unclear whether the c-kit phosphorylation imaged in Figure 8-7 is the result of hyper-extended receptor activation or the formation of newly activated complexes. The prolonged activation of c-kit in response to cellulose-localized C B D - S C F was noted. Marshall et al. (1995) suggested that the prolonged activation of E R K noted for N G F stimulation of PC-12 cells, may allow time for the active molecule to translocate to the nuclear compartment and interact with transcription factors. Thus, the duration of c-kit receptor activation might induce alternative responses by modulating the duration of activation of downstream signaling molecules. The mechanism of receptor phosphorylation stabilization and its effect on cell stimulation should therefore receive further study. We suggest two potential mechanisms to explain the extended activation of c-kit in response to cellulose-localized C B D - S C F : High CBD affinity for cellulose hinders c-kit patching W e have made the point that the noncovalent attachment of C B D to cellulose has the advantage of supplying surface-localized as opposed to surface-immobilized factors. However, we found that the dimeric nature of the S C F molecule produced a double C B D molecular species with significantly reduced surface mobility. The patching (aggregation) of receptors in the cell membrane following ligand binding is an important step in both signal transduction and receptor 207 internalization (Broudy, 1997). If the resistance to lateral motion of the C B D is high enough, it may hinder the directed transport of activated receptors into patches. However, patches of c-kit were noted in cell membranes in all imaging studies, both with B M C C fibrils and V. ventricosa cellulose sheets. Therefore, although the patching of receptors may have been slowed, c-kit does patch in response to cellulose-localized C B D - S C F . Furthermore, this implies that the C B D - S C F may be mobile under the motile force of the cytoskeleton-ligand complex. High CBD affinity for cellulose hinders receptor internalization Perhaps the most plausible explanation for the prolonged phosphorylation of c-kit is the high affinity and essentially irreversible association of C B D s with crystalline cellulose. This suggests that some impediment to c-kit internalization may have been produced - recall that the S C F dimer contains two C B D s . If receptor down-regulation occurs following receptor endocytosis, c-kit stabilization in the membrane would produce a prolonged signal. This is consistent with our observation of c-kit polarization, strong attachment of cells to treated cellulose, and B M C C fibril internalization. Imaging studies should be performed to determine whether c-kit is still phosphorylated on internalized B M C C fibrils. The receptor-ligand patches imaged at the cell-cellulose sheet interface, and the lack of significant levels of internalized c-kit (Figure 8-6), indicate that the rate of receptor internalization is likely controlling the kinetics of c-kit signaling by cellulose-presented C B D - S C F . For many receptor tyrosine kinases, ligand binding is followed by receptor dimerization and subsequent trans-autophosphorylation (see Will iams and Shoelson, 1993 for a review). Phosphorylated tyrosine residues form docking sites for src homology-2 (SH2) domain-containing proteins (Gish et al, 1995). These proteins include members of the PI3-kinase, P L C y and scr family kinases, and G A P , She and Grb2 in the ras pathway. The formation of stable c-kit 208 complexes on the cellulose surface should permit the direct confocal imaging of these complexes. A s was demonstrated, the cellulose matrix makes an effective solid phase for capturing receptors and, potentially, receptor-associated molecules. Such an approach should permit the spatial-temporal dissection of the signal transduction cascade at the cell membrane. 209 Chapter 9 Conclusions and Recommendations We had hypothesized that CBD-cytokine fusion proteins and cellulose-based tissue culture matrices would facilitate the spatial regulation of growth factor stimulation in cell cultures. C B D s from cellulases make effective affinity tags for the cellulose-localization of fusion proteins; chimeric proteins of C B D s and growth factors retain the properties of both fusion protein domains. Furthermore, although binding of a C B D to cellulose is effectively irreversible under cell culture conditions, we have shown that the C B D is free to diffuse in 2 dimensions across the cellulose surface. These attributes of the C B D suggest that C B D fusion proteins are not simply immobilized on a cellulose surface, but rather are localized at it. Indeed, the 2-dimensional mobility of CBD-containing proteins, suggests that CBD-cytokine fusions may be an effective mimic of growth factors presented in the membranes of stromal cells. C B D fusion protein technology facilitates the binding of bioactive cytokines to cellulose materials, and has permitted the analysis of several aspects of cell stimulation by non-soluble growth factors. A significant advantage of the C B D fusion protein system is that it permits the stimulation of factor-dependent cells with localized growth factor, essentially free of non-factor-derived interactions between the cell and the matrix. Furthermore, the tight binding of C B D -cytokine fusions to crystalline cellulose limits the spatial availability of cytokines and provides a convenient means to retain growth factors within perfusion tissue culture vessels. Indeed, cellulose-bound C B D - S C F localizes activated c-kit receptors to the cellulose interface. The spatially polarized growth factor signal stimulated receptor polarization in the cell membrane and cell adherence to the cellulose matrix. In addition, cellulose-surface presentation of the C B D -S C F modulated c-kit dephosphorylation kinetics, thus potentially modulating the overall response of the cell to the S C F signal. 210 The hematopoietic microenvironment is highly dynamic (Mayani et al, 1992). Similarly, we found that for B6SutA and M 0 7 e cells, the interaction of cells with the cellulose matrix can be very dynamic. Both of these factor-dependent cell lines are motile. For the culture of motile cells, the selection of the appropriate cellulose matrix morphology (e.g. Av ice l particles, B M C C fibrils, or V. ventricosa cellulose sheets) depends upon the desired geometry of cell stimulation. Conversely, the culture of nonmotile cells w i l l require the consideration of mixing conditions within the bioreactor to ensure cell-cellulose contact. However, B M C C fibrils remained well dispersed within the culture medium even in relatively static cell cultures. The lack of C B D - S C F surface diffusion does not equate to the immobility which would result from covalent linkage to the cellulose. For example, the dimer might be mobile under the directed force of receptor aggregation following ligand binding. Rather, it is l ikely that the binding stability of the C B D - S C F : S C F - C B D dimer is such that sufficient surface contacts are not broken during molecular "fluctuations" to permit significant surface diffusion. The surface mobility of other CBD-containing proteins suggest that other CBD-cytokine fusion proteins, which do not form dimers on the cellulose surface, should diffuse in 2-D while adsorbed to the cellulose surface. Our biophysical characterizations have shown that the C B D - S C F is effectively bound irreversibly to crystalline cellulose under cell culture conditions. This result implies that cells must interact directly with cellulose-bound C B D - S C F . The irreversible binding simplifies interpretation of cell culture results and confirms that C B D - S C F produced in E. coli does not stimulate cells by slow release from cellulose during cell culture. C B D - S C F encounters with its receptor may therefore be limited by (a) the cell membrane surface area contacting the cellulose, 211 (b) the diffusion of c-kit within the cell membrane, or (c) by membrane motion over the cellulose surface during cell movement. A further cellulose matrix selection criteria must also be the ease of recovery of the cellulose matrix and ideally all remaining CBD-cytokine fusions, from a cell culture prior to in vivo application of the cultured cells. This issue was not considered here. CBD-mediated localization of fusion proteins to various forms of cellulose is an adjuvant in the development of specific immune responses, both against the C B D and the fusion protein partner ( U B C Cellulase Group). In this study, biocompatibility considerations were limited to the application of CBD-mediated protein localization to cellulose for in vitro applications. Additionally, the question of the suitability for transplantation of cells grown in culture with growth factor-CBD fusion proteins, was not considered. The complete recovery of cellulose and C B D s from long-term cell cultures needs to be demonstrated before CBD-mediated growth factor immobilization for the cultivation of factor-dependent cells for transplantation can proceed. Finally, problems with the protease sensitivity of C B D fusion proteins at the P/T linker need to be addressed. This might be done by performing mutagenesis studies and fragment sequencing to identify and remove potential sites for protease attack. It must be noted that the C B D fusion proteins considered in this work were stable following binding to cellulose. This suggests that the molecules themselves are stable, but are susceptible to as yet unidentified E. coli proteases. In addition, the difficulty encountered when eluting E. co/i-produced cytokine-C B D fusion proteins (IL-3 and SCF) suggests that the C B D is not well suited as an affinity tag for all protein purifications. Rather, the high affinity and essentially irreversible binding of the C B D fusions, make C B D fusion protein technology an ideal approach for many protein immobilization applications. 212 213 Chapter 10 References 1. Abney, J. R., Scalettar, B . A . , and Owicki , J. C . (1989). Self diffusion of interacting membrane proteins. Biophys J 55, 817-33. 2. Adachi , S., Eb i , Y . , Nishikawa, S., Hayashi, S., Yamazaki, M . , Kasugai, T., Yamamura, T., Nomura, S., and Kitamura, Y . (1992). Necessity of extracellular domain of W (c-kit) receptors for attachment of murine cultured mast cells to fibroblasts. Blood 79, 650-6. 3. A l a i , M . , M u i , A . L . , Cutler, R. L . , Bustelo, X . R., Barbacid, M . , and Krystal, G . (1992). Steel factor stimulates the tyrosine phosphorylation of the proto- oncogene product, p95vav, in human hemopoietic cells. J B i o l Chem 267, 18021-5. 4. A l o n , R., Cahalon, L . , Hershkoviz, R., Elbaz, D . , Reizis, B . , Wallach, D . , Akiyama, S. K . , Yamada, K . M . , and Lider, O. (1994). TNF-alpha binds to the N-terminal domain of fibronectin and augments the beta 1-integrin-mediated adhesion of CD4+ T lymphocytes to the glycoprotein. J Immunol 152, 1304-13. 5. Asonuma, K . , and Vacanti, J. P. (1992). Cel l transplantation as replacement therapy for the future. Crit Care Nurs C l i n North A m 4, 249-54. 6. Avanzi , G . C , Br i zz i , M . F. , Giannotti, J. , Ciarletta, A . , Yang, Y . C , Pegoraro, L . , and Clark, S. C . (1990). M-07e human leukemic factor-dependent cell line provides a rapid and sensitive bioassay for the human cytokines G M - C S F and IL-3. J Ce l l Physiol 145, 458-64. 7. Axelrod, D . , Koppel, D . E . , Schlessinger, J., Elson, E . , and Webb, W . W . (1976). Mobi l i ty measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16, 1055-69. 214 8. Axelrod, D . , Ravdin, P., Koppel, D . E . , Schlessinger, J., Webb, W . W. , Elson, E . L . , and Podleski, T. R. (1976). Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. Proc Natl Acad Sci U S A 73, 4594-8. 9. Baghestanian, M . , Agis , H . , Bevec, D . , Bankl, H . C , Hofbauer, R., Kress, H . G . , Butterfield, J. H . , Muller , M . R., Ashman, L . K . , Fureder, W. , Wil lheim, M . , Lechner, K . , and Valent, P. (1996). Stem cell factor-induced downregulation of c-kit in human lung mast cells and H M C - 1 mast cells. Exp Hematol 24, 1377-86. 10. Basile, C . , and Drueke, T. (1989). Dialysis membrane biocompatibility. Nephron 52, 113-8. 11. B e l l , E . (1991). Tissue engineering: a perspective. J Ce l l Biochem45, 239-41. 12. Berg, H . C , Purcell, E . M . (1977). Physics of chemoreception. Biophys J 20, 193-219 13. Berg, H . C . (1993). Random Walks in Biology. Princeton: Princeton University Press. 14. Blackwell , J. , and Kolpak, F. J. (1975). The cellulose microfibril as an imperfect array of elementary fibrils. Macromolecules 8, 322-6. 15. Bray, M . R., Johnson, P. E . , Gilkes, N . R., Mcintosh, L . P., Kilburn, D . G . , and Warren, R. A . (1996). Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification. Protein Sci 5, 2311-8. 16. B r i zz i , M . F. , Avanzi , G . C , and Pegoraro, L . (1991). Hematopoietic growth factor receptors. Int J Ce l l Cloning 9, 274-300. 17. B r i zz i , M . F. , Garbarino, G . , Rossi, P. R., Pagliardi, G . L . , Arduino, C , Avanzi , G . C , and Pegoraro, L . (1993). Interleukin 3 stimulates proliferation and triggers endothelial- leukocyte adhesion molecule 1 gene activation of human endothelial cells. J C l i n Invest 91, 2887-92. 18. Broudy, V . C . (1997). Stem cell factor and hematopoiesis. Blood 90, 1345-64. 215 19. Broudy, V . C , L i n , N . L . , Priestley, G . V . , Nocka, K . , and Wolf, N . S. (1996). Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen. Blood 88, 75-81. 20. Broxmeyer, H . E . , Hangoc, G . , Cooper, S., Ribeiro, R. C , Graves, V . , Yoder, M . , Wagner, J., Vadhan-Raj, S., Benninger, L . , Rubinstein, P., and et al. (1992). Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci U S A 89, 4109-13. 21. Broxmeyer, H . E . , and Vadhan-Raj, S. (1989). Preclinical and clinical studies with the hematopoietic colony- stimulating factors and related interleukins. Immunol Res 8, 185-201. 22. Brunner, G . , Gabrilove, J. , Rifkin, D . B . , and Wilson, E . L . (1991). Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan. J Ce l l B i o l 114, 1275-83. 23. Caldwell , J., Locey, B . , Clarke, M . F. , Emerson, S. G . , and Palsson, B . O. (1991). Influence of medium exchange schedules on metabolic, growth, and G M - C S F . Biotechnol Prog 7, 2-8. 24. Cavallo, M . G . , Pozz i l l i , P., and Thorpe, R. (1994). Cytokines and autoimmunity. C l i n Exp Immunol 96, 1-7. 25. Charnick, S.B., Lauffenburger, D . A . (1990). Mathematical analysis of cell-target encounter rates in three dimensions. Effect of chemotaxis. Biophys J 5 1009-23 26. Chen, C. S., Mrksich, M . , Huang, S., Whitesides, G . M . , and Ingber, D . E . (1997). Geometric control of cell life and death. Science 276, 1425-8. 216 27. Chen, G . , Ito, Y . , and Imanishi, Y . (1997). Photo-immobilization of epidermal growth factor enhances its mitogenic effect by artificial juxtacrine signaling. Biochim Biophys Acta 1358, 200-8. 28. Chung, J. C , Sciaky, N . , and Gross, D . J. (1997). Heterogeneity of epidermal growth factor binding kinetics on individual cells. Biophys J 73, 1089-102. 29. Cima, L . G . , Vacanti, J. P., Vacanti, C , Ingber, D . , Mooney, D . , and Langer, R. (1991). Tissue engineering by cell transplantation using degradable polymer substrates. J Biomech Eng 113, 143-51. 30. Clark-Lewis, I., Aebersold, R., Ziltener, H . , Schrader, J. W. , Hood, L . E . , and Kent, S. B . (1986). Automated chemical synthesis of a protein growth factor for hemopoietic cells, interleukin-3. Science 231, 134-9. 31. Colotta, F. , Bussolino, F. , Polentarutti, N . , Guglielmetti, A . , Sironi, M . , Bocchietto, E . , De Rossi, M . , and Mantovani, A . (1993). Differential expression of the common beta and specific alpha chains of the receptors for G M - C S F , IL-3, and IL-5 in endothelial cells. Exp Ce l l Res 206, 311-7. 32. Conneally, E . , Eaves, C . J., and Humphries, R. K . (1998). Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential. Blood 91, 3487-93. 33. Coulombel, L . , Eaves, C , Kalousek, D . , Gupta, C , and Eaves, A . (1985). Long-term marrow culture of cells from patients with acute myelogenous leukemia. Selection in favor of normal phenotypes in some but not all cases. J C l i n Invest 75, 961-9. 34. Crane, G . M . , Ishaug, S. L . , and Mikos , A . G . (1995). Bone tissue engineering. Nat M e d 1, 1322-4. 217 35. Crapper, R. M . , Clark-Lewis, I., and Schrader, J. W . (1984). The in vivo functions and properties of persisting cell-stimulating factor. Immunology 53, 33-42. 36. Creagh, A . L . , Ong, E . , Jervis, E . , Kilburn, D . G . , and Haynes, C . A . (1996). Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas fimi to insoluble microcrystalline cellulose is entropically driven. Proc Natl Acad Sci U S A 93, 12229-34. 37. Cronkite, E . P. (1988). Analytical review of structure and regulation of hemopoiesis. Blood Cells 14(2-3), 313-328. 38. Crum, E . D . , and Kaplan, D . R. (1991). In vivo activity of solid phase interleukin 2. Cancer Res 51, 875-9. 39. Cuatrecasas, P. (1969). Interaction of insulin with the cell membrane: the primary action of insulin. Proc Natl Acad Sci U S A 63, 450-7. 40. Damude, H . G . , Withers, S. G . , Kilburn, D . G . , Mi l le r , R. C , Jr., and Warren, R. A . (1995). Site-directed mutation of the putative catalytic residues of endoglucanase C e n A from Cellulomonas fimi. Biochemistry 34, 2220-4. 41. Davis, M . W . , and Vacanti, J. P. (1996). Toward development of an implantable tissue engineered liver. Biomaterials 17, 365-72. 42. Dayer, J. M . , Ricard-Blum, S., Kaufmann, M . T., and Herbage, D . (1986). Type I X collagen is a potent inducer of P G E 2 and interleukin 1 production by human monocyte macrophages. F E B S Lett 198, 208-12. 43. De Smedt, S. C , Meyvis , T. K . L . , Demeester, J., Van Oostveldt, P., Blonk, J. C. G . , and Hennink, W . E . (1997). Diffusion of macromolecules in dextran methacrylate solutions and gels as studied by confocal scanning laser microscopy. Macromolecules 30, 4863-4870. 218 44. Dexter, T. M . , Al len , T. D . , and Lajtha, L . G . (1977). Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cel l Physiol 91, 335-44. 45. Dexter, T. M . , Coutinho, L . H . , Spooncer, E . , Heyworth, C. M . , Daniel, C . P., Schiro, R., Chang, J. , and Al len , T. D . (1990). Stromal cells in haemopoiesis. Ciba Found Symp 148, 76-86; discussion 86-95. 46. Dexter, T. M . , Spooncer, E . , Schofield, R., Lord, B . I., and Simmons, P. (1984). Haemopoietic stem cells and the problem of self-renewal. Blood Cells 10, 315-39. 47. D i n , N . , Forsythe, I. J., Burtnick, L . D. , Gilkes, N . R., Mil ler , R. C , Jr., Warren, R. A . , and Kilburn, D . G . (1994). The cellulose-binding domain of endoglucanase A (CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding. M o l Microbio l 77,747-55. 48. D in , N . , Gilkes, N . R., T., B . , Mi l le r , R. C , Jr., Warren, R. A . , and Kilburn, D . G . (1991). Non-hydrolytic Disruption of Cellulsoe Fibers by the Binding Domain of a Bacterial Cellulase. Bio/Technology 9, 1096-1099. 49. Ding, A . H . , Porteu, F., Sanchez, E . , and Nathan, C. F. (1990). Downregulation of tumor necrosis factor receptors on macrophages and endothelial cells by microtubule depolymerizing agents. J Exp M e d 171, 715-27. 50. Doheny G . (1996). U . B . C . Masters Thesis. 51. Downing, J. R., Roussel, M . F. , and Sherr, C. J. (1989). Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms. M o l Ce l l B i o l 9, 2890-6. 52. Drubin, D . G . , and Nelson, W . J. (1996). Origins of cell polarity. Ce l l 84, 335-44. 219 53. Dunbar, C . E . , Smith, D . A . , Kimbal l , J., Garrison, L . , Nienhuis, A . W. , and Young, N . S. (1991). Treatment of Diamond-Blackfan anaemia with haematopoietic growth factors, granulocyte-macrophage colony stimulating factor and interleukin 3: sustained remissions following IL-3 [see comments]. B r J Haematol 79, 316-21. 54. Eaves, A . C. , and Eaves, C. J. (1988). Maintenance and proliferation control of primitive hemopoietic progenitors in long-term cultures of human marrow cells. Blood Cells 14, 355-68. 55. Eaves, A . C , Eaves, C . J., Phillips, G . L . , and Barnett, M . J. (1993). Culture purging in leukemia: past, present, and future. Leuk Lymphoma 11, 259-63. 56. Eaves, C. J. , Cashman, J. D . , Kay, R. J., Dougherty, G . J., Otsuka, T., Gaboury, L . A . , Hogge, D . E . , Lansdorp, P. M . , Eaves, A . C , and Humphries, R. K . (1991). Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures, n . Analysis of positive and negative regulators produced by stromal cells within the adherent layer. Blood 78, 110-7. 57. Eaves, C . J., Cashman, J. D. , Sutherland, H . J., Otsuka, T., Humphries, R. K . , Hogge, D . E . , Lansdorp, P. L . , and Eaves, A . C. (1991). Molecular analysis of primitive hematopoietic cell proliferation control mechanisms. A n n N Y Acad Sci 628, 298-306. 58. Edelman, E . R., Mathiowitz, E . , Langer, R., and Klagsbrun, M . (1991). Controlled and modulated release of basic fibroblast growth factor. Biomaterials 12, 619-26. 59. Edgington, S. M . (1992). 3-D biotech: tissue engineering. Biotechnology (N Y ) 10, 855-60. 60: Edgington, S. M . (1994). A new force in biotech: tissue engineering. Understanding how mechanical force combines with cytokines and other growth factors may help shape future biotech drugs. Biotechnology (N Y ) 12, 361-4. 220 61. Eierman, D . F. , Johnson, C. E . , and Haskil l , J. S. (1989). Human monocyte inflammatory mediator gene expression is selectively regulated by adherence substrates. J Immunol 142, 1970-6. 62. Elam, J. H . , and Elam, M . (1993). Surface modification of intravenous catheters to reduce local tissue reactions. Biomaterials 14, 861-4. 63. Emerson, S.G., Palsson, B .O . , Clarke, M . F . (1991) The construction of high efficiency human bone marrow tissue ex vivo. J Ce l l Biochem 45, 268-72. 64. Enenstein, J., Waleh, N . S., and Kramer, R. H . (1992). Basic F G F and TGF-beta differentially modulate integrin expression of human microvascular endothelial cells. Exp Ce l l Res 203, 499-503. 65. Ertel, S. I., Ratner, B . D . , Kaul , A . , Schway, M . B . , and Horbett, T. A . (1994). In vitro study of the intrinsic toxicity of synthetic surfaces to cells. J Biomed Mater Res 28, 667-75. 66. Farrar, W . L . , Thomas, T. P., and Anderson, W . B . (1985). Altered cytosol/membrane enzyme redistribution on interleukin-3 activation of protein kinase C. Nature 315, 235-7. 67. Fielden, K . E . , Newton, J. M . , O'Brien, P., and Rowe, R. C. (1988). Thermal studies on the interaction of water and microcrystalline cellulose. J Pharm Pharmacol 40, 674-8. 68. Fraser, C . C , Szilvassy, S. J., Eaves, C. J., and Humphries, R. K . (1992). Proliferation of totipotent hematopoietic stem cells in vitro with retention of long-term competitive in vivo reconstituting ability. Proc Natl Acad Sci U S A 89, 1968-72. 69. Friesel, R. E . , and Maciag, T. (1995). Molecular mechanisms of angiogenesis: fibroblast growth factor signal transduction. Faseb J 9, 919-25. 221 70. Fukunaga, R., Seto, Y . , Mizushima, S., and Nagata, S. (1990). Three different m R N A s encoding human granulocyte colony-stimulating factor receptor. Proc Natl Acad Sci U S A 87, 8702-6. 71. Fukushima, N . , and Ohkawa, H . (1995). Hematopoietic stem cells and microenvironment: the proliferation and differentiation of stromal cells. Crit Rev Oncol Hematol 20, 255-70. 72. Gabay, C. , Silacci, P., Genin, B . , Mentha, G . , Le Coultre, C. , and Guerne, P. A . (1995). Soluble interleukin-6 receptor strongly increases the production of acute-phase protein by hepatoma cells but exerts minimal changes on human primary hepatocytes. Eur J Immunol 25, 2378-83. 73. Gadella, T. W . , Jr., and Jovin, T. M . (1995). Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. J Ce l l B i o l 129, 1543-58. 74. Gardner, K . H . , and Blackwell , J. (1971). The substructure of the cellulose microfibrils from the cell walls of the algae Valonia ventricosa. J Ultrastruct Res 36, 725-31. 75. Gibson, F. M . , Scopes, J., Daly, S., Ba l l , S. E . , and Gordon-Smith, E . C . (1994). In vitro response of normal and aplastic anemia bone marrow to mast cell growth factor and in combination with granulocyte-macrophage colony-stimulating factor and interleukin-3. Exp Hematol 22, 302-12. 76. Gilkes, N . R., Henrissat, B . , Kilburn, D . G . , Mi l le r , R. C , Jr., and Warren, R. A . (1991). Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbio l Rev 55, 303-15. 222 77. Gilkes, N . R., Jervis, E . , Henrissat, B . , Tekant, B . , Mi l le r , R. C , Jr., Warren, R. A . , and Kilburn, D . G . (1992). The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose. J B i o l Chem 267, 6743-9. 78. Gilkes, N . R., Kilburn, D . G . , Mi l le r , R. C. , Jr., Warren, R. A . , Sugiyama, J., Chanzy, H . , and Henrissat, B . (1993). Visualization of the adsorption of a bacterial endo-beta-l,4-glucanase and its isolated cellulose-binding domain to crystalline cellulose. Int J B i o l Macromol 15, 347-51. 79. Gilkes, N . R., Langsford, M . L . , Kilburn, D . G . , Mi l le r , R. C , Jr., and Warren, R. A . (1984). Mode of action and substrate specificities of cellulases from cloned bacterial genes. J B i o l Chem 259, 10455-9. 80. Gilkes, N . R. , Warren, R. A . , Mi l le r , R. C , Jr., and Kilburn, D . G . (1988). Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. J B i o l Chem 263, 10401-7. 81. Gi l l i s , S., and Watson, J. (1981). Interleukin-2 induction of hapten-specific cytolytic T cells in nude mice. J Immunol 126, 1245-8. 82. Gish, G . , Larose, L . , Shen, R., and Pawson, T. (1995). Biochemical analysis of SH2 domain-mediated protein interactions. Methods Enzymol 254, 503-23. 83. Goodall , G . J. , Bagley, C . J. , Vadas, M . A . , and Lopez, A . F . (1993). A model for the interaction of the G M - C S F , IL-3 and IL-5 receptors with their ligands. Growth Factors 8, 87-97. 84. Gordon, M . Y . , Riley, G . P., Watt, S. M . , and Greaves, M . F. (1987). Compartmentalization of a haematopoietic growth factor ( G M - C S F ) by glycosaminoglycans in the bone marrow microenvironment. Nature 326, 403-5. 223 85. Grabbe, J., Welker, P., Moller , A . , Dippel, E . , Ashman, L . K . , and Czarnetzki, B . M . (1994). Comparative cytokine release from human monocytes, monocyte-derived immature mast cells, and a human mast cell line (HMC-1) . J Invest Dermatol 103, 504-8. 86. Graham, R. W . , Greenwood, J. M . , Warren, R. A . , Kilburn, D . G . , and Trimbur, D . E . (1995). The pTugA and pTugAS vectors for high-level expression of cloned genes in Escherichia coli . Gene 758,51-4. 87. Greenwood, J . M . (1993). U . B . C . Ph.D. Thesis. 88. Greenwood, J. M . , Gilkes, N . R., Kilburn, D . G . , Mi l le r , R. C , Jr., and Warren, R. A . (1989). Fusion to an endoglucanase allows alkaline phosphatase to bind to cellulose [published erratum appears in F E B S Lett 1990 Feb 12;261(1):217]. F E B S Lett 244, 127-31. 89. Greenwood, J. M . , Ong, E . , Gilkes, N . R., Warren, R. A . , Mil ler , R. C , Jr., and Kilburn, D . G . (1992). Cellulose-binding domains: potential for purification of complex proteins. Protein Eng 5, 361-5. 90. Guo, C , Dower, S. K . , Holowka, D . , and Baird, B . (1995). Fluorescence resonance energy transfer reveals interleukin (IL)-1- dependent aggregation of IL-1 type I receptors that correlates with receptor activation. J B i o l Chem 270, 27562-8. 91. Hansbrough, J. F. , Cooper, M . L . , Cohen, R., Spielvogel, R., Greenleaf, G . , Bartel, R. L . , and Naughton, G . (1992). Evaluation of a biodegradable matrix containing cultured human fibroblasts as a dermal replacement beneath meshed skin grafts on athymic mice. Surgery 111, 438-46. 92. Harpel, J. G . , Metz, C. N . , Kojima, S., and Rifkin, D . B . (1992). Control of transforming growth factor-beta activity: latency vs. activation. Prog Growth Factor Res 4, 321-35. 224 93. Hasenwinkle, D . , Jervis, E . , Kops, O., L i u , C , Lesnicki, G . , Haynes, C. A . , and Kilburn, D . G . (1997). Very high-level production and export in Escherichia coli of a cellulose binding domain for use in a generic secretion-affinity fusion system. Biotechnol Bioeng 55, 854-863. 94. Haynes, C . A . , and Norde, W . (1995). Structures and stabilities of adsorbed proteins. J Col lo id Interface Sci 169, 313-328. 95. Healy, K . E . , Lorn, B . , and Hockberger, P. E . (1994). Spatial distribution of mammalian cells dictated by material surface chemistry. Biotechnol Bioeng 43, 792-800. 96. Hecht P., A . K . (1992). Extracellular proteases and embryonic pattern formation. Trends cell, biol . 2, 197-202. 97. Heldin, C . H . , Betsholtz, C , Johnsson, A . , Nister, M . , Ek, B . , Ronnstrand, L . , Wasteson, A . , and Westermark, B . (1985). Platelet-derived growth factor: mechanism of action and relation to oncogenes. J Ce l l Sci Suppl 3, 65-76. 98. Henis, Y . I., Yaron, T., Lamed, R., Rishpon, J., Sahar, E . , and Katchalski-Katzir, E . (1988). Mobi l i ty of enzymes on insoluble substrates: the beta-amylase-starch gel system. Biopolymers 27, 123-38. 99. Henrissat, B . , Claeyssens, M . , Tomme, P., Lemesle, L . , and Mornon, J. P. (1989). Cellulase families revealed by hydrophobic cluster analysis. Gene 81, 83-95. 100. Hertle, M . D. , Jones, P. H . , Groves, R. W . , Hudson, D . L . , and Watt, F . M . (1995). Integrin expression by human epidermal keratinocytes can be modulated by interferon-gamma, transforming growth factor-beta, tumor necrosis factor-alpha, and culture on a dermal equivalent. J Invest Dermatol 104, 260-5. 101. H i b i , M . , Murakami, M . , Saito, M . , Hirano, T., Taga, T., and Kishimoto, T. (1990). Molecular cloning and expression of an IL-6 signal transducer, gpl30. Cel l 63, 1149-57. 225 102. H i b i , M . , Nakajima, K . , and Hirano, T. (1996). IL-6 cytokine family and signal transduction: a model of the cytokine system. J M o l M e d 74, 1-12. 103. Hogge, D . E . , Cashman, J. D . , Humphries, R. K . , and Eaves, C. J. (1991). Differential and synergistic effects of human granulocyte-macrophage colony-stimulating factor and human granulocyte colony-stimulating factor on hematopoiesis in human long-term marrow cultures. Blood 77, 493-9. 104. Hooton, J. W . , Gibbs, C , and Paetkau, V . (1985). Interaction of interleukin 2 with cells: quantitative analysis of effects. J Immunol 135, 2464-73. 105. Hora, M . S., Rana, R. K . , Nunberg, J. H . , Tice, T. R., Gilley, R. M . , and Hudson, M . E . (1990). Controlled release of interleukin-2 from biodegradable microspheres. Biotechnology (N Y ) 8, 755-8. 106. Horwitz, J. I., Toner, M . , Tompkins, R. G . , and Yarmush, M . L . (1993). Immobilized IL-2 preserves the viability of an IL-2 dependent cell line. M o l Immunol 30, 1041-8. 107. Hsu, Y . R., Narhi, L . O., Spahr, C , Langley, K . E . , and L u , H . S. (1996). In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization. Protein Sci 5, 1165-73. 108. Hsu, Y . R., W u , G . M . , Mendiaz, E . A . , Syed, R., Wypych, J., Toso, R., Mann, M . B . , Boone, T. C , Narhi, L . O., L u , H . S., and Langley, K . E . (1997). The majority of stem cell factor exists as monomer under physiological conditions. Implications for dimerization mediating biological activity. J B i o l Chem 272, 6406-15. 109. Hubbell, J. A . , Massia, S. P., Desai, N . P., and Drumheller, P. D . (1991). Endothelial cell-selective materials for tissue engineering in the vascular graft via a new receptor. Biotechnology (N Y ) 9, 568-72. 226 110. Hughes, P. F. , Thacker, J. D . , Hogge, D . , Sutherland, H . J., Thomas, T. E . , Lansdorp, P. M . , Eaves, C . J. , and Humphries, R. K . (1992). Retroviral gene transfer to primitive normal and leukemic hematopoietic cells using clinically applicable procedures. J C l i n Invest 89, 1817-24. 111. Hwang, Y . , and Ediger, M . D . (1996). Enhanced translational diffusion of rubrene and tetracene in polysulfone. J Polym Sci Part B 34, 2853-2861. 112. Uile, J. N . , Keller, J. , Oroszlan, S., Henderson, L . E . , Copeland, T. D . , Fitch, F., Prystowsky, M . B . , Goldwasser, E . , Schrader, J. W. , Palaszynski, E . , Dy, M . , and Lebel, B . (1983). Biologic properties of homogeneous interleukin 3. I. Demonstration of W E H I - 3 growth factor activity, mast cell growth factor activity, p cell- stimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity. J Immunol 131, 282-7. 113. Indrova, M . , Pajtasz-Piasecka, E . , Radzikowski, C , and Bubenik, J. (1997). C T L L assay: comparison of two methods for IL-2 determination. Fol ia B i o l 43, 45-7. 114. Ingber, D . E . (1992). Extracellular matrix as a solid-state regulator in angiogenesis: identification of new targets for anti-cancer therapy. Semin Cancer B i o l 3, 57-63. 115. Ingber, D . E . (1993). The riddle of morphogenesis: a question of solution chemistry or molecular cell engineering? Ce l l 75, 1249-52. 116. Ingber, D . E . , and Folkman, J. (1989). How does extracellular matrix control capillary morphogenesis? Ce l l 58, 803-5. 117. Ito, Y . , Chen, G . , and Imanishi, Y . (1996). Photoimmobilization of insulin onto polystyrene dishes for protein-free cell culture. Biotechnol Prog 12, 700-2. 118. Ito, Y . , Kondo, S., Chen, G . , and Imanishi, Y . (1997). Patterned artificial juxtacrine stimulation of cells by covalently immobilized insulin. F E B S Lett 403, 159-62., 227 119. Ito, Y . , L i u , S. Q., and Imanishi, Y . (1991). Enhancement of cell growth on growth factor-immobilized polymer fi lm. Biomaterials 12, 449-53. 120. Jacobson, K . , Derzko, Z . , W u , E . S., Hou, Y . , and Poste, G . (1976). Measurement of the lateral mobility of cell surface components in single, l iving cells by fluorescence recovery after photobleaching. J Supramol Struct 5, 565(417)-576(428). 121. Jacobson, K . , Ishihara, A . , and Inman, R. (1987). Lateral diffusion of proteins in membranes. Annu Rev Physiol 49, 163-75. 122. Jones, M . D. , Narhi, L . O., Chang, W . C , and L u , H . S. (1996). Refolding and oxidation of recombinant human stem cell factor produced in Escherichia coli . J B i o l Chem 271, 11301-8. 123. Kaplan, D . R. (1991). Solid phase interleukin 2. M o l Immunol 28, 1255-61. 124. Kaplan, G . , Britton, W . J., Hancock, G . E . , Theuvenet, W . J. , Smith, K . A . , Job, C . K . , Roche, P. W . , Mol loy , A . , Burkhardt, R., Barker, J., and et al. (1991). The systemic influence of recombinant interleukin 2 on the manifestations of lepromatous leprosy. J Exp M e d 173, 993-1006. 125. Katchalski-Katzir, E . , Rishpon, J., Sahar, E . , Lamed, R., and Henis, Y . I. (1985). Enzyme diffusion and action on soluble and insoluble substrate biopolymers. Biopolymers 24, 257-77'. 126. Kaufman, E . N . , and Jain, R. K . (1991). Measurement of mass transport and reaction parameters in bulk solution using photobleaching. Reaction limited binding regime. Biophys J 60, 596-610. 127. Kaufman, E . N . , and Jain, R. K . (1990). Quantification of transport and binding parameters using fluorescence recovery after photobleaching. Potential for in vivo applications. Biophys J 58, 873-85. 228 128. Kinashi, T., and Springer, T. A . (1994). Steel factor and c-kit regulate cell-matrix adhesion. Blood 83, 1033-8. 129. Kittler, E . L . , McGrath, H . , Temeles, D . , Crittenden, R. B . , Kister, V . K . , and Quesenberry, P. J. (1992). Biologic significance of constitutive and subliminal growth factor production by bone marrow stroma. Blood 79, 3168-78. 130. Klagsbrun, M . (1990). The affinity of fibroblast growth factors (FGFs) for heparin; F G F -heparan sulfate interactions in cells and extracellular matrix. Curr Opin Ce l l B i o l 2, 857-63. 131. K le in , G . (1995). The extracellular matrix of the hematopoietic microenvironment. Experientia 51, 914-26. 132. Klingemann, H . G . , Neerunjun, J., Schwulera, U . , and Ziltener, H . J. (1993). Culture of normal and leukemic bone marrow in interleukin-2: analysis of cell activation, cell proliferation, and cytokine production. Leukemia 7, 1389-93. 133. Knospe, W . H . , Husseini, S. G . , and Fried, W . (1989). Hematopoiesis on cellulose ester membranes. X I . Induction of new bone and a hematopoietic microenvironment by matrix factors secreted by marrow stromal cells. Blood 74, 66-70. 134. Knowles, G . C , and McCul loch , C. A . (1992). Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods. J Histochem Cytochem40, 1605-12. 135. Kodama, H . , Nose, M . , Niida, S., and Nishikawa, S. (1994). Involvement of the c-kit receptor in the adhesion of hematopoietic stem cells to stromal cells. Exp Hematol 22, 979-84. 136. Koller , M . R., Bender, J. G . , Mi l le r , W . M . , and Papoutsakis, E . T. (1993). Expansion of primitive human hematopoietic progenitors in a perfusion bioreactor system with IL-3, IL-6, and stem cell factor. Biotechnology ( N Y ) 11, 358-63. 229 137. Koller , M . R., Bender, J. G . , Mil ler , W . M . , and Papoutsakis, E . T. (1992). Reduced oxygen tension increases hematopoiesis in long-term culture of human stem and progenitor cells from cord blood and bone marrow. Exp Hematol 20, 264-70. 138. Koller , M . R., Bradley, M . S., and Palsson, B . O. (1995). Growth factor consumption and production in perfusion cultures of human bone marrow correlate with specific cell production. Exp Hematol 23, 1275-83. 139. Koller , M . R., Emerson, S. G . , and Palsson, B . O. (1993). Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures. Blood 82, 378-84. 140. Koller , M . R., Manchel, I., and Palsson, B . O. (1997). Importance of parenchymal:stromal cell ratio for the ex vivo reconstitution of human hematopoiesis. Stem Cells 15, 305-13. 141. Koller , M . R., and Palsson, B . O. (1993). Tissue engineering: Reconstitution of human hematopoiesis ex vivo. Biotechnol Bioeng 42, 909-930. 142. Korbling, M . , and Champlin, R. (1996). Peripheral blood progenitor cell transplantation: a replacement for marrow auto- or allografts. Stem Cells 14, 185-95. 143. Kubitscheck, U . , Tschodrich-Rotter, M . , Wedekind, P., and Peters, R. (1996). Two-photon scanning microphotolysis for three-dimensional data storage and biological transport measurements. J Microsc 182, 225-33. 144. Kuh l , P. R., and Griffith-Cima, L . G . (1996). Tethered epidermal growth factor as a paradigm for growth factor- induced stimulation from the solid phase [published erratum appears in Nat M e d 1997 Jan;3(l):93]. Nat M e d 2, 1022-7. 145. Kurosawa, K . , Miyazawa, K . , Gotoh, A . , Katagiri, T., Nishimaki, J. , Ashman, L . K . , and Toyama, K . (1996). Immobilized anti-KIT monoclonal antibody induces ligand-independent 230 dimerization and activation of Steel factor receptor: biologic similarity with membrane-bound form of Steel factor rather than its soluble form. Blood 87, 2235-43. 146. Kurosawa, K . , Miyazawa, K . , Gotoh, A . , Katagiri, T., Nishimaki, J., Ashman, L . K . , and Toyama, K . (1996). Immobilized anti-KTT monoclonal antibody induces ligand-independent dimerization and activation of Steel factor receptor: biologic similarity, with membrane-bound form of Steel factor rather than its soluble form. Blood 87, 2235-43. 147. Laluppa, J. A . , McAdams, T. A . , Papoutsakis, E . T., and Mil le r , W . M . (1997). Culture materials affect ex vivo expansion of hematopoietic progenitor cells. J Biomed Mater Res 36, 347-59. 148. Langer, R. (1996). Controlled release of a therapeutic protein. Nat M e d 2, 742-3. 149. Langer, R. (1997). Tissue engineering: a new field and its challenges. Pharm Res 14, 840-1. 150. Langer, R., and Moses, M . (1991). Biocompatible controlled release polymers for delivery of polypeptides and growth factors. J Ce l l Biochem 45, 340-5. 151. Langer, R., and Vacanti, J. P. (1993). Tissue engineering. Science 260, 920-6. 152. Langley, K . E . , Mendiaz, E . A . , L i u , N . , Narhi, L . O., Zeni, L . , Parseghian, C. M . , Clogston, C . L . , Leslie, I., Pope, J. A . , L u , H . S., and et al. (1994). Properties of variant forms of human stem cell factor recombinantly expressed in Escherichia coli . Arch Biochem Biophys 311, 55-61. 153. Langsford, M . L . , Gilkes, N . R., Singh, B . , Moser, B . , Mi l le r , R. C , Jr., Warren, R. A . , and Kilburn, D . G . (1987). Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains. F E B S Lett 225, 163-7. 231 154. Lansdorp, P. M . , and Dragowska, W . (1993). Maintenance of hematopoiesis in serum-free bone marrow cultures involves sequential recruitment of quiescent progenitors. Exp Hematol 21, 1321-7. 155. Lemmon, M . A . , Pinchasi, D. , Zhou, M . , Lax, L , and Schlessinger, J. (1997). K i t receptor dimerization is driven by bivalent binding of stem cell factor. J B i o l Chem 272, 6311-7. 156. Lenarsky, C. (1995). Immune recovery after bone marrow transplantation. Curr Opin Hematol 2, 409-12. 157. Lev, S., Yarden, Y . , and Givo l , D . (1992). Dimerization and activation of the kit receptor by monovalent and bivalent binding of the stem cell factor. J B i o l Chem 267, 15970-7. 158. Linder, M . , Mattinen, M . L . , Kontteli, M . , Lindeberg, G . , Stahlberg, J., Drakenberg, T., Reinikainen, T., Pettersson, G . , and Annila, A . (1995). Identification of functionally important amino acids in the cellulose- binding domain of Trichoderma reesei cellobiohydrolase I. Protein Sci 4, 1056-64. 159. L i u , J. J. , Chen, B . S., Tsai, T. F. , W u , Y . J., Pang, V . F. , Hsieh, A . , Hsieh, J. H . , and Chang, T. H . (1991). Long term and large-scale cultivation of human hepatoma Hep G 2 cells in hollow fiber bioreactor. Cultivation of human hepatoma Hep G2 in hollow fiber bioreactor. Cytotechnology 5, 129-39. 160. L i u , L . , Cutler, R. L . , M u i , A . L . , and Krystal, G . (1994). Steel factor stimulates the serine/threonine phosphorylation of the interleukin-3 receptor. J B i o l Chem 269, 16774-9. 161. L i u , S. Q., Ito, Y . , and Imanishi, Y . (1992). Ce l l growth on immobilized cell-growth factor; 4: Interaction of fibroblast cells with insulin immobilized on poly(methyl methacrylate) membrane. J Biochem Biophys Methods 25, 139-48. 232 162. Ljungquist, P., Wasteson, A . , and Magnusson, K . E . (1989). Lateral diffusion of plasma membrane receptors labelled with either platelet-derived growth factor (PDGF) or wheat germ agglutinin ( W G A ) in human polymorphonuclear leukocytes and fibroblasts. Biosci Rep 9, 63-73. 163. Ljungquist-Hoddelius, P., Li rva l l , M . , Wasteson, A . , and Magnusson, K . E . (1991). Lateral diffusion of P D G F beta-receptors in human fibroblasts. Biosci Rep 11, 43-52. 164. Long, M . W . , Briddell , R., Walter, A . W. , Bruno, E . , and Hoffman, R. (1992). Human hematopoietic stem cell adherence to cytokines and matrix molecules. J C l i n Invest 90, 251-5. 165. Lopina, S. T., W u , G . , Merr i l l , E . W. , and Griffith-Cima, L . (1996). Hepatocyte culture on carbohydrate-modified star polyethylene oxide hydrogels. Biomaterials 17, 559-69. 166. Lowenthal, J. W . , Corthesy, P., Tougne, C , Lees, R., MacDonald, H . R., and Nabholz, M . (1985). High and low affinity IL 2 receptors: analysis by IL 2 dissociation rate and reactivity with monoclonal anti-receptor antibody PC61. J Immunol 135, 3988-94. 167. Lowenthal, J. W . , MacDonald, H . R., and Iacopetta, B . J. (1986). Intracellular pathway of interleukin 2 following receptor-mediated endocytosis. Eur J Immunol 16, 1461-3. 168. Lowry, P. A . , and Tabbara, I. A . (1992). Peripheral hematopoietic stem cell transplantation: current concepts. Exp Hematol 20, 937-42. 169. L u , H . S., Chang, W . C , Mendiaz, E . A . , Mann, M . B . , Langley, K . E . , and Hsu, Y . R. (1995). Spontaneous dissociation-association of monomers of the human-stem-cell- factor dimer. Biochem J 305, 563-8. 170. L u , L . , Xiao , M . , Shen, R. N . , Grigsby, S., and Broxmeyer, H . E . (1993). Enrichment, characterization, and responsiveness of single primitive CD34 human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential. Blood 81, 41-8. 233 171. Majumdar, M . K . , Everett, E . T., Xiao, X . , Cooper, R., Langley, K . , Kapur, R., V i k , T., and Wil l iams, D . A . (1996). Xenogeneic expression of human stem cell factor in transgenic mice mimics codominant c-kit mutations. Blood 87, 3203-11. 172. Mandolfo, S., Tetta, C , David, S., Gervasio, R., Ognibene, D . , Wratten, M . L . , Tessore, E . , and Imbasciati, E . (1997). In vitro and in vivo biocompatibility of substituted cellulose and synthetic membranes. Int J Ar t i f Organs 20, 603-9. 173. Manfredi, R., Re, M . C , Furlini, G . , Gorini , R., and Chiodo, F. (1997). In vivo effects of recombinant human granulocyte-macrophage colony- stimulating factor ( r H u G M - C S F ) , alone and associated with zidovudine, on H l Y - l replication. New Microbiol 20, 345-50. 174. Marks, M . G. , Doil lon, C , and Silver, F. H . (1991). Effects of fibroblasts and basic fibroblast growth factor on facilitation of dermal wound healing by type I collagen matrices. J Biomed Mater Res 25, 683-96. 175. Matsui, Y . , Toksoz, D . , Nishikawa, S., Nishikawa, S., Will iams, D . , Zsebo, K . , and Hogan, B . L . (1991). Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture. Nature 353, 750-2. 176. Mayani, H . , Guilbert, L . J., and Janowska-Wieczorek, A . (1992). Biology of the hemopoietic microenvironment. Eur J Haematol 49, 225-33. 177. McGhee, J. D . , and Hippel, P. H . v. (1974). Theoretical aspects of DNA-protein interactions: co-operative and non- co-operative binding of large ligands to a one-dimensional homogeneous lattice. J M o l B i o l 86, 469-89. 178. M c K a y , D . A . , Kusel, J. R., and Wilkinson, P. C. (1991). Studies of chemotactic factor-induced polarity in human neutrophils. L ip id mobility, receptor distribution and the time-sequence of polarization. J Ce l l Sci 100,473-9. 234 179. McKinstry , W . J., L i , C . L . , Rasko, J. E . , Nicola, N . A . , Johnson, G . R., and Metcalf, D . (1997). Cytokine receptor expression on hematopoietic stem and progenitor cells. Blood 89, 65-71. 180. Meininger, C. J., Yano, H . , Rottapel, R., Bernstein, A . , Zsebo, K . M . , and Zetter, B . R. (1992). The c-kit receptor ligand functions as a mast cell chemoattractant. Blood 79, 958-63. 181. Meinke, A . , Damude, H . G . , Tomme, P., Kwan, E . , Kilburn, D . G . , Mi l le r , R. C , Jr., Warren, R. A . , and Gilkes, N . R. (1995). Enhancement of the endo-beta-l,4-glucanase activity of an exocellobiohydrolase by deletion of a surface loop. J B i o l Chem 270, 4383-6. 182. Meinke, A . , Gilkes, N . R., Kilburn, D . G . , Mi l le r , R. C , Jr., and Warren, R. A . (1993). Cellulose-binding polypeptides from Cellulomonas fimi: endoglucanase D (CenD), a family A beta-l,4-glucanase. J Bacteriol 175, 1910-8. 183. Metcalf, D . (1992). The hemopoietic regulators—an embarrassment of riches. Bioessays 14, 799-805. 184. Miao , H . Q., Ishai-Michaeli, R., Atzmon, R., Peretz, T., and Vlodavsky, I. (1996). Sulfate moieties in the subendothelial extracellular matrix are involved in basic fibroblast growth factor sequestration, dimerization, and stimulation of cell proliferation. J B i o l Chem 271, 4879-86. 185. Mikos , A . G . , Sarakinos, G . , Leite, S. M . , Vacanti, J. P., and Langer, R. (1993). Laminated three-dimensional biodegradable foams for use in tissue engineering. Biomaterials 14, 323-30. 186. Minton, A . P. (1989). Lateral diffusion of membrane proteins in protein-rich membranes. A simple hard particle model for concentration dependence of the two- dimensional diffusion coefficient. Biophys J 55, 805-8. 235 187. Miyajima, A . (1992). Molecular structure of the IL-3, G M - C S F and IL-5 receptors. Int J Ce l l Cloning 10, 126-34. 188. Miyajima, A . , Hara, T., and Kitamura, T. (1992). Common subunits of cytokine receptors and the functional redundancy of cytokines. Trends Biochem Sci 17, 378-82. 189. Miyajima, A . , M u i , A . L . , Ogorochi, T., and Sakamaki, K . (1993). Receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5. Blood 82, 1960-74. 190. Miyazawa, K . , Shimomura, T., and Kitamura, N . (1996). Activation of hepatocyte growth factor in the injured tissues is mediated by hepatocyte growth factor activator. J B i o l Chem 271, 3615-8. 191. Miyazawa, K . , Wil l iams, D . A . , Gotoh, A . , Nishimaki, J., Broxmeyer, H . E . , and Toyama, K . (1995). Membrane-bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form. Blood 85, 641-9. 192. Moore, M . A . , and Hoskins, I. (1994). E x vivo expansion of cord blood-derived stem cells and progenitors. Blood Cells 20, 468-79. 193. Morris , A . J., Turnbull, J. E . , Riley, G . P., Gordon, M . Y . , and Gallagher, J. T. (1991). Production of heparan sulphate proteoglycans by human bone marrow stromal cells. J Ce l l Sci 99, 149-56. 194. Morstyn, G . , and Burgess, A . W . (1988). Hemopoietic growth factors: a review. Cancer Res 48, 5624-37. 195. Mosley, B . , Beckmann, M . P., March, C. J., Idzerda, R. L . , Gimpel, S. D . , VandenBos, T., Friend, D . , Alpert, A . , Anderson, D . , Jackson, J., and et al. (1989). The murine interleukin-4 236 receptor: molecular cloning and characterization of secreted and membrane bound forms. Cel l 59, 335-48. 196. M u i , A . L . , Murthy, S. C , Sorensen, P. H . , and Krystal, G . (1990). The mechanism of action of murine interleukin-3: current status. Prog C l i n B i o l Res 352, 169-78. 197. Mujais, S. K . , Ivanovich, P., Bereza, L . A . , and Vidovich, M . (1995). Biocompatibility and the clinical choice of dialysis membranes. Contrib Nephrol 113, 101-9. 198. Murata, M . , Yano, T., Yoshino, I., Togami, M . , Sogabe, M . , Yasumoto, K . , Sugimachi, K . , Kimura, G . , and Nomoto, K . (1991). Development of a new culture system for human lymphokine-activated killer cells: comparison with a conventional static culture method. Cytotechnology 7, 75-83. 199. Murray, J. B . , Brown, L . , Langer, R., and Klagsburn, M . (1983). A micro sustained release system for epidermal growth factor. In Vitro 19, 743-8. 200. Murthy, S. C , Eaves, C. J., and Krystal, G . (1989). A simple three-step purification procedure for interleukin 3 involving absorption to fixed cells. Exp Hematol 17, 997-1003. 201. Murthy, S. C , M u i , A . L . , and Krystal, G . (1990). Characterization of the interleukin 3 receptor. Exp Hematol 18, 11-7. 202. Murthy, S. C , Sorensen, P. H . , M u i , A . L . , and Krystal, G . (1989). Interleukin-3 down-regulates its own receptor. Blood 73, 1180-7. 203. Naeim, F. , Moatamed, F. , and Sahimi, M . (1996). Morphogenesis of the bone marrow: fractal structures and diffusion- limited growth. Blood 87, 5027-31. 204. Nathan, C , and Sanchez, E . (1990). Tumor necrosis factor and CD11/CD18 (beta 2) integrins act synergistically to lower c A M P in human neutrophils. J Ce l l B i o l 111, 2171-81. 205. Nathan, C , and Sporn, M . (1991). Cytokines in context. J Cel l B i o l 113, 981-6. 237 206. Nelson, B . H . , Lord, J. D. , and Greenberg, P. D . (1994). Cytoplasmic domains of the interleukin-2 receptor beta and gamma chains mediate the signal for T-cell proliferation. Nature 369, 333-6. 207. Nemunaitis, J. (1993). Growth factors in allogeneic transplantation. Semin Oncol 20, 96-101. 208. Nemunaitis, J. , and Rosenfeld, C. (1993). Mobilization of peripheral stem cells for transplantation. J Hematother 2, 351-5. 209. Nemunaitis, J. J. (1992). R h G M - C S F in bone marrow transplantation: experience in pediatric patients. M e d Pediatr Oncol Suppl 2, 31-3. 210. Nicola , N . A . (1994). Cytokine pleiotropy and redundancy: a view from the receptor. Stem Cells (Dayt) 12, 3-12; discussion 12-4. 211. Nicola , N . A . (1989). Hemopoietic cell growth factors and their receptors. Annu Rev Biochem 58, 45-77. 212. Nordon, R. E . , Haylock, D . N . , Gaudry, L . , and Schindhelm, K . (1996). Hollow-fibre affinity cell separation system for CD34+ cell enrichment. Cytometry 24, 340-7. 213. Nunberg, J. H . , Doyle, M . V . , York, S. M . , and York, C. J. (1989). Interleukin 2 acts as an adjuvant to increase the potency of inactivated rabies virus vaccine. Proc Natl Acad Sci U S A 86, 4240-3. 214. Okumura, N . , Tsuji, K . , Ebihara, Y . , Tanaka, I., Sawai, N . , Koike, K . , Komiyama, A . , and Nakahata, T. (1996). Chemotactic and chemokinetic activities of stem cell factor on murine hematopoietic progenitor cells. Blood 87, 4100-8. 215. Olsson, I., Gullberg, U . , Lantz, M . , and Richter, J. (1992). The receptors for regulatory molecules of hematopoiesis. Eur J Haematol 48, 1-9. 238 216. Olsson, I., Lantz, M . , Nilsson, E . , Peetre, C , Thysell, H . , Grubb, A . , and Adolf, G . (1989). Isolation and characterization of a tumor necrosis factor binding protein from urine. Eur J Haematol 42, 270-5. 217. Ong, E . , Al imont i , J. B . , Greenwood, J. M . , Mi l le r , R. C. , Jr., Warren, R. A . , and Kilburn, D . G . (1995). Purification of human interleukin-2 using the cellulose-binding domain of a prokaryotic cellulase. Bioseparation 5, 95-104. 218. Ong, E . , Gilkes, N . R., Mi l le r , R. C. , Jr., Warren, A . J., and Kilburn, D . G . (1991). Enzyme immobilization using a cellulose-binding domain: properties of a beta-glucosidase fusion protein. Enzyme Microb Technol 13, 59-65. 219. Ong, E . , Kilburn, D . G . , Mi l le r , R. C , Jr., and Warren, R. A . (1994). Streptomyces lividans glycosylates the linker region of a beta-1,4- glycanase from Cellulomonas fimi. J Bacteriol 776,999-1008. 220. Osawa, M . , Hanada, K . , Hamada, H . , and Nakauchi, H . (1996). Long-term lymphohematopoietic reconstitution by a single CD34- low/negative hematopoietic stem cell. Science 273, 242-5. 221. Otsuka, T., Thacker, J. D . , Eaves, C . J., and Hogge, D . E . (1991). Differential effects of microenvironmentally presented interleukin 3 versus soluble growth factor on primitive human hematopoietic cells. J C l i n Invest 88, Ml-22. 222. Otsuka, T., Thacker, J. D . , and Hogge, D . E . (1991). The effects of interleukin 6 and interleukin 3 on early hematopoietic events in long-term cultures of human marrow. Exp Hematol 19, 1042-8. 239 223. Pandiella, A . , Bosenberg, M . W . , Huang, E . J., Besmer, P., and Massague, J. (1992). Cleavage of membrane-anchored growth factors involves distinct protease activities regulated through common mechanisms. J B i o l Chem 267, 24028-33. 224. Park, L . S., and Gi l l i s , S. (1990). Characterization of hematopoietic growth factor receptors. Prog C l i n B i o l Res 352, 189-96. 225. Petzer, A . L . , Eaves, C . J., Barnett, M . J., and Eaves, A . C . (1997). Selective expansion of primitive normal hematopoietic cells in cytokine- supplemented cultures of purified cells from patients with chronic myeloid leukemia. Blood 90, 64-9. 226. Petzer, A . L . , Hogge, D . E . , Landsdorp, P. M . , Reid, D . S., and Eaves, C . J. (1996). Self-renewal of primitive human hematopoietic cells (long-term-culture- initiating cells) in vitro and their expansion in defined medium. Proc Natl Acad Sci U S A 93, 1470-4. 227. Petzer, A . L . , Zandstra, P. W. , Piret, J. M . , and Eaves, C . J. (1996). Differential cytokine effects on primitive (CD34+CD38-) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin. J Exp M e d 183, 2551-8. 228. Philo, J. S., Wen, J., Wypych, J., Schwartz, M . G. , Mendiaz, E . A . , and Langley, K . E . (1996). Human stem cell factor dimer forms a complex with two molecules of the extracellular domain of its receptor, Ki t . J B i o l Chem 271, 6895-902. 229. Pierson, B . A . , Europa, A . F. , Hu , W . S., and Mil le r , J. S. (1996). Production of human natural killer cells for adoptive immunotherapy using a computer-controlled stirred-tank bioreactor. J Hematother 5,475-83. 230. Putnam, A . J. , and Mooney, D . J. (1996). Tissue engineering using synthetic extracellular matrices. Nat M e d 2, 824-6. 240 231. Qiang, S., Yaoting, Y . , Hongyin, L . , and Klinkmann, H . (1997). Comparative evaluation of different membranes for the construction of an artificial liver support system. Int J Ar t i f Organs 20, 119-24. 232. Reddy, C . C , Niyogi , S. K . , Wells, A . , Wiley, H . S., and Lauffenburger, D . A . (1996). Engineering epidermal growth factor for enhanced mitogenic potency [In Process Citation]. Nat Biotechnol 14, 1696-9. 233. Reddy, C . C , Wells, A . , and Lauffenburger, D . A . (1994). Proliferative response of fibroblasts expressing internalization- deficient epidermal growth factor (EGF) receptors is altered via differential E G F depletion effect. Biotechnol Prog 10, 377-84. 234. Redei, I., Waller, E . K . , Holland, H . K . , Devine, S. M . , and Wingard, J. R. (1997). Successful engraftment after primary graft failure in aplastic anemia using G - C S F mobilized peripheral stem cell transfusions. Bone Marrow Transplant 19, 175-7. 235. Reith, A . D . , Rottapel, R., Giddens, E . , Brady, C , Forrester, L . , and Bernstein, A . (1990). W mutant mice with mild or severe developmental defects contain distinct point mutations in the kinase domain of the c-kit receptor. Genes Dev 4, 390-400. 236. Rinkes, I. H . , Toner, M . , Tompkins, R. G . , and Yarmush, M . L . (1994). A n extracorporeal microscopy perfusion chamber for on-line studies of environmental effects on cultured hepatocytes. J Biomech Eng 116, 135-9. 237. Robb, R. J. , Munck, A . , and Smith, K . A . (1981). T cell growth factor receptors. Quantitation, specificity, and biological relevance. J Exp M e d 154, 1455-74. 238. Roberts, R., Gallagher, J., Spooncer, E . , Al len , T. D . , Bloomfield, F. , and Dexter, T. M . (1988). Heparan sulphate bound growth factors: a mechanism for stromal cell mediated haemopoiesis. Nature 332, 376-8. 241 239. Roy, F. , DeBlois , C , and Doil lon, C. J. (1993). Extracellular matrix analogs as carriers for growth factors: in vitro fibroblast behavior. J Biomed Mater Res 27, 389-97. 240. Ruoslahti, E . , and Yamaguchi, Y . (1991). Proteoglycans as modulators of growth factor activities. Ce l l 64, 867-9. 241. Sanchez, A . , Gupta, R. K . , Alonso, M . J., Siber, G . R., and Langer, R. (1996). Pulsed controlled-released system for potential use in vaccine delivery. J Pharm Sci 85, 547-52. 242. Sasaki, K . , Ikeda, K . , Ogami, K . , Takahara, J., and Irino, S. (1995). Cell-to-cell interaction of cytokine-dependent myeloblastic line constitutively expressing membrane-bound stem cell factor abrogates cytokine dependency partially through granulocyte-macrophage colony- stimulating factor production. Blood 85, 1220-8. 243. Scalettar, B . A . , Abney, J. R., and Owicki , J. C . (1988). Theoretical comparison of the self diffusion and mutual diffusion of interacting membrane proteins. Proc Natl Acad Sci U S A 85, 6726-30. 244. Schrader, J. W . (1991). Peptide regulatory factors and optimization of vaccines. M o l Immunol 28, 295-9. 245. Schrader, J. W. , Bartlett, P. F. , Clark-Lewis, I., and Boyd, A . W . (1981). Lymphoid differentiation in vitro. Ciba Found Symp 84, 130-60. 246. Schuppan, D . , and Ruhl, M . (1994). Matrix in signal transduction and growth factor modulation. Braz J M e d B i o l Res 27, 2125-41. 247. Schwartz, R. M . , Emerson, S. G . , Clarke, M . F. , and Palsson, B . O. (1991). In vitro myelopoiesis stimulated by rapid medium exchange and supplementation with hematopoietic growth factors. Blood 78, 3155-61. 242 248. Sensebe, L . , Deschaseaux, M . , L i , J., Herve, P., and Charbord, P. (1997). The broad spectrum of cytokine gene expression by myoid cells from the human marrow microenvironment. Stem Cells 15, 133-43. 249. Shaw, P. (1994). Deconvolution in 3-D optical microscopy. Histochem J 26, 687-94. 250. Sheardown, H . , Wedge, C , Chou, L . , Apel , R., Rootman, D . S., and Cheng, Y . L . (1993). Continuous epidermal growth factor delivery in corneal epithelial wound healing. Invest Ophthalmol V i s Sci 34, 3593-600. 251. Shimizu, Y . , Ashman, L . K . , Du, Z . , and Schwartz, L . B . (1996). Internalization of K i t together with stem cell factor on human fetal liver-derived mast cells: new protein and R N A synthesis are required for reappearance of Ki t . J Immunol 156, 3443-9. 252. Shinoka, T., Breuer, C . K . , Tanel, R. E . , Zund, G . , Miura, T., M a , P. X . , Langer, R., Vacanti, J. P., and Mayer, J. E . , Jr. (1995). Tissue engineering heart valves: valve leaflet replacement study in a lamb model. A n n Thorac Surg 60, S513-6. 253. Siczkowski , M . , Robertson, D . , and Gordon, M . Y . (1992). Synthesis and deposition of glycosaminoglycans in the murine hemopoietic stromal line S17: modulators of the hemopoietic microenvironment. Exp Hematol 20, 1285-90. 254. Slanicka Krieger, M . , Nissen, C , Manz, C . Y . , Toksoz, D . , Lyman, S. D . , and Wodnaf-Fi l ipowicz, A . (1998). The membrane-bound isoform of stem cell factor synergizes with soluble flt3 ligand in supporting early hematopoietic cells in long-term cultures of normal and aplastic anemia bone marrow. Exp Hematol 26, 365-73. 255. Smith, K . A . (1980). T-cell growth factor. Immunol Rev 51, 337-57. 243 256. Somasundaram, R., and Schuppan, D . (1996). Type I, U , m, IV, V , and V I collagens serve as extracellular ligands for the isoforms of platelet-derived growth factor ( A A , B B , and A B ) . J B i o l Chem 271, 26884-91. 257. Sorensen, P., M u i , A . L . , and Krystal, G . (1989). Interleukin-3 stimulates the tyrosine phosphorylation of the 140- kilodalton interleukin-3 receptor. J B i o l Chem 264, 19253-8. 258. Spooncer, E . , and Dexter, T. M . (1984). Long-term bone marrow cultures. B i b l Haematol, 366-83. 259. Stoclet, J. C , Andriantsitohaina, R., N , L . h., Martinez, C , Germain, L . , and Auger, F. (1996). Use of human vessels and human vascular smooth muscle cells in pharmacology. Ce l l B i o l Toxicol 12, 223-5. 260. Stout, A . L . , and Axelrod, D . (1995). Spontaneous recovery of fluorescence by photobleached surface-adsorbed proteins. Photochem Photobiol 62, 239-44. 261. Sundaram, S., Irvine, L . , Courtney, J. M . , and Lowe, G . D . (1991). Modification of the influence of biomaterials on contact activation. Int J Ar t i f Organs 14, 729-31. 262. Sutherland, H . J., Eaves, C. J., Lansdorp, P. M . , Phillips, G . L . , and Hogge, D . E . (1994). Kinetics of committed and primitive blood progenitor mobilization after chemotherapy and growth factor treatment and their use in autotransplants. Blood 83, 3808-14. 263. Sutherland, H . J., Eaves, C. J., Lansdorp, P. M . , Thacker, J. D . , and Hogge, D . E . (1991). Differential regulation of primitive human hematopoietic cells in long- term cultures maintained on genetically engineered murine stromal cells. Blood 78, 666-72. 264. Sutherland, H . J., Hogge, D . E . , Cook, D . , and Eaves, C . J. (1993). Alternative mechanisms with and without steel factor support primitive human hematopoiesis. B lood 81, 1465-70. 244 265. Sutherland, H . J., Hogge, D . E . , and Eaves, C. J. (1993). Growth factor regulation of the maintenance and differentiation of human long-term culture-initiating cells (LTC-IC) . Leukemia 7, S122-5. 266. Sutherland, H . J. , Lansdorp, P. M . , Henkelman, D . H . , Eaves, A . C , and Eaves, C. J. (1990). Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Natl Acad Sci U S A 87, 3584-8. 267. Taipale, J., and Keski-Oja, J. (1997). Growth factors in the extracellular matrix. Faseb J 77,51-9. 268. Taniguchi, T., and Minami , Y . (1993). The IL-2/JL-2 receptor system: a current overview. Ce l l 73, 5-8. 269. Temeles, D . S., McGrath, H . E . , Kittler, E . L . , Shadduck, R. K . , Kister, V . K . , Crittenden, R. B . , Turner, B . L . , and Quesenberry, P. J. (1993). Cytokine expression from bone marrow derived macrophages. Exp Hematol 21, 388-93. 270. Thorens, B . , Mermod, J. J., and Vassalli, P. (1987). Phagocytosis and inflammatory stimuli induce G M - C S F m R N A in macrophages through posttranscriptional regulation. Ce l l 48, 671-9. 271. T i l l , J. E . , and McCul loch , E . A . (1980). Hemopoietic stem cell differentiation. Biochim Biophys Acta 605, 431-59. 272. Tilton, R. D . , Gast, A . P., and Robertson, C. R. (1990). Surface diffusion of interacting proteins. Effect of concentration on the lateral mobility of adsorbed bovine serum albumin. Biophys J 58, 1321-6. 273. Toksoz, D . , Zsebo, K . M . , Smith, K . A . , Hu , S., Brankow, D. , Suggs, S. V . , Martin, F. H . , and Wil l iams, D . A . (1992). Support of human hematopoiesis in long-term bone marrow cultures 245 by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor. Proc Natl Acad Sci U S A 89, 7350-4. 274. Tomme, P., Creagh, A . L . , Kilburn, D . G . , and Haynes, C. A . (1996). Interaction of polysaccharides with the N-terminal cellulose-binding domain of Cellulomonas fimi CenC. 1. Binding specificity and calorimetric analysis. Biochemistry 35, 13885-94. 275. Tomme, P., Driver, D . P., Amandoron, E . A . , Mil ler , R. C , Jr., Antony, R., Warren, J., and Kilburn, D . G . (1995). Comparison of a fungal (family I) and bacterial (family U) cellulose-binding domain. J Bacteriol 177, 4356-63. 276. Tomme, P., Gilkes, N . R., Guarna, M . M . , Haynes, C . A . , Hasenwinkle, D . , Jervis, E . , Johnson, P., Mcintosh, L . , Warren, R. A . , and Kilburn, D . G . (1996). Cellulose-binding domains. Versatile affinity tags for inexpensive large-scale purification, concentration, and immobilization of fusion proteins. A n n N Y Acad Sci 799, 418-24. 277. Tomme, P., Warren, R. A . , and Gilkes, N . R. (1995). Cellulose hydrolysis by bacteria and fungi. A d v Microb Physiol 37, 1-81. 278. Toniatti, C , Cabibbo, A . , Sporena, E . , Salvati, A . L . , Cerretani, M . , Serafini, S., Lahm, A . , Cortese, R., and Ciliberto, G . (1996). Engineering human interleukin-6 to obtain variants with strongly enhanced bioactivity. Embo J 75, 2726-37. 279. Torigoe, T., O'Connor, R., Santoli, D . , and Reed, J. C. (1992). Interleukin-3 regulates the activity of the L Y N protein-tyrosine kinase in myeloid-committed leukemic cell lines. Blood 80, 617-24. 280. Traverse, S., Gomez, N . , Paterson, H . , Marshall, C , and Cohen, P. (1992). Sustained activation of the mitogen-activated protein ( M A P ) kinase cascade may be required for 246 differentiation of P C 12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem J 288, 351-5. 281. Tsay, T. T., and Jacobson, K . A . (1991). Spatial Fourier analysis of video photobleaching measurements. Principles and optimization. Biophys J 60, 360-8. 282. Turner, A . M . , Bennett, L . G. , L i n , N . L . , Wypych, J., Bartley, T. D . , Hunt, R. W . , Atkins, H . L . , Langley, K . E . , Parker, V . , Martin, F. , and et al. (1995). Identification and characterization of a soluble c-kit receptor produced by human hematopoietic cell lines. Blood 85, 2052-8. 283. Vadhan-Raj, S. (1994). PIXY321 (GM-CSF/TL-3 fusion protein): biology and early clinical development. Stem Cells (Dayt) 12, 253-61. 284. Valentini, R. F. , Vargo, T. G . , Gardella, J. A . , Jr., and Aebischer, P. (1992). Electrically charged polymeric substrates enhance nerve fibre outgrowth in vitro. Biomaterials 13, 183-90. 285. Verfaillie, C , Hurley, R., Bhatia, R., and McCarthy, J. B . (1994). Role of bone marrow matrix in normal and abnormal hematopoiesis. Crit Rev Oncol Hematol 16, 201-24. 286. Verfaillie, C . M . , and Catanzaro, P. (1996). Direct contact with stroma inhibits proliferation of human long-term culture initiating cells. Leukemia 10, 498-504. 287. V o n Hippel, P. H . , and McGhee, J. D . (1972). DNA-protein interactions. Annu Rev Biochem 47,231-300. 288. Vondrys, P., Simova, J. , Takacova, S., Jandlova, T., and Bubenik, J. (1997). Recombinant interleukin-2 acts as an adjuvant and helps to increase the efficacy of tumour vaccines. Fol ia B i o l 43, 39-40. 289. Vujanovic, N . L . , Rabinowich, H . , Lee, Y . J., Jost, L . , Herberman, R. B . , and Whiteside, T. L . (1993). Distinct phenotypic and functional characteristics of human natural killer cells obtained by rapid interleukin 2-induced adherence to plastic. Ce l l Immunol 151, 133-57. 247 290. Vunjak-Novakovic, G . , Obradovic, B . , Martin, I., Bursac, P. M . , Langer, R., and Freed, L . E . (1998). Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering. Biotechnol Prog 14, 193-202. 291. Wald , H . L . , Sarakinos, G . , Lyman, M . D. , Mikos , A . G . , Vacanti, J. P., and Langer, R. (1993). Ce l l seeding in porous transplantation devices. Biomaterials 14, 270-8. 292. Wang, T. Y . , and W u , J. H . (1992). A continuous perfusion bioreactor for long-term bone marrow culture. A n n N Y Acad Sci 665, 274-84. 293. Wang, Y . L . (1985). Exchange of actin subunits at the leading edge of l iving fibroblasts: possible role of treadmilling. J Ce l l B i o l 101, 597-602. 294. Watrous, D . A . , and Andrews, B . S. (1989). The metabolism and immunology of bone. Semin Arthritis Rheum 19, 45-65. 295. Whetton, A . D . , Monk, P. N . , Consalvey, S. D. , and Downes, C. P. (1986). The haemopoietic growth factors interleukin 3 and colony stimulating factor-1 stimulate proliferation but do not induce inositol l ipid breakdown in murine bone-marrow-derived macrophages. Embo J 5, 3281-6. 296. White, J. G . , Amos, W . B . , and Fordham, M . (1987). A n evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J Ce l l B i o l 105, 41-8. 297. Wierzba, A . , Reichl, U . , Turner, R. F. B . , Antony, R., Warren, J., and Kilburn, D . G . (1995). Adhesion of mammalian cells to a recombinant attachment factor, C B D / R G D , analyzed by image analysis. Biotechnol Bioeng 46, 185-193. 298. Wil l iams, K . P., and Shoelson, S. E . (1993). Cooperative self-assembly of SH2 domain fragments restores phosphopeptide binding. Biochemistry 32, 11279-84. 248 299. Wil l iams, L . T. (1989). Signal transduction by the platelet-derived growth factor receptor. Science 243, 1564-70. 300. Wright, D . G . , LaRussa, V . F., Salvado, A . J., and Knight, R. D . (1989). Abnormal responses of myeloid progenitor cells to granulocyte- macrophage colony-stimulating factor in human cyclic neutropenia. J C l i n Invest 83, 1414-8. 301. Wrighton, N . C . , Farrell, F . X . , Chang, R., Kashyap, A . K . , Barbone. F.P. , Mulcahy, L .S . , Johnson, D . L . , Barrett, R .W. , Jolliffe, L . K . , Dower, W.J . (1996) Small peptides as potent mimetics of the protein hormone erythropoietin. Science 26, 458-64. 302. X u , G . Y . , Ong, E . , Gilkes, N . R., Kilburn, D . G . , Muhandiram, D . R., Harris-Brandts, M . , Carver, J. P., Kay, L . E . , and Harvey, T. S. (1995). Solution structure of a cellulose-binding domain from Cellulomonas fimi by nuclear magnetic resonance spectroscopy. Biochemistry 34, 6993-7009. 303. Yannas, I. V . , Burke, J. F. , Orgi l l , D . P., and Skrabut, E . M . (1982). Wound tissue can utilize a polymeric template to synthesize a functional extension of skin. Science 215, 174-6. 304. Yayon, A . , and Klagsbrun, M . (1990). Autocrine regulation of cell growth and transformation by basic fibroblast growth factor. Cancer Metastasis Rev 9, 191-202. 305. Yee, N . S., Hsiau, C . W. , Serve, H . , Vosseller, K . , and Besmer, P. (1994). Mechanism of down-regulation of c-kit receptor. Roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase, and protein kinase C. J B i o l Chem 269, 31991-8. 306. Yee, N . S., Langen, H . , and Besmer, P. (1993). Mechanism of kit ligand, phorbol ester, and calcium-induced down- regulation of c-kit receptors in mast cells. J B i o l Chem 268, 14189-201. 307. Zandstra, P .W. (1997). U . B . C . Ph.D. Thesis. 249 308. Zandstra, P. W . , Conneally, E . , Petzer, A . L . , Piret, J. M . , and Eaves, C. J. (1997b). Cytokine manipulation of primitive human hematopoietic cell self- renewal. Proc Natl Acad Sci U S A 94, 4698-703. 309. Zandstra, P. W . , Eaves, C. J., and Piret, J. M . (1994). Expansion of hematopoietic progenitor cell populations in stirred suspension bioreactors of normal human bone marrow cells. Biotechnology (N Y ) 12, 909-14. 310. Ziltener, H . J., Clark-Lewis, I., de St. Groth, B . F. , Orban, P. C , Hood, L . E . , Kent, S. B . , and Schrader, J. W . (1988). Monoclonal antipeptide antibodies recognize IL-3 and neutralize its bioactivity in vivo. J Immunol 140, 1182-7. 311. Ziltener, H . J., Fazekas de St. Groth, B . , Leslie, K . B . , and Schrader, J. W . (1988). Mult iple glycosylated forms of T cell-derived interleukin 3 (DL-3). Heterogeneity of EL-3 from physiological and nonphysiological sources. J B i o l Chem 263, 14511-7. 312. Zuckerman K . S., Prince C. W. , and Gay S., The Hemopoietic Extracellular Matrix, Travassoli M . ed. The Handbook of the Hematopoietic Microenvironment, Humana Press Inc., 399-432, 1989. 

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