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Characterization of the unc-23 gene, an HSP-1 chaperone regulator, in Caenorhabditis elegans Rahmani Gorji, Poupak 2002

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Characterization of the unc-23 gene, an HSP-1 chaperone regulator, in Caenorhabditis elegans. by Poupak Rahmani Gorji B .Sc , The University of British Columbia, 1995 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIRMENT OF THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A JUNE 2001 © Poupak Rahmani Gorji, 2001 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 DE-6 (2/88) Abstract Mutations in the unc-23 gene in the free living nematode, Caenorhabditis elegans, result in detachment and dystrophy of the anterior body wall musculature and a bent-head phenotype when grown on solid substrate (Waterston et al, 1980). This phenotype is not observed when animals are grown in liquid culture (Bullerjahn and Riddle, pers. comm.) Neither muscle cell growth and positioning nor myofilament assembly is affected in liquid grown unc-23 animals. Muscle cell attachment, however, is affected since a small amount of stress applied by rolling the animals under the coverslip results in detachment of the muscle cells from the hypodermis. The results of immunological staining with antibodies to basement membrane and hypodermal components suggest that the primary defect in unc-23 animals is located within the hypodermis. UNC-23 encodes a cytoplasmic protein with a domain similar to the mammalian chaperone regulator BAG-2 (BCL2-associated athanogene 2). Human BAG-2 and C. elegans UNC-23 share 40% amino acid identity and 62% similarity over the B A G domain and its upstream region. In mammals, the B A G family of chaperone regulators contain a conserved 45 amino acid region near their C termini (the B A G domain) that binds Hsp70/Hsc70 and control their chaperone activity (Takayama et al, 1999). UNC-23 is first expressed in only a few cells in 1.5 fold stage embryos. As animals develop and grow, UNC-23 expression expands and eventually includes several tissues. In adult animals, UNC-23 is expressed in the pharynx, the body wall muscle cells, the hypodermis, H cells, and the vulva. In body wall muscle cells, UNC-23 is localized in a i i pattern similar to that of the transmembrane protein, (3 integrin, suggesting that it is associated with the proteins that make up the dense bodies and M lines. In the hypodermis, UNC-23 is present throughout the tissue with the exception of the nuclei and is distributed in a pattern reminiscent of the intermediate filaments. A yeast two-hybrid screen identified the ATPase domain of the non-inducible heat shock protein 70, HSP-1, as an interacting partner with the COOH terminus of U N C -23. This in vitro result was confirmed in vivo with the identification of two dominant suppressors of unc-23(e25) as alleles of the hsp-1 gene in C. elegans. The molecular lesions that result in HSP-1 suppressor activity are missense mutations located within the ATPase domain of the HSP-1 molecule. In vitro studies of BAG-2 in mammals suggest that this protein is a negative regulator of Hsc70. It is predicted that UNC-23 in C. elegans is also a negative regulator of the HSP-1 chaperone. The results from suppression analysis and the phenotypic characterization of unc-23 mutants suggest that the UNC-23-HSP-1 interaction is required for the maintenance of hypodermal integrity and muscle cell attachment during mechanical stress in C. elegans. i n Table of contents Abstract ii Table of contents iv List of Tables vii List of Figures viii List of Abbreviations viii Acknowledgement xii Dedication _xiii Chapter 1. Introduction 1 Anatomical description of tissues involved in production and transmission of contractile force in C. elegans 7 Description of mutants that affect cell-ECM attachment in C. elegans. 13 Chapter 2. Material and Methods 19 Nematode maintenance and culture conditions 19 Nematode strains 20 Immunofluorescence staining and microscopy 21 i v PCR deficiency and polymorphism mapping 24 GFP constructs 25 Microinjections and complementation tests with transgenic strains 26 Sequence analysis of unc-23 28 unc-23(e25) suppressor screen 29 Genetic characterization and mapping of suppressors 30 Sequence analysis of dominant suppressors 31 Yeast two-hybrid constructs, transformations, and screening 32 Chapter 3. Results 35 Loss ofhypodermal integrity causes muscle detachment in unc-23 mutants 35 unc-23 encodes a protein with a domain similar to the mammalian Bcl2-associated athanogene 2(BAG-2), a chaperone regulator 50 UNC-23 is expressed in muscle and hypodermis 58 Screening, mapping, and genetic characterization of unc-23(e25) suppressors 62 UNC-23 interacts with HSP-1 in yeast two-hybrid analysis 65 Sequence analysis of the dominant suppressors 70 Chapter 4. Discussion 72 unc-23 encodes a protein with a domain similar to the mammalian Bcl-2-associated athanogene 2, a chaperone regulator 72 UNC-23 is expressed in the hypodermis and the body wall muscle cells and is required for hypodermal integrity 73 UNC-23 directly interacts with the ATPase domain ofHSP-1, the non-inducible heat shock protein 70 77 BAG-1 is an Hsc70 regulator 80 UNC-23-HSP-1 interaction is required for maintenance of hypodermal integrity during mechanical stress 85 References 90 vi List of Tables Table 1. Characterization of the unc-23 alleles. 39 Table 2. Sequenced alleles of unc-23 and their molecular lesions. 55 Table 3. Mapping and genetic characterization of the unc-23 suppressors. 64 v i i List of Figures Figure 1. Diagrammatic representation of C. elegans muscle. 8 Figure 2. Polarized light images of wild type and unc-23(e25) animals. 37 Figure 3. GM1 immunofluoresence staining of wild type and unc-23(e25) animals. 44 Figure 4. MH4 immunofluoresence staining of wild type and unc-23(e25) animals. 46 Figure 5. MH5 immunofluoresence staining of wild type and unc-23(e25) animals. 48 Figure 6. The genetic and physical map of the unc-23 region. 53 Figure 7. The unc-23 gene structure. 54 Figure 8. Alignment of C. elegans UNC-23 and human BAG-2 proteins. 56 Figure 9. Spatial distribution of UNC-23 ::GFP in unc-23(e25) rescued mutants._ 60 Figure 10. Schematic representation of the unc-23 gene and the HSP-1 protein. _ 66 Figure 11. Alignment of C. elegans HSP-1 and mammalian Hsc70 protein sequences. 68 Figure 12. A schematic representation of the hsp-1 gene. 71 Figure 13. The ribbon model of the three dimensional structure of the ATPase domain of the Hsc70 protein. 79 Figure 14. Model of Hsc70 reaction cycle in presence of its regulating cofactors. 84 Figure 15. Anterior body wall muscle cell arrangement in C. elegans. 87 viii List of Abbreviations A b A antibody buffer A Ade" Adenine minus bp basepairs B A G Bcl2-associated athanogene CHIP carboxyl terminus of Hsc70-interacting protein D A B C O 1,4-Diazabicyclo[2.2.2]octane Df deficiency dpy dumpy DTT 1,4-Dithio-DL-threitol E C M extracellular matrix EDTA Ethylenediminetetraacetate E M electron Microscopy EMS Ethylmethanesulfonate FI filial generation one F2 filial generation two FITC Fluorescein Isothiocyanate F O A 5-Fluoroorotic acid GFP green fluorescent protein GM1 rabbit polyclonal sera recognizing the basement membrane proteoglycan, UNC-52/perlecan Hip Hsc70-interacting protein His" Histidine minus ix heat shock protein 70 heat shock cognate 70 kilobase kilodalton Larval development stages 1 to 4 lethal long worm washing buffer mouse monoclonal sera recognizing the intermediate filaments in the hypodermis of C. elegans mouse monoclonal sera, recognizing the hemidesmosomes in the hypodermis of C. elegans milliliter millimolar muscle-attachment defective muscle-positioning defective C. elegans wild type Bristol strain Sodium National Center for Biotechnology Information nanogram nematode growth media leaky uracil-requiring strain of E. coli ORF open reading frame Po parental generation PBS phosphate buffered saline PCR polymerase chain reaction PEG Polyethylene Glycol pers. comm. personal communication R N A i R N A interference rol roller, helically twisted body. Animals roll when moving. rpm revolutions per minute SD synthetic medium plus dextrose TBS Tris buffered saline TBS-T Tris buffered saline + 0.1% Tween 20 Tel transposable element (transposon Caenorhabditis) TMP Trimethylpsoralen TRSC Texas Red Sulfonyl Chloride TTB Tris Triton buffer pi microliter pg microgram unc uncoordinated UTR untranslated region vab variable abnormal morphology Y P D yeast extract + peptone+ dextrose xi Acknowledgement I would like to extend my most sincere thanks and appreciation to my supervisor Dr. Donald Moerman for his guidance, support, and for giving me the opportunity to learn to do science. My five years in the Moerman lab will always be one of the most wonderful experiences of my life. I would like to thank members of the Moerman lab, past and present, including Dr. Teresa Rogalski, Dr. Greg Mullen, Dr. Ken Norman, Dr. Hiroshi Qadota, Shaun Cordes, Blazej Szczygielski, Danelle Devenport, Jason Bush, Katy Cullin, Rebecca Newbury, my friend Dr. Colin Thacker, the members of the Vancouver C. elegans Reverse Genetics Core Facility and my supervisory committee: Dr. Vanessa Auld, Dr. John Gosline, Dr. Linda Matsuuchi, and Dr. Wayne Vogl for their patience and guidance. xii Dedication This thesis is dedicated with love and thanks to my parents and my sister who have always been there for me. xiii Chapter 1. Introduction Cell-cell and cell-extracellular matrix (ECM) interactions play a critical role in development of multicellular organisms. These interactions have been implicated in many developmental processes including morphogenesis (Kramer et al, 1988, Mittenthal and Jacobson, 1990; Costa et al. 1998). During morphogenesis, the E C M , a complex network of intricate macromolecules, is involved in two distinct and yet interconnected functions. The first is to provide cells with a multitude of chemical and molecular instructions such as growth factors and morphogens. The second function is to provide a structural framework to give the mechanical stability necessary for maintenance of tissue shape. Mechanisms involved in morphogenesis include chemical or molecular communications and mechanical forces. Mechanical forces play a critical role in morphogenesis and tissue formation. A n excellent example of a requirement for mechanical forces during morphogenesis is the development of the C. elegans embryo. Morphogenesis in C. elegans involves the transformation of a round ball of cells into a long thin worm. This transformation entails large-scale cell shape changes, cell spreading and cell migrations, all of which require mechanical forces (reviewed in Chin-Sang and Chisholm, 2000). In C. elegans, some of the morphogenetic movements during embryogenesis involve epithelial sheath cells. Epithelial sheath cells (also known as hypodermis) begin their movement at the dorsal side of the embryo and spread to the ventral midline where they enclose the embryo within an epidermal cell layer (Priess and Hirsh, 1986). Upon enclosure of the embryo, lateral epidermal cells form circumferential 1 actin filaments that contract and squeeze the bean shaped embryo into a thin long larva within the eggshell (Williams-Masson et al, 1997). Constriction of cells into a larva not only requires the contraction of the epithelial actin filaments, but also involves the contraction of underlying body wall muscle cells (Williams and Waterston, 1994). The muscle contractile force is transmitted to the epidermis through a specialized extracellular matrix, known as the basement membrane. Once the embryo takes form, the cuticular extracellular matrix, which is the exoskeleton of the worm, is deposited by the epidermis to help maintain the newly formed body shape. The fundamental requirements for mechanical forces during morphogenesis in C. elegans were ascertained through identification and characterization of a group of mutations that affect mechanical force production or transmission during morphogenesis in C. elegans. Mutations that affect actin based motility of the epithelial cells result in the absence of cell movement and failure to enclose the embryo within the epidermal sheath cells (Costa et al, 1998). Analyses of elongation defective embryos have also revealed a group of genes that regulate the actin-based contractions required for elongation of the embryo (Wissmann et al, 1997 and 1999). In these embryos, hypodermal enclosure occurs but embryos fail to elongate due to their inability to regulate the contraction of circumferential actin filaments. Mutants that lack muscle contraction or fail to transmit the contractile force to the epidermis are also unable to elongate properly and arrest as two fold embryos (Rogalski et al. 1993; Williams and Waterston, 1994; Hresko, et al. 1999). The requirement for both muscle myofilament and epithelial actin filament contractions during morphogenesis points not only to the indispensability of mechanical forces for morphogenesis, but also to the need for co-2 ordination of morphogenetic events across adjacent tissues. In addition to the above examples, study of morphogenesis in C. elegans has further clarified the role of cell-E C M interactions in morphogenesis and maintenance of body shape. Mutations that affect components of the basement membrane in C. elegans are unable to elongate and arrest as two fold embryos (Rogalski et al, 1993). Mutations that affect the function or structural components of the cuticle result in dramatic changes in the body shape (Kramer et al, 1988; Kramer and Johnson, 1993; Von Mende et al, 1988). The newly developed larvae are unable to maintain their shape due to changes in the composition or function of the cuticular extracellular matrix (also reviewed in Kramer, 1997). Requirement for mechanical forces during embryogenesis is not limited to the C. elegans embryo. The process of gastrulation, which is common to multicellular embryos, requires mechanical forces that cause some cell layers to fold outwards while others bend and invaginate (Beloussov et al, 1975). Pattern and tissue formation also involves mechanical forces. During the formation of neural tube for example, mechanical forces are required for bending of the epithelial tissues and cell shape changes (Gordon and Brodland, 1987 cited in Ingber et al, 1994). Mechanical forces are not only required during morphogenetic stages of development, but they are also essential to maintenance and homeostasis of tissues post-embryonically. Organisms experience internal (contractile and hemodynamic) and external (movement and gravity) mechanical forces on a daily basis. These mechanical forces are important in maintenance of tissue homeostasis. Lack of gravity (Johnson, 1998) or prolonged bed rest (Bloomfield, 1997), for example, induce muscle, bone, and tendon atrophy. Mechanical stresses also challenge the integrity of cells and ECMs. 3 Shear stress experienced by the vascular endothelial cells due to blood flow and force transduction of muscle cells during movement are examples of mechanical stresses that challenge the integrity of tissues. To keep their shape and maintain their integrity, therefore, cells within a tissue must be capable of adaptive responses to various types of mechanical stresses. This adaptation is achieved through the ability of cells to control the amount and composition of their E C M based on the magnitude of mechanical stresses that acts on the tissue (Hay, 1992; Macki et al, 1992; Webb et al, 1997) and through the rearrangement of cytoskeleton and anchoring junctions (Ingber, 1997). To bear different types of mechanical stresses such as tension, compression or shear, tissues produce highly specialized matrices such as skin, bone, and cartilage (Hay, 1992). Cells are able to control the deposition, composition and maintenance of molecules that make up their surrounding E C M , based on the type and magnitude of the mechanical stresses that act on the tissue (Chiquet, 1999). Tendon fibroblasts, for example, respond to tension by producing and depositing large amount of collagen I molecules into their surrounding E C M (Quinones et al, 1986). These same cells, when under compression, transdifferentiate into fibrocartilage. They down regulate collagen I production and start to produce cartilage components (Benjamin and Ralphs, 1998). Cellular responses to mechanical forces are not limited to structural or molecular rearrangement of the E C M alone. Cells under mechanical stress have also been shown to respond by remodeling their cytoskeleton and changing the distribution of the structural components that associate with the cytoskeleton (Ingber, 1997). Like E C M , the compositions of the biopolymers (i.e. microfilaments, microtubules, and intermediate filaments) that make up the cytoskeleton are tailored to accommodate the mechanical 4 demand that is put on the cell (Fuchs and Cleveland, 1998). The ability to rearrange the E C M and the cytoskeleton in face of mechanical stresses is an essential response, which allows the cell to maintain its shape and withstand deformative forces that compromise cellular integrity. As discussed earlier, embryonic morphogenetic events involve many mechanical forces that shape the embryo to its proper form. The mechanisms that are involved in maintenance of cellular integrity during embryogenesis are different from the cellular responses observed post-embryonically. Mutations that affect the ability to maintain cell attachment and integrity during morphogenesis result in embryonic lethality (Goh and Bogaert, 1991; Costa et al, 1998). Mutations that affect the regulatory or structural components involved in adaptive responses discussed above do not affect embryonic development, rather they result in fragility and separation of the tissue and have late phenotypic onsets and rapid progression. The primary site of tissue fragility and separation in these mutations usually indicates the location of the affected molecule. Some of the best known examples of diseases that are caused by these mutations include mechanobullous disorders of the skin and muscular dystrophy. Although we are aware that mechanisms for adaptive responses to different types of mechanical forces exist, we know very little about how cells detect and convert mechanical signals into biological responses. The ensuing signaling and regulatory events that result in maintenance of cellular attachments and integrity of tissues during mechanical stress are also not well known or understood. I am interested in mechanisms and molecules involved in maintenance of cell-E C M adhesion in tissues that are under physiological stress due to muscle attachment and 5 movement. For this purpose, I have chosen the free-living nematode Caenorhabditis elegans to study this biological question. Caenorhabditis elegans is a small (1 mm) free living soil nematode that is easily reared on E. coli in laboratories. The life cycle of this nematode is only 2.5 days at 25°, and it includes a period of embryonic development and hatching followed by four larval stages (LI to L4), and adulthood, which is characterized by sexual maturity (Wood, 1988). The most common sexual form of C. elegans is a hermaphrodite (XX), which is capable of self-fertilization. Males (XO) may also be produced for use in genetic studies. In C. elegans the anatomy, cell lineage, and position of all somatic cells have been determined (Sulston and Horvitz, 1977; Sulston et al. 1983, White et al. 1986). C. elegans is easily mutagenized and its genetic analysis has led to a detailed genetic map (Brenner, 1974). It also produces large populations of progeny, which is favorable for genetic and biochemical analysis. C. elegans has a small genome that has been fully sequenced (C. elegans Sequencing Consortium, 1998) and to date the majority of biological functions examined in this soil nematode have proven to be conserved in larger and more complex organisms. Recently it has been shown that 40% of the genes of this small nematode are shared with humans (Rubin et al., 2000). These attributes make C. elegans a valuable genetic model to study many biological questions, including cell-ECM adhesion maintenance. Since this work pertains to maintenance of cell-ECM attachment during movement in C. elegans, I will focus my attention on the anatomical description of tissues that are involved in the production and transmission of contractile force. 6 Anatomical description of tissues involved in production and transmission of contractile force in C. elegans In C. elegans, body wall muscle cells are arranged into four quadrants, two dorsal and two ventral (Figure 1, C). Muscle quadrants are two cells wide and run anterior to posterior along the length of the animal (Figure 1, A). C. elegans body wall muscles are very similar to vertebrate-striated muscle. The contractile unit, called a sarcomere, is delineated on both ends by a dense body, a structure analogous to Z-lines in vertebrate muscle (Figure 1, E). Actin-containing thin filaments extend in both directions from the dense body and inter-digitate over part of their length with myosin-containing thick filaments (Figure 1, E) (Francis and Waterston, 1985). The thick and thin filaments are anchored to the plasma membrane by M lines and dense bodies, respectively (Francis et al. 1985). Dense bodies extend the entire depth of the lattice, anchoring the thin filaments to the membrane through a-actinin, vinculin, and talin (Figure 1, G). Integrin, the transmembrane receptor, is a component of the base of the dense bodies and M lines (Francis and Waterston, 1991; Gettner et al. 1994), and anchors these structures to the underlying specialized extracellular matrix, known as the basement membrane (Figure 1, G). This attachment is mediated by perlecan, a basement membrane proteoglycan that covers the whole of the basement membrane and is also concentrated over the dense bodies and M-lines (Francis and Waterston, 1991; Rogalski et al. 1993). 7 Figure 1. Diagrammatic representation of C. elegans muscle. A) A drawing of an adult C. elegans hermaphrodite. Anterior is to the left, dorsal is up. The body wall muscle cells are shown as rhomboid shaped cells that extend from the anterior to the posterior along the length of the animal. The pharynx is the grinding apparatus involved in breaking down the food. B) A cartoon showing the top view of the muscle cells. The small white dots correspond to the dense bodies. C) A cross-section of the worm. C. elegans have four muscle quadrants, two located dorsally(top) and two ventrally(bottom). This cross-section is showing the four muscle quadrants in the worm. D) A n enlarged view of a muscle cell. The dense bodies and M-lines are shown. Muscle cells are polarized. The myofilament lattice forms at the cell membrane facing the hypodermis. The nucleus and the mitochondria fill out the portion of the cell not taken up by the myofilament lattice. E) A cartoon of the thick and thin filaments in the sarcomere of the muscle cells. The dense bodies anchor actin thin filaments. The M lines anchor myosin thick filaments. The two filaments inter-digitate over part of their length to slide past each other to produce contractile force. F) Underneath the muscle cells is a highly specialized basement membrane where perlecan is located. Muscle cells are connected through the basement membrane and the hypodermis to the cuticle. The hemidesmosomes and intermediate filaments, which are responsible for transmission of force are located within the hypodermis along with myotactin , the transmembrane receptor that localizes to the hypodermal cell membrane facing the basement membrane. G) The proposed arrangement of the molecules involved in linkage of the thin filaments to the muscle cell membrane is shown here. The transmembrane receptors a and f3 integrin are present both at the base of the dense bodies and M lines (not shown). 8 3 Myotactin, a hypodermal transmembrane receptor is involved in the attachment of the basement membrane to the hypodermis (Hresko et al, 1999). Detailed anatomical studies on C. elegans have revealed two major extracellular matrices that are involved in C. elegans motility: the basement membrane and the cuticle (Figure 1, F). The basement membrane covers the pharynx, intestine, gonad, and body wall muscle cells, and is required for muscle myofilament assembly. The basement membrane plays an essential role in transmission of contractile force from muscle to cuticle and consequently in nematode movement (Francis and Waterston, 1991; Rogalski et al. 1993). The cuticle is required for embryonic morphogenesis and motility throughout development, and provides the exoskeleton that covers the outside of the animal (Kramer, 1997). In between the basement membrane and the cuticle is the hypodermis, which is the nematode's equivalent of skin (Figure 1, F). The hypodermis not only plays an important role in transmission of force from the muscle to the cuticle but is also responsible for deposition of the cuticle and perhaps some basement membrane proteins (Francis and Waterston, 1991; Kramer, 1997; Hresko etal, 1994). Hemidesmosomes, which are the cell-ECM anchoring junctions for the intermediate filaments, are located at both the basement membrane and cuticular face of the hypodermis (Francis and Waterston, 1991; Hresko et al, 1994) (Figure 1, F). Hemidesmosomes provide the structural and physical linkage between the basement membrane and the cuticle and are therefore direct mediators of muscle to cuticle attachment. The association between the apical and basal hemidesmosomes is made by intermediate filaments, which not only connect the hemidesmosomes but also provide cellular integrity during force transmission (Francis and Waterston, 1991) (Figure 1, F). 10 Since the hypodermis is directly involved in transfer of contractile force from muscle to cuticle, it affords us a great opportunity to study cell-ECM attachment maintenance during muscle contraction. There are several advantages to using the hypodermis as a model tissue. The hypodermis and muscle cells go through several biologically unique processes, post-embryonically. New myoblasts, for example, are incorporated into the muscle quadrants at the end of the L I larval stage. This incorporation results in remodeling of the already established attachment structures between muscle cells and muscle cells and the hypodermis. In addition to incorporation of new myoblasts, the hypodermis and the muscle cells grow extensively during larval growth. Muscle cells in a newly hatched L I contain only two sarcomeres. By adult stage, the number of sarcomeres in the muscle cells have risen from 2 to 8-10 and the muscle cells have grown in both depth and length. Furthermore, at the end of each larval stage, C. elegans undergo a molt during which a new cuticle is formed and the old cuticle is shed (Singh and Sulston, 1978). When molting, animals enter a lethargic state during which movement and pharyngeal pumping is suppressed and the connections between the hypodermis and the cuticle are broken until a new cuticle is formed (Singh and Sulston, 1978). It is suggested that any muscle remodeling that occurs post-embryonically happens during this lethargic state, when muscle contraction is suppressed (Plenefisch et al, 2000). As the animal and its muscle cells grow, so does the force of contraction required for nematode motility. The hypodermal tissue, therefore, should be capable of not only transmitting the contractile force but also recognizing the increase in mechanical forces as the animal develops. We are, therefore, able to investigate the mechanisms 11 involved in cellular adaptability in face of increased mechanical force as well as the mechanisms involved in force transmission. Another advantage to using the hypodermis as a model tissue is the number of cell-ECM attachment-affecting mutants that have been identified to date. To better understand the mechanism involved in maintenance of cell-ECM adhesion and cellular integrity during mechanical stress, several mutations that affect this pathway have been identified in C. elegans (Plenefisch et al, 2000; Waterston et al, 1980). These mutations have a very distinct phenotype, which allows for their easy identification in genetic screens (Plenefisch et al, 2000). Mutations that affect cell-ECM attachment have loss of muscle, attachment (Mua) or an ^coordinated (Unc) phenotype. This phenotype manifests itself in early larval development and involves progressive detachment of the muscle cells from the underlying hypodermis. Some of the genes identified to date appear to be involved in cell-ECM adhesion. These genes include unc-23 (Waterston et al, 1980), mua-1, 2, 3, 4, 5, 6, 7, 9, 10 and vab-10 (variable abnormal) (Plenefisch etal, 2000). The following is a brief description of each gene and if known, their gene product. 12 Description of mutants that affect cell-ECM attachment in C. elegans. As mentioned above, motility in C. elegans involves transmission of the contractile force of the body wall muscle cells to the cuticle. This transmission is achieved via a continuous physical linkage between the muscle and the cuticle. Mutations that disrupt part of this linkage result in fragility and separation of tissues during normal growth and movement. The following is a description of genes that affect the maintenance of cell-ECM interaction in C. elegans and result in failure of tissue attachment during muscle contraction. Mutations in the mua-1 gene results in progressive detachment of muscle cells from each other and from the hypodermis (J. Plenefisch, pers. comm.). Mua-1 is predicted to encode a zinc finger transcription factor and is suggested to be involved in regulating the expression of structural proteins needed within muscle attachment sites during growth and mechanical stress (J. Plenefisch, pers. comm.). Mutations in the mua-3 gene result in progressive detachment of part or all of the body wall muscle cells from the underlying hypodermis (J. Plenefisch, pers. comm.). Mua-3 encodes a transmembrane receptor that is located at the apical and basal faces of the hypodermis and co-localizes with mua-6 and with the hemidesmosomal complexes in the hypodermis. Mua-6 encodes the cytoplasmic intermediate filament protein, IFA2 (M. Hresko et al, pers. comm.). Mua-6 co-localizes with the hemidesmosomes in the hypodermis and it is believed that mua-6 is able to interact directly or indirectly with two hypodermal transmembrane proteins, myotactin and mua-3 (M. Hresko, pers. comm.). 13 Mua-10 mutations result in detachment of the head musculature in adult C. elegans (J. Plenefisch, pers. comm.). Mua-10 is different from other mua mutants in that the dystrophic phenotype is not observed during larval development and only manifests in the adults. Mua-4 mutations also result in dystrophic detachment of body wall muscle cells in C. elegans (J. Plenefisch, pers. comm.). The products of either gene are yet to be determined. Vab-10, another gene with muscle detachment phenotype has recently been identified to encode the different members of the plakin protein family through alternative splicing of its transcript. The different isoforms share a common amino-terminus that encodes a predicted actin-binding domain but differ in their carboxy-termini (J. Bosher et al.,; V . Hapiak et al.,; M . Hresko et al, pers. comm.). The isoforms can be divided into two groups of proteins. One, encodes a protein related to the human plectin, while the other is related to the human macrophin and Drosophila kakapo. In mammals, plectin is suggested to function as a linker between the intermediate filaments, microtubules and microfilaments (McLean et al, 1996). Plectin is also involved in anchoring the intermediate filaments to desmosomes and hemidesmosomes (McLean et al, 1996). Human macrophin and Drosophila kakapo are actin binding and actin cross-linking proteins (Okuda et al, 1999; Gregory and Brown, 1998). Vab-10 is expressed in the hypodermis and localizes to the areas of contact between the hypodermis and the body wall muscle cells. The plectin and the kakapo-specific isoforms of vab-10 are suggested to perform different functions (J . Bosher et al, pers. comm.). Removal of the plectin isoform results in embryonic lethality in which the muscle cells are positioned correctly but fail to attach, while in embryos that lack the kakapo-like isoform can 14 elongate and hatch, but the resulting larvae are misshapen and lumpy and usually do not survive. It is not yet clear which of the isoforms of vab-10 is responsible for the late larval muscle detachment phenotype. Unc-23, which is the focus of this thesis, is also one of the identified genes that affects cell-ECM adhesion maintenance. Mutations affecting the unc-23 gene were first isolated in a forward genetic screen designed to identify visible mutants (Brenner, 1974). Among the original 619 isolated mutants, 410 were uncoordinated (Unc) and only unc-23 exhibited a progressive dystrophy of musculature that was confined to the head of the animal. A detailed analysis of this unique phenotype was later conducted by Waterston et al. (1980). They described the progressive dystrophy of the head musculature, which causes the bent-head phenotype in unc-23 animals and examined the affected area using electron microscopy. The high resolution E M images show that mutations in the unc-23 gene result in loss of hypodermal integrity and detachment of the muscle cells from the hypodermis. Unc-23, due to its unique phenotype and ease of maintenance as a mutant, has been used extensively as a genetic marker in the C. elegans community. The use of unc-23 as a genetic marker led to the discovery of another unique phenotypic feature of unc-23 (Bullerjahn and Riddle, pers. Comm.). During a genetic mapping experiment, which required growth of worms in liquid culture, Bullerjahn and Riddle used unc-23 as a genetic marker. Upon transfer of worms homozygous for an unc-23 mutation from liquid to agar plates, they discovered that their genetic marker no longer exhibited the bent-head phenotype. After allowing the worms to grow for a day on plates, gradually animals started to exhibit the bent-head phenotype. This observation suggested that growth of 15 unc-23 mutants in liquid culture results in temporary suppression of the bent-head phenotype. After transfer of the worms to agar plates and given sufficient time the muscle detachment and dystrophy will occur and the bent-head phenotype is again observed. Unc-23 mutants are, therefore, able to exhibits a wild type or mutant phenotype depending on their growth conditions. The primary phenotypic characterization of unc-23 by Waterston et al. (1980) suggested that unc-23 defects result in loss of hypodermal integrity, and muscle attachment during larval development in C. elegans. The discovery of detachment suppression, when unc-23 animals are grown in liquid, pointed to unc-23 as being a muscle-hypodermal attachment defect, rather than a failure in myofilament assembly or muscle positioning. These observations led me to hypothesize that the unc-23 gene encodes a structural or a regulatory protein that is involved in maintenance of hypodermal integrity and muscle cell attachment in C. elegans. To test this hypothesis, I determined the cellular basis of the muscle cell attachment defect by examining the basement membrane and the hypodermis, the two tissues that are affected by muscle cell detachment, and found that the primary defect is located within the hypodermis. To find the unc-23 gene and characterize its gene product, I set out to clone the unc-23 gene in C. elegans. I took advantage of the C. elegans sequenced genome to determine the position of the unc-23 gene on the physical map. Once the physical region in which the unc-23 gene is located was determined, I used an array of unc-23 alleles to look for polymorphisms. This polymorphism mapping resulted in the identification of an open reading frame, H14N18.1. Sequencing of unc-23 alleles and discovery of molecular lesions within this ORF confirmed that H14N18.1 is the unc-23 gene. I characterized the 16 expression pattern of the UNC-23 protein, using a functional UNC-23: :GFP fusion construct that rescues the unc-23 bent head phenotype. To further analyze a developmental pathway or to find different members of a structural complex, it is desirable to identify new interacting genes. One method of obtaining additional members of a pathway or a complex is to isolate suppressor mutations of an already identified mutant. In viruses and bacteria, suppression analysis has proven to be powerful in identifying interacting genes. In a suppression screen, one is looking for new mutations that alleviate the effects of the original mutation, such that a milder or a wild type phenotype is observed. Suppressor mutants usually act by compensating for the lost function of the original mutation, or affect subunit interactions in multiple protein assemblies (Jarvik and Botstein, 1975). Other types of suppressors provide alternative physiological pathways ( Kuwano et al, 1969) and some suppressors change the overall physiological balance of the cell and thus compensate for the original mutation (Floor, 1970). One of the attributes of C. elegans that makes it a powerful genetic system is the large number of progeny produced by a single worm. One healthy hermaphrodite is capable of producing up to 300 progeny. This is a particularly important feature of C. elegans biology when considering using this organism for genetic suppression analysis. Suppressor analysis in C. elegans has led to the discovery of many new genes and has resulted in the identification of interacting members of several different biological pathways (Inoue and Thomas, 2000; Gieseler et al, 2000; Nishiwaki and Miwa, 1998; Conradt and Horvitz, 1998). Although suppressor screens can result in identification of new genes, it is not possible to determine direct interactions from genetic suppression analysis alone. A 17 particularly powerful approach to determine direct interaction between proteins is yeast two-hybrid analysis (Golemis et al, 1994). Yeast two-hybrid screens involve a series of selections through which proteins that specifically interact with the protein of interest are isolated from a library (Golemis et al., 1994). Yeast two-hybrid analysis may help to determine the function of a particular protein by identifying novel proteins with which it interacts (Takayama et al, 1997). It also allows us to specifically test interactions between two proteins for which there is a prior reason to expect direct interaction (Wang etal, 1996). To find UNC-23 interacting proteins, I took advantage of these two powerful methods, the yeast two hybrid system and genetic suppression analysis. Using these two techniques, I identified the non-inducible heat shock protein 70, HSP-1 as an UNC-23 interacting protein. In the following chapters, I will describe the phenotypic characterization of unc-23 mutants and the identification of the unc-23 gene product. I will describe the UNC-23 expression pattern and relate the experiments that led me to conclude that UNC-23 interacts with the non-inducible heat shock protein 70, HSP-1. Finally, I will propose a model as to how UNC-23 is involved in maintenance of hypodermal integrity and hypodermal-ECM attachment. 18 Chapter 2. Material and Methods Nematode maintenance and culture conditions Nematodes were grown and maintained on N G M (3 g NaCl, 17 g agar, 2.5 g peptone, 1 ml of 5mg/ml cholesterol, 1 ml of 1M CaCl 2 , 1 ml of 1 M MgSC>4 and 25 ml of 1 M potassium phosphate [pH 6] in 975 ml of dH 2 0) plates streaked with Escherichia coli (OP50 strain), using standard laboratory techniques described by Brenner (1974). Liquid cultures were prepared as follows: Day one, a 5 ml culture of L Broth with OP50 was grown at 37°C overnight. Day two, 1 ml of the above bacterial growth was added to 300 ml culture of L Broth and allowed to grow over night. Day three, overnight culture was centrifuged for 5 minutes at 6000 rpm in a Beckman J2-21 centrifuge. The supernatant was then discarded and the pellet weighed. For every 5 gram of pellet, 100 ml of S medium (0.1 m NaCl, 0.05 M potassium phosphate, 0.002 mg/ml cholesterol, 1 m M potassium citrate, 3 m M CaCL;, 3 m M MgSC>4, 5 m M disodium EDTA, 2.5 m M FeS0 4 .7H 2 0, 1 m M MnCl 2 . 4H 2 0 , 1 m M ZnS0 4 .7H 2 0, 0.1 m M CuS0 4 . 5H 2 0 in dH 2 0) was added. The mixture was vortexed and the culture transferred to an autoclaved flask. Worms to be grown in liquid culture were washed off the plates with M9 buffer (22 m M K H 2 P 0 4 , 38 m M Na 2 HP0 4 , 85 m M NaCl, 1 m M M g S 0 4 in dH 2 0) and transferred to the liquid culture. Liquid cultures were maintained at 20°C and aerated by shaking at 200 rpm. 19 Nematode strains The standard nomenclature as described by Horvitz et al. (1979) was followed in this study. The N2 Bristol strain was used as the wild type strain. Unless otherwise indicated, all genetic experiments were carried out at 20°C. Strains used in this study are as follows: Wild type N2, CB25: unc-23(e25); CB255: unc-23(e255); CB324: unc-23(e324)\ CB988: unc-23(e988); CB2154: unc-23(e2154); DM0801: unc-23(ra801); DM0806: unc-23(ra806); unc-23(rhl92); BC2617: dpy-18(e364)/eTl III; mDfl/eTl V; BC3474: dpy-18(e364)/eTl III; unc-46(el77) sDf47/let-500(s2165) eTl V; BC3954: dpy-18(e364)/eTl III; unc-46(el77) sD/71/let-500(s2165) eTl; DM5109: mDfl/dpy-ll(e224); DM5110: sD/71/dpy-ll(e224). The following strains were constructed for this study (see following sections for methods of construction): DM7012; DM7013; DM7014; DM7015; DM7020; DM7021; DM7022; DM7023; DM7024; DM7025, DM7029. To determine the breakpoints for deficiencies mDfland sDf71, two strains DM5109 and DM5110 were constructed to produce arrested embryos that are homozygous for each deficiency, respectively. These strains were constructed as follows: unc-23(e25)/+ males were crossed into BC3954: dpy-18(e364)/eTl III; unc-46(el77) sDf71/let-500(s2165) eTl Vand BC2617: dpy-18(e364)/eTl III; mDfl/eTl V 20 hermaphrodites. Dpy-1 l(e224)/+ males were crossed into the FI progeny with an unc-23 phenotype {dpy-18(e364)/+; unc-46(el77) sDf71or mDfl/unc-23(e25)} and single wild-type hermaphrodite progeny were picked to new plates. These hermaphrodites were allowed to self-cross and their progeny were scored for presence of dpy-1 l(e224), arrested deficiency homozygous embryos, and wild-type heterozygous animals carrying the deficiency. Strains were maintained by picking wild-type hermaphrodites. Some strains were kindly provided by: D. L. Baillie at Simon Fraser University (Vancouver, British Columbia), J. Hodgkin at M R C laboratory (Cambridge, England), M . Nonet at Washington University (St. Louis, Missouri); The Caenorhabditis elegans Genetic Center (CGC) at the University of Minnesota (Minneapolis, Minnesota), The C. elegans Reverse Genetics Core Facility at the University of British Columbia (Vancouver, British Columbia) Immunofluorescence staining and microscopy Adult hermaphrodites were stained as described by Finney and Ruvkin (1990) with some modifications. Worms were washed off 3-4 small N G M plates with M9 buffer and pelleted by gentle centrifugation in glass tubes. Liquid culture animals were first spun gently to remove the culture then washed in M9 buffer twice. Worms were then suspended in 4% sucrose, 1 m M EDTA, gently rocked for 15-30 minutes, and afterward pelleted by gentle centrifugation to remove most of the sucrose solution, leaving worms in a small volume of 300pl. An equal volume of 2x Ruvkin fixation buffer [ 160 m M KC1, 40 m M NaCl, 20 m M disodium ethylene glycol bis ((3-aminoethyl ethe^-A^AP-21 tetraacetic acid (Na 2 EGTA), 10 m M spermidine-HCl, 30 m M Na 1,4-piperazinediethanesulfonic acid (Pipes), pH 7.4] with 50% methanol was added and the content of the tube were mixed gently. Formaldehyde, to a final concentration of 2%, was added and worms were frozen at -80°C for minimum of 30 minutes. When ready to proceed, tubes were thawed under cold tap water, mixed gently, and incubated on ice for 30 minutes. Worms were washed in Tris-Triton buffer (TTB: 100 m M Tris-Cl pH 7.4, 1 m M EDTA, 1% Triton X-100) twice and re-suspended in TTB + 2% (3-mercaptoethanol for 2 hours at 37°C with gentle rocking. The (3-mercaptoethanol treatment reduces the disulfide bonds in the cuticle of the worm. To complete the reduction reaction, worms were washed in l x B 0 3 buffer ( 50 mM H 3 B 0 3 pH 9.5, 2.5 m M NaOH) + 0.01% Triton x-100 once, then re-suspended in l x B 0 3 buffer + 0.01% Triton X-100 + 10 m M dithiothreitol for 15 minutes at room temperature while rocking gently. To oxidize the -SH groups, worms were washed once in lx B 0 3 buffer + 0.01% Triton X-100 and re-suspended in l x B 0 3 buffer + 0.01% Triton X-100 +0.3% hydrogen peroxide for 15 minutes at room temperature with gentle rocking. Samples were washed again in l x B 0 3 buffer + 0.01% Triton X-100 and then once in AbA buffer (PBS + 0.01% Triton X-100 + 2% milk powder + 0.05% sodium azide) for 15 minutes at room temperature. For immunofluorescence staining, worms were pelleted by gentle centrifugation and re-suspended in fresh A b A buffer. Primary antibodies were added and samples were incubated overnight at 15°C. The next day, after several washes in AbA buffer, worms were pelleted by gentle centrifugation and re-suspended in fresh AbA. Secondary antibodies were added and worms were incubated overnight at 15°C. After extensive 22 washing in A b A and PBS buffers worms were pelleted by gentle centrifugation and then re-suspended in mounting media. For immunofluorescence staining, rabbit polyclonal sera, GM1 (G. P. Mullen pers. Comm., Moerman et al, 1996), was diluted 1:50, while the mouse monoclonal antibodies MH4, and MH5 (Francis and Waterston, 1985) were diluted 1:200. The secondary antibodies, FITC-labeled donkey anti-mouse IgG F(ab')2 and TRSC-labeled donkey anti-rabbit IgG F(ab')2 (Jackson ImmunoReseach Laboratories; West Grove, PA, USA) were diluted 1:200. Confocal images were captured on a BioRad M R C 600 system, using the CoMOS 7.0a application. Optical sections of adult worms were collected at 0.2p intervals (Z steps) for the full depth of the specimen. Z-series were imported to NIH image 1.60 for viewing and projections. For final images, projections were imported into Adobe Photoshop (Versions 4.0 and 5.0) and images were printed on Codonics NP-1600 printer. Worms examined under polarized light were removed from N G M plates, placed in a drop of M9 buffer on a slide, and then immobilized by placing a coverslip over them. Polarized microscopy was done on a Zeiss Axiophot Photomicroscope (Carl Zeiss D-7082 Oberkochen). Images were captured on 35 mm Tmax 400 film. 23 PCR deficiency and polymorphism mapping The breakpoints of two deficiencies sDf71 and mDfl were determined in the following manner: A series of primers were designed to specific ORFs located on sequenced cosmids in the region of unc-23. Arrested deficiency homozygous embryos obtained from strains DM5109 and DM5110 were used as D N A templates in PCR reaction that used the above primers. If a set of primers failed to amplify the fragment of interest, then that region was considered to be deleted by that particular deficiency. If primers amplified the correct size fragment, then that region of the cosmid was not deleted by the deficiency. To do the PCR, 2-3 embryos or 2 adult hermaphrodites (positive control) were placed in 3|il of lysis buffer (50 m M KC1, 10 m M Tris (pH 8.0), 2.5 m M M g C l 2 , 0.45% Tween-20, 0.45% NP-40, 60|J,g/ml proteinase K) in the lid of a 0.5 ml eppendorf tube with drop of mineral oil. The tubes were briefly spun in a microfuge and then incubated in a thermocycler for 45 minutes at 60°C to allow lysis, then 15 minutes at 95 °C to inactivate the proteinase K. These lysates were used directly to do standard PCR as described in Rogalski et al. (1993,1995). Each tube includes 10 m M dNTPs, 25 pmol forward primer, 25 pmol reverse primer, 2.5 ul standard 10X PCR reaction buffer (200 m M Tris-HCl (pH 8.4) and 500 mM KC1), 25 m M M g C l 2 , and 2.5 units of Taq polymerase (Life Technologies, Burlington, Ontario). A l l PCR reaction mixtures were amplified in a Perkin-Elmer-Cetus 480 thermocycler under the following conditions: 30 seconds at 95°C, 60 seconds at annealing temperature (55°C-58°C) and 90 seconds at polymerization temperature of 72°C for 34 cycles. Presence or absence of 24 D N A fragments were determined by running PCR products on standard 1% agarose gels stained with Ethidium Bromide. Fragments were viewed under ultra violet light. Polymorphisms associated with particular unc-23 mutations were identified in a similar manner. PCR reactions were performed with D N A from N2 (positive control), unc-23 mutants. Primers were designed to amplify the H14N18.1, H14N18.2 and H14N18.3 ORFs (please refer to Figure 6 in chapter 3). Any strain that failed to amplify or amplified a different size fragment than the wild type carried a polymorphism. GFP constructs Constructs encoding fusion proteins containing unc-23 and the green fluorescent protein (GFP) were made as described by Hobert et a/.(1999). To fuse the unc-23 promoter and coding region to the green fluoresent protein gene, two independent PCR reactions were performed. In one, the unc-23 promoter (-2166 to A T G start codon) and coding region (ATG start codon to end of exon five minus the T A G stop codon) were amplified using a 5' primer specific to the promoter and a 3' hybrid (40mer) primer. The first 20 base pairs of the hybrid primer were identical to the last 20 base pairs of coding region of unc-23, minus the stop codon. The sequence of the last 20 base pairs were identical to the first 20 base pairs of GFP. In the second PCR reaction, the GFP portion from plasmid pPD95.75 was amplified using a 5'hybrid primer that was the reverse complement of the unc-23 3' hybrid primer. The 3' primer used in this reaction was identical to the 3' un-translated region (UTR) of the GFP. The two fragments obtained were purified using the QIAquick PCR purification Kit (Cat. # 28104. QIAGEN Inc. Mississauga, Ontario) and used as template in a third PCR reaction in which the two 25 fragments are fused together, using internal primers that were specific to the unc-23 promoter and the GFP 3' UTR. The unc-23 promoter used in this and other constructs started at -2166 upstream of the A T G start codon. This region contains all of the 5' upstream region of the unc-23 gene plus a portion of the 3' UTR of the HI4N18.2 gene which is located to the left of unc-23 (Figure 6, chapter 6). For spatial expression studies, the same method was used to fuse the myo-3 and jam-1 promoters to the full length unc-25::GFP fragments. PCR reactions as described above were performed using the Expand High Fidelity PCR system (Roche Molecular Biochemicals, Cat. no. 1 732 641. Mannheim, Germany) and Expand Long Template PCR System (Roche Molecular Biochemicals, Cat. No. 1 681 834. Mannheim, Germany). The GFP plasmids used in this study were kindly provided by A. Fire, S. Xu, J. Ahnn, and G. Seydoux from the Carnegie Institute of Washington (Baltimore, Maryland). Microinjections and complementation tests with transgenic strains Injections were performed as described by Mello and Fire (1995). N2 hermaphrodites were injected with the pRF#4 plasmid (86 r|g/pl), which carries the dominant marker roller, rol-6(sul006), and either cosmid D N A (8-12 r|g/pl), or GFP constructs. Roller worms have helically twisted bodies, so that animals roll during movement. FI roller hermaphrodites were obtained and any that produced roller progeny were kept as transgenic strains. Presence of cosmids in transgenic worms was confirmed by PCR, using primers specific to the cosmid vectors. Presence of GFP fragments was confirmed by observation of GFP expression under an immunofluorescence microscope. 26 To determine whether the GFP fusion constructs or cosmids are capable of rescuing the unc-23 phenotype, unc-23/+ males were mated to the transgenic roller hermaphrodites. Single FI roller hermaphrodites were put on plates and their progeny were scored for presence of unc-23 bent-head phenotype. If any F2 roller progeny from these plates exhibited the unc-23 bent-head phenotype, the GFP fusion construct or cosmid was not capable of rescue. However, if all the roller progeny exhibited a wild type phenotype, then the GFP fusion construct or cosmid was capable of rescue. Cosmids were kindly provide by Alan Coulson (The Sanger Center, Welcome Trust Genome Campus, Hinxton, UK) . Using the technique described above the following cosmid and GFP fusion transgenic strains were made for this study: DM7012: +/+; raExl2 [T27E4(V) + pRF#4{rol-6(sul 006dm)}]. DM7013: +/+; raExl3 [F07B7(V) + C01B7(V) + pKF#4{rol-6(sul 006dm)}]. DM7014: +/+; raExl4 [T19A5(V) + pRF#4{rol-6(sul 006dm)}]. DM7015: +/+; raExl5 [R03H4(V) + K06C4(V) + yXS#4{rol-6(sul006dm)}]. DM7029: +/+; raEx29 [ F59E11 + pKF#4{rol-6(sul006dm)}]. DM7020: +/+; raEx20 [full length unc-23::G¥V (10r|g/ul) + pPD95.75 (33rig/u.l) +pRF#4{rol-6(sul006dm) (78rig/|il)}]. DM7021: +/+ ; raEx21 [promoter unc-23::GFP+NLS (12rig/ul) + pPD95.67 (15rig/ixl) + pT(F#4{wl-6(sul006dm) (S6r\g/\i\)}]. DM7022: unc-23(e25); raEx20. DM7024: +/+; raEx 22 [myo-3::unc-23-GF? (12r|g/|il) + pRF#4{rol-6(sul006dm) (86r,g/ul)}]. DM7025: unc-23(e25); raEx22. 27 Sequence analysis of unc-23 The polymerase chain reaction (Barstead et al, 1991; Rogalski et al., 1995) was used to amplify the following homozygous alleles of unc-23: e25, e324, e988, ra801, ra806, and rhl92. Three sets of primers that give overlapping fragments were used to amplify genomic D N A from each of the above alleles. Fragments were prepared for sequencing using a QIAquick PCR purification Kit (Catalog #28104. QIAGEN Inc. Mississauga, Ontario). A l l of exons 2,3,4, and the majority of exon 5 (except for the last 25 bps) were sequenced (Figure7, chapter 3). Exon one is only 12 bps and since for all sequenced alleles, the molecular lesions were found to be in the downstream regions, this portion of unc-23 was not sequenced. Several unc-23 cDNAs were obtained as XZapII clones from the C. elegans cDNA project (kindly provided by Dr. Y . Kohara, National Institute of Genetics, Mishima, Japan). The Bluescript plasmid containing the cDNA insert was excised and transformed into XL1 blue cells (Stratagene. PDI Bioscience Inc. Aurora, Ontario). Plasmid D N A was prepared for sequencing using the Qiagen miniprep kit (catalog # 27106. QIAGEN Inc. Mississauga, Ontario). The full-length cDNA was sequenced to independently confirm the exon-intron boundaries predicted by the Genefinder program. A l l sequencing reactions were performed by the Nucleic Acid/Protein Service (NAPS) unit at the University of British Columbia. Proteins with sequence similarity to UNC-23 were identified using the NCBI blast search algorithm (Altschul et al. 1997). The Clustal W program (MacVector 6.0, Oxford Molecular Group) was used for protein sequence alignment and comparison. 28 unc-23(e25) suppressor screen The e25 allele of unc-23 was used to conduct the suppressor screen as described by Sulston and Hodgkin (1988). The screen was carried out as follows: 5 large N G M plates containing L4 and adult unc-23 (e25) animals were washed with M9 buffer three times. Animals were mutagenized in 50 m M Ethylmethanesulfonate (EMS) for four hours at 20°C. After mutagenesis, animals were washed 3x in M9 buffer and then allowed to recover for 3 hours on large N G M plates. After recovery, a total of 1050 EMS mutagenized worms (Po), were distributed on 70 large N G M plates and allowed to lay progeny over 6 days at 20°C. Both the FI and F2 progeny of P 0 mutagenized worms were examined for well-moving animals that lacked the bent-head phenotype. Individual wild type worms were transferred to new plates and their progeny were screened for presence of unc-23 and wild type phenotype. Homozygous suppressor strains were established by picking wild type animals that failed to produce the unc-23 bent-head phenotype. 29 Genetic characterization and mapping of suppressors To determine whether the suppressors were dominant or recessive unc-23(e25)/+ males were crossed into suppressor homozygous strains and their progeny were checked for presence or absence of unc-23 phenotype. Dominant suppressors do not show the bent head phenotype in the F I progeny. Recessive suppressors fail to suppress the bent head phenotype in the F I brood. 1/2 of the progeny from this cross w i l l present the bent-head phenotype. To determine whether the suppressors are inter- or intragenic, wi ld type N 2 males were crossed into homozygous suppressors and the F I progeny were self-crossed. The F2 worms were scored for presence or absence of unc-23 phenotype. 3 / l 6 of the recessive and 1/16 of the dominant intergenic suppressors are expected to present the unc-23 phenotype, while none-of the progeny from the intragenic suppressors w i l l show the bent-head phenotype. To map the suppressor loci the following chromosome markers were used: dpy-5(e61)I; dpy-10(el28)11; lon-l(el85) III; dpy-1 l(e224) V; lon-2(e678) X; PD4792: N2+ myo-2::GFP (IV). To make double mutants with unc-23(e25) and dpy or G F P markers, unc-23(e25)/+ males were crossed to homozygous dpy or G F P markers and individual wi ld type progeny were transferred to new plates. The F2 progeny were scored for presence of unc-23(e25); dpy or G F P double mutants. The following double mutant strains were constructed for mapping as described above: unc-23(e25);dpy-5(e61), unc-23(e25);dpy-10(e!28), unc-23(e25);lon-l(el85), unc-23(e25);dpy-ll(e224), unc-23(e25);lon-2(e678), unc-23 (e25);PD4792. 30 To map the suppressors, unc-23(e25); dpy/+ males were crossed into homozygous suppressors. FI unc-23 (recessive suppressor) or wild type (dominant suppressor) progeny were individually picked to new plates and allowed to self. In case of recessive suppressors, the F2 progeny were scored for presence or absence of the dpy markers. For dominant suppressors, the F2 progeny were scored for presence or absence of the unc-23 bent-head phenotype. In both cases, absence of the scoring phenotype (i.e. dpy for recessive and unc for dominant) indicated that the marker is on the same linkage group as the suppressing locus. Presence of the marker, however, confirms lack of linkage between the two loci. Sequence analysis of dominant suppressors The polymerase chain reaction was used to amplify two of the dominant suppressors of unc-23(e25), hsp-l(ra807) and hsp-l(ra808), for sequence analysis. Suppressors hsp-l(ra807) and hsp-l(ra808) were used as template to amplify overlapping fragments that span the whole of the hsp-1 gene. Fragments were prepared for sequencing using QIAquick PCR purification kit (Catalog #28104. QIAGEN Inc. Mississauga, Ontario). Sequencing was performed by the Nucleic Acid/Protein Services (NAPS) Unit at the University of British Columbia. The Clustal W program (MacVector, Oxford Molecular Group) was used for protein sequence alignment and comparison. 31 Yeast two-hybrid constructs, transformations, and screening pDM#220 (pGBDU-C3 + unc-23 bait) and pDM#221 (pGAD-C3 + unc-23 bait) constructs were made as follows. The PWO D N A Polymerase kit (Roche Molecular Biochemicals, Catalog #1644 947. Mannheim, Germany) was used to amplify exon 3 to 5 (614 bp) of unc-23 cDNA (please refer to figure 10 in chapter-3), according to recommendation of the manufacturer (http://biochem.roche.com/pack-insert/1644947a.pdf). PCR amplified fragments were inserted in to a pCR-BLUNT vector (Invitrogen, Carlsbad, California) as described by the manufacturer (http://www.invitrogen.com). The ligation mixture was used to transform DH5a competent cells (Life Technologies, catalog #18265-017. Burlington, Ontario) and transformed colonies were obtained after overnight growth on Kanamycin selection plates. After overnight growth in 2 ml L Broth + Kanamycin, D N A was obtained from the cultured cells using the Qiagen miniprep kit (catalog # 27106). The bait was released from the pCR-BLUNT vector using the EcoR I restriction sites and re-inserted into the pGBDU-C3 and pGAD-C3 vectors' EcoR I restriction sites (James et al., 1996). The orientation of bait was determined by PCR reactions, which used primers specific to unc-23 and vector. The PCR reaction failed to amplify a 769 bp fragment, if insertion was not in the correct orientation. To confirm sequence integrity of the bait, several of the constructs that were confirmed to have the bait in correct orientation were sequenced by the NAPS unit at the University of British Columbia. Yeast strain PJ69-4A (James et al, 1996) was used to make yDM#220 and yDM#221 yeast strains. Competent cells from the yeast PJ69-4A strain were made as follows: PJ69-4A yeast cells were grown in Y P D media (1% bacto-yeast extract, 2% bacto-peptone, 2% 32 dextrose in dH.20) at 25°C for two nights while shaking. A 50 ml Y P D culture was inoculated with all of 2 ml and incubated at 25°C for seven hours. The culture was spun down at 3000 rpm for five minutes. Supernatant was discarded and cells were re-suspended in 5 ml Lithium Acetate (0.1 M) and incubated at 25°C for one hour. Competent cells were centrifuged for five minutes at 3000 rpm and re-suspended in 1ml Lithium Acetate (0.1M). The transformation reaction was set up as follows: 10 pi of salmon sperm D N A , 10 pi of either pDM#220 or pDM#221, 100 pi of PJ69-4A competent cells and 230 pi of 50% PEG (Polyethylene Glycol 3350 in Tris EDTA) were mixed and kept at room temperature. After one-hour transformation mixture was spread on selection plates and incubated at 25°C for 4-5 days. Transformed cells were given the yDM#220 (pGBDU-C3 + unc-23 bait) and yDM#221 (pGAD-C3 + unc-23 bait) designations. Library transformation was carried out as described above with some modifications. yDM#220 strain was used to prepare competent cells as explained above. Transformation mixture was prepared as follows: 10 pi Salmon sperm DNA, 5 pi library D N A , 300 pi competent cells, and 700 pi 50% PEG. Transformation mixture was left at room temperature for one hour and then heat shocked at 42°C for 30 minutes with occasional mixing. Following heat shock, 100 pi of SD medium (0.67 % Bacto-yeast extract nitrogen base without amino acids, 2% dextrose in dF^O) was added to the cells. Cells were spread on histidine minus (His") selection plates in 10 or 100 pi volumes and incubated at 25°C for 4-5 days. Transformation efficiency was determined by diluting the original transformation mixture lOOx and plating 10 pi of the dilution on proper 33 selection plates. After 5 days, the number of colonies on these plates where counted and the transformation efficiency was determined. yDM#220 was used as bait to screen a mixed stage C. elegans c D N A library (kindly provided by Dr. B . Barstead, Oklahoma Medical Research Foundation. Oklahoma City, O K ) as described by James et al. (1996). Briefly, colonies obtained on His* selection plates were transferred to adenine minus (Ade") plates to be re-screened for their positive interaction. The established colonies on Ade" plates were transferred to third selection plate containing 5-Fluoroorotic Acid (FOA) to promote loss of bait plasmid. Once the bait plasmid was lost, the prey plasmid was recovered and re-transformed into yDM#220 to confirm positive interaction. Plasmids that showed interaction with yDM#220 were sequenced by the N A P S unit at The University of British Columbia. Sequences obtained were examined against the C. elegans genomic sequence available in the data base. 34 Chapter 3. Results Loss of hypodermal integrity causes muscle detachment in unc-23 mutants Mutations in the unc-23 gene result in progressive dystrophy of the anterior body wall musculature. During mid-larval development, muscle cells in the head region begin to detach from the hypodermis (Waterston et al. 1980), resulting in adults with heads that are noticeably thinner than wild type animals. The resulting loss of control of the head is observed as a bent-head when the animal tries to move forward. Detachment of muscle cells from the hypodermis begins with the anterior-most cells and is usually confined to the head of the animal (Figure 2, B). Occasionally, muscle cell detachment extends beyond the terminal bulb of the pharynx, but never past the vulva. The extent of muscle detachment is highly variable among different alleles, ranging from severe to almost wild type. Other less penetrant phenotypes observed in unc-23 mutants include occasional protrusion of vulva, egg laying defects, mild constipation due to interruption of defecation cycle and rarely, small blisters at the tip of the nose. The pharynx of unc-23 mutant animals functions normally. In severe alleles of unc-23, where muscle cell detachment has resulted in collapse of surrounding tissues on the pharynx, however, careless transfer of the worms can result in exudation of the pharynx from the mouth. To date there are seventeen recessive alleles of unc-23 identified. The most severe allele, e25, is the reference allele of unc-23 and the first allele to be isolated (Brenner, 1974). Unc-23(e25) was used extensively in this study for the characterization 35 and analysis of the gene. Ten of the unc-23 alleles were examined for severity of muscle detachment, nose blistering and vulva protrusion. The result of this characterization is summarized in Table 1. The severity of the detachment described in table 1 is based on the extent of anterior muscle cell detachment. In severe alleles, muscle detachments extend just beyond the terminal bulb of the pharynx and involve both the dorsal and ventral quadrants. In these animals, the upper tissues completely collapse on top of the pharynx, resulting in complete loss of head movement and a true bent-head phenotype. In mild alleles, the detachments do not extend past the anterior bulb of the pharynx. Only the tip of the nose is affected and although there is muscle detachment, the animal has partial control of its head movement. In mild alleles, for the most part, no head dragging is observed. Bullerjahn and Riddle (pers. comm.) observed that unc-23 mutant animals, grown in liquid culture, fail to exhibit the bent-head phenotype. The suppression of the muscle cell detachment phenotype lasts up to 48 hours after animals are placed on solid medium. This experiment was repeated to examine the implications of this temporary suppression on the arrangement of the myofilament lattice and muscle cell positioning (Figure 2, C). In liquid grown worms, anterior muscle cells are positioned correctly and myofilament assembly is normal but muscle cells are easily detached by mechanical stress. Application of external force, such as putting worms a under coverslip and gently rolling 36 Figure 2. Polarized light images of wild type and unc-23(e25) animals. A) Polarized light image of a wild-type adult hermaphrodite (anterior is to the right in all three panels). In C. elegans, the body wall muscle cells extend from the tip of the nose to the posterior end of the animal. The striation observed is due to alternation of thick and thin filament-containing regions in the sarcomere. The pharynx, which is the grinding apparatus of the worm can be seen in this image. The bulbous structures represent the anterior (Ant. Bulb) and posterior or terminal bulb (Pos. bulb) of the pharynx. B) Polarized light image of a plate grown unc-23(e25) adult hermaphrodite. The most anterior muscle cells have detached from the tip of the nose and retracted posteriorly. Note that the head in this animal is thinner than the wild type. Arrows point to the regions where muscle cells have stopped detaching. Posterior to the site of detachment, the light and dark pattern of thick and thin filaments can clearly be seen. C) Polarized light image of an unc-23(e25) animal grown in liquid culture. The muscle quadrants extend to the tip of the nose and no detachment is observed. Animals grown in liquid culture are usually thinner than plate grown animals. The liquid grown unc-23 worm shown here is, therefore, thinner than its plate grown counterparts. As is shown in this image, muscle cells are positioned correctly and extend all the way to the tip of the nose. The light and dark pattern, representing the thick and thin filament organization of myofilament lattice in sarcomere are also well organized and show no defects. Scale =10 uM 37 36 Table 1. Characterization of the unc-23 alleles. * Based on the severity of the bent-head phenotype. 1 Brenner (1974) 2 Vancouver Reverse Genetics Core Facility Allele Phenotype* Protruding Vulva Blister at tip of nose Induced by e25 Severe (n=100) 30 2 EMS (Brenner) i e255 Mild (n=52) 0 0 EMS (Brenner) e324 Severe (n=26) 15 0 EMS (Brenner) e462 Mild (n=33) 0 0 EMS (Brenner) e478 Mild/Wild-type (n=66) 0 0 EMS (Brenner) ra801 Mild/Wild type (n=40) 0 0 TMP (VRGCF) 2 ra806 Mild/wild type (n=45) 0 0 TMP (VRGCF) e988 Severe/Mild (n=55) 11 0 EMS (Brenner) ell82 Mild (n=48) 0 0 EMS (Brenner) e2154 Mild/Wild-type (n=70) 0 1 Mutator strain (Mancillas and Waterston, pers. Comm.) 39 them, can results in detachment of muscle quadrants from the underlying hypodermis. Once detached, muscle cells continue to contract for a short period of time. The force of contraction is enough to cause further detachment of more posterior cells. Once the contractions cease, muscle detachment also stops. Subjecting wild-type animals to the same treatment, however, does not result in any muscle cell detachment. In fact, wild type animals can withstand a great deal more force than the mutant animals examined above. Wild type or mutant worms that are grown in liquid culture are usually thinner than their plate grown counterparts (compare A and C in figure 2). Worms in liquid culture thrash about rather than move in smooth sinuous movement seen with plate grown worms. The temporary muscle detachment suppression observed with the liquid grown unc-23(e25) mutants and the manifestation of this phenotype after transfer to agar plates, suggests that there are different forces acting on muscle and hypodermal attachments in these two growth situations. One possible difference may be in the increased shear force exerted on the anterior muscle cells as the animals forage for food on plates. Even a casual observation of worms on plates shows that their heads are quite actively engaged in testing the surrounding environment. This type of head movement seems to be absent in liquid grown cultures. In liquid grown animals, muscle cell positioning and myofilament assembly occurs correctly, but in the face of increased mechanical force muscle cells detach. Observations with liquid grown animals suggest that the unc-23 phenotype reflects a defect in muscle cell attachment rather than in muscle cell positioning or myofilament 40 assembly. To determine the effect of muscle cell detachment on the surrounding tissues, wild type, liquid, and plate grown unc-23 (e25) animals were stained with antibodies that recognize components of the basement membrane and attachment structures within the hypodermis. The nematode homologue of the basement membrane proteoglycan, U N C -52/perlecan, was identified in our lab by Rogalski et al. (1993). Perlecan is distributed over the whole of the underlying basement membrane and is concentrated over the dense bodies and M-lines (Figure 3, A). Perlecan plays an essential role in myofilament lattice assembly. Animals that lack perlecan fail to assemble their myofilament lattice and expire as two fold embryos. Viable alleles of unc-52, however, are able to move as young larvae, but as they grow, they become progressively paralyzed. This paralysis is due to gradual loss of myofilament linkage to the muscle membrane and disruption of myofilament lattice (Rogalski et al, 1993). To examine the effect of muscle cell detachment and to determine if the location of the weak attachment maps to the basement membrane, the overall organization of perlecan in the unc-23(e25) animals was examined. Examination of the plate grown unc-23(e25) animals with GM1, a polyclonal sera that recognizes UNC-52/perlecan in C. elegans, revealed that perlecan detaches along with the most anterior muscle cells and is completely absent from the areas where muscle cell have detached (Figure 3, B). The distribution of perlecan in the rest of the animal ranges from normal to slightly disorganized in areas of stress such as the vulva. Unc-23 (e25) liquid grown mutants stained with GM1, however, show a wild type distribution and localization for perlecan (Figure 3, C). This suggests that deposition and localization of perlecan in the basement 41 membrane occurs in unc-23 mutant animals but that the basement membrane detaches along with the muscle in plate grown animals. In C. elegans, hypodermis is directly involved in transmission of contractile force from muscle to the cuticle. Hemidesmosomes, which are the cell-ECM anchoring junctions for the intermediate filaments within the hypodermis, are direct mediators of muscle to cuticle attachment (Francis and Waterston, 1991). To examine the effect of muscle cell detachment on the anchoring junctions in the hypodermis, unc-23 (e25) animals were stained with MH4 and MH5 antibodies. The MH4 antibody recognizes the intermediate filaments within the hypodermis. The MH5 antibody recognizes a member of the plakin protein family that localizes to the hemidesmosomal complex in the hypodermis (Francis and Waterston, 1985; J. Bosher et al, V . Hapik et al, M . Hresko et al, pers. comm.). In wild type worms the intermediate filaments appear as repeating and equally spaced bands that run circumferentially from one edge of a muscle quadrant to the other (Figure 4, A). The wild type pattern of the plakin protein member in the hemidesmosomes is more irregular and appear as series of small dots that not only cover the whole of the muscle quadrants but are also more intense in areas where muscle cells contact each other (Figure 5, A . arrow). Examination of MH4 staining revealed that there is a good deal of variability in intermediate filament organization in unc-23 animals grown on plates (Figure 4, B). The disruption in MH4 staining observed in affected areas can range from mild to full detachment. Unc-23 (e25) liquid grown mutants stained with MH4 show no disorganization or detachment and resemble wild type animals (Figure 4, C). Analogous to MH4, the MH5 pattern is variably affected by the detachment of muscle cells in unc-42 23 plate grown mutants. The disruption of the MH5 staining pattern also ranges from fairly mild to complete detachment (Figure 5, B). unc-23(e25) liquid grown mutants stained with MH5, however, do not show any detachment or disorganization (data not shown). Liquid grown unc-23 animals appear similar to the wild type animals. This implies that defects in UNC-23 do not affect any aspect of muscle proliferation, migration, growth, or initial attachment to the underlying matrices. Upon application of mechanical stress, however, muscle detaches whole, tearing away the underlying basement membrane and disrupting the hypodermis. In mutants that affect tissue integrity and maintenance, the primary site of tissue fragility and separation usually indicates the location of the affected molecule. The antibody staining studies point to the hypodermis as the tissue affected in unc-23 mutant animals. This is in agreement with the earlier study by Waterston et al. (1980). The E M images obtained by Waterston et al. (1980), clearly indicates that the hypodermis is the site of tissue fragility and detachment. The occasional blisters observed in severe alleles of unc-23, therefore, can be explained to be the areas where hypodermis has ruptured and has filled with fluid. 43 Figure 3. GM1 immunofluoresence staining of wild type and unc-23 (e25) animals. A) Wild type adult hermaphrodite stained with GM1, a polyclonal antibody that recognizes UNC-52/perlecan, a basement membrane proteoglycan in C. elegans. Perlecan is distributed over the whole of the basement membrane and is localized to the dense bodies and M lines. Note the staining of basement membrane surrounding the pharynx. The anterior and posterior bulbs of the pharynx are shown. Anterior is to the right in the three panels. B) Plate grown unc-23(e25) adult hermaphrodite stained with GM1. The detachment and retraction of the anterior muscle cells has resulted in detachment and disorganization of perlecan in the basement membrane. Arrow points to patches of perlecan left behind after muscle detachment. Note that in the pharynx, the organization and localization of perlecan is not affected. Both the anterior and posterior bulbs of the pharynx are clearly seen in this image. C) Liquid grown unc-23(e25) adult hermaphrodite stained with GM1. No evidence of disorganization or detachment is present. Perlecan is deposited by the muscle cells and has localized in absence of UNC-23. Scale =10 | i M . 44 4 5 Figure 4. MH4 immunofluoresence staining of wild type and unc-23(e25) animals. A) A n adult wild type hermaphrodite stained with the MH4 antibody, which recognizes the intermediate filaments of the hypodermis (anterior is to the right in all three panels). Intermediate filaments are patterned in regularly spaced bands that extend from one edge of the muscle quadrant to the other. Intermediate filaments are also present in the pharynx. The anterior and posterior bulbs of the pharynx are shown. To allow comparison, the arrow in A points to the same general area as the arrow in B. B) A plate grown unc-23(e25) adult hermaphrodite stained with the MH4 antibody. In plate grown animals, muscle detachment results in disorganization and or complete detachment of the intermediate filaments in the hypodermis. Arrows point out the areas where intermediate filaments have detached along with the muscle cells. Pharynx is not affected. C) Unc-23(e25) liquid grown animals stained with the MH4 antibody. No detachment or disorganization is observed in liquid grown animals. In absence of UNC-23, intermediate filaments are produced and localized to their proper location in the hypodermis. Scale =10 pM. 46 Pos. bulb Pos. bulb Ant. bulb Ant. bulb M3-Figure 5. MH5 immunofluoresence staining of wild type and unc-23(e25) animals. A) A n adult wild type hermaphrodite stained with the MH5 antibody, which recognizes a member of the plakin family in the worm (anterior is to the right in both panels). The MH5 antigen is a component of the hemidesmosomes in the hypodermis and localizes as a series of irregular dots that are more intense over the areas where muscle cells contact one another (arrow). This member of the plakin family is also present in the pharynx. The anterior and posterior bulbs of the pharynx are shown. B) A plate grown unc-23(e25) adult hermaphrodite stained with the MH5 antibody. In plate grown animals, the extent of detachment and disorganization of MH5 antigen is highly variable. In some animals muscle detachment results in complete detachment of the MH5 antigen, while in others severe to mild disorganization and even at times fairly wild type patterns can be seen. Arrows point to the areas where muscle detachment has resulted in detachment and aggregation of MH5 antigen. Pharynx is not affected in unc-23 mutants. The anterior and posterior bulbs of the pharynx are shown. Scale =10 u.M. 48 4 ^ unc-23 encodes a protein with a domain similar to the mammalian Bcl2-associated athanogene 2(BAG-2), a chaperone regulator The unc-23 gene maps to a region of chromosome V that is uncovered by several deficiencies (Figure 6). To place the unc-23 gene on the physical map, PCR deficiency mapping strategy was used to determine the breakpoints of the two deficiencies sDf71 and mDfl, which uncover unc-23. The left breakpoint of sD/71 is in cosmid F25G6 and its right breakpoint lies just to the right of cosmid R03H4. The left break point of mDfl lies to the left of cosmid ZC190 . This data determined that the region containing the unc-23 gene is between cosmids R03H4 and ZC190, on the physical map (Figure 6). Pools of cosmids from this region were introduced into wild type adult hermaphrodites by microinjection (Fire, 1986; Mello etal., 1991) and the stable transgenic strains with stable arrays were tested for their ability to rescue the unc-23 bent head phenotype. The unc-23 phenotype was rescued with a transgenic array (kindly provided by A. Schaefer and Dr. M . Nonet) carrying four overlapping cosmids R03H4, F59E11, T19A5 and H14N18 (Figure 6). When tested individually cosmids R03H4, F59E11 and T19A5 failed to rescue the unc-23 phenotype. I was unable to obtain a strain carrying the H14N18 transgenic array, therefore, I was unable to test and determine if this array was capable of rescue. As an alternative approach to study this cosmid, I decided to look for polymorphisms in the H14N18 interval. A rich array of unc-23 alleles including 8 EMS, 2 putative Tel transposon insertion, and two TMP induced alleles were used in this study. Primers were designed 50 for three of the open reading frames H14N18.1, .3, and .4 (Figure 6) and the polymerase chain reaction was used to amplify the open reading frames using genomic D N A from the unc-23 mutants as template. The ORFs, H14N18.3 and H14N18.4, did not show any polymorphism. The polymerase chain reactions, which amplified the HI4N 18.1 open reading frame, however, showed two distinct polymorphisms for the ra801 and ra806 alleles of unc-23. ra801 and ra806 amplified as smaller fragments, while the fragment from the e2154 allele failed to amplify. To confirm that H14N18.1 is the unc-23 gene, the ra801, ra806, e25, e988, e324, and rh.192 alleles of unc-23 were sequenced. A l l six alleles show sequence alterations, which confirmed that the HI4N 18.1 open reading frame is in fact the unc-23 gene. The most severe allele, unc-23(e25), is a missense mutation that alters glutamic acid residue 297 into a lysine (Figure 7 and 8). e324 is a nonsense mutation located in exon five that affects the glutamine residue 397 (Figure 7 and 8). e988 is also a nonsense mutation affecting amino acid residue 186 in exon two (Figure 7 and 8). The positions of the two nonsense mutations allow for expression of a truncated UNC-23 protein in both alleles. The ra801 mutation is a 117 bps in frame deletion in exon two, while ra806 is a 155 bps out of frame deletion in the same exon that results in a sixteen amino acid long polypeptide and it probably represents a null allele of unc-23(¥igure 7 and 8). Rhl92 is a four bps insertion, which results in a frame shift and termination in exon two (Figure 7 and 8). Table 2 summarizes the sequence alterations of unc-23 alleles sequenced to date. The ACeDB (A C. elegans Data Base) program (Eckman and Durbin, 1995) describes the unc-23 gene as encoding 5 exons spreading over 2660 base pairs (Figure 7). Based on this program the coding region of unc-23 is 1377 base pairs and it translates into a 458 amino 51 acid protein. To examine the ACeDB prediction, an unc-23 cDNA (Y. Kohara, pers. comm.) was sequenced, which independently confirmed the exon-intron boundaries predicted by the Genefinder program. The B L A S T server at NIH identified BAG-2, as a human protein with significant similarity to the UNC-23 protein sequence (Takayama et al. 1999) (Figure 8). Human BAG-2 is a 211 amino acid protein that is a member of the B A G family of chaperone regulators. In humans, the B A G family of chaperone regulators contain a conserved 45 amino acid region, the B A G domain, near their C termini, which binds Hsp70/Hsc70 and controls their chaperone activity (Takayama et al. 1999). To date, five members of this family, BAG-1 to BAG-5, have been identified. The C. elegans UNC-23 and human BAG-2, share 40% amino acid identity and 62% similarity over the B A G domain and its upstream region (Figure 8). UNC-23 contains 458 amino acids and the region of similarity shared between mammalian BAG-2 and UNC-23 is confined to the carboxy-terminus of UNC-23. The amino-terminal of UNC-23 is unique and is not similar to any sequence available in the database. The B A G domain of UNC-23 starts at residue 427 and extends to the end of the protein (Figure 8). The B A G domain in the human BAG-2 protein starts at residue 164 and ends at residue 196 (Figure 8). 52 § HH o 3 i B H PH o PH < h-H u HH PH u HH H o I 00 ca I f OX) xi O P H (U Cl ^ ox)' cu a § -3 -Q a -K X! o C .S2 o JO p. 9 ca o O . 2 "5 C cn <D CD s CD Xi H d o '5b CD I o a CD o o ca CD T 3 CD > H £ & C O CD 1 CD o CD T 3 g 0 t CD J D CD u -3-K O T3 CD T3 S ^3 2 « .in W 13 CD CD 1 - 1 c 00 I CD 5 CD 00 I CO o o O OX) •c CD Xi o Os O N § ° s cn o o V, £ a a 8 I O t 3 O !> c« CD O XI cn CD = cn CD 'o Cl CD < + H Co cn I M o a I H ca -° 2 ^ £ ox) cn 'C CD c 3 & O CD O J=l o 8 ^ o « g <oa .2 a CO XI C H o Cl CD •»—1 2 « ca CD o ^ > "| 2 Cl CD CD > ox) 33 <D 1 / 3 J=l H s « CD ^ > H CU CD CD 3 o GO ca o xl cn O ca a cn bf) .3 a >. o Cl CD CD Cl CD ox) 0-1 1 Cj Cl K • g cn « 2 o CN DC O to S CD T 3 O Cl CD t a r S u -*-» o d CD S 3 Table 2 Sequenced alleles of unc-23 and their molecular lesions. Allele Type of mutation Residue changes Exon affected Phenotype severity e25 Missense E297K G A A (glu) to A A A (lys) 3 very severe e324 Nonsense Q397stop C A A (gin) to T A A (stop) 5 very severe e988 Nonsense R186stop C G A (arg) to T G A (stop) 2 severe ra801 Deletion 117 bps deletion. Deletes amino acid residues 17 to 55. New Leucine made at site of deletion. 2 mild ra806 Deletion 155 bps deletion, frame shift and termination. Deletes amino acid residues 6 to 57. Null allele. 2 mild rhl92 Insertion G G A A insertion at residue 221. Frame shift and termination. 2 severe/mild 55 Figure 8. Alignment of C. elegans UNC-23 and human BAG-2 proteins. C. elegans UNC-23 and human BAG-2 share 40% amino acid identity (shown as boxed residues) and 62% amino acid similarity (highlighted residues). UNC-23 is 458 amino acids compared to BAG-2, which contains 211 amino acid. BAG-2 is most similar to the carboxy-terminus of UNC-23. The amino terminus of UNC-23 is novel and does not recognize any sequence available in the database. The B A G domain of UNC-23 starts at residue 427 and extends to the end of the protein (blue arrows). The B A G domain in the human BAG-2 protein starts at residue 164 and ends at residue 196. Positions of the unc-23 alleles are also shown relative to the protein sequence. The affected residues are circled. e25, the most severe allele of unc-23 alters the glutamic acid residue 297 to a lysine. This glutamic acid residue is conserved in UNC-23 and BAG-2. e324 and e988 alleles affect glutamine 397 and arginine 186, respectively, and alter them to stop codons. rhl92 is a four base pairs insertion in the lysine residue 221 (red arrow points to the point of insertion) and results in frame shift and termination. Brackets indicate the position of deletion alleles, ra801 and ra806, relative to the UNC-23 protein. ra801 is a 117 bps deletion that eliminates amino acid residues 17 to 55. ra806 is a 155 bps deletion that takes out amino acid residues 6 to 57 and results in a frame shift and termination. 56 (0 W o <*> r n W Pi u o a w EH o « H rt EH 3 H H EH W > W - rf: co 2; CO Ii. PS > < > s in CO H < H " E H << Hi rw Q 'WE z a s H H w H H H o s co OS E its EH u w w J J « OS OS OS OS OS l>S KS K H H H 04 w bS K o> a a Q Q Q o W < < < u u co s rfl co' • OS a 1 1 K a ~ a a H .. . H H w & K K S 3 U H H OS Hi OS 2 a P. H E 3 H IH En EH H H m a <;' > > > K|A|A A|A|T > CO < l> >l> a M I • « H > > > OS OS EH EH EH j a o | M M j H H > • | H H | o EH EH K PS CO O H I Q to ra ra CO Q X o u U 2 u < < E j S3 w Q o i-H o O «* in iH HJ CJ C •H CD 4-1 CN O 0 u o O & < ft < 09 ro cn CN a O) a a CJ u UN K § X S I UNC-23 is expressed in muscle and hypodermis To examine the spatial and temporal expression pattern of UNC-23, a fusion construct encoding the unc-23 gene and green fluorescent protein (unc-2 3 ::GFP) was made. A stable transgenic line expressing a functional UNC-23 ::GFP protein from a transgenic array was established. This transgene, which included the unc-23 promoter and structural element, was able to fully rescue the unc-23 bent-head phenotype, when introduced into unc-23(e25) mutant worms. This indicates that the fusion protein construct is functional and that the expression pattern and sub-cellular localization of this protein is similar to the endogenous UNC-23 protein. Based on the observation of the GFP fusion protein, UNC-23 is first detected in 1.5 fold stage embryos. At this time, only a few unidentified cells express UNC-23. Expression continues through embryogenesis and by three-fold stage, it has expanded to include muscle, hypodermis, and pharynx. At this point, it is unclear whether UNC-23 has any sub-cellular localization during embryogenesis. The UNC-23::GFP protein is expressed in the pharynx, body wall muscle cells, hypodermis, H cells, and the vulva in adult animals. In body wall muscle cells, UNC-23 is localized to the dense bodies and M lines, in a pattern similar to that of the transmembrane protein, P integrin (Williams and Waterston, 1994) (Figure 9, B and C ). In the hypodermis, UNC-23 is distributed over the whole of the tissue, except the nuclei, and is also localized in a pattern reminiscent of the intermediate filaments (Francis and Waterston, 1991) (Figure 9, A). 58 UNC-23 is present in both the body wall muscles and the hypodermis. To determine the tissue responsible for the muscle cell attachment defects in the unc-23 mutants, full-length unc-23::G¥V constructs using either myo-3, a body wall muscle specific promoter, or jam-1, a hypodermal specific promoter were made. The idea was to express UNC-23 in only one of these tissues and determine if either expression pattern could rescue the bent head phenotype in unc-23 mutant animals. A stable transgenic strain that carries the myo-3::unc23-GFP construct was established. UNC-23::GFP expressed from this construct localizes to the dense bodies and M lines, which is identical to the expression pattern of UNC-23 in animals that carry the functional fusion construct. The level of protein expression is comparable to that of the functional fusion construct. This construct, however, is not capable of rescuing the muscle detachment phenotype. The inability to rescue the bent head phenotype through UNC-23 expression in the muscle cells agrees with the earlier cell biological observations, which suggested that the unc-23 defect is located within the hypodermis. My attempts at obtaining stable transgenic lines, carrying the jam-l:\unc-23::GFP construct, have so far been unsuccessful. Therefore, it is unclear whether the expression of UNC-23 in the hypodermis alone is sufficient to rescue the unc-23 muscle detachment phenotype. 59 Figure 9. Spatial distribution of UNC-23::GFP in unc-23(e25) rescued mutants. A) UNC-23 ::GFP is present in a variety of tissues including the body wall muscle cells and the hypodermis. Both the hypodermal and the muscle expression of UNC-23 can be seen in this image. Anterior is to the left. The hypodermal expression between the dorsal and ventral quadrants can be clearly seen here. The arrowhead points to the lateral seam cells. Hypodermal cells are multinucleated. The tilted arrow in the hypodermis points to a nucleus that completely lacks UNC-23 expression. UNC-23 is present in the whole of the hypodermis and displays a localization pattern that is very similar to the pattern of intermediate filaments described before. The horizontal arrow points out an example of this localization in the hypodermis. UNC-23 is also expressed in muscle cells and localizes with the dense bodies and M lines. The vertical arrow points out a dense body in a dorsal muscle cell. B) UNC-23 expression pattern in the head of the animal. Anterior is to the left. In this image both the muscle and the hypodermal localization of UNC-23 can be seen. The vertical arrow indicates a dense body, while the horizontal arrow points out UNC-23 localization in the hypodermis. C) A magnified view of UNC-23 localization in muscle cells. The arrow points to a dense body, while the arrowhead points out an M line. 60 61 Screening, mapping, and genetic characterization of unc-23(e25) suppressors To identify unc-23 interacting genes a suppressor screen was devised using the allele unc-23 (e25). The progeny of EMS mutagenized unc-23 (e25) worms were examined for the presence of well-moving animals that did not display the bent-head phenotype. In order to obtain dominant as well as recessive suppressors, the FI and F2 progeny of mutagenized animals were examined. Seven independently isolated suppressors were obtained (Table 3, see description of suppressor name designations) and from the analysis of three of these, it was revealed that hsp-l(ra807) and hsp-l(ra808) are dominant suppressors of unc-23 (e25) and 21-1 is a recessive suppressor. A l l three suppressors are unlinked to unc-23 and all fully suppress the unc-23 bent-head phenotype, which means that no muscle detachment is observed in suppressed animals. When removed from an e25 mutant background these three suppressors have a wild type phenotype. Genetic mapping has placed the hsp-l(ra807) and hsp-l(ra808) suppressors on chromosome IV. The 21-1 suppressor may also map to chromosome IV, but this result is tentative and needs to be confirmed. During genetic characterization of these three suppressors, it became apparent that all three are temperature sensitive. Suppressor animals when grown and maintained at 15°C have a wild type phenotype, but the majority of the same suppressors, when grown at 20°C, expire as L3 larvae or become sterile adults. A small number of animals escape the temperature sensitivity and lay eggs that hatch. At 25°C, only very few of the suppressors survive. Most of the animals expire as L3 larvae. The animals that survive to adulthood are sterile. While it is not 62 clear yet whether the temperature sensitive phenotype is an intrinsic property of these mutations, it does seem to be more than a coincidence that all.three suppressor mutations impart a temperature sensitive phenotype. 30A-2, 1B-3, and 36-F2 are recessive suppressors of the bent head phenotype and all are unlinked to unc-23. Of the seven suppressors, 40-F2 is the only one that does not fully suppress the unc-23 bent-head phenotype. In an unc-23 genetic background, 40-F2 suppresses the bent head phenotype in about 70% of the animals. The remaining 30% show the bent-head phenotype but with less severity than the unc-23(e25) animals, which means that the muscle detachment does not extend beyond the anterior bulb of the pharynx. 63 Table 3. Mapping and genetic characterization of the unc-23 suppressors. * Suppressors, hsp-l(ra807) and hsp-l(ra808), are assigned proper gene names because through this study, they have been identified to correspond to the non-inducible heat shock protein 70 in C. elegans (see "sequence analysis of dominant suppressors" in the result section). Other suppressors will also be assigned proper gene names once their corresponding genes are identified. Suppressor Inter/intragenic Dominant/ Recessive Linkage group hsp-l(ra807)* Intergenic Dominant IV hsp-l(ra808) Intergenic Dominant IV 21-1 Intergenic Recessive IV (?) 30A-2 Intergenic Recessive ? 1B-3 Intergenic Recessive ? 36-F2 Intergenic Recessive ? 40-F2 Intergenic ? ? 64 UNC-23 interacts with HSP-1 in yeast two-hybrid analysis yDM#220, which is the UNC-23 region homologous to the mammalian BAG-2 (Figure 10, A), was used as bait to screen a mixed stage C. elegans cDNA library using a yeast two-hybrid screen. The purpose of this screen was to identify UNC-23 interacting proteins. From the primary screen, 97 interacting clones were identified and 73 of these clones were further examined. Of the 73 interacting clones, 36 were re-introduced into yeast for confirmation of interaction. Ten of the re-introduced clones showed an interaction with UNC-23 and were sequenced. A l l ten were found to be copies of the non-inducible heat shock protein 70, HSP-1. This data identifies HSP-1 as an UNC-23 interacting protein and is in agreement with mammalian studies, which also identify BAG-2 as an Hsc70 interacting protein (Takayama et al., 1999). The deduced minimal region of HSP-1 required for an interaction with UNC-23 comprises amino acid residues 189 to 419, which corresponds to the ATPase domain of the HSP-1 protein (Figure 10, B). Heat shock proteins are highly conserved across species as illustrated in Figure 11. The mammalian Hsc70 and the C. elegans HSP-1 proteins are 79% identical and 84% similar. 65 Figure 10. Schematic representation of the unc-23 gene and the HSP-1 protein. A) Position of yDM#220 construct used as bait in yeast two-hybrid screen relative to the unc-23 gene is shown here. The yDM#220 region corresponds to the area where UNC-23 is most homologous to the mammalian BAG-2. B) Schematic representation of the HSP-1 protein. The ATPase domain and the peptide-binding domain are shown here. The red boxes represent portions of HSP-1 protein encoded by the cDNAs, which were obtained through the yeast two-hybrid screen. The deduced minimal region of HSP-1 necessary for interaction with UNC-23 comprise amino acid residues 189 to 419, which correspond to the ATPase domain of the HSP-1 protein. 66 o -f-SO oo c a B o O • i-H • r-i +-> , PH B o Q PH H < o o "* so o 00 tN SO tN Figure 11. Alignment of C. elegans HSP-1 and mammalian Hsc70 protein sequences. C. elegans HSP-1 and mammalian Hsc70 share 79% amino acid identity (shown as boxed residues) and 84% amino acid similarity (highlighted residues). Amino acid residues 1-386 make up the ATPase domain of the heat shock proteins (enclosed within the red box). The substrate binding domain comprises of residues 384 to 543, while residues 542 to 646 correspond to the 10 KDa variable region of the heat shock proteins. The minimal region required for UNC-23 interaction is shown by two blue arrows. 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The hsp-1 gene in C. elegans, maps to the right end of chromosome IV. Interestingly, the two dominant suppressors, hsp-1 (ra807) and hsp-1 (ra808), isolated in the e25 suppressor screen also map to linkage group IV. Since it seemed quite possible that the suppression activity could be due to alterations in the hsp-1, this gene in each mutant strain was sequenced. Indeed, sequencing of the hsp-1 gene in both mutant strains revealed that hsp-1 (ra807) and hsp-1 (ra808) both harbor molecular lesions in the ATPase domain of this gene (Figure 11 and 12). The hsp-1 gene is 2221 base pairs and contains four exons (Figure 12). The hsp-l(ra807) molecular lesion is a missense C to T transition, which results in the alteration of alanine 379 to valine and maps to exon three (Figure 11 and 12). The molecular lesion in hsp-1 (ra808) is a G to A missense mutation, which alters the aspartic acid residue 233 to an asparagine (Figure 11). This mutation is located in exon two of the hsp-1 gene . (Figure 12). Both mutations are located within the minimal region required for HSP-1 interaction with UNC-23 (Figure 11 and 12) and are conserved residues. A schematic representation of the hsp-1 gene and the position of the mutations are shown in Figure 11 and 12. 70 g ° 0 rs .s o Q CD to cd a o q S-H a in c .2 _CD | o CD o d o co o a. CD g EU d eu g 1 +-> o d o . i-H eu CO CD S-H OH eu d o 4> rH t8 «g CO s 2 o u - £ -5 G *> d o 2 & ?5 -a Cd eu * a GO .5 M CO g 1 •a to CD <D 3 1 OH 5 3 « g OH O eu '+3 21 a ^ eu <3 I 2 H O <J Cd CD to -B "H o £ 2 M cd <-i, .2 'B a to -5 2 0 'co pd tu H C O (O tu +-> Q Lo « ^ 8 i > cd eu H d o to I O CL ""I eu - d H 5 s •a g 8P"1 6 -I OH °* to cj g «M cd O d o eu 3 — eu CO C+H o d o to cd s CD ej CO tN to o <u CO cd to oo "cd C5 CD CO a 1 OH cd cd 00 a Is q cd 00 co CD U .2 3 S £ OH Id S3 - G cd 43 cd 1^ T3 CD o a co cd <U eu T3 7-1 Chapter 4. Discussion unc-23 encodes a protein with a domain similar to the mammalian Bcl-2-associated athanogene 2, a chaperone regulator The C. elegans unc-23 gene encodes a member of the Bcl-2 associated athanogene (BAG) family of molecular chaperone regulators. In mammals, the B A G family of chaperone regulators contain a conserved 45 amino acid region (BAG domain) near their C termini, which binds Hsp70/Hsc70 and controls their chaperone activity (Takayama et al. 1999). To date, five members of this family, BAG-1 to BAG-5 , have been identified (Takayama et al. 1999). Except for the B A G domain, no significant homology exists between the different members of the B A G family. UNC-23 is most similar to the mammalian BAG-2 protein. The UNC-23 and BAG-2 proteins share 40% amino acid identity and 62% similarity over the B A G domain and its upstream region. The shared region of similarity between mammalian BAG-2 and UNC-23 is confined to the carboxy-terminus of UNC-23. The amino terminus of UNC-23, consisting of amino acids 1 to 260, is unique and is not similar to any sequence within the NCBI database. The amino terminus of UNC-23 is rich in glutamine and proline amino acids, but this does not give any clue to the function of this part of the protein. A search of the C. elegans genome has revealed another B A G domain containing protein. This protein, encoded by the F57B10.11 gene, is most similar to mammalian BAG-1 . C. elegans F57B10.11 and BAG-1 share 24 % identity and 45% similarity over the B A G domain and its upstream region. Similar to BAG-1 , C. elegans F57B10.11 protein also contains a ubiquitin-like domain at its N terminus. Except for the B A G domain, sequence 72 comparison of UNC-23 and F57B10.11 proteins reveals no significant homology between the two proteins. Preliminary characterization of a possible null allele of F57B10.11 has revealed an increase of 20% in the number of progeny as the only obvious phenotype (E. Nollen and R. Morimoto, pers. comm.). A search of the Saccharomyces cerevisiae and Drosophila melanogaster genomes revealed no proteins with any significant similarity to UNC-23. UNC-23 is expressed in the hypodermis and the body wall muscle cells and is required for hypodermal integrity UNC-23 expression is first detected in only a few cells in 1.5 fold stage embryos. By L I stage of development, UNC-23 expression has expanded to include the pharynx, body wall muscle cells and the hypodermis. In the adult animals, UNC-23 is expressed in the pharynx, the body wall muscle cells, the hypodermis, H cells, and the vulva. In body wall muscle cells, UNC-23 is localized in a pattern similar to that of the transmembrane protein, (3 integrin, suggesting that it is associated with the dense bodies and M lines, (Gettner et al, 1995; Williams and Waterston, 1994). In the hypodermis, UNC-23 is distributed throughout the tissue, with the exception of the nuclei, and is distributed in a pattern reminiscent of the intermediate filaments (Francis and Waterston, 1991). Phenotypic analysis of unc-23 mutants suggests that UNC-23 is required for maintenance of muscle cell attachment during post-embryonic development. Mutants 73 that affect the unc-23 gene result in progressive detachment and dystrophy of the anterior body wall muscle cells that starts around the L2 stage of development. Muscle detachment results in animals that have a bent-head phenotype. The bent-head phenotype of unc-23 mutants can be temporarily suppressed in animals that are grown in liquid culture. Once the worms are transferred onto solid medium, however, the progressive detachment of the anterior muscle cells begins and within a short period of time animals exhibit the bent-head phenotype. Muscle cells in liquid grown mutants are capable of myofilament assembly, growth, contraction, and attachment. This attachment, however, is weak, since applied external force or movement of worms on agar plates results in muscle detachment. These observations indicate that UNC-23 does not affect migration, growth, myofilament assembly, or attachment of the muscle cells to the underlying basement membrane. The lack of effect on muscle cell development in unc-23 mutants and the association of UNC-23 with adhesion complexes within muscle and the attachment structures within the hypodermis suggests that UNC-23 acts in some manner to regulate cell attachment and maintain hypodermal integrity. In mutants where tissue integrity and maintenance are affected, the primary site of tissue fragility and separation probably indicates the location of the affected molecule. In unc-23 mutant worms, muscle cells detach whole from the underlying tissues. No detachment, fracturing, shearing, or separation of muscle cells from each other is observed. Although UNC-23 is present at the dense bodies and M lines, no weakness, or detachment is observed at these sites in unc-23 mutants. In fact, restricted expression of UNC-23 to the muscle cells alone is not sufficient to rescue the bent-head phenotype. This suggests that the site of separation and fragility is in the attachment of muscle cells 74 to the underlying hypodermis rather than between the muscle cells or at site of myofilament attachment. Immunofluorescence staining of unc-23 liquid and plate grown animals with antibodies recognizing components of the underlying basement membrane and the hypodermis indicates that the hypodermis is the affected tissue in unc-23 mutant animals. The hemidesmosomes and the intermediate filament networks are clearly disrupted in unc-23 mutants. These observations are in agreement with ah earlier study by Waterston et al. (1980), which used electron microscopy to examine the hypodermal-muscle junction in detail. Waterston et al. (1980) observed a " delamination" and local ruptures within the hypodermis. The critical experiment to see if the exclusive expression of UNC-23 in the hypodermis is sufficient to rescue the muscle detachment phenotype in unc-23 mutant animals has not yet been done. It is possible that expression of UNC-23 in the hypodermis alone will not be enough to rescue the mutant phenotype. It is possible that UNC-23 is required in adhesion complexes on both sides of the E C M . The severity of the bent-head phenotype observed in unc-23 mutants is highly variable and allele specific. The most severe allele of unc-23, e25, has a missense mutation in exon three. This mutation affects a glutamic acid residue that is conserved in mammals and C. elegans (Figure 8). Other severe alleles of unc-23 include e324 and e988. e324 is a nonsense mutation that is located in exon five, almost immediately before the B A G domain. e988 is also a nonsense mutation affecting amino acid residue 187 in exon two. The positions of the nonsense mutations allow for expression of a truncated UNC-23 protein in both alleles (Figure 8). The three mentioned alleles impart a much more severe bent-head phenotype than ra801 or ra806 alleles of unc-23. The ra801 mutation is an in frame deletion in exon two, the novel region of UNC-23, and 75 does not affect the B A G domain or the area of homology with BAG-2 (Figure 8). Ra806 is an out of frame deletion in exon 2 that results in a sixteen amino acid long polypeptide and it probably represents a null allele of unc-23 (Figure 8). The majority of ra801 or ra806 mutants fail to display the bent-head phenotype and largely resemble wild type animals. The phenotype of the small percentage of animals that do display the bent-head are less severe. Examination of the mutant phenotypes and the molecular lesions affecting each of the alleles suggest a correlation between the region of UNC-23 affected and the severity of phenotype observed. Partial deletion of the unique region of UNC-23 or complete loss of UNC-23 is not as damaging to maintenance of muscle cell attachment since in ra801 or ra806 mutants the majority of the animals look wild type. The more severe alleles contain the amino-terminus but lack the B A G domain or harbor a mutation in a conserved residue within the homologous region, which suggest that for proper function, UNC-23 requires the B A G domain and its upstream region. 76 UNC-23 directly interacts with the ATPase domain of HSP-1, the non-inducible heat shock protein 70 The yeast two hybrid analysis, using the UNC-23 B A G domain as bait against a C. elegans cDNA library identified the non-inducible heat shock protein 70 or HSP-1 as a pairing partner for UNC-23. The HSP-1 minimal region required for interaction with UNC-23 corresponds to the ATPase domain of this protein. This result confirms that UNC-23 in C. elegans, like its mammalian counterpart, interacts with the heat shock protein 70. In mammals, BAG-1 , BAG-2, and BAG-3 are not capable of forming homo-or hetero-dimers (Takayama et al, 1999). Yeast two-hybrid tests designed to examine the ability of UNC-23 to form homo-dimers indicated that the region of UNC-23 used as bait is unable to interact with itself. The full-length UNC-23 protein was not examined to see i f it is capable of homo-dimer formation. The yeast two hybrid studies are highly suggestive of a direct interaction between UNC-23 and HSP-1. The results of the genetic suppression screen, which identified HSP-1 as a suppressor of unc-23(e25) mutation, supports this conclusion. The two HSP-1 dominant suppressors, ra807 and ra808, are missense mutations that affect the ATPase domain (Figure 13) and are located within the minimal region required for the in vitro interaction of HSP-1 with UNC-23. Hsp-1 (ra807) and hsp-1 (ra808), when removed from the e25 mutant background have a wild type phenotype. The R N A i progeny of the hsp-1 gene in C. elegans (Piano, pers. comm.) are, however, embryonic lethal. This suggests that hsp-1(ra807) and hsp-l(ra808) mutations probably do not result in 77 complete loss of HSP-1 activity. Rather, they may specifically affect the UNC-23-ATPase dependant activity of HSP-1. If UNC-23 functions as a negative regulator of HSP-1 in a manner similar to BAG-2, then the mutations that affect the HSP-1 ATPase domain may curtail the ATPase activity. These altered HSP-1 proteins would then act as if bound to UNC-23. Another possibility is that the unc-23 (e25) mutation may result in a conformational change in the tertiary structure of UNC-23, so that the HSP-1-UNC-23 interaction is severely affected. Hsp-1 (ra807) and hsp-1 (ra808) mutations may also result in conformational changes that now allow UNC-23 to interact with the HSP-1 ATPase domain. It is possible to test the validity of the above predictions by examining the effect of a null allele of unc-23 in combination with these HSP-1 suppressor alleles. This test would allow us to determine whether suppression requires the presence of U N C -23 in some form or whether HSP-1 alterations are epistatic to UNC-23 function. 78 BAG-1 is an Hsc70 regulator The most intensively studied member of the B A G family of chaperone regulators is BAG-1 . BAG-1 was first identified as a Bcl-2 binding protein (Takayama et al, 1995) that when co-expressed with Bcl-2 increases the anti-apoptotic ability of cells. Since its identification as a Bcl-2 binding protein, BAG-1 has been implicated in many cellular interactions, including the binding of hepatocyte growth factor receptors (Bardelli et al, 1996), glucocorticoid receptors (Kanelakis et al, 1999), retinoic acid receptors (Liu et al, 1998), Raf-1 kinase (Wang et al, 1996), and targeting to the proteasome (Luders et al, 2000). The heat shock cognate protein 70, Hsc70, was identified by Takayama et al. (1997) to be a direct binding partner of BAG-1 . BAG-1 binds the ATPase domain of Hsc70 and modulates the Hsc70 ATPase activity. The protein structure of Hsc70 is conserved and can be divided into three functional segments. The N-terminus of Hsc70 consists of a 44 K D a domain that binds ATP, while the middle segment is an 18 K D a substrate binding domain. The 10 KDa C-terminus forms the third region of the molecule and it may act as a lid to the substrate-binding cleft. This third domain also contains highly conserved motifs that determine the functional specificity of the Hsc70 (Kiang and Tsokos, 1998; Demand et al, 1998) (Figure 8). Substrate binding is controlled by binding of the nucleotide to the amino-terminal ATPase domain, ATP hydrolysis and nucleotide exchange (McCarty et al, 1995; Rudiger et al, 1997). The ATP bound form of Hsc70 has low overall affinity for substrate, while the A D P bound 80 form binds the substrate firmly. The cycling of Hsc70 between the different nucleotide states is regulated by chaperone co-factors that bind Hsc70 and modulate its ATPase activity. The affinity of the chaperone for a specific substrate, therefore, depends on the combination of co-factors interacting with the Hsc70 chaperone. Efficient binding to substrate requires binding of the Hsp40 co-factor to the 10 KDa carboxy-terminal of Hsc70 chaperone. The Hsp40 co-factor stimulates the conversion of ATP-bound Hsc70 to ADP, resulting in slow but stable association of the chaperone with a substrate. The Hsp40-stimulated ATPase activity of Hsc70 is inhibited by the chaperone co-factor CHIP (carboxyl terminus of Hsc70-interacting protein). CHIP blocks the forward reaction of the Hsc70 substrate-binding cycle by interacting with the 10 KDa Carboxy-terminus of the Hsc70. CHIP decreases the net ATPase activity of Hsc70 and therefore reduces the chaperone-substrate binding efficiency. The ADP and substrate-bound conformation of Hsc70, which is achieved through binding of the Hsp40, is, however, further stabilized by binding of another cofactor, Hip (Hsc70-interacting protein) (Hohfeld et al, 1995; Nollen et al, 2001). Hip binds the ATPase domain of Hsc70, stabilizes the substrate/chaperone complex, and helps maintain the substrate at the substrate binding cleft until Hsc70 can interact with other chaperone machinery. BAG-1 also binds the ATPase domain of the Hsc70 chaperone, but its effect on the chaperone activity is opposite to that of Hip. BAG-1 stimulates the ADP release from the chaperone and promotes release of substrate (Nollen et al, 2001; Nollen et al, 2000; Hohfeld and Jentsch, 1997) (Figure 14). Takayama et al (1999) identified four other B A G containing proteins, BAG-2 to BAG-5 , through yeast two-hybrid screening with the ATPase domain of Hsc70. These B A G family members do not share any homology except for the B A G domain in their 81 carboxy-terminus. Takayama et al. (1999) showed that the B A G domain and the upstream region of the BAG-2 and BAG-3 interact with the ATPase domain of Hsc70 in vitro and in vivo. BAG-2 and BAG-3 are probably negative regulators of the Hsc70 chaperone activity and addition of Hip completely negates the inhibitory effect of these two proteins. This suggests that the suppression of Hsc70 activity by B A G proteins is reversible and that antagonistic abilities of Hip, which were demonstrated for BAG-1 (Nollen et al, 2001), also extend to include BAG-2 and BAG-3. Some in vitro reports on effects of BAG-1 on Hsc70 agree that BAG-1 is probably a negative regulator of Hsc70 (Bimston et al, 1998; Nollen et al, 2000). B A G -1 interaction with hsc70 in vivo, however, can result in both inhibition and enhancement of the chaperone-dependent events, depending on the substrates involved (Takayama et al, 1997). This suggests that the in vivo function and effect of BAG-1 on Hsc70 may depend on the specific substrate and the cellular context under which the two proteins are interacting. Therefore, the effect of BAG-1 regulation on the chaperoning activity, chaperone-substrate complex, and the folding state of released substrate remains unclear (Hohfeld, 1998; Takayama et al, 1997). Some of the functional perplexity of the B A G family members may be resolved if these proteins could be studied in a model organism. This study of UNC-23 in C. elegans offers a great opportunity to examine the possible functions of UNC-23's B A G domain within a biologically relevant context. Characterization of the phenotype observed in unc-23 mutants has allowed us to determine that UNC-23's function is required specifically for maintenance of hypodermal integrity and muscle cell attachment in C. elegans. The results from the liquid culture also suggest that UNC-23 is part of a biological pathway that responds to mechanical 82 stress during growth. Characterization of F57B 10.11, which is the other B A G domain-containing gene in C. elegans, and its comparison with UNC-23 can help us better understand the biological role of the B A G domains and the functional significance of their unique N-termini. 83 substrate O Hsp40 A D P A T P Hip Hsc70 BAG-1 1 A b P A T P Figure 14. Model of Hsc70 reaction cycle in presence of its regulating cofactors. The ATP-bound Hsc70 has low affinity for substrate, while the ADP-bound form binds substrate firmly. The conversion of ATP to ADP is stimulated by the Hsp40 cofactor which promotes substrate binding. Chaperone/substrate complex is further stabilized by binding of cofactor Hip to the ATPase domain of Hsc70. BAG-1 stimulates the release of ADP from the chaperone and therefore promotes the release of substrate. BAG-1 and Hip both bind the ATPase domain of Hsc70 in a manner antagonistic to each other. 24-UNC-23-HSP-1 interaction is required for maintenance of hypodermal integrity during mechanical stress Identification of mutations that affect the maintenance of the hypodermal integrity and muscle cell attachment during larval development suggest that a biological mechanism exists that responds to cell growth and the increase in contractile force (Plenefisch et al, 2000; Waterston, et al, 1980). The phenotypes of the majority of muscle attachment {Mud) mutations identified to date manifest themselves during larval development and do not affect embryonic muscle attachment. Based on the phenotype of the mua genes, these genes most likely encode regulatory or structural proteins required for maintenance of integrity and attachment during growth and mechanical stress. UNC-23, as a chaperone regulator, is an example of a regulatory protein required for the maintenance of hypodermal integrity. UNC-23 is the first HSP-1 cofactor identified in C. elegans. Since mutations in unc-23 result in loss of integrity of the hypodermis and muscle detachment, it is reasonable to predict that the interaction of UNC-23 with HSP-1 is required in biological pathways that respond to increasing contractile force by reinforcing the integrity of the hypodermis. This prediction is supported by the results obtained from the liquid culture and by the genetic suppression of unc-23 (e25). Unc-23(e25) animals that are grown in liquid culture do not display the bent-head phenotype. The muscle and hypodermal cell growth and attachment that occur in liquid grown animals is comparable to that of wild type worms. Animals that are grown in 85 liquid culture do not experience the shear force that the plate-grown animals experience as they forage for food. This suggests that the integrity of the hypodermis is not challenged in liquid grown worms because there is less mechanical stress. Once animals are transferred to a solid substrate, they use their head muscles extensively to test the environment and forage for food (Figure 15). The use of the anterior muscle cells and the shear force exerted during movement initiates the muscle detachment and loss of hypodermal integrity. It has been suggested that muscle contraction is an enhancer i f not the cause of muscle detachment in mua mutants studied to date (Plenefisch et al, 2000). The mutation, unc-54(el90), which lacks the major body wall muscle myosin heavy chain (Epstein et al., 1974) causes complete paralysis. A double mutant of el90 in combination with selected mua mutations greatly suppresses the muscle detachment defect normally imparted by these mua mutations. In cases of mua-l(rhl60) and mua-10(rh267) this suppression is complete (Plenefisch et al, 2000). The HSP-1 genetic suppression of UNC-23 established that these two proteins interact in vivo and that they are important in biological pathways that participate in maintenance of hypodermal integrity and muscle attachment. Hsc70 chaperones have been shown to interact with several cytoskeletal proteins including intermediate filaments (Napolitano et al, 1985 cited in Liao et al, 1995) and microtubules (Napolitano et al, 1985; Ohtsuka et al, 1985; Green and Liem, 1989 cited in Liao et al, 1995). In addition, hsc70s are also involved in folding, stabilization, assembly, and targeting of multi-protein complexes such as the steroid hormone receptors (reviewed in Cheung and Smith, 2000). 86 Figure 15. Anterior body wall muscle cell arrangement in C. elegans. A ) Ventral view of the anterior body wall muscle cells (Anterior is to the left). The first four anterior cells are designted as the head muscle cells. The next four cells correspond to the neck muscles and the rest of the cells make up the body wall muscle cells Body wall muscle cells can be classified according to their source of innervation. The first four anterior muscle cells in each quadrant are innervated by the motor neurons from the nerve ring. The next four muscle cells of the neck are innervated by motor neurons of the nerve ring and the ventral nerve cord. The remaining muscles are innervated exclusively by the motor neurons from the ventral nerve cord. The motor neurons of the nerve cord innervate either the dorsal or the ventral muscle cells. The body movement is, therefore, limited to the dorsoventral plane only. The head, however, is able to make lateral as well as dorsoventral movements. Muscle cell arrangement in the head region of the animal is also more compacted when compared to the rest of the body. B) Lateral view of the anterior body wall muscle cells. It is likely that HSP-1 performs similar cellular functions in C. elegans. In response to increasing contractile force, new proteins may be required to build up existing or newly formed attachment structures in the hypodermis. HSP-1 might be involved in folding, assembling, and chaperoning these newly made proteins to their locations. Likewise, in response to an increase in contractile force, certain proteins essential for the maintenance of c e l l - E C M attachments might be produced in larger amounts. Production of large amount of intermediate filaments, for example, would be required at newly made attachment sites and for the reinforcement of existing attachment structures to help maintain the integrity of the hypodermis. HSP-1 might bind the intermediate filaments to prevent their aggregation and chaperone them to their proper location. The chaperoning functions of HSP-1 are not limited to interaction with intermediate filaments. Membrane bound receptors and structural proteins that make up the attachment structures and or bind the intermediate filaments are other possible substrates for HSP-1. If UNC-23 functions as a negative regulator of HSP-1, in the same manner as B A G - 2 , then one might predict that the interaction of UNC-23 with HSP-1 is required for the release of the substrate from the binding cleft of HSP-1. In other words, in the absence of UNC-23 , HSP-1 is unable to release its substrate in response to mechanical stresses experienced by the hypodermis. The sub-cellular localization of UNC-23 in the hypodermis is pertinent to this type of model, since its distribution is reminiscent of the distribution of the intermediate filaments within the hypodermis. The predicted role of UNC-23 as a regulator that promotes HSP-1 substrate release, agrees with the localization pattern observed in the hypodermis. If UNC-23 promotes the release of substrates specific to 88 attachment and maintenance of integrity, it is reasonable for it to be located within areas where its function is required. Given the number of biological pathways that HSP-1 may be involved in, it is not surprising that hsp-1 R N A interference results in embryonic lethality (Piano, pers. comm.). What is interesting is that mutations in unc-23 do not result in a similar phenotype. Mutations in unc-23 only affect the hypodermal integrity and muscle cell attachment of the anterior body wall muscle cells. This suggests that the interaction of UNC-23 with HSP-1 provides specificity for HSP-1 function in the biological pathway(s) that are involved in maintenance of muscle attachment during development. 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