THE ROLE OF CYTOPLASMIC PROTRUSIONS IN THE INTERCELLULAR ADHESION OF RAT LEUKEMIA CELLS (LINE IRC 741) by DOMINIC KWOK-WO YIP B. Sc., University of Br i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1972 In present ing th i s thes is in pa r t i a l f u l f i lmen t o f the requirements fo r an advanced degree at the Un ive rs i t y of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying of th i s thes i s for scho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i c a t i on of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of The Un ivers i ty of B r i t i s h Columbia Vancouver 8, Canada - i i -ABSTRACT I. The function, structure and response to environmental factors of a cytoplasmic protrusion found in rat leukemia cells IRC 741 were investigated. A greater r i g i d i t y and adhesiveness of the protrusions, as compared to the main c e l l body, was demonstrated by time-lapse cinematography, and this functional difference was correlated with localized ultrastructural differences in the cytoplasm and on the c e l l surface, and with higher negative surface-charge density, as shown by c e l l electrophoresis. The formation or maintenance of the cytoplasmic protrusions depended on adequate nutritional conditions, and was interfered with by diminished intercellular .contact,, by environmental temperatures below 37°C, by alkaline pH and by calcium-ion depletion. The protrusion appears to represent a type of adhesive organelle not previously described in either cancer cells or normal c e l l s . II. In the course of the above work, a method was developed whereby the differential staining of viable and nonviable unfixed c e l l s , as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining i s due, at least in part, to structural differences that are retained following aldehyde-fixation. - i i i -TABLE OF CONTENTS Page INTRODUCTION 1 PART I The role of Cytoplasmic Protrusions in the Intercellular Adhesion of Rat Leukemia Cells (Line IRC 741) . 2 MATERIALS AND METHODS 8 1. General 8 2. Electron Microscopy 8 3. Histochemical Methods - Light Microscopy 9 4. Electron-microscopic Cytochemistry 10 5. Cell Electrophoresis ....11 6. Time-lapse Cinematography ..11 7. The Induction and Maintenance of Protrusions .12 RESULTS 16 1. Histochemistry 16 2. Electron Microscopic Cytochemistry .16 3. Enzymatic Digestion of Ultrathin Sections 19 4. Cell Electrophoresis ....19 5. Cinematography • 24 6. The Induction and Maintenance of the Protrusion 27 DISCUSSION 37 Cytochemistry and Electrocytochemistry 38 Cell Electrophoresis 40 Cinematography ......43 The Induction and Maintenance of Protrusion 49 - iv -Page PART II The Dye-Exclusion Test for Cell V i a b i l i t y : Persistance of Differential Staining Following Fixation 56 INTRODUCTION 57 MATERIALS AND METHODS .. ..58 Dyes 58 Cell Cultures .60 Fixation and Counting 60 RESULTS 62 DISCUSSION ...69 SUMMARY 75 BIBLIOGRAPHY 77 APPENDICES .' 84 1. Preparation of Cells for Microscopy ..................................85 2. The Chemistry of EDTA . ...86 - v -LIST OF TABLES Table Page I. Dyes Used for V i a b i l i t y Determinations, Arranged in Ascending Order of Molecular weights 59 II. The Number of Stained Cells (Per Thousand) .„ in Four Overgrown Cultures of IRC Cells, Prior to, and Following Glutaraldehyde Fixation 63 III. Effect of Storage Following Fixation on the Subsequent Staining of Cells with Alcian Blue 64 - vi -LIST OF FIGURES Figure Page 1 Living IRC cells in culture 4 2 Electron micrograph of IRC c e l l clump 5 3 The change of the proportion of pear-shaped c e l l s with c e l l density and culturing .time .13 4 Reaction of KCN/10% HCl with sections of IRC cel l s stained with colloidal iron 17 5 Electron micrographs of IRC c e l l s stained with colloidal iron 18 6 Trypsinized IRC cells stained with colloidal iron 20 7 Electron micrographs of epon sections immersed in phosphate buffer and in protease/phosphate buffer 21 8 Electron micrographs of epon sections immersed for 1 hr in 0.1 N HCl and in pepsin/0.1 N HCl 22 9 The change in orientation of the protrusion of IRC ce l l s with the change of the e l e c t r i c a l polarity in c e l l electrophoresis 23 10 The interaction of a single pear-shaped c e l l with an aggregate of three c e l l s 26 11 . The change of proportion of viable pear-shaped cel l s with c e l l density and culturing time ...28 12 Changes in the proportion of pear-shaped cells with changes in the degree of c e l l contact ...30 - v i i -Figure Page 13, Change in the proportion of pear-shaped ce l l s 14 15 ' with changes in environmental temperature .31, 32 16, 17 Proportion of pear-shaped ce l l s in c e l l samples suspended in Hanks' BSS, Moscona's BSS and various concentrations of EDTA at pH 6.5, 7.4 and 8. 34, 35 18 "': Effect of calcium-depletion on the proportion of pear-shaped ce l l s over 3 hours 36 19 Interaction of electron-dense particles .47 20 Rates of diffusion into fixed c e l l s of seven dyes, over one hour 66 21 I n i t i a l s t a b i l i t y of the proportion of alcian-blue stained c e l l s with continued exposure to the dye 67 22 Changes in the proportion of alcian-blue stained cells over 48 hours of exposure to the dye 67 23 The molecular structures and molecular weights of the dyes used in the present study 73 24 The formation of metal-ion-EDTA complex 87 25 The structure of EDTA in aqueous solution 87 26 Titration of EDTA with KOH 87 27 Moles of EDTA required to bind one mole of calcium ions at different pH's 87 - v i i i -ACKNOWLEDGEMENT I w i s h t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o : Dr. R. L. Noble, D i r e c t o r o f the Cancer Research C e n t r e , f o r t h e o p p o r t u n i t y and f a c i l i t i e s to c a r r y o u t t h i s r e s e a r c h . Dr. N. A u e r s p e r g , Department o f Zoology and Cancer R e s e a r c h C e n t r e , f o r h e r a d v i c e , g u i d a n c e and encouragement t h r o u g h o u t t h i s i n v e s t i g a t i o n . D r s . A. B. A c t o n and P. F o r d , Department o f Zoology, f o r t h e i r h e l p , a d v i c e , i n s t r u c t i v e comments and r e v i e w o f the m a n u s c r i p t . Dr. G. E. Dower and Mr. W. Z i e g l e r , Department o f Pharmacology, f o r t h e i r p e r m i s s i o n and i n s t r u c t i o n s i n the use o f t h e cinema-t o g r a p h i c equipment. Dr. P. S. V a s s a r , Department o f P a t h o l o g y , f o r h i s p e r m i s s i o n and i n s t r u c t i o n s i n the use o f the a p p a r a t u s f o r - c e l l e l e c t r o p h o r e s i s . . D u r i n g the t e n u r e o f t h i s i n v e s t i g a t i o n , t h e a u t h o r was t h e r e c i p i e n t o f a s t u d e n t s h i p from the N a t i o n a l Cancer I n s t i t u t e o f Canada. The r e s e a r c h was s u p p o r t e d by a g r a n t from t h e N a t i o n a l Cancer I n s t i t u t e o f Canada. - 1 -INTRODUCTION The role of the c e l l surface i n development and i n cellular interactions has been receiving increasing attention (Weiss, 1967; Carr et a l . , 1970; Chedd, 1972). Among the surface properties of normal and malignant cells that have been studied extensively are the structural factors that underlie cellular adhesiveness., the maintenance of the ri g i d i t y of the plasma membrane and the basis for c e l l asymmetry. The malignant behaviour of tumour ce l l s , such as invasiveness, lack of contact inhibition (Abercrombie, 1966), or decrease of adhesiveness (Coman, 1944), have been thought to be due to modifications of surface properties. Although research has failed to demonstrate any consistent change i n the c e l l surface that i s unique to cancer (Weiss, 1967), there are many quantitative differences in surface properties between tumour ce l l s and normal c e l l s . Among others, lack of communication between tumour cells (Loewenstein and Kanno, 1966), a decreased requirement of calcium ions i n metabolism (Gilbert, 1972), and often a relative increase of electrophoretic mobility between tumour ce l l s and their normal counterparts (Ambrose, 1966), have been observed. Some demonstrable surface features, although not unique to cancer ce l l s , have been found to accompany malignant transformation. Examples of these are an increase i n the rate of c e l l coat synthesis and c e l l coat thickness (Majlucci et a l . , 1972) and in cellular bubbling (Price, 1967). Structural changes of the surface, specific to certain types of transformed c e l l s , resulting i n the emergence of tumour-specific antigens (Klein and Cochran, 1971) and in the agglutinatability - 2 -by Concanavalin A (Inbar et a l . , 1972), have also been reported. In studying these problems, i t i s often d i f f i c u l t to correlate the behaviour of cells with the characteristics of the c e l l surface. Cells from solid tissues, or from monolayer culture, which may have structurally and functionally distinct regions on the c e l l surface, usually have to be enzymatically dissociated before the study (Moscona, 1962) and this alters their surface properties. On the other hand, cells which grow in suspension cultures are characteristically symmetrical in shape and surface properties, and here one of the main problems in trying to correlate different functions with specific regions has been the disperse nature of structural and functional heterogeneities on the c e l l surface (Weiss, 1967). Topologic differences on the c e l l surface in chemical composition, electric charges or conformations of the plasma membrane, and the related distribution of sites of adhesive, locomotor, or metabolic a c t i v i t i e s , frequently are d i f f i c u l t to identify. This is true also for a l l leukemic and leukocytic suspension cultures studied to date (de Harven, 1967; Chandra et a l . , 1968). It is the aim of the present project to analyze the relationship between the shape, surface structure and adhesive functions of leukemic cells IRC 741 (Dunning and Curtis, 1957), as these cells have been found to have unusual properties which might make i t possible to correlate such surface characteristics more easily than with other existing models (Yip, 1970). When grown in stationary suspension culture, a proportion of these cells was pear-shaped, with a cytoplasmic - 3 -protrusion by which they preferentially adhere to each other (Figure 1). Ultrastrueturally, the asymmetric c e l l s showed an accumulation of organelles near the protrusion. The c e l l surface over the protrusion o had no m i c r o v i l l i , but was covered with 200-400 A electron-dense particles embedded in a thick c e l l coat. The c e l l surface over the main c e l l body had m i c r o v i l l i but no particles and a thin c e l l coat (Figure 2). Thus, i n contrast to other c e l l s , which in suspension culture show no consistent asymmetry of c e l l shape or surface, two structurally and functionally distinct parts could be identified on the IRC cells even at the light-microscopic level, and these provide an opportunity to relate specific adhesive and locomotor characteristics of the cells to specific areas on their surface and to characterize these areas chemically and morphologically, without resorting to enzymatic digestion. Much of the quantitation in this study involved c e l l counts and determinations of c e l l shapes and c e l l v i a b i l i t y . The existing dye-exclusion test for c e l l - v i a b i l i t y (Hanks and Wallace, 1958) i s performed on l i v i n g c e l l populations which change continuously, and the test i s therefore only valid i f done immediately. To avoid the interruptions of the main experimental work, as well as the va r i a b i l i t y in timing, introduced by the counting of large numbers of samples by this technique, a new staining method was developed which permitted the dye-exclusion test as well as the determination of c e l l shapes to be performed on fixed c e l l samples subsequent to the main experimental work. Without this method, many of the observations on the properties of the leukemic c e l l s , described in Part I of this - A -Figure 1. Living IRC cells in culture, protrusions with accumulation of organelles (Pn). Anoptral phase microscopy, x 4,000. - 5 -Figure 2. Electron micrograph showing IRC c e l l clump. Adhesion by protrusions with extracellular particles (ECP), ECP separated from plasma membrane by electron-lucent area. Thick arrow, area of close adhesion; broken arrow, area of loose adhesion. T-microtubule; F-microfilaments; V-microvillus. x 13,500. - 6 -thesis, would not have been possible. The development of this new test i s presented as Part II of this thesis. - 7 -PART I THE ROLE OF CYTOPLASMIC PROTRUSIONS IN THE INTERCELLULAR ADHESION OF RAT LEUKEMIA CELLS (LINE IRC 741) - 8 -MATERIALS AND METHODS 1. General (a) The Cell Line IRC 741 is a spontaneous transplantable rat monocytic leukemia which originated in 1955 (Dunning and Curtis, 1957) and has been maintained at U.B.C. both as an ascites tumour in rats and as a tissue culture c e l l line for about seven years. The present study dealt with the cultured c e l l s . (b) Tissue Culture The leukemic cells were maintained in stationary suspension culture at 37°C in Fischer's medium with 10% horse serum, 100 units/ml of p e n i c i l l i n and 100 jug/ml of streptomycin. Cells were transplanted every three days , when the c e l l density had reached about 6xl0^/ml, which is close to the maximum density without significant c e l l degeneration. 4 New c e l l cultures were set up at a density of 2x10 /ml. The doubling time of IRC cells i s about 12 hours. 2. Electron Microscopy (a) Fixation Unless stated otherwise, cells in culture medium were i n i t i a l l y fixed by adding 0.1% glutaraldehyde in phosphate buffer (Pease, 1964) to an equal volume of c e l l suspension in medium at pH 7.3 and at room temperature. After 5 minutes, the cells were centrifuged for four minutes. The recovered cells were then covered with 2.5% glutaraldehyde in buffer for another 30 minutes, washed - 9 -twice in the same buffer, fixed with buffered 1% OsO^ and again washed twice with buffer. (b) Embedding For electron microscopy, cells fixed as described above were embedded in 5% agar (Glauert, 1965), dehydrated through 70%, 90% and 100% alcohol and embedded in epon according to the routine procedure (Appendix 1). (c) Sectioning Sections, 0.4 microns thick, were cut with a MT-2 Porter-Blum ultramicrotome, stained with 1% toluidine blue/1% borax and observed with the light microscope. For electron microscopy, silver-gold sections were mounted on carbon-coated grids , stained with uranyl acetate (Watson, 1958) and lead citrate (Venable and Coggeshall, 1965) and examined with a Hitachi HS-7S electron microscope. 3. Histochemical Methods - Light Microscopy In order to find possible differences in protein composition or acid mucosubstance distribution between the c e l l surface outlining the protrusion and that outlining the rest of the c e l l body, several methods were used. Cell smears were prepared by fixation with absolute alcohol. Epon sections (0.5-10 /J) were cut from material prepared for electron microscopy as described above. (a) Acidic and Basic Proteins For the following, both smears and epon sections were used. Toluidine blue is a basic dye which in acid solution (pH 4.2 to 5.6) stains acidic residues blue (Chayen et a l . ,1969). Solochrome - 10 -cyanine in acid solution (pH 1.4 to 2.1) stains acidic proteins blue and basic proteins red (Pearse, 1961). Fast green i s another acidic dye which stains basic proteins green (Chayen et a l . , 1969). The preparation of solutions and the staining procedures were according to those outlined by the authors except for the solochrome cyanine method, where a slight modification of the original method was required for penetration of the stain into epon sections: very thin sections (about 0.4 ji) and hot solutions had to be used and hot water was required for subsequent differentiation, (b) Acid Mucosubstances IRC c e l l s , either untreated or pretreated with 0.12% trypsin/ BSS for 30 min. at pH 7.2 and 37°C, were fixed as for electron microscopy, stained overnight with colloidal iron (Rinehart et a l . , 1951) at pH 1.9, washed with 25% acetic acid at pH2 .2 and then processed and embedded as for electron microscopy (Wetzel et a l . , 1966). Epon sections, 0.5-1.0 ju thick, were cut and treated with 10% potassium ferrocyanide in 10% HCl for 60 minutes (Tanaka and Berschauer, 1969). 4. Electron-microscopic Cytochemistry (a) Acid Mucosubstances Ultrathin sections were cut from material prepared as described in 3(b), and were examined with an electron microscope without further staining. (b) Enzymatic Digestion of Ultrathin sections Uncoated gold grids (300 mesh) bearing silver epon sections were immersed in 3 to 4 ml of 5% H ^ at 37°C for 10 to 20 minutes (Anderson and Andre, 1968). After the oxidation, the grids were - 11 -thoroughly washed in d i s t i l l e d water and then were immersed in 1 to 2 ml of one of the following solutions at 37°C to 40°C for 1 to 4 hours: 1. Protease (Bovine pancreatic, partially purified, Nutritional Biochemicals Corp.) 0.03% solution in phosphate buffer at pH 6.8 (Douglas et a l . , 1970). Control : in same buffer at pH 6.8. 2. Pepsin (3X crystalline, N.B.C.), 0.2 to 0.5% i n 0.1 N HC1. Control : in 0.1 N HC1 (Anderson and Andre, 1968). 5. Cell Electrophoresis Since the electron cytochemical study showed that the surface of the cellular protrusion had a heavier layer of acid mucosubstances than the rest of the c e l l surface, experiments were carried out to establish whether these two parts of the cells also differ in their electrophoretic mobility. IRC ce l l s , washed with and suspended in medium without serum, were transferred to a cylindrical chamber equipped with Ag/AgCl electrodes (Vassar et a l . , 1969) at 25°C. The movements and the orientation of the protrusion and the c e l l body in an electric f i e l d were observed with the b u i l t - i n microscope. 6. Time-lapse Cinematography IRC cells were transferred to Sykes-Moore chambers (Sykes and Moore, 1959) which were placed on the stage of an inverted Wild microscope built inside a Prior incubator at 37°C. Pictures were taken with Kodak double-X negative black and white 16 mm film in - 12 -a Bolex movie camera at speeds varying from 1 to 10 frames per minute. 7. The Induction and Maintenance,of Protrusions In a stationary culture the proportion of pear-shaped cells 4 increases steadily until the density reaches about 20x10 /ml, and then drops as the culture ages (Yip, 1970 , Figure 3). Since this increase in c e l l density is associated with an increase in the degree of c e l l contact, the exhaustion of nutrients supply, the accumulation of waste products and changes of pH, these factors were investigated separately to determine their roles in the i n i t i a t i o n or maintenance of protrusions of IRC c e l l s . (a) Effect of Renewal of the Growth Medium Cells from a single population , diluted to lxlO^/ml, were divided among two groups of tubes. Both groups were placed on a rol l e r drum (4 rpm) and incubated at 37°C. Every 24 hours, four tubes from each group were fixed with glutaraldehyde while the remaining tubes were centrifuged at low speed. The medium in one group of tubes was renewed "daily for 4 days while the cells i n the control group were resuspended in their original medium. In this series of experiments, as well as in a l l subsequent experiments , the proportion of viable cells with protrusions and of non-viable cells was determined from the fixed samples by the alcian blue method described in Part II of this thesis. About 1,000 cells were counted for each sample. (b) Effect of Cell Contact To study the relationship of the degree of c e l l contact to the formation of protrusions, cells were grown, in addition to the - 13 -Figure 3. The change of the proportion of pear-shaped cells with c e l l density and culturing time. Proportions were estimated by drawing samples daily from five independent 4 cultures, a l l started at a density of 1x10 /ml , medium unchanged. 5 1 1 1 1 1 1 " T 2 0 40 6 0 CELL DENSITY! X 104/ ml) 2 4 4 8 72 96 CULTURING TIME ( HOURS ) 3 - 14 -standard stationary cultures , in spinner cultures and in gyratory shaker cultures. At moderate speeds (70-80 rpm) in the gyratory shaker cultures, the c e l l contact was increased over that in stationary cultures, while i n spinner cultures i t was reduced (Moscona , 1961; Henkart and Humphreys, 1970). Samples were drawn daily, fixed with glutaraldehyde, and the proportion of pear_shaped and dead cells was determined. The cells cultured in the gyratory shaker showed a consistent increase i n the proportion of pear-shaped cells (maximum 50-60% compared with 30-50% in the stationary suspension culture) and therefore the shaker system was employed as the culturing method for the subsequent experiments described below. Cells , grown in standard culture, were 4 transferred at a density of 1x10 cells/ml , at 20 ml of c e l l suspension per 50-ml Erlenmeyer flask, incubated on a gyratory shaker (70-80 rpm) 4 and were used after 48 hours (approximately 12x10 cells/ml) when the proportion of pear-shaped cells reached i t s maximum (Figure 12). (c) Effect of Temperature Cells grown in gyratory shaker cultures were mixed thoroughly in a 37°C water bath and then were transferred to spinner culture flasks (40 mis of cells per flask) on magnetic stirrers at either 4°C, 25°C (room temperature) or 37°C. At various time intervals, samples were drawn and fixed immediately with 2.5% glutaraldehyde for the determination of the proportions of viable cells with protrusions and of non-viable c e l l s . (d) Effect of pH and of Divalent Ions Cells from gyratory shaker cultures were mixed thoroughly and transferred, in 8-ml aliquots, into small test tubes i n a 37°C water - 15 -bath. After centrifugation in an incubator at 37°C, the supernatant was removed and the cells resuspended in the following solutions for various periods of time and pH's (measured with a Beckman pH-meter). 1. Hanks' balanced salt solution, 2. Calcium and magnesium free Hanks' solution (Moscona's BSS), 3. Moscona's BSS containing Versene (etylene-diamine-tetraacetic tetrasodium dihydrate, (Nutritional Biochemicals Corp.) at various concentrations and pH's. - 16 -RESULTS L. Histochemistry No difference in staining for acidic and basic proteins was found between the surface outlining the protrusion and that outlining the rest of the c e l l s . Sections containing colloidal-iron stained c e l l s treated with ferrocyanide showed the presence of mucosubstances over the whole c e l l surface, but stained particularly heavily at the area surrounding the protrusions. Control cells unstained with iron showed no reaction with ferrocyanide (Figure 4). 2. Electrojn Microscopic: Cytochemistry In response to colloidal iron staining for acid mucosubstances a rather sparse layer of iron particles was observed in the c e l l coat overlaying the main c e l l body (Figure 5). It was quite similar in appearance and distribution to the iron staining of the c e l l coat of the normal macrophage (Carr et a l . , 1970). Within the c e l l coat overlaying the protrusion, however, there was a heavier deposition of iron particles in the matrix between the extracellular particles , but the particles themselves did not stain. On the surface of m i c r o v i l l i and the particles, there were filamentous structures which were not visible after staining with lead citrate and uranyl acetate alone. Treatment of l i v e cells with 0.12% crystalline trypsin, in balanced salt solution at pH 7.2 and 37°C for 30 minutes prior to the above treatment, removed the extracellular particles and matrix from the surface of the protrusion. However , the c e l l coat over - 17 -Figure 4. Reaction of 10% KCN/10% HCl w i t h epon s e c t i o n s of IRC c e l l s f i x e d w i t h glutaraldehyde, w i t h (A) and without (B) c o l l o i d a l i r o n s t a i n i n g p r i o r to embedding. In A, the areas surrounding p r o t r u s i o n s were s t a i n e d more h e a v i l y , x 1,200. - 18 -Figure 5. Electron micrographs of IRC cells stained with colloidal iron. A. The protrusion of a pear-shaped c e l l i s covered o with a c e l l coat, 3,000-4,000 A thick (J)* Unstained extracellular particles (P) are embedded in an iron-positive matrix. The surface of the remaining part of the c e l l is covered with a thin layer of iron particles (arrow), x 36, 000. B. Filamentous structures (arrows) are present on the surface of mi c r o v i l l i and the ECP. x 46,000. - 19 -the remaining c e l l surface was s t i l l stained with iron (Figure 6). 3. Enzymatic Digestion of Ultrathin Sections Ultrathin sections mounted on copper grids , after enzymatic treatments, were often covered with a coat of particles, possibly due to a reaction of the copper with the chemicals, because i t did not occur on sections mounted on gold grids. The following results were obtained with gold grids. Treatment with protease/phosphate buffer at pH 6.8 (Figure 7B), 0.1 N HC1 (Figure 8A) and pepsin/0.1 N HC1 at pH 1.2-1.5 (Figure 8B,C) at 37°C for one hour a l l removed the amorphous matrix from among the extracellular particles. This change was not observed after treatment with the buffer alone. The particles themselves became more irregularly shaped with definite electron-lucent round areas in the centre. After four hours, the mitochondrial matrix and some of the non-particulate cytoplasm and nucleoplasm were removed. However, the morphology of the extracellular particles remained unchanged, and the ribosomes , endoplasmic-reticulum, microtubules and the various membranes also remained intact. No differences between the results of the 3 treatments were observed. 4. Cell Electrophoresis The electrophoretic movement of the cells is illustrated in Figure 9. When there was no electric potential applied through the medium, the asymmetric cells settled down slowly in response to gravity with the protrusion on the top (Figure 9A). When a voltage gradient of about 50 to 60 V was applied, the cells were attracted to - 20 -Figure 6. T r y p s i n i z e d IRC c e l l s s t a i n e d w i t h c o l l o i d a l i r o n . Most c e l l s were round but c e l l s w i t h p r o t r u s i o n s were a l s o present. The amount of e x t r a c e l l u l a r p a r t i c l e s (ECP) l e f t on the p r o t r u s i o n was v a r i a b l e . A and B are two micrographs of two s e c t i o n s of the same p r o t r u s i o n . A small d e p o s i t i o n of ECP i s present i n the area denoted by an arrow i n A but not i n the s i m i l a r area i n B. A l a r g e r amount of ECP was s p l i t o f f the p r o t r u s i o n . A t h i n l a y e r of c o l l o i d a l i r o n i s evident on the surface of the p r o t r u s i o n , the c e l l body and the e x t r a c e l l u l a r p a r t i c l e s ( P ) . A, x 15,600. B, x 18,000. - 21 -Figure 7. Electron micrographs of epon sections immersed for 1 hour in phosphate buffer at pH 6.8 (A) and in 0.03% protease/phosphate buffer (B). Poststained with lead citrate and uranyl acetate, x 32,000. The amorphous matrix among the extracellular particles was removed in B. The electron-lucent round areas in the centre of the particles became more apparent after digestion (B) . x 32 ,000. - 22 -F i g u r e 8. E l e c t r o n m i c r o g r a p h s of epon s e c t i o n s immersed f o r 1 hour i n 0.1 N HCl (A) and i n pepsin/0.1 N HCl (B,C). R e s u l t s were s i m i l a r to those d e s c r i b e d i n F i g u r e 7(B). A, x 4,600. B, x 1 ,600. C, x 50 p00. - 23 -Figure 9. The change in orientation of the protrusion of IRC cells with the change of the electrical polarity in c e l l electrophoresis. A, no electrical current ; B,C and D ill u s t r a t e changes of polarity. Large broken arrow, gravitational force; large solid arrow, electrical attractive force; small arrow, resultant direction of c e l l movement. 9 - 24 -the anode in a direction which was the resultant of the gravitational and the electrical attractive forces. This i s the usual picture in c e l l electrophoresis (Weiss , 1967). However, in the present case, the interesting observation was that the protrusion of the IRC c e l l s , as the whole c e l l was moving, gradually turned further towards the anode than the c e l l body. Thus the protrusion would turn l e f t or right in response to changes in the polarity of the current (Figure 9B, C and D). This peculiar behaviour of the protrusion was recognized in over 90% (34/37) of the observed pear_shaped cells on two separate occasions. The degreee of turning, i.e., the angle of deflection from the v e r t i c a l , seemed to vary with the size and shape of the protrusion ; and the time for assuming the maximum angle, although also variable, was around one minute. 5. Cinemato graphy (a) Cell Movement Gliding movement (Bhisey and Freed, 1971) was the characteristic movement in IRC c e l l locomotion. This particular movement was associated with the formation of a ruffled membrane at the leading edge of the cytoplasmic mass while the remainder of the c e l l mass moved in a rather rigid fashion. The microspikes appeared and disappeared rapidly on every part of the leading side, as observed with the 40X phase objective. In contrast, the protrusion, which was usually at the back, remained stable at the same site although i t s s i - z e v a r i e d a l l the time. There were no microspikes on i t s surface. Occasionally , a single pear-shaped c e l l might round up and then reform i t s protrusion. The pear-shaped - 25 -c e l l s were al s o able to move sideways or w i t h the p r o t r u s i o n at the le a d i n g s i d e , e s p e c i a l l y when c e l l s were coming together. O c c a s i o n a l l y , ameboid movement, which in v o l v e d s u b s t a n t i a l cytoplasmic streaming i n t o pseudopods, r e s u l t i n g i n elongating and shortening of the c e l l body, was observed i n some IRC c e l l s . The c e l l s u s u a l l y moved very s l o w l y , at about 0.1 p/sec. However, i n c e r t a i n cases, such as when two daughter c e l l s separated, when c e l l s were going to j o i n together by t h e i r p r o t r u s i o n s o r separate from adhesion at t h e i r p r o t r u s i o n s , the c e l l s might suddenly increase t h e i r speeds to 1.7 p/sec, which could be r e a l i z e d without d i f f i c u l t y by d i r e c t microscopic o b s e r v a t i o n (Figure 1 0 ) . (b) C e l l Adhesion I n t e r c e l l u l a r c o n t a c t , r e s u l t i n g from c o l l i s i o n s during random c e l l movement, almost i n v a r i a b l y r e s u l t e d i n f a i r l y s t a b l e i n t e r c e l l u l a r a s s o c i a t i o n s . When pear-shaped c e l l s came together, they tended to a t t a c h to one another d i r e c t l y by t h e i r p r o t r u s i o n s i f t h e i r p r o t r u s i o n s were f a c i n g one aiother at the time of i n i t i a l c o n t a c t . I f the p r o t r u s i o n of one c e l l met the c e l l body of another pear-shaped c e l l , they might f i r s t adhere together by the p r o t r u s i o n -c e l l body c o n t a c t , then the c e l l w i t h the p r o t r u s i o n might search around the other c e l l u n t i l f i n a l l y both p r o t r u s i o n s j o i n e d together. Adhesion by the pr o t r u s i o n s was not always immediate. A c e l l j o i n i n g a c e l l clump might undergo forward and backward movements s i m i l a r to a parking c a r , before f i n a l l y a r e l a t i v e l y s t a b l e p r o t r u s i o n - p r o t r u s i o n adhesion was formed (Figure 1 0 ) . This type of adhesion was s t a b l e - 26 -Figure 10. The interaction of a single pear-shaped c e l l with an aggregate of three cells i s illustrated by these composite diagrams made by tracing the images on the screen of a film editor. The numbers indicate the positions of the approaching c e l l in different frames. The time interval between consecutive numbers is 6 seconds. In A, a single c e l l advances with i t s protrusion in front at various speeds (1 to 3, 0.5 ju/sec; 3 to 4 , 1.2u/sec ; 4 to 5 , 1.7 ^i/sec). Then the protrusion turns towards the adhering protrusions until i t joins them (B). But the adhesion i s not yet stable. The newcomer separates and rejoins the group several times (C to E) until f i n a l l y a stable association is accomplished which lasts about one minute (not shown). F depicts the same c e l l moving away from the group. The speed was approximately 0.1 ju/sec. These diagrams illustrate that a pear-shaped c e l l can move in a l l directions at speeds varying from 0.1 p/sec to 1.7 yu/sec. They also suggest that the IRC cells "prefer" to adhere by their protrusions. - 2 7 -in spite of the vigorous motion of the c e l l s . However, occasionally, one of the ce l l s in an aggregate might separate from the rest and move away. Cells in aggregates also could divide. The protrusion seemed to disappear and the whole c e l l became larger and round. After division, both daughter cells stayed together and one of them s t i l l attached to other c e l l s at the same site where the protrusion had been observed before i f disappeared during prophase. Daughter oells immediately after division did not have protrusions. (c) Cell Aggregation Single c e l l s moved around and aggregates of two or three c e l l s were formed as the ce l l s collided. These small aggregates were also able to move as a whole while adhering by the protrusions, and the ruffled membranes were conspicous on the leading side of the leading ce l l s of the group. Small aggregates in turn formed larger c e l l clumps. Single c e l l s joining and , occasionaly, leaving the c e l l colonies were also observed. 6. The Induction and Maintenance of the Protrusion (a) Effect of Renewal of the Growth Medium There seemed to be l i t t l e difference in the proportion of pear-shaped c e l l s grown with or without daily renewal of culture medium 4 unti l after 48 hours of culturing or at a c e l l density of about 5x10 /ml (Figure 11). The proportion of pear-shaped ce l l s in the cultures with 4 unchanged medium reached about 35% at 25x10 /ml (48-72 hours) cells and then dropped with the culturing time u n t i l , at 96 hours, most cells Figure 11: The change of proportion of viable pear-shaped cells with c e l l density (live and dead) and culturing time in groups of cultures with (A) and without (•) daily renewal of medium. Each symbol represents a separate culture. |,proportions of pear-shaped cells in the group without daily renewal of medium co after 96 hours of culturing. PROPORTION OF PEAR-SHAPED CELLS ( X 0.1) — K ) U> *»* L/l • • - 29 -were dead and only few showed protrusions. In contrast, the proportion of pear-shaped ce l l s in the group grown with daily renewal of medium 4 increased steadily to about 50% when the c e l l density was about 60x10 /ml at 72 hours, before i t dropped slowly as the culture continued, suggesting that beyond a certain c e l l density, the formation or maintenance of protrusions might be limited by nutritional factors. (b) Effect of Intercellular Contact The growth patterns of ce l l s maintained in gyratory, stationary and spinner cultures, illustrated in Figure 12, indicate that the formation of protrusions was proportional to intercellular contact. At low c e l l densities, the maximum proportion of pear-shaped c e l l s was consistently highest in gyratory cultures and lowest i n spinner cultures. The proportion dropped most slowly in the stationary cultures as the cultures aged. (c) Effect of Temperature In response to the change of environmental temperature from 37°C to either 25°C (Figure 13) or 4°C (Figure 14), the proportion of pear-shaped ce l l s dropped over one hour, when i t levelled off and was maintained at a rather steady level as long as 4 hours. If at this point the bottles were placed back into a 37°C environment, the proportion of pear-shaped c e l l s slowly went up, sometimes approaching the original level within 1 hour. When samples from a single c e l l population were placed in either 4°C or 25°C, the reduction in the proportion of pear-shaped ce l l s was similar in both temperatures (Figure 15). Furthermore, the recovery of the original proportion of pear-shaped cells was s t i l l observed after 5 hours of temperature depression. - 30 -Figure 12: Change in the proportion of pear-shaped cells with changes in the degree of c e l l contact. - control stationary culture; - gyratory culture; - spinner culture. o o m r — r — m c/> —I X o o & o PROPORTION OF PEAR-SHAPED CELLS ( X 0.1) I C O C n • Figure 13. Change in the proportion of pear-shaped cells with changes in environmental temperature. A, control at 37°C; at 256C, after 4 hours, returned to 37°C. Each curve represents a spinner culture. Figure 14. Change in the proportion of pear-shaped cells with changes in environmental temperature. • , control at 37°C; 9 , at 4°C, after 4 hours, returned to 37°C. Each curve represents a spinner culture. Figure 15. Change in the proportion of pear-shaped ce l l s with changes in environmental temperature. 4 cultures from a single c e l l population, kept at 37°C, were placed separately in either 25°C or 4°C. After 5 hours, a l l were returned to the 37°C environment. Each curve represents a spinner culture. Thick arrow indicates the i n i t i a l proportion of pear-shaped c e l l s . PROPORTION OF PEAR-SHAPED CELLS ( X 0.1) - 33 -(d) Effect of pH and EDTA The effects of Hanks' BSS, Moscona's BSS and EDTA solution at various pH's are represented in Figure 16. The groups not significantly different from one another are enclosed by IECtangles in Figure 17. In a l l solutions except the 2% EDTA there was a significant drop i n the proportion of pear-shaped ce l l s at pH 8 from that at pH 7.4. There was also a slight increase in the proportion of pear-shaped c e l l s at the acid pH. With respect to the effect of the concentration of EDTA, the lowering of the proportion by 2 % EDTA was remarkable and nicely separated from the others. In contrast, the difference in the effects of other solutions at pH 5.6 and 7.4 was not so clear-cut. Although st a t i s t i c s separates them into groups (Figure 17), the results were only based on 4 samples per group which seems inadequate for results that are close. Moreover, in the acid pH, many cells of various Irregular shapes were observed, which might have diminished the accuracy of the counts of pear-shaped c e l l s . However, the important observation in this series of experiments was that the pH by i t s e l f has an effect upon the protrusion. The rate of change in the proportion of pear-shaped ce l l s was proportional to the concentration of BDTA as demonstrated in Figure 18. In 2% EDTA, the proportion dropped to below 10 after 30 minutes. After 40 to 45 minutes, the difference between the effects of 0.2 and 0.02% EDTA became clearly discernible. After 3 hours, the effects of Hanks' BSS, Moscona's BSS, 0.02% and 0.2% EDTA were very variable, probably due to nutritional factors. - 34 -Figure 16. Proportion of pear-shaped cells in c e l l samples suspended in Hanks' BBS, Moscona's BBS and various concentrations of EDTA at pH 6.5, 7.4 and 8. Arrow and asterick indicate the i n i t i a l proportion at pH 7.4. Time of treatment, 15 minutes. Each dot represents the mean of four samples. 1 6 Figure 17. Same as Figure 16. The means which are not significantly different from one another are enclosed by rectangles. Analysis of Variance (Sokal and Rohlf, 1969) and Duncan's New Multiple Range Test (Steel and Torrie, 1960). 17 Figure 18. Effect of cation-depletion on the proportion of pear-shaped cells over 3 hours. The points in each group of four represent samples treated separately. TIME (HOURS) 1 8 DISCUSSION Cytoplasmic protrusions are common structures in animal c e l l s . They may be transient, as, for example, in cells undergoing ameboid movement where the pseudopods are formed by multidirectional streaming of the cytoplasm. Cellular bubbling, or blebbing, i.e. the rapid projection and retraction of the cytoplasm, represents another type of transient protrusion which.is not related to locomotion (Price, 1967) However, the cytoplasmic protrusion found in IRC c e l l s appears to differ in many aspects from those found in other c e l l s . Although i t does not seem to be a permanently differentiated structure, i t seems more stable than either the pseudopods observed in ameboid movement or the cellular blebs. Moreover, i t is not a locomotor organelle nor a sign of degeneration, but rather seems primarily a structure used for intercellular adhesion. In addition, this structure i s associated with a localized, unusual and complex type of c e l l coat. Extracellular materials (ECM) have been of major interest in studies on growth, developmental interactions and differentiation in multicellular systems (Moscona, 1962). Grobstein (1961) demonstrated that intercellular factors were responsible for the transmission of morphogenetic information during induction. On the other hand, Moscona was interested in demonstrating whether or not extracellular materials are responsible for c e l l aggregation. The results seemed to be positive He (Mosconaj 1962a) found that enzymatically dissociated c e l l s reaggregated rapidly at 38°C, but failed to do so at 25°C. If rotated rapidly, c e l l s at 38°C liberated to the medium a material containing - 38 -mucoprotein which was isolated and shown to be able to promote c e l l aggregation at 25°C. This cell-binding material was further demonstrated to show a certain amount of tissue sp e c i f i c i t y (Moscona, 1962a) and to be dependent on RNA and protein synthesis (Moscona and Moscona, 1963). Experiments with malignant ce l l s also gave similar results. Mouse malignant c e l l s formed aggregates in gyratory cultures at 6°C after mechanical dissociation but not after trypsinization (Pessac and A l l i o t , 1970). A l l these observations suggest the importance of ECM i n c e l l adhesion. An adhesive organelle, associated with a modified c e l l coat, like the one observed in the IRG c e l l s , has not been reported in other normal and malignant c e l l s . In the following, different aspects of the surface properties and shape of IRC c e l l s are discussed under appropriate sub-headings. Cytochemistry and Electron-cytochemistry The collodial iron method Indicates the presence of acid mucosubstances over the whole surface of the IRC cells as well as i n the matrix embedding the extracellular particles. In comparison with related normal c e l l s , i.e. macrophages (Carr e_t a l . , 1970), no increase in cell-surface mucosubstances could be demonstrated in these leukemic cel l s by this method, except for the localized region over the protrusion. The c e l l coat over the main c e l l body was v i s i b l e only after iron staining, while on the other hand, the matrix was discernible after uranyl acetate and lead citrate staining alone. This might be due to either quantitative or qualitative differences in components of - 39 -the coat over the two parts of the c e l l surface. The filamentous structures revealed by colloidal iron on m i c r o v i l l i and the c e l l coat over the protrusion resemble those demonstrated in carcinoma c e l l s (Auersperg, 1969), and in human trophoblasts (Bradbury et a l . , 1970), but have apparently not been reported i n other cells of hemopoietic origin. No differences in the staining for either acidic or basic proteins were demonstrated between the surface of the protrusion and that of the c e l l body. However, i t is possible that existing differences were too small, either quantitatively or qualitatively, to be detected by the methods employed in the present study. The removal, by trypsin treatment, of the thick coat covering the protrusion of l i v e c e l l s and the digestion of the matrix from epon sections with protease indicate the presence of a proteinaceous moiety, in addition to acid residues, in the matrix embedding the electron-dense particles. However, as the cells after trypsin digestion were stained by the colloidal iron, there was s t i l l a layer of mucoproteins l e f t behind after the trypsin treatment. This finding i s consistent with those obtained by Gasic and Loebel (1966) and Carr eit a l . , (1970). The former authors observed that the staining for s i a l i c acids in sections of fixed mouse ascites tumour cells was actually enhanced after trypsin digestion. In contrast to the matrix, the electron-dense particles seemed unstained by the colloidal iron, and protease-, HC1- and pepsin-resistant, and the ring-shaped structures of the particles after enzymatic digestion suggest a protease- and pepsin-resistant framework. - 40 -Since the matrix i s susceptible to both trypsin and the bovine pancreatic protease, which contains 140 units/mg of trypsin, i t could be suggested that the matrix contains a proteinaceous moiety rich in arginine and lysine. However, since both 0.1 N HCl and pepsin/0.1 N HCl removed the matrix, i t i s d i f f i c u l t to evaluate the action of pepsin upon the matrix. The removal of matrix by HCl i s of interest because i t indicates that the proteins or other structures -in the matrix are susceptible to weak acid treatment, although a stronger acid solution at higher temperatures (e.g. excess 6N HCl at 100°C to 120°C for 10 to 24 hours) i s usually heeded to achieve the complete hydrolysis of proteins (Lehninger, 1970). Although the results show that electron-dense particles are protease-, HCl- and/or pepsin-resistant, the results do not prove that the particles are not proteinaceous in nature. It i s well known that different kinds of proteinaceous structures are susceptible to different types of proteolytic enzymes (Anderson and Andre, 1968; Douglas et a l . , 1970); and in the present study, only protease and pepsin were used. The finding that the mitochondrial matrix was removed after 4 hours treatment of pepsin while the the cytoplasmic microtubules and some other protein-containing structures remained intact is similar to that obtained by Anderson and Andre (1968). Of course, the framework of the electron-dense particles could also consist of nucleic acids or l i p i d s , for example. Cell Electrophoresis The observation that the protrusion turned further towards the - 41 -anode than did the c e l l body indicates that i t has a higher electrophoretic mobility. This may indicate that the average surface-charge density i s higher in this particular area (Weiss, 1967). However, the electrophoretic mobility of a particle i s also influenced by i t s shape and size. To rule out the possibility that the protrusion was more strongly attracted because of Its smaller size, some theoretical considerations are necessary. For an e l e c t r i c a l l y charged sphere of radius a, the density of charge can be related to ^ , the viscosity of the suspending medium; u, the electrophoretic mobility in u/sec/volt/cm; and k, where 1/k is the Debye-Huckel parameter which indicates the "effective" thickness of the diffuse electrical double layer surrounding the c e l l surface, by the following equation (Tenforde, 1970): • 3 988 860 180 844 368 125 50 •rl 4 . 976 920 160 905 357 130 66 mean 983.0 910.5 163.3 906.5 361.5 136.8 57.8 a - No significant difference at the p< 0.05 level among the seven non-fixed groups and including the fixed group stained with alcian blue ( s t a t i s t i c a l analysis by analysis of variance (Sokal and Rohlf, 1969) plus Duncan's new multiple range test (Steel and Torrie, 1960). - 64 -TABLE III EFFECT OF STORAGE FOLLOWING FIXATION ON THE SUBSEQUENT STAINING OF CELLS WITH ALCIAN BLUE 3 Storage after fixation Numb er of stained cells (per thousand) Time Solution " ' • .. ^ 1 hour 24 hours 1 week 1 month Phosphate buffer - 64.3 - 3.21 80.8 - 10.00 149.3 - 24.60 c Glutaraldehyde fixative 45.8 - 3.95 49.09^ 5.47d 49.5 - 3.69d 80.6 - 8.20 a - Means - S.D., quadruplicate samples, overgrown IRC c e l l s , b - Millonig's buffer (Pease, 1964). c - Equal volumes of 2.5% glutaraldehyde/phosphate buffer and of culture medium, d - Not significantly different from 1 hour specimen. - 65 -buffer immediately before staining gave sim i l a r counts for one week, although there was an increase i n the proportion of stained c e l l s after one month. The rate of penetration by various dyes into glutaraldehyde-fixed c e l l s was examined to determine, i n p a r t i c u l a r , how stable the proportion of stained c e l l s was following the addition of dyes. Figure 20 i l l u s t r a t e s the results obtained with a c e l l suspension where pr i o r to f i x a t i o n , only a small proportion (.7%) of c e l l s was. nonviable by dye exclusion. Following f i x a t i o n , safranin, with a molecular weight of 351, stained almost a l l c e l l s within 5 minutes. In contrast, the number of c e l l s stained with a l c i a n blue (M.W. 1341) a f t e r 5 minutes was not s i g n i f i c a n t l y d i f f e r e n t from that of the unfixed controls. The other dyes diffused into the c e l l s at intermediate rates. A more detailed study of a l c i a n blue staining showed that the proportion of c e l l s stained at 5 minutes remained constant for another 15 minutes (Figure '21). Then there was a small increase i n the number of stained c e l l s and a f t e r a few hours, a steady l e v e l was reached which did not change substantially for 2 days (Figure 22), and, as shown by other s i m i l a r experiments, not even after 4 days. Renewal of the dye after 2 days did not change th i s pattern. On the basis of these re s u l t s , the following routine procedure was adopted: 1. A 2.0-5.0 ml sample of a c e l l suspension i s transferred to a centrifuge tube. 2. An equal amount of cold 2.5% glutaraldehyde-phosphate buffer (pH 7.4) i s added and f i x a t i o n continued for one hour i n an i c e bath. Subsequently the mixture can be stored at 4°C for up to one week. - 66 -Figure 20. Rates of d i f f u s i o n i n t o f i x e d c e l l s of seven dyes, over one -4 hour. Overgrown, g l u t a r a l d e h y d e - f i x e d IRC c e l l s i n 2.26 x 10 m of dye/phosphate b u f f e r . Arrow i n d i c a t e s number of trypan-blue s t a i n e d c e l l s / t h o u s a n d i n the u n f i x e d c o n t r o l sample. 2 0 4 0 6 0 STAINING TIME (MINUTES) 2 0 - 67 -Figure 21. I n i t i a l s t a b i l i t y of the proportion of alcian- b l u e stained c e l l s with continued exposure to the dye. 4 - N i t r o q u i n o l i n e - l -oxide-treated, glutaraldehyde-fixed IRC c e l l s i n 0.03% (2.26 x -4 10 m) a l c i a n blue/phosphate b u f f e r . Each point represents the mean of seven samples. Arrow in d i c a t e s number of trypan-blue stained cells/thousand i n the unfixed c o n t r o l sample. Figure 22. Changes i n the proportion of al c i a n - b l u e stained c e l l s over 48 hours of exposure to the dye. Overgrown, glutaraldehyde-fixed IRC c e l l s i n 0.03% a l c i a n blue/phosphate b u f f e r . Each point represents the mean of four samples. Arrow i n d i c a t e s number of trypan-blue stained cells/thousand i n the unfixed control sample. 2 5 0 - ,