effect of temperature on the mutual adhesion of ... - Semantic Scholar

J. Cell Sci. 8, 751-765 (1970

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Printed in Great Britain

EFFECT OF TEMPERATURE ON THE MUTUAL ADHESION OF PREAGGREGATION CELLS OF THE SLIME MOULD, DICTYOSTELIUM DISCOIDEUM D. R. GARROD AND G. V. R. BORN Department of Biology as Applied to Medicine, The Middlesex Hospital Medical School, London, W. 1, England, and Department of Pharmacology, Royal College of Surgeons of England, London, W.C.2, England

SUMMARY The mutual adhesion of slime-mould cells was investigated at 24 and 1 °C in stirred and unstirred medium. Adhesion was induced by adding sodium chloride and followed by recording the diminution in optical density of the cell suspension associated with the formation of cell aggregates. It was found that: (1) when cells, previously stored at 1 °C, were stirred in distilled water at 24 CC the optical density of the suspension increased; (2) when sodium chloride was added after this increase, the optical density fell as cells adhered to each other; (3) both the increase in optical density and subsequent adhesion were reversibly inhibited at 1 °C; and (4) the rise in optical density was a prerequisite for adhesion. Comparison of cell shape and volume at the 2 temperatures suggested that the rise in optical density was due to (a) extension of pseudopodia and a consequent change from a spherical to an irregular shape, and (A) an increase in mean cell volume of about 17 %. Observations on the formation of adhesions in still medium at 24 °C showed that initial contacts were formed either by microspikes or by rounded parts of the cell surface. The cell surfaces then flattened against each other, thereby increasing the area of contact. No flattening occurred at 1 °C. Microspikes were present on the surfaces of both cold and warm cells and contained longitudinal fibrils. It is suggested that adhesion of these cells is inhibited in the cold in stirred medium because the cells are unable to expand areas of mutual contact. Cold adhesions are, therefore, weak and easily disrupted by shearing forces. The inability to expand adhesive contacts may be due to the inhibition of cell motility at low temperature. INTRODUCTION

Previous investigations on the effect of temperature on the formation of cell adhesions have led to confusion. Whether certain types of cells form mutual adhesions at low temperatures clearly depends on the experimental conditions. Several different mechanisms have been suggested to explain the failure of cells to adhere at low temperatures where this has been observed. It is generally agreed that dissociated chick embryo cells do not reaggregate at low temperature in shaker culture in the presence of serum (Moscona, 1961 a, b; Steinberg, 1962; Curtis & Greaves, 1965). The following reasons have been suggested for this, (i) At low temperatures the cells are unable to synthesize an intercellular binding 48-2

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D. R. Garrod and G. V. R. Born

material required for adhesion (Moscona, 1961a, b). (ii) Serum contains an adhesioninhibiting protein which the cells cannot break down at low temperature; this suggestion was based on the observation that cells did reaggregate at low temperature in serum-free medium (Curtis & Greaves, 1965). (iii) Cell-surface motile activity may be inhibited at low temperature and this may hinder the initiation of mutual adhesions (Steinberg, 1962). Sponge cells, dissociated in the absence of calcium and magnesium, failed to adhere at low temperature in shaken suspension (Humphreys, 1963; Moscona, 1963, 1968). In still medium, on the other hand, the cells did reaggregate at low temperature (Curtis, 1962a). These results have also been interpreted differently. Clearly, more work is required to establish how temperature affects cell adhesion. In this paper, we report experiments on the effects of temperature on the mutual adhesion (i.e. the formation of multicellular aggregates) of cells of the slime mould Dictyostelium discoideum in the pre-aggregation stage of their life-cycle (see Bonner, 1967). The use of these cells is advantageous because suspensions of single cells can be obtained without damaging chemical treatment, e.g. with trypsin or EDTA, and because adhesion experiments can be performed in simple saline media. Previously we have shown that slime-mould cells, suspended in distilled water, can be caused to adhere to each other by adding simple salts (Born & Garrod, 1968). As the cells approach the chemotactic aggregation stage of their life-cycle (Bonner, 1967), the concentration of salt required to cause adhesion diminishes; this is associated with a decrease in their surface charge density (Garrod & Gingell, 1970). Further, we have presented evidence which suggests that the colloid theory of cell adhesion (Curtis, 19626) cannot account for the adhesion of slime-mould cells (Gingell & Garrod, 1969; Gingell, Garrod & Palmer, 1969; Garrod, 1969). Here we show that these cells form adhesions less readily in the cold (0-2 °C) than at room temperature (22-24 °C) and provide evidence that this difference is due, at least in part, to the inhibition of cell motility at the lower temperature. MATERIALS AND METHODS Preparation of cells The slime mould Dictyostelium discoideum was grown at 22 °C on S.M. agar (Sussman, 1966) in association with Escherichia coli B/r. The cells were harvested in cold distilled water just before the spontaneous aggregation stage and washed free of bacteria by centrifugation at about i5Og. Resuspended in distilled water, the cells were pipetted on to the surface of Whatman No. 50 filter papers resting on Millipore support pads which were saturated with distilled water; in this way they were stored until required for use. For the experiments the cells were washed off the filter paper and suspended in distilled water at about 1 °C. Cell adhesion experiments The tendency of the cells to adhere to each other was followed by an optical-density method (Born & Garrod, 1968) developed for investigating platelet aggregation (Born, 1962a, b), as follows. One-millilitre samples of cell suspension were pipetted into a small glass test-tube in the ' aggregometer' apparatus. Water circulating through a metal jacket surrounded the tube to control the temperature of the cell suspension. The suspension was stirred by a small magnetic stirrer rapidly enough (1000 rev/min) to ensure that the collision rate between the cells was

Effect of temperature on cell adhesion

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non-limiting (Born & Cross, 1963). A beam of light was passed through the tube and the light transmission recorded continuously on a pen-recorder. When the cells adhered to each other they formed aggregates; as a result, the light transmission increased. With platelets, the optical density of their suspension depends on the number, size and packing density of the aggregates (Born & Hume, 1967). In the present experiments, the dependence of the decrease in optical density on the formation of cell clumps was verified microscopically. In still medium, adhesion tests were done as follows. One millilitre of cell suspension, previously stored in iced water, was pipetted into a test-tube which was maintained at either 1 °C or about 24 °C, i.e. room temperature. To allow the cells to warm up to the higher temperature they stood at that temperature for 5 min before the experiment. One millilitre of sodium chloride solution was added at the required temperature and concentration. After 5 min the tube was gently shaken to resuspend cells and clumps and a sample was taken with a Pasteur pipette. The number of single cells remaining in the suspension was counted and expressed as a percentage of the original single cell concentration. Strictly speaking, the medium was not completely still but it was much less agitated than in the aggregometer. Cell volume determinations These were done as described for platelets (Born, 1970). The extracellular volume in sediments of packed cells was measured with radioactive substances which did not penetrate the cells and were not adsorbed by them (see Table 1, p. 756). Electron microscopy After harvesting, the cells were suspended in a solution approximating in ionic concentration to half-strength Bonner's solution (Bonner, 1947) at about 1 °C or at 24 °C. The cells were fixed by adding an equal volume of 2 % glutaraldehyde solution in 002 M cacodylate buffer at pH 7-05 so that thefinalconcentration of glutaraldehyde was 1 %. After 30 min fixation at the appropriate temperature the cells were centrifuged and resuspended in the cacodylate buffer; this was repeated 3 times over a 2-h period. Post-fixation was with 05 % osmium tetroxide in o-oi M cacodylate buffer for 10 min. The cells were dehydrated through an ethanol series, treated with propylene oxide for 15 min and embedded in Araldite. Thin sections were cut on a Huxley ultramicrotome, stained with lead citrate and examined with either an AEI EM 6 or a Philips EM 300 electron microscope.

RESULTS Adhesion experiments Adhesion was followed in the aggregometer at 24 °C and 1 °C using cells which had been suspended in distilled water at about 1 °C for at least 30 min. When a sample of such a suspension of single cells was transferred to the aggregometer tube at 24 °C the light transmission decreased progressively (Fig. 1). This decrease usually ended after 3-5 min although, with suspensions which had been maintained at 1 °C for 2 h or longer, the decrease lasted for 10-12 min. When o-oi ml of 1 M sodium chloride was added after the decrease, the light transmission increased immediately and steeply (Fig. i), denoting the rapid formation of cell aggregates. In contrast, at 1 °C the initial decrease in light transmission did not occur, and no cell adhesion was caused by the addition of the same volume of 1 M sodium chloride which caused adhesion at 24 °C (Fig. 2). Subsequent addition of 0-02 ml of 1 M sodium chloride again failed to cause adhesion. However, when the temperature was then raised to 24 °C there was a rapid and steep fall in light transmission which was followed by an increase denoting adhesion (Fig. 2). 48-3


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These results show that the saline-induced adhesion of the cells was reversibly inhibited at low temperature in stirred medium. Furthermore, adhesion was always preceded by a spontaneous decrease in light transmission which occurred when cells at low temperature were warmed to 24 °C. It seemed possible that the spontaneous decrease in light transmission was a manifestation of an event essential to adhesion. To test this possibility a suspension of cells which had been kept in iced water was transferred to the aggregometer at 24 °C and o-oi ml of 1 M sodium chloride added immediately (Fig. 3). This did not cause the cells to adhere; instead, the light trans-

Time, mln

Fig. 1. Tracing of record showing changes in light transmission during an aggregometer experiment at 24 °C. In this case the spontaneous decrease in light transmission flattened off about 4'5 min after placing the cells in the tube. Sodium chloride (final concentration io~a M) was added at the arrow and adhesion, denoted by a rapid increase in light transmission, began immediately. (The sudden rise in the curve at the arrow was caused by the introduction of a pipette into the light path.) Light transmission in arbitrary units.

i\

I

I _J

I

I

18

Time, min Fig. 2. Tracing from aggregometer experiment at 1 °C. There was no spontaneous decrease in light transmission and no adhesion was caused by addition of sodium chloride (io~* M) at a. At b, the temperature was raised to 24 °C, causing a rapid decrease in light transmission followed by a slower increase denoting adhesion. Light transmission in arbitrary units.


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mission decreased spontaneously for almost as long as when the cells were stirred in distilled water alone. Only after light transmission had decreased considerably did adhesion begin. It seemed likely, therefore, that the spontaneous decrease in light transmission represented some change in the cells which was associated with warming them from the cold and which was essential for their adhesion. Adhesion was followed also in still medium at the same 2 temperatures and at sodium chloride concentrations between 5 x io" 4 and 2 x icr 2 M. In these experiments there were quantitative variations with different batches of cells - a difficulty experienced previously with these cells (Born & Garrod, 1968; Garrod & Gingell, 1970).

Time, mln

Fig. 3. Tracing of aggregometer experiment at 24 °C. Sodium chloride (io" 1 M) was added (arrow) immediately after placing cells in the tube. Light transmission decreased for about 2 min, then adhesion began. Light transmission in arbitrary units.

5x10-" 10"3

2x10"2 NaCI conc.M

5x10" NaCI conc.M

Fig. 4. Results of 2 adhesion experiments in still medium at 1 °C ( • ) and 24 °C ( • ) . Ordinates: number of single cells remaining at end of experiment expressed as percentage of number originally present. Abscissae: sodium chloride concentration on log scale. Initial single-cell concentrations for A and B were 563 and 3-16 x io'/ml, respectively. For further details see text. 48-4

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Qualitatively the results were always similar (Fig. 4), justifying 2 general conclusions. First, unlike what happened in stirred medium, in still medium adhesion did occur in the cold. Secondly, although the number of single cells decreased with increasing ionic strength at both temperatures, the decrease was slightly smaller in the cold. Microscopic examination showed that the warmer cells formed closely packed aggregates, whereas the colder cells adhered loosely in chains. The increase in optical density observed when cells were warmed in the aggregometer was most probably caused by an increase in volume and/or a change in shape. Each possibility was investigated. Cell volume

In 4 experiments, using 3 different extracellular space markers, the mean cell volume was 646 /tm3 at 1 °C and 756 /im5 at 24 °C, i.e. 17% greater at the higher temperature (Table 1). Table 1. Comparison of cell volume at 1 and 24 °C Extracellular space marker ADP-8-[14C] ADP-8-[14C] Hydroxymethyl-[14C]-inulin Human serum albumin (1S1I)

3 Mean cell volume O'm ) at i °C 24 °C

Increase (%) at higher temperature

610

710

675

780

17 16

640 660

765 77°

19 17

Cell shape

Cells stored in ice-cold distilled water were spherical. When stirred in distilled water at 24 °C they changed their shape by extending pseudopodia. This suggested that differences in cell shape and activity accounted for the effect of temperature on the adhesive properties of the cells. We therefore investigated the morphology of the cells at different temperatures and the formation of intercellular adhesions by Nomarski interference and electron microscopy. This had to be done in still medium only, because stable adhesions were not formed in the cold in stirred medium. Pre-aggregation cells were suspended in distilled water at either 1 or 24 °C and fixed in suspension with 1 % glutaraldehyde in distilled water. Nomarski interference microscopy showed that cells fixed in the cold were rounded (Fig. 5) and had many small microspikes evenly distributed over the cell surface. Cells fixed at the higher temperature had irregular outlines, pseudopodia and a few long microspikes which were usually grouped together (Fig. 6). The morphology of warm and cold cells was also compared by electron microscopy. As was seen with the light microscope cold cells were rounded (Fig. 7) whereas the warm cells had irregular outlines (Fig. 8), and there were more microspikes on the cold cells than on the warm. In both cold and warm cells the microspikes had a fine structure of longitudinal fibrils and the microspike cytoplasm was more electron-dense than that of the rest of


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the cell (Fig. 9). The fibrils did not extend beyond the base of the microspikes, except in one picture (Fig. 10), where they extended into the cell body. Some microspikes were in contact with the surfaces of other cells (Fig. 11). Formation of adiiesions

The sequence of events involved in the formation of intercellular adhesions was investigated as follows. Pre-aggregation cells were harvested as before and stored in ice-cold distilled water; i-ml samples of the suspension were warmed to room temperature for 5 min. Then 1 ml of 2 x io~3 M sodium chloride solution was added to each sample and after 0-25, 0-5, i, 2 and 4 min different samples were fixed by the addition of 2 ml of 2 % glutaraldehyde in distilled water. Each sample was examined by Nomarski interference microscopy. At 24 °C, up to 1 min after the addition of saline, contacts between cells were of 2 types: either microspikes extended between the surfaces of the cells (Fig. 12) or hemispherical parts of the surfaces of adjacent cells were in close apposition (Fig. 13), with little or no flattening of these surfaces. In preparations fixed 2 min after adding saline, many of the cell contacts had become flattened (Fig. 14), so that the areas of contact were increased. Around these flattened areas there were often pseudopodia and/or microspikes (Fig. 15). Some microspikes extended from one cell to the surface of the other beyond the regions of broad contact. Similar preparations made in the cold showed very little flattening of the cell surfaces in the areas of contact, even 10 min after the addition of saline.

DISCUSSION

In the cold, slime-mould cells in the pre-aggregation phase formed stable mutual adhesions in still medium but not when stirred. When, in stirred medium, the temperature was raised from 1 to 24 °C, the cells rapidly adhered to each other. These results suggest that it is not necessary for the cells to synthesize an adhesive material in order to adhere. Instead it seems that, although adhesions can be formed in the cold, they are not strong enough to resist disruption by the shearing force applied by rapid stirring. We suggest that this is because the adhesions formed in the cold were of smaller area than those formed in the warm. The smaller areas of adhesion between cold cells was presumably due to their inability to flatten against each other because cold inhibits cell motility. In the warm there was considerable flattening of cells in contact, which increased the areas of mutual adhesion. If we make the assumption that the energy per unit area of adhesion between cold cells is not greater than that between warm cells, it follows that the total energy' of adhesion between cold cells is less than the total energy of adhesion between warm cells. The cells were spherical in the cold but extended pseudopodia when warmed. This change in cell shape was probably partly responsible for the rise in optical density which occurred when a cell suspension, previously stored in the cold, was stirred in the aggregometer at room temperature. It is known that the optical density of suspensions

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of blood platelets increases when the cells change their shape in a somewhat similar way without increasing their volume (Born, 1970). With slime-mould cells, adhesion was always preceded by this rise in optical density. It seems reasonable to conclude therefore that motile activity is required for the expansion of the area of mutual contact between cells. However, it would be naive to suppose that this is the only factor involved, because adhesiveness depends also on the energy of adhesion per unit area and on cell deformability. If the energy of adhesion per unit area were less between cold cells than between warm cells, and/or cold cells were less deformable than warm cells, adhesions between cold cells would not only be less easily formed than those between warm cells but, once formed, would be more easily disrupted. The spontaneous rise in optical density may also have been partly caused by the measured increase in cell volume. Because of the impossibility of describing cell shape mathematically, we were unable to assess the relative contributions of volume increase and of shape change to the observed increase in optical density. Furthermore, it is uncertain how the increase in cell volume affects cell adhesion. An increase in cell volume would probably stretch the cell surface, which may explain why there were fewer microspikes on the surfaces of warm cells than cold cells. That the volume increase produces no surface configurational change at the molecular level may be suggested from the observation that the zeta potentials and surface charge densities of these cells at 1 and 24 °C are identical (D. R. Garrod and D. Gingell, unpublished observations). It is interesting that both warm and cold cells possessed surface microspikes. Microspikes would favour the formation of intercellular adhesions by diminishing the electrostatic repulsive energy barrier between negatively charged cell surfaces which are approaching each other because the repulsive energy between charged particles in suspension is directly proportional to their radius of curvature (Bangham & Pethica, i960; Pethica, 1961; Lesseps, 1963). Our results suggest that microspikes are involved in the initial formation of adhesive contacts. Furthermore, the diminished adhesion of the cells in the cold is not due to the absence of microspikes from their surfaces. Microspikes are present also on the surface of slime-mould cells when in a concentration of EDTA which inhibits their adhesion (Garrod, 1969). When an adhesion is initiated by microspikes it may be that they contract to pull the cell surfaces together and so increase the area of adhesion. This contractile activity could be brought about by the fibrils that are present in the microspikes (Taylor, 1966). We thank Professor L. Wolpert for reading the manuscript, and Mrs Christine Howatson, Mr M. A. Gregory and Mr P. A. Farnsworth for assistance. The work was carried out while D.R.G. was in receipt of a Science Research Council Research Studentship. REFERENCES A. D. & PETHICA, B. A. (i960). The adhesiveness of cells and the nature of the chemical groups at their surfaces. Proc. R. Soc. Edinb. 28, 43-52. BONNER, J. T. (1947). Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold, Dictyostelium discoideum. J. exp. Zool. 106, 1-26. BONNER, J. T. (1967). The Cellular Slime Molds. Princeton, New Jersey: Princeton University Press.

BANGHAM,


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BORN, G. V. R. (1962a). Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature, Lond. 194, 927-929. BORN, G. V. R. (1962A). Quantitative investigations into the aggregation of blood platelets. J. PhysioL, Lond. 162, 67-68 P. BORN, G. V. R. (1970). Observations on the change in shape of blood platelets brought about by adenosine diphosphate. J. PhysioL, Lond. 209, 487-511. BORN, G. V. R. & CROSS, M. J. (1963). The aggregation of blood platelets. J. PhysioL, Lond. 168, 178-195. BORN, G. V. R. & GARROD, D. R. (1968). Photometric demonstration of aggregation of slime mould cells showing effects of temperature and ionic strength. Nature, Lond. 220, 616—618. BORN, G. V. R. & HUME, M. (1967). Effects of the number and size of platelet aggregates on the optical density of plasma. Nature, Lond. 215, 1027-1029. CURTIS, A. S. G. (1962a). Pattern and mechanism in the reaggregation of sponges. Nature, Lond. zoo, 1235-1236. CURTIS, A. S. G. (19626). Cell contact and adhesion. Biol. Rev. 37, 82-129. CURTIS, A. S. G. & GREAVES, M. F. (1965). The inhibition of cell aggregation by a pure serum protein. J. Embryol. exp. Morph. 13, 309-326. GARROD, D. R. (1969). Cell Movement and Adhesion in Dictyostelium discoideum. Ph.D. thesis, University of London. GARROD, D. R. & GINGELL, D. (1970). A progressive change in the electrophoretic mobility of preaggregation cells of the slime mould, Dictyostelium discoideum. J. Cell Sci. 6, 277-284. GINGELL, D. & GARROD, D. R. (1969). Effect of EDTA on electrophoretic mobility of slime mould cells and its relationship to current theories of cell adhesion. Nature, Lond. 221, 192193GINGELL, D., GARROD, D. R. & PALMER, J. F. (1969). Divalent cations and cell adhesion. In Biological Council Symposia on Drug Action: Calcium and Cellular Function, pp. 59—64. London: Churchill. HUMPHREYS, T. (1963). Chemical dissolution and in vitro reconstruction of sponge cell adhesions. I. Isolation and functional demonstration of the components involved. Devi Biol. 8, 27-47. LESSEPS, R. J. (1963). Cell surface projections: their role in the aggregation of embryonic chick cells as revealed by electron microscopy. J. exp. Zool. 153, 171-182. MOSCONA, A. A. (1961a). Rotation mediated histogenetic aggregation of dissociated cells: a quantifiable approach to cell interactions in vitro. Expl Cell Res. 22, 455-475. MOSCONA, A. A. (1961 b). Effect of temperature on adhesion to glass and histogenetic cohesion of dissociated cells. Nature, Lond. 190, 408-409. MOSCONA, A. A. (1963). Studies on cell aggregation: demonstration of materials with a selective cell-binding activity. Proc. natn. Acad. Sci U.S.A. 49, 742-747. MOSCONA, A. A. (1968). Cell aggregation: properties of specific cell-ligands and their role in the formation of multicellular systems. Devi Biol. 18, 250-277. PETHICA, B. A. (1961). The physical chemistry of cell adhesion. Expl Cell Res. (Suppl.), 8, 123140. STEINBERG, M. S. (1962). The role of temperature in the control of aggregation of dissociated embryonic cells. Expl Cell Res. 28, 1-10. SUSSMAN, M. (1966). Biochemical and genetic methods in the study of cellular slime mold development. In Methods in Cell Physiology, vol. 2 (ed. D. M. Prescott), pp. 397-410. New York: Academic Press. TAYLOR, A. C. (1966). Microtubules in the microspike3 and cortical cytoplasm of isolated cells. J. Cell Biol. 28, 155-168.

{Received 10 November 1970)

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Fig. 5. Cells fixed in suspension at 1 °C. Fig. 6. Cells fixed in suspension at 24 °C.

w

Effect of temperature on cell adhesion •-. • ,

, 10//m ,

8

*

•

*

ca. 10//m Fig. 7. Electron micrograph of cells fixed in suspension at i °C. Longitudinal and transverse sections of microspikes can be seen near the cells' surfaces. Fig. 8. Composite electron micrograph of cells fixed in suspension at 24 °C.

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Fig. 9. Electron micrograph of surface microspike of cell fixed in suspension at 1 °C. The fibrillar organization of the dense microspike cytoplasm can be seen. Fig. 10. Electron micrograph of surface microspikes of cellfixedin suspension at 24 °C. The fibrillar elements of the microspike extend into the cytoplasm of the cell body at the left of the picture. Fig. 11. Electron micrograph of microspike in contact with the surface of another cell.

Ejfect of temperature on cell adhesion

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0-1

11

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Fig. 12. Microspike contact (arrow) between 2 cellsfixed0-25 min after the addition of sodium chloride to a cell suspension in distilled water at 24 °C. Fig. 13. Rounded contacts between cellsfixedin suspension 1 min after sodium chloride addition at 24 °C. Fig. 14. Cluster of cells fixed in suspension 2 min after the addition of sodium chloride at 24 CC, showing expansion of the area of mutual contact. Fig. 15. As in Fig. 14, but showing pseudopodia and microspikes at the edges of the broad areas of contact between cells.


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