J. Cell Sci. 8, 751-765 (1970 751Printed 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. BORNDepartment of Biology as Applied to Medicine,The Middlesex Hospital Medical School, London, W. 1, England, andDepartment of Pharmacology, Royal College of Surgeons of England, London, W.C.2,England

SUMMARYThe 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 recordingthe diminution in optical density of the cell suspension associated with the formation of cellaggregates. It was found that: (1) when cells, previously stored at 1 °C, were stirred in distilledwater at 24 CC the optical density of the suspension increased; (2) when sodium chloride wasadded after this increase, the optical density fell as cells adhered to each other; (3) both theincrease 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 opticaldensity was due to (a) extension of pseudopodia and a consequent change from a spherical toan 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 initialcontacts were formed either by microspikes or by rounded parts of the cell surface. The cellsurfaces then flattened against each other, thereby increasing the area of contact. No flatteningoccurred at 1 °C. Microspikes were present on the surfaces of both cold and warm cells andcontained longitudinal fibrils.

It is suggested that adhesion of these cells is inhibited in the cold in stirred medium becausethe cells are unable to expand areas of mutual contact. Cold adhesions are, therefore, weak andeasily disrupted by shearing forces. The inability to expand adhesive contacts may be due to theinhibition of cell motility at low temperature.

INTRODUCTION

Previous investigations on the effect of temperature on the formation of cell adhesionshave led to confusion. Whether certain types of cells form mutual adhesions at lowtemperatures clearly depends on the experimental conditions. Several differentmechanisms have been suggested to explain the failure of cells to adhere at low tem-peratures where this has been observed.

It is generally agreed that dissociated chick embryo cells do not reaggregate at lowtemperature 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

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

material required for adhesion (Moscona, 1961a, b). (ii) Serum contains an adhesion-inhibiting protein which the cells cannot break down at low temperature; this sugges-tion was based on the observation that cells did reaggregate at low temperature inserum-free medium (Curtis & Greaves, 1965). (iii) Cell-surface motile activity may beinhibited 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 adhereat low temperature in shaken suspension (Humphreys, 1963; Moscona, 1963, 1968). Instill 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 mutualadhesion (i.e. the formation of multicellular aggregates) of cells of the slime mouldDictyostelium 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 canbe obtained without damaging chemical treatment, e.g. with trypsin or EDTA, andbecause adhesion experiments can be performed in simple saline media.

Previously we have shown that slime-mould cells, suspended in distilled water, canbe caused to adhere to each other by adding simple salts (Born & Garrod, 1968). Asthe cells approach the chemotactic aggregation stage of their life-cycle (Bonner, 1967),the concentration of salt required to cause adhesion diminishes; this is associated witha decrease in their surface charge density (Garrod & Gingell, 1970). Further, we havepresented 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 formadhesions less readily in the cold (0-2 °C) than at room temperature (22-24 °C) andprovide evidence that this difference is due, at least in part, to the inhibition of cellmotility 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 justbefore the spontaneous aggregation stage and washed free of bacteria by centrifugation at abouti5Og. Resuspended in distilled water, the cells were pipetted on to the surface of WhatmanNo. 50 filter papers resting on Millipore support pads which were saturated with distilledwater; in this way they were stored until required for use. For the experiments the cells werewashed 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), asfollows. 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 tocontrol the temperature of the cell suspension. The suspension was stirred by a small magneticstirrer rapidly enough (1000 rev/min) to ensure that the collision rate between the cells was

Effect of temperature on cell adhesion 753

non-limiting (Born & Cross, 1963). A beam of light was passed through the tube and the lighttransmission recorded continuously on a pen-recorder. When the cells adhered to each otherthey formed aggregates; as a result, the light transmission increased. With platelets, the opticaldensity 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 opticaldensity on the formation of cell clumps was verified microscopically.

In still medium, adhesion tests were done as follows. One millilitre of cell suspension, pre-viously stored in iced water, was pipetted into a test-tube which was maintained at either 1 °Cor about 24 °C, i.e. room temperature. To allow the cells to warm up to the higher temperaturethey stood at that temperature for 5 min before the experiment. One millilitre of sodium chloridesolution was added at the required temperature and concentration. After 5 min the tube wasgently 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 percen-tage of the original single cell concentration. Strictly speaking, the medium was not completelystill 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 insediments of packed cells was measured with radioactive substances which did not penetratethe 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 concentrationto half-strength Bonner's solution (Bonner, 1947) at about 1 °C or at 24 °C. The cells werefixed by adding an equal volume of 2 % glutaraldehyde solution in 002 M cacodylate buffer atpH 7-05 so that the final concentration of glutaraldehyde was 1 %. After 30 min fixation at theappropriate 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 ino-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 ona Huxley ultramicrotome, stained with lead citrate and examined with either an AEI EM 6 ora Philips EM 300 electron microscope.

RESULTS

Adhesion experiments

Adhesion was followed in the aggregometer at 24 °C and 1 °C using cells which hadbeen suspended in distilled water at about 1 °C for at least 30 min. When a sample ofsuch a suspension of single cells was transferred to the aggregometer tube at 24 °C thelight transmission decreased progressively (Fig. 1). This decrease usually ended after3-5 min although, with suspensions which had been maintained at 1 °C for 2 h orlonger, the decrease lasted for 10-12 min. When o-oi ml of 1 M sodium chloride wasadded 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 initialdecrease in light transmission did not occur, and no cell adhesion was caused by theaddition 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 causeadhesion. However, when the temperature was then raised to 24 °C there was a rapidand steep fall in light transmission which was followed by an increase denoting adhesion(Fig. 2).

48-3

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

These results show that the saline-induced adhesion of the cells was reversiblyinhibited at low temperature in stirred medium. Furthermore, adhesion was alwayspreceded by a spontaneous decrease in light transmission which occurred when cellsat low temperature were warmed to 24 °C. It seemed possible that the spontaneousdecrease 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 wastransferred to the aggregometer at 24 °C and o-oi ml of 1 M sodium chloride addedimmediately (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 aggregometerexperiment at 24 °C. In this case the spontaneous decrease in light transmission flat-tened off about 4'5 min after placing the cells in the tube. Sodium chloride (finalconcentration io~a M) was added at the arrow and adhesion, denoted by a rapid in-crease in light transmission, began immediately. (The sudden rise in the curve at thearrow was caused by the introduction of a pipette into the light path.) Light trans-mission in arbitrary units.

i\

I I_J I I

18Time, min

Fig. 2. Tracing from aggregometer experiment at 1 °C. There was no spontaneousdecrease in light transmission and no adhesion was caused by addition of sodiumchloride (io~* M) at a. At b, the temperature was raised to 24 °C, causing a rapid de-crease in light transmission followed by a slower increase denoting adhesion. Lighttransmission in arbitrary units.

Effect of temperature on cell adhesion 755

mission decreased spontaneously for almost as long as when the cells were stirred indistilled water alone. Only after light transmission had decreased considerably didadhesion begin. It seemed likely, therefore, that the spontaneous decrease in lighttransmission represented some change in the cells which was associated with warmingthem from the cold and which was essential for their adhesion.

Adhesion was followed also in still medium at the same 2 temperatures and atsodium chloride concentrations between 5 x io"4 and 2 x icr2 M. In these experimentsthere were quantitative variations with different batches of cells - a difficulty experi-enced 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) wasadded (arrow) immediately after placing cells in the tube. Light transmission de-creased for about 2 min, then adhesion began. Light transmission in arbitrary units.

5x10-" 10"3 2x10"2 5x10"NaCI conc.M 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 per-centage of number originally present. Abscissae: sodium chloride concentration onlog scale. Initial single-cell concentrations for A and B were 563 and 3-16 x io'/ml,respectively. For further details see text.

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

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 inthe cold. Secondly, although the number of single cells decreased with increasingionic strength at both temperatures, the decrease was slightly smaller in the cold.Microscopic examination showed that the warmer cells formed closely packed aggre-gates, whereas the colder cells adhered loosely in chains.

The increase in optical density observed when cells were warmed in the aggrego-meter 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 cellvolume was 646 /tm3 at 1 °C and 756 /im5 at 24 °C, i.e. 17% greater at the highertemperature (Table 1).

Table 1. Comparison of cell volume at 1 and 24 °C

Extracellularspace marker

ADP-8-[14C]ADP-8-[14C]Hydroxymethyl-[14C]-inulinHuman serum albumin (1S1I)

Mean cell volumei °C

6 1 0

675640660

O'm3) at24 °C

7 1 0780

76577°

Increase (%)at higher

temperature

1716

1917

Cell shape

Cells stored in ice-cold distilled water were spherical. When stirred in distilledwater at 24 °C they changed their shape by extending pseudopodia. This suggestedthat differences in cell shape and activity accounted for the effect of temperature onthe adhesive properties of the cells. We therefore investigated the morphology of thecells at different temperatures and the formation of intercellular adhesions by Nomarskiinterference 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 andfixed in suspension with 1 % glutaraldehyde in distilled water. Nomarski interferencemicroscopy showed that cells fixed in the cold were rounded (Fig. 5) and had manysmall microspikes evenly distributed over the cell surface. Cells fixed at the highertemperature had irregular outlines, pseudopodia and a few long microspikes whichwere 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 thewarm cells had irregular outlines (Fig. 8), and there were more microspikes on thecold cells than on the warm.

In both cold and warm cells the microspikes had a fine structure of longitudinalfibrils and the microspike cytoplasm was more electron-dense than that of the rest of

Effect of temperature on cell adhesion 757

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

Formation of adiiesions

The sequence of events involved in the formation of intercellular adhesions wasinvestigated as follows. Pre-aggregation cells were harvested as before and stored inice-cold distilled water; i-ml samples of the suspension were warmed to room tem-perature for 5 min. Then 1 ml of 2 x io~3 M sodium chloride solution was added toeach sample and after 0-25, 0-5, i, 2 and 4 min different samples were fixed by theaddition of 2 ml of 2 % glutaraldehyde in distilled water. Each sample was examinedby Nomarski interference microscopy.

At 24 °C, up to 1 min after the addition of saline, contacts between cells were of2 types: either microspikes extended between the surfaces of the cells (Fig. 12) orhemispherical 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 addingsaline, many of the cell contacts had become flattened (Fig. 14), so that the areas ofcontact were increased. Around these flattened areas there were often pseudopodiaand/or microspikes (Fig. 15). Some microspikes extended from one cell to the surfaceof the other beyond the regions of broad contact.

Similar preparations made in the cold showed very little flattening of the cell surfacesin 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 mutualadhesions in still medium but not when stirred. When, in stirred medium, the tem-perature was raised from 1 to 24 °C, the cells rapidly adhered to each other. Theseresults suggest that it is not necessary for the cells to synthesize an adhesive materialin order to adhere. Instead it seems that, although adhesions can be formed in thecold, they are not strong enough to resist disruption by the shearing force applied byrapid stirring. We suggest that this is because the adhesions formed in the cold wereof smaller area than those formed in the warm. The smaller areas of adhesion betweencold cells was presumably due to their inability to flatten against each other becausecold inhibits cell motility.

In the warm there was considerable flattening of cells in contact, which increased theareas of mutual adhesion. If we make the assumption that the energy per unit area ofadhesion between cold cells is not greater than that between warm cells, it follows thatthe total energy' of adhesion between cold cells is less than the total energy of adhesionbetween warm cells.

The cells were spherical in the cold but extended pseudopodia when warmed. Thischange in cell shape was probably partly responsible for the rise in optical densitywhich occurred when a cell suspension, previously stored in the cold, was stirred in theaggregometer at room temperature. It is known that the optical density of suspensions

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

of blood platelets increases when the cells change their shape in a somewhat similarway without increasing their volume (Born, 1970). With slime-mould cells, adhesionwas always preceded by this rise in optical density. It seems reasonable to concludetherefore that motile activity is required for the expansion of the area of mutual con-tact between cells. However, it would be naive to suppose that this is the only factorinvolved, because adhesiveness depends also on the energy of adhesion per unit areaand on cell deformability. If the energy of adhesion per unit area were less betweencold cells than between warm cells, and/or cold cells were less deformable than warmcells, adhesions between cold cells would not only be less easily formed than thosebetween warm cells but, once formed, would be more easily disrupted.

The spontaneous rise in optical density may also have been partly caused by themeasured increase in cell volume. Because of the impossibility of describing cell shapemathematically, we were unable to assess the relative contributions of volume increaseand of shape change to the observed increase in optical density. Furthermore, it isuncertain how the increase in cell volume affects cell adhesion.

An increase in cell volume would probably stretch the cell surface, which mayexplain 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 molecularlevel may be suggested from the observation that the zeta potentials and surface chargedensities 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. Micro-spikes would favour the formation of intercellular adhesions by diminishing theelectrostatic repulsive energy barrier between negatively charged cell surfaces whichare approaching each other because the repulsive energy between charged particlesin suspension is directly proportional to their radius of curvature (Bangham & Pethica,i960; Pethica, 1961; Lesseps, 1963). Our results suggest that microspikes are involvedin the initial formation of adhesive contacts. Furthermore, the diminished adhesionof 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 concen-tration of EDTA which inhibits their adhesion (Garrod, 1969). When an adhesion isinitiated by microspikes it may be that they contract to pull the cell surfaces togetherand so increase the area of adhesion. This contractile activity could be brought aboutby 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 whileD.R.G. was in receipt of a Science Research Council Research Studentship.

REFERENCES

BANGHAM, A. D. & PETHICA, B. A. (i960). The adhesiveness of cells and the nature of thechemical 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 thedevelopment of the slime mold, Dictyostelium discoideum. J. exp. Zool. 106, 1-26.

BONNER, J. T. (1967). The Cellular Slime Molds. Princeton, New Jersey: Princeton UniversityPress.

Effect of temperature on cell adhesion 759

BORN, G. V. R. (1962a). Aggregation of blood platelets by adenosine diphosphate and itsreversal. 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 aboutby 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 slimemould 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 theoptical 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, 192-193-

GINGELL, D., GARROD, D. R. & PALMER, J. F. (1969). Divalent cations and cell adhesion. InBiological 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 chickcells 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 cohesionof dissociated cells. Nature, Lond. 190, 408-409.

MOSCONA, A. A. (1963). Studies on cell aggregation: demonstration of materials with a selectivecell-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 inthe formation of multicellular systems. Devi Biol. 18, 250-277.

PETHICA, B. A. (1961). The physical chemistry of cell adhesion. Expl Cell Res. (Suppl.), 8, 123-140.

STEINBERG, M. S. (1962). The role of temperature in the control of aggregation of dissociatedembryonic cells. Expl Cell Res. 28, 1-10.

SUSSMAN, M. (1966). Biochemical and genetic methods in the study of cellular slime molddevelopment. In Methods in Cell Physiology, vol. 2 (ed. D. M. Prescott), pp. 397-410. NewYork: 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)

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

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

wEffect of temperature on cell adhesion

•-. • ,

, 10//m ,

8

* • *

ca.10//m

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

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

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 cell fixed in suspension at 24 °C.The fibrillar elements of the microspike extend into the cytoplasm of the cell body atthe left of the picture.Fig. 11. Electron micrograph of microspike in contact with the surface of another cell.

Ejfect of temperature on cell adhesion 763

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

Fig. 12. Microspike contact (arrow) between 2 cells fixed 0-25 min after the addition ofsodium chloride to a cell suspension in distilled water at 24 °C.Fig. 13. Rounded contacts between cells fixed in suspension 1 min after sodium chlorideaddition at 24 °C.Fig. 14. Cluster of cells fixed in suspension 2 min after the addition of sodiumchloride 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 thebroad areas of contact between cells.

Effect of temperature on cell adhesion •765