J. Cell Sci. 16, 385-399 (i974) 385Printed in Great Britain

LIMPET HAEMOCYTES

III. EFFECTS OF CYTOCHALASIN B AND COLCHICINEON CELL SPREADING AND AGGREGATION

G. E.JONES

Biology Department, Queen Elizabeth College, London WS, England

AND T. PARTRIDGE

Department of Experimental Pathology, Charing Cross HospitalMedical School, London W6, England

SUMMARY

Limpet blood cells are suitable for the study of a number of interdependent features of cellbehaviour; microspike formation, spreading and locomotion on a solid substrate, and aggre-gation. The effects of cytochalasin B (CCB) and colchicine on each of these activities is studiedwith a view (a) to determining with which functions these 2 reagents interfere; and (6) togaining information on the relationships between these functions in normal cell behaviour.

Rapid spike formation and fast aggregation in a shaker system, which are normal features ofthe behaviour of amoebocytes in limpet blood or in seawater, are totally but reversibly in-hibited by the presence of low concentrations of CCB (e.g. 0-5 /ig/cm3). Similarly, the spreadingof amoebocytes on to a solid substrate is greatly inhibited by CCB, both in rate and extent,and the rapid locomotion on a solid substrate of normal amoebocytes is completely abolishedby CCB. The first observable effects of CCB on spread amoebocytes are loss of optical integrityof the spikes and retraction of parts of the anterior cell margin. The loss of distinctivecell shape, the inhibition of spreading on to a glass surface and the lack of motility on such asurface may all be immediate consequences of the disruption of spike structure.

CCB inhibits aggregation but does not disrupt preformed contacts between cells, whichsuggests that it acts on an early stage in the formation of stable contact rather than on theadhesiveness per unit area of contacting cell surface. A possible link between the effects ofCCB on spikes and on aggregation is the proposal that low-diameter projections are neededto establish initial contact between cells. Alternatively, the spikes may be required for therapid spreading of cells, not only on to glass but also over the surfaces of other cells, enablingthem to increase their mutual contact area very rapidly and thus stabilize an adhesion.

Amoebocytes maintained in the presence of 50 /tg/cm1 colchicine for a number of hoursgradually lose their bipolar form and the entire cell margin becomes occupied by the spike-supported lamella which normally constitutes the leading edge of the cell. Thus microtubulesare probably not necessary for the skeletal functions of spikes or for their roles in spreadingand aggregation, but they do appear to play a part in the control of spike orientation.

Macrophages, cells lacking obvious spikes and showing little sign of bipolarity, appearunaffected by CCB or colchicine and spread normally on to a glass slide in the presence ofeither reagent. This, together with the limited spreading of amoebocytes in CCB, suggeststhat at least 2 distinct mechanisms may operate in the spreading of cells on to a solid substrate.

INTRODUCTION

Colchicine and cytochalasin B have been used widely in attempts to interfere in afairly specific way with a number of processes involving motility of cells (Spooner,Yamada & Wessells, 1971). Among other effects, cytochalasin B (CCB) has been

23 C E L 16

386 G. E.Jones and T. Partridge

shown to inhibit the aggregation of blood platelets (White, 1971; Haslam, 1972;Kay & Fudenberg, 1973), to inhibit the spreading (Weiss, 1972) and locomotion(Wessells, Spooner & Luduena, 1973) of cells on a solid substrate and to alter cellshape, particularly by preventing the formation and maintenance of microspikes(Wessells et al. 1971). Colchicine too affects the shape, spreading and solid substratelocomotion of some cells (Vasiliev et al. 1970; Spooner et al. 1971). In view of thesuggested associations between cell shape, especially the presence of microspikes,cell adhesion (Pethica, 1961; Weiss, 1972), and locomotion (Taylor & Robbins, 1963;Partridge & Davies, 1974), it would be interesting to correlate the effects of CCB andcolchicine on each of these cellular activities. The systems previously used to studythe effects of these agents on such aspects of cell behaviour are so disparate thatcomparisons between them are not very useful. For this reason we have investigatedthe effects of CCB and colchicine on the development and maintenance of microspikes,on aggregation and on the cell-substrate relationships of limpet blood cells. This isa particularly convenient system, for the haemocytes display rapid aggregation andsubstrate spreading. The great majority of the cells, the amoebocytes, developprominent spikes which appear to be large microspikes (Davies & Partridge, 1972)and which participate in aggregation, spreading and locomotion (Partridge & Davies,1974). Conversely, a small proportion of cells, macrophages, spread on a glass sub-strate without forming spikes. Thus we have 2 different models of cell spreading inthis system.

MATERIALS AND METHODS

Blood was withdrawn from the pallial veins of limpets {Patella vulgata) as described pre-viously (Davies & Partridge, 1972) and preparations plated on to glass slides in humid chambers(Partridge & Davies, 1974) were used to study the spreading and locomotion in variousmedia.

For aggregation experiments the blood was drawn into an equal quantity of EDTA-seawater.The cells were pelleted by centrifugation and resuspended in various aggregation media inwhich they were shaken rapidly (10 strokes/s) on a Griffin flask shaker for 5 min. This treat-ment caused normal limpet haemocytes in seawater or serum to aggregate to their endpoint atroom temperature (Davies & Partridge, 1972). Haemocytometer counts were made of the totalparticle number (any number of contacting cells = 1 particle) per graticule square at thebeginning of aggregation (N°) and after 5 min (N*). The percentage aggregation was calculated

o.% agg. =

The media used were constituted as follows:Artificial seawater: NaCl 23-44 g, KC1 0-725 g, Na,SO4 390 g, CaCls.6H.O 2-20 g,MgC1..6H,O 1064 g.EDTA-seawater: NaCl 1720 g, KC1 0-725 g, NajSO4 3-90 g, EDTA 37-20 g.CMF-seawater: NaCl 2890 g, KC1 0-725 g, Na2SO4 3-90 g.Each of the above solutions was made up to 1 dm3 with glass-distilled water and the pHadjusted to 820 with 05 mol dm~3 NaOH.

Cytochalasin B was obtained from ICI Ltd., and made up in a stock solution in DMSO at aconcentration of 0-5 mg/cm3. A Hamilton microlitre syringe 702-N was used to dispensealiquots from the stock solution as required. The DMSO used in the making of the stock

Effects of cytochalasin B and colchicine mi haemocytes 387

solution was obtained from the same source as the DMSO used in the controls (Koch-LightLaboratories Ltd.). Colchicine was obtained from BDH and made up to a concentration of50 /ig/cm3 in artificial seawater.

RESULTS

The formation of spikes, which normally occurs during the first 3 min afterwithdrawal of amoebocytes from the limpet (Fig. 2) (Davies & Partridge, 1972), iscompletely inhibited by addition to the withdrawn blood of an equal quantity ofseawater containing CCB (Fig. 3). The cells become lobulated into a number ofsmooth-edged blebs and lamellae, which results in a somewhat mulberry-like shape.This effect is equally evident at all CCB concentrations tested (0-0625-1-0/Jg/cm3).Similarly, fully formed spikes on suspended cells collapse rapidly when exposed toCCB. Addition of seawater containing DMSO at a concentration of 1 rnm'/cm3

is without effect on the form of suspended cells.Amoebocytes suspended in serum or seawater containing CCB are greatly in-

hibited with regard to their ability to spread on to a glass surface. Individual amoebo-cytes in serum, artificial seawater or a mixture of the two, containing 1 mm3/cm3

DMSO spread completely in 15-30 min. In contrast, suspensions in media containing0-0625-0-5 /tg/cm3 CCB have barely begun to spread within the first hour and require2-3 h to spread to their full extent. In addition, the spreading in the presence ofCCB (Fig. 4) is unlike that seen in untreated cells (Fig. 5) in that the extending lamellaecontain no obvious spikes. Within the range of concentrations used, there is little orno dose effect of CCB on spreading.

Cells which have been allowed to spread in serum show no obvious changes whenthe medium is replaced by artificial seawater containing 1 mm3/cm3 DMSO for periodsof several hours and such cells move normally on the glass substrate. Within 1 minof addition of seawater containing CCB to a preparation of spread amoebocytes,effects on the spikes and cell margins are observed. These changes are usually sorapid that they are difficult to photograph, but in some cases, where the coverslipis closely applied to the slide, the penetration of replacement media into the pre-paration occurs slowly and the sequence of effects on the spread cells is retarded. Insuch cases it is possible to obtain a better series of micrographs (Figs. 8-12) whichillustrate the nature of the changes but do not reflect the speed with which thesechanges can occur. Where they project beyond the margin of the cell, the spikesbecome beaded or irregular in shape and reduced in phase density. Simultaneously,the phase-dense proximal regions of the spike which lie within the lamellar portionsof the cells become indistinct, break up into irregularly shaped structures, or becomeinvisible (Fig. 9). Subsequently the extended margins of the cells retract. Sometimesthis retraction is quite pronounced and the cells contract into tight clumps, leavinglong tenuous retraction fibrils trailing back to attachment points of the originalspike tips (Fig. 10, cell a). In other cases the retraction is less marked, and consistsof the withdrawal of the lamellar web between the spikes. The spikes themselvesremain as retraction fibrils which are rather flaccid as judged by their movement incurrents in the medium. In less well spread cells the lamellar web merges with the

25-2

388 G. E. Jones and T. Partridge

bases of the spikes to give a polygonal outline to the cell. No net translocation ofamoebocytes on the substrate occurs in CCB-containing media, apart from thosemovements interpreted as retraction.

Replacement of media containing CCB with fresh seawater containing i mm3/cm3

DMSO results in a rapid reversal of the effects in either suspended or spread amoebo-cytes. Within a few minutes new spikes begin to form (Fig. 11) and normal spreadingoccurs within 30 min (Fig. 12).

Effect of colchicine on spreading and cell shape

Suspension of haemocytes in seawater containing 50 /

Effects of cytochalasin B and colchicine on haemocytes 389

Effect of CCB and colchicine on macrophages

Macrophages are observed to spread normally and to remain spread in the presenceof up to i -o /tg/cm3 CCB (the highest concentration used). Likewise 50 /tg/cm3

colchicine produces no noticeable effect on these cells.

Effect of CCB on aggregation of haemocytes

Considerable variation was found between individual aggregation experiments butthe main effects were quite consistent between experiments and the following examplesare representative of the results obtained.

Table 2. The effects of various concentrations of CCB on the aggregation of limpethaemocytes shaken in artificial seawater. (P values based on 't' test between particlenumbers at o and 5 min.)

Total particle no. ± S.E. perhaemocytometer square at

times:

Suspension medium o min 5 rnin % aggregation

1 mnV/cm3 DMSOin artificial seawater

0-125 /tg/cm3

CCB in artificialseawater

025 /tg/cm3 CCBin artificial seawater

0-5 /tg/cm3 CCBin artificial seawater

44'4±2-S

44'6 ±2-3

42-2± I- I

38-8±i-5

18-4 ±0-5

34-8 ± i -8

3 I - 8 ± I - 2

37-8 ±0-9

58-6

2 1 9

2 4 6

2 6

39° G. E. Jones and T. Partridge

centrifugation and resuspension between counts 2 and 3 may be due to adhesion ofcells to the centrifuge tube or to one another during centrifugation.

A1

1 5 cm1 BLOOD WITHDRAWN INTO 1-5 cm5

EDTA-SEAWATER. DIVIDED INTO THREE1-cm1 PORTIONS

PELLETED BY CENTRIFUGATION. RESUSPENDED IN:

CI

1 mm'/cm" DMSO

In artificial seawater

(OS cm')

COUNT 1

0-5 /(g/cm1 CCB

In artificial seawater

(0-5 cm1)

COUNT 1

0-5 //g/cm1 CCB

in artificial seawater

(OS cm1)

COUNT 1

SHAKEN FOR 5 MIN

COUNT 2

JCOUNT 2

ICOUNT 2

PELLETED BY CENTRIFUGATION. RESUSPENDED IN:

I1 mm'/tm1 DMSO

in artificial seawater

(OS cm1)

COUNT 3

0-5 //g/cm1 CCB

In artificial seawater

(OS cm1)

COUNT 3

1 mm'/tm1 DMSO

in artificial seawater

(OS cm1)

COUNT 3

COUNT 4

SHAKEN FORS MIN

COUNT 4 COUNT 4

Fig. 1. Scheme of experiment to test the reversibility of the inhibition caused by CCBon the aggregation of limpet haemocytes shaken in seawater.

To test the effect of CCB on established cell contacts, aggregates previouslyformed by shaking a suspension of haemocytes in seawater for 5 min, were suspendedin 0-5 /tg/cm3 CCB in seawater and agitated again on the shaker. Counts made onsamples taken at zero time and after 5 and 10 min shaking did not reveal any significantchange in total particle number, indicating that no disaggregation caused by break-down of established contacts between cells had occurred.

Effects of cytochalasin B and colchicine on haemocytes 391

Effect of colchicine on aggregation

The effects of colchicine on spread cells were evident only after about 1 h. There-fore haemocytes were maintained in suspension in CMF-seawater or in 50 /tg/cm3

colchicine in CMF-seawater for 1 h. They were then pelleted by centrifugation andresuspended in seawater and in 50/6g/cm3 colchicine in seawater respectively, foraggregation by shaking in the normal way. The results of a sample experiment givenin Table 4 show that colchicine has no significant effect on the aggregation ofhaemocytes.

Table 3. Counts 1-4 and percentage aggregation after each period of shaking in experimentillustrated in Fig. 1. (P values are based on lt' tests between particle counts 1 and 2 andcounts 3 and 4.)

Total particle no. atCOUNT I±S.E .

Total particle no. atC O U N T 2 ± S . E .

% aggregation 1-2P

Total particle no. atC O U N T 3 ± S . E .

Total particle no. atC 0 U N T 4 ± S . E .

% aggregation 3-4P

A

262 ± 1-2

40 ±O-6

847P>0'3

60 ±07

2-9±o-s

5i-70-005 >/>>o-ooi

Table 4. The effect of colchicine on the aggregation of limpet haemocytes shaken inartificial seawater. (P values are based on 't' tests between particle counts at o and 5 min.)

Total particle no. at0 min±S.E.

Total particle no. at5 min±s.E.

% aggregationP

Haemocytes kept for 1 hin CMF-seawater, thentransferred to artificialseawater and shaken

for s min

2I-2±O'8

10-4 ±0-9

S°9

392 G. E, Jones and T. Partridge

DISCUSSION

In the moderately short-term experiments conducted, colchicine was found to havea limited effect on the spreading of amoebocytes, consisting of a gradual loss ofpolarity over the first few hours. This is in accord with the observations of Vasilievet al. (1970) on fibroblasts, which gradually lost oriented movement and morpho-logical polarity when cultured in colchicine. It appears that the microtubular systemin amoebocytes is probably concerned with regulation of the orientation of thespikes rather than with their structure or their function in spreading and aggregation.

CCB at very low concentrations was found to have rapid and profound effects onthe formation and maintenance of spikes, and on the aggregation, spreading andsolid substrate locomotion of the amoebocytes. The action on all of these functionswas very rapid and almost completely reversible on removal of CCB, observationswhich suggest that major disruption of cellular mechanisms is not involved, and thatthe phenomena resulting from CCB treatment of amoebocytes may be the result ofinterference with a single system.

Several observations point to the spikes as the CCB-sensitive organelles whose dis-ruption is responsible for most if not all of the changes found. The earliest observedeffect of CCB on spread cells is on the optical integrity of these structures. Althoughthe change in form of the spikes where they project beyond the margin of the lamellacould be attributed to the collapse of deformable structures following a reduction ofcell-substrate adhesiveness, the simultaneous disruption of the portion of the spikelying within the lamella is not amenable to such an explanation and seems to indicatethat the structure of the spike itself is affected. Furthermore, the retraction of cellswhich follows the disruption of the spikes provides no evidence of loss of adhesiveness,for the tips of the spikes retain their attachment to the substrate and the adhesionsbetween the cells remain intact. Rather, the retraction appears to result from a lossof rigidity of the lamellipodial portions of the cells due perhaps to the loss of supportivespikes (Partridge & Davies, 1974). The fact that the spike-supported lamellae ofamoebocytes in suspension collapse in the presence of CCB is also in agreement withthis view.

Electron-microscopic observations have revealed that microspikes are based on astructure of microfilaments (Goldman & Follett, 1969; Wessells et al. 1971; Allison,1973) and initial investigations suggest that the spikes of limpet amoebocytes possessa similar structure. The suggestion that the activity of CCB is attributable to an effecton spike structure is, therefore, in accordance with the proposal of Wessells et al.(1971) that CCB disrupts the 5-nm microfilament lattice system. This hypothesis isdisputed (Carter, 1972; Goldman, 1972) and there is strong evidence that CCB actson cell surface components involved in the movement of molecules across membranes(Mizel & Wilson, 1972; Plagemann & Estensen, 1972). However, these 2 hypothesesare not incompatible: a hydrophobic molecule like CCB (Rothweiler & Tamm, 1966)which may disturb the structure of lipid membranes could interfere with the insertionof microfilaments into them, and thus disorganize microfilament-based structures andfunctions.

Effects of cytochalasin B and colchicine on haemocytes 393

Spreading of amoebocytes on to a glass substrate is greatly inhibited by CCB withregard both to rate and final extent of spreading. Similarly, the locomotion of thesecells, which seems to depend on the same basic mechanism as spreading (Partridge &Davies, 1974), was completely inhibited by CCB. This is probably a reflexion of theimportant role of spikes in the normal process of advance of the leading edge ofamoebocytes over a solid substrate. However, the fact that amoebocytes do spread inthe presence of CCB, albeit slowly and to a reduced extent, suggests that some othermechanism plays a part in the process. This may be a surface tension effect as proposedby Carter (1967) or a physicochemical interaction based on the DLVO theory forlyophobic colloids, a theory of cell adhesion favoured by Curtis (1973) and Gingell(1971). It is of interest in this respect that the macrophages spread normally in thepresence of CCB; these cells do not display spikes and it is possible that they spreadby means of the mechanism which is responsible for the partial spreading of amoebo-cytes in CCB.

It appears, from the aggregation experiments, that CCB interferes with the mech-anism whereby single cells develop initial adhesions rather than with the stability ofexisting adhesive contacts between cell surfaces. Aggregates, in which cells haveformed stable adhesive contacts, are not dispersed by concentrations of CCB andconditions of shear which completely inhibit the aggregation of single cells. Theinhibition could arise from the suggested inability of cells to form close contactsexcept by means of low-diameter probes (Pethica, 1961; Weiss, 1964), that is, bymeans of microspikes. On the other hand, it seems possible that cell surfaces doapproach one another closely enough to form adhesions, because amoebocytes areable to adhere to a glass surface in the presence of CCB. In this case, the inhibitionof aggregation by CCB could be explained by the low rate of spreading of amoebocytesin the absence of spike-supported lamellae, for the initial contact would have to berapidly stabilized, for example, by an increase in contacting area, to resist the highshear forces in shaken suspensions (Davies & Partridge, 1972). These alternativehypotheses of the mechanism of inhibition of aggregation by CCB are under investi-gation (Partridge & Jones, in preparation). There is some circumstantial evidence infavour of the view that the inhibition of aggregation induced by CCB operates by itseffect on spikes and the rate of stabilization of contact. Thus platelet aggregation,which is a fast aggregating system involving the production of spikes, is inhibitedby CCB (White, 1971; Haslam, 1972; Kay & Fudenberg, 1973), whereas the com-paratively slow aggregation of dissociated chick tissue cells which do not formprominent spikes, is not inhibited by CCB (Armstrong & Parenti, 1972; Steinberg &Wiseman, 1972).

It appears that the effects of CCB on the spreading and locomotion of amoebocyteson a glass surface and on the aggregation of cells in shaken suspensions can beattributed to the action of CCB on the integrity of the spikes, which seem to playimportant roles in these aspects of cell behaviour. Thus, the amoebocyte may repre-sent one extreme of spike-dependent cell behaviour while the macrophage falls at theother end of the range.

On this basis an investigation is currently in progress on the ultrastructure of the

394 G. E. Jones and T. Partridge

2 cell types, with regard especially to the role played by the spikes in the adhesionand locomotion of the amoebocyte. The effects of colchicine and CCB on theseprocesses should provide some indication of the mechanisms by which the spikesoperate in these aspects of cell behaviour.

REFERENCES

ALLISON, A. C. (1973). Microfilaments and microtubules in cell movement. In Locomotion ofTissue Cells, Ciba Fdn Symp. 14, pp. 109-143.

ARMSTRONG, P. B. & PARENT:, D. (1972). Cell sorting in the presence of cytochalasin B.J.CellBiol. 55, 542-553-

CARTER, S. B. (1967). Haptotaxis and the mechanism of cell motility. Nature, Lond. 213,256-261.

CARTER, S. B. (1972). The cytochalasins as research tools in cytology. Endeavour 31, 77-82.CURTIS, A. S. G. (i960). Area and volume measurements by random sampling methods.

Med. biol. Illust. 10, 261-266.CURTIS, A. S. G. (1973). Cell adhesion. Prog. Biophys. molec. Biol. 27, 317-386.DAVIES, P. S. & PARTRIDGE, T. (1972). Limpet haemocytes. I. Studies on aggregation and spike

formation. J. Cell Sci. I I , 757-770.GINGELL, D. (1971). Computed force and energy of membrane interaction. J. tlieor. Biol.

30, 121-149.GOLDMAN, R. D. (1972). The effects of cytochalasin B on the microfilaments of baby hamster

kidney (BHK-21) cells. J. Cell Biol. 52, 246-254.GOLDMAN, R. D. & FOLLETT, E. A. C. (1969). The structure of the major cell process of isolated

BHK21 fibroblasts. Expl Cell Res. 57, 263-276.HASLAM, R. J. (1972). Inhibition of blood platelet function by cytochalasins: effects on

thrombosthenin and glucose metabolism. Biochem. J. 127, 34 P.KAY, M. M. B. & FUDENBERG, H. H. (1973). Inhibition and reversal of platelet activation by

cytochalasin B or colcemid. Nature, Lond. 244, 288-289.MIZEL, S. B. & WILSON, L. (1972). Inhibition of the transport of several hexoses in mammalian

cells by cytochalasin B. J. biol. Client. 241, 4102-4105.PARTRIDGE, T. & DAVIES, P. S. (1974). Limpet haemocytes. II. The role of spikes in locomotion

and spreading. J. Cell Sci. 14, 319-330.PETHICA, B. A. (1961). The physical chemistry of cell adhesion. ExplCellRes., Suppl. 8, 123-140.PLAGEMANN, P. G. W. & ESTENSEN, R. D. (1972). Cytochalasin B. VI. Competitive inhibition

of nucleoside transport by cultured Novikoff rat hepatoma cells. J. Cell Biol. 55, 179-185.ROTHWEILER, W. & TAMM, C H . (1966). Isolation and structure of Phomin. Experievtia 22,

750-752.SPOONER, B. S., YAMADA, K. & WESSELLS, N. K. (1971). Microfilaments and cell locomotion.

J. Cell Biol. 49, 595-°13-STEINBERG, M. S. & WISEMAN, L. L. (1972). Do morphogenetic tissue rearrangements require

active cell movement? The reversible inhibition of cell sorting and tissue spreading byCCB. J. Cell Biol. 55, 606-615.

TAYLOR, A. C. & ROBBINS, E. (1963). Observations on microextensions from the surface ofisolated vertebrate cells. Devi Biol. 7, 660-673.

VASILIEV, JU. M., GELFAND, I. M., DOMNIA, L. V., IVANOVA, O. Y., KOMM, S. G. & OLSHEV-

SKAJA, L. V. (1970). Effect of colcemid on the locomotory behaviour of fibroblasts.J. Embryol. exp. Morph. 24, 625-640.

WEISS, L. (1964). Cellular locomotive pressure in relation to initial cell contacts. .7. tlieor. Biol.6, 275-281.

WEISS, L. (1972). Studies on cellular adhesion in tissue culture. XII . Some effects of cyto-chalasins and colchicine. Expl Cell Res. 74, 21-26.

WESSELLS, N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. O., LUDUENA, M. A., TAYLOR, E. L.,

WRENN, J. T. & YAMADA, K. M. (1971). Microfilaments in cellular and developmentalprocesses. Science, N.Y. 171, 135-143.

Effects of cytochalasin B and colchicine on haemocytes 395

WESSELLS, N. K., SPOONER, B. S. & LUDUENA, M.A.(i973).Surfacemovements, microfilamentsand cell locomotion. In Locomotion of Tissue Cells, Ciba Fdn Symp. 14, pp. 53-77.

WHITE, J. G. (1971). Platelet microtubules and microfilaments: effects of cytochalasin B onstructure and function. In Platelet Aggregation (ed. J. Caen), pp. 15-52. Paris: Masson etCie.

(Received 26 March 1974)

396 G. E. Jones and T. Partridge

Fig. 2. Limpet haemocytes suspended in a mixture of serum and artificial seawater(1: i), showing the spikes (s) which develop within a few minutes of withdrawal fromthe limpet.Fig. 3. Limpet haemocytes in suspension in a 1:1 mixture of serum and artificialseawater containing 025 /4g/cm3 cytochalasin B. Spikes do not develop in the presenceof cytochalasin B but smooth, rounded protrusions do form, giving the cell a mul-berry-like appearance in some cases.Fig. 4. Limpet haemocytes plated on to a glass slide in artificial seawater containing00625 ffi/cm3 of cytochalasin B and kept for 1 h. Even at so low a concentration ofcytochalasin B, spikes are absent. Spreading on to the substrate of smooth-edgedlamellar projections of the cells (p) occurs very slowly in comparison with the rapidspreading of control cells and similarly the extent of spreading is greatly reduced incomparison with that of the spike-supported lamellae of the amoebocytes in thefollowing figure.Fig. 5. Limpet haemocytes which have been allowed to spread for 1 h on a glass slidefrom a medium consisting of artificial seawater containing 1 mm'/cm3 DMSO. Theamoebocytes are well spread and show the normal bipolar morphology and spike-sup-ported lamellae of this cell type. A macrophage (m) is also present and although partiallyobscured by the phase halo of an amoebocyte, it is seen to have no obvious polarity andto have a smooth margin.

Figs. 6, 7. The effects of colchicine on spread amoebocytes. Fig. 6 shows amoebo-cytes which have been kept for 1 h in 50 /tg/cm3 colchicine in artificial seawater. Thecells show some signs of loss of bipolarity in that the spike-supported lamellae extendfrom an unusually large proportion of the cell margin. Fig. 7 illustrates the almosttotal loss of bipolar morphology after 4 h in colchicine.

Effects of cytochalasin B and colchidne on haemocytes

398 G. E. Jones and T. Partridge

Figs. 8-12. Sequence of micrographs of a group of 4 amoebocytes showing theeffects of addition of 0-125 /'g/cm3 cytochalasin B and the reversal of these effects onreplacement of the medium by artificial seawater.

Fig. 8. Group of amoebocytes in serum, fully spread on to a glass slide.Fig. 9. Six minutes after beginning to draw a drop of artificial seawater containing

0-125 /*g/cm3 cytochalasin B under the coverslip. Some retraction of the margin of thecell has occurred and simultaneously the spikes within the lamella have disappeared,lost phase density or broken up.

Fig. 10. After 15 min in cytochalasin B the spikes within the lamellae have de-generated further and retraction of the frontal lamellae is more marked. One cell (a)has retracted completely from the substrate leaving long retraction fibrils (rf) derivedfrom spikes which have retained their terminal attachments to the substrate. An inter-mediate stage of formation of retraction fibrils is seen in the case of the partiallyretracted cell.

Fig. 11. Fresh artificial seawater is being drawn under the coverslip; 3-5 min afterbeginning replacement of the medium with fresh artificial seawater the first new spikeformation is seen at the anterior margins of 2 of the cells (ro) and the lamellae havebegun to expand (compare with preceding figure).

Fig. 12. After 25 min recovery in artificial seawater the lamellae are well spreadand contain well developed spikes.

Effects of cytochalasin B and colchidne on haemocytes 399