SOME CLUES AS TO THE FORMATION OF PROTRUSIONS BY...

J. Cell Sci. 26, 139-150 (1977) 139Printed in Great Britain

SOME CLUES AS TO THE FORMATION OF

PROTRUSIONS BY FUNDULUS DEEP CELLS1

C. TICKLE* AND J. P. TRINKAUSf• Department 0/ Biology as Applied to Medicine, The Middlesex Hospital MedicalSchool, London WiP 6DB, U.K.,f Department of Biology, Yale University, New Haven, Connecticut 06570, U.S.A. and•f Marine Biological Laboratory, Woods Hole, Massachusetts 02543, U.S.A.

SUMMARY

One of the ways in which Fundulus deep cells move in vivo is by putting out long, fingerlikeprotrusions. This involves a change in the shape of the cell as a whole, with cytoplasmic flow,and is not just a local phenomenon. Moreover, particles on the cell surface move toward aprotrusion as it is forming, suggesting surface flow. The role of surface flow is discussed both ona gross level and in respect to molecular fluidity. Long, stable protrusions can be pulled fromcells by the application of negative pressure at a constant rate and these behave in a similarway to those formed during cell locomotion. Such long protrusions must be structured. Theimportance of contractile properties of the cytoplasm in the formation of protrusions wasstudied by treating cells with media that modify cellular contractility.

INTRODUCTION

A major problem of development is how cells move. It is now known from the studyof a number of different embryos and larvae that this is often accomplished by cellsextending protrusions of their surface which adhere and pull the cells along. Fundulusdeep cells form blebs and lobopodia, filopodia and lamellipodia (Trinkaus, 1973);sea-urchin mesenchyme cells and cells in the tunic of sea squirts bleb and extendlong filopodia (Gustafson & Wolpert, 1967; Izzard, 1974); and fibroblasts in the tailfin of an anuran larva and in the chick cornea extend long filopodia (Clark, 1912;Bard & Hay, 1975).

When a cell puts out a protrusion, this could involve a change in overall cell shape,with accompanying flow of both cell surface and cytoplasm, or a local change in thecell surface at the point where the protrusion is to be formed. Such a local surfacechange would also have to be followed by cytoplasmic flow in order to stabilizethe protrusion. If surface flow is involved, this could be from unfolding of the plasmamembrane (Wolpert & Gingell, 1969; Follett & Goldman, 1970; Erickson & Trinkaus,1976) or simply from adjacent smooth parts of the cell surface (Abercrombie, Heays-man & Pegrum, 1970; Frye & Edidin, 1970; Harris, 1973). A local surface changecould involve unfolding or assembly of new membrane at the point where the pro-trusion is being formed (Abercrombie et al. 1970; Bray, 1973; Harris, 1973).

1 We wish to dedicate this paper to Professor Katsuma Dan, a pioneer in the study of thebehaviour of the cell surface during changes in cell form, on the occasion of his seventiethbirthday.

140 C. Tickle andj. P. Trinkaus

Formation of a protrusion could, in principle, be due to stretching of the plasmamembrane. However, the one membrane that has been studied in detail appears tostretch little if at all. The surface area of the human erythrocyte remains constantthroughout the process of osmotic haemolysis (Canham & Parkinson, 1970; see alsoJay, 1973). In any case, whether the changes are solely local or not, the microfila-mentous cortex of the cytoplasm is probably important. It has been suggested, forexample, that protrusions form only in places where locally the cortex has becomeweakened (Harris, 1973), perhaps by detaching from the plasma membrane.

One way of studying the formation of protrusions of the cell surface is to createthem artificially. This may be done by applying negative pressure to the cell surfaceby means of a micropipette. When this is done to Fundulus deep cells, protrusionsare formed which greatly resemble blebs. Moreover, they form more readily on thesurface of the motile gastrula cells than on non-motile cells from blastulae (Tickle &Trinkaus, 1973); a parallelism which strengthens the thought that these artificialprotrusions are similar to those formed normally. There is an important difference,however. In our previous study, artificial protrusions broke up into globules when thelength of the protrusion exceeded its circumference, in accord with the principle ofPlateau (see D'Arcy Thompson, 1942, pp. 377-388). Rand & Burton (1964) madesimilar observations on red blood cells and concluded that the protrusions pulledwere acting as if they were water, with high tension at the surface. But normal lobo-podia put out by deep cells in vivo are frequently longer than their circumference(e.g. figs. 16 and 20, Trinkaus, 1973). It is clear that these protrusions are not simplyelongated blebs.

In order to shed light on how cells form surface protrusions in connexion withlocomotion, we have examined further both normal protrusions formed by deep cellsin vivo, and artificial protrusions formed upon the application of negative pressure tocell surface. The former were observed with time-lapse cinemicrography. The latterwere observed directly, in conjunction with marking of the cell surface, changing themethod of applying negative pressure, and treatment with media that affect cyto-plasmic contractility.

MATERIALS AND METHODS

Fertilized eggs of Fundulus heteroclitus were staged according to the series of Armstrong &Child (1965) and the chorion was removed surgically (Trinkaus, 1967). The surface behaviourof deep cells was filmed in vivo within blastoderms of whole eggs of blastula and gastrulastages with time-lapse cinemicrography (see Trinkaus, 1973, for details of technique). Forstudying the characteristics of artificial protrusions, deep cells were isolated in vitro in a simplenutrient medium and protrusions were pulled from them into a micropipette (inner diameter6 fim) by the application of negative pressure. This technique has been previously describedin detail (Tickle & Trinkaus, 1973).

Carbon marking of the cell surface was accomplished by spewing finely divided granules ofblood carbon (Merck) into the cell culture. We are confident that the carbon particles weobserved were firmly stuck to the cell surface because we used only those that were not dis-lodged by jets of medium being blown over the cells. Calcium-free medium was simply double-strength Holtfreter's solution lacking CaCla. Medium with augmented calcium was made bydoubling the concentration of CaCl, in the double-strength Holtfreter's solution, which is thesaline base for the nutrient culture medium. Cytochalasin B dissolved in dimethylsulphoxide

Formation of cell protrusions 141

(DMSO) was used in a final concentration of 10 /tg/ml cytochalasin B (1 % DMSO) in culturemedium. DMSO alone in culture medium at the same concentration had no effect on thecontractility of the yolk syncytial layer oiFundulus eggs (Betchaku & Trinkaus, in preparation).

RESULTS

Cytoplasmic flow during protrusion formation

Four gastrula cells (stage 17-19) were examined in films of deep cells moving duringdevelopment. The cells were chosen because they were clearly visible and in focusin this 3-dimensional system, while forming or resorbing protrusions. Measurementswere made either from stills of the films or on tracings made by projecting the film onto a screen and drawing round the cell outlines (see Table 1).

Table 1. Dimensions* of cells and protrusions of gastrula deep cells in vivo

TimefCell 1

0

4 0

9 0

130

Cell 20

2 0

34547494

1 1 4

Cell 30

321 1 2

Cell 40

30

68

Horizontaldiameter

25-0

22-5

2 2 5

2 2 5

1 9 220-824-024-020-82 1 42 0 8

i8-75i7'51 5 0

15-0

n-66i3'3

Cell bodyA

Verticaldiameter

2375

22-5

2 1 52 2 5

14-41 6 81 6 81 9 21 9 21 6 0

17-6

187517-5I7-5

1 6 61 6 61 5 0

RX

12-19

1125

10-9411-25

8 49-4

IO-2io-81 0 0

9-359 6

9388758 1 3

7-97 0 77-08

Length

(«)(*)(.a)

(b)(a)(a)

io-o7'5

11255-0

1 7 520-0

27-22 4 8I9-21 6 0

I2-O4 83-2

24-752 5 0

2 0

158-3

Cell protrusion

Width Circu

875io-oio-o1 0 0

io-oio-o

4-84-84 84 84-89 6

IO-2

5-o675

5 0

6-38-3

inference^

262731-4231-423I-4231-4231-42

15-08150815081508150830-173205

1 5 723-55

I5-7194925-77

• All dimensions are in fim.f In seconds.X Mean radius of the cell body, not including the protrusion.§ Calculated from the width of the protrusion, assuming that the protrusion is a cylinder.

Even very crude measurements, not taking into account changes in depth of thecell, show that the formation of a protrusion tends to decrease the size of the cellbody, both estimated in 2 dimensions. For instance, in cell 1 we can see from the

CEL 26


measurements that as one of the protrusions (a) grows longer, the dimensions of thecell decrease, even though the other protrusion (b) is being resorbed. Cell 2 hasinitially a long protrusion. This is resorbed during the observation period and thecell body increases in size. In cell 3 we see the reverse process. Here the cell is puttingout a protrusion during the observation period and the cell body decreases in size.Cell 4 is another case of protrusion resorption. Here, however, as the protrusionshortens it thickens and is accompanied by a decrease in the size of the cell body. Ingeneral, therefore, there must be cytoplasmic flow during the formation and retractionof protrusions of the cell surface.

Table 2. Dimensions of cells and protrusions pulled from gastrula deep cells in vitro

Cell 1 Cell 2 Cell 3 Cell 4 Cell 5

• Cell diameter 30 24 18 36 30 36 24 36 24 39 27§ Protrusion length o 45 60 o 45 o 78 o 54 o 75

• All dimensions are in fim.§ The protrusions were pulled into micropipettes with an inner diameter 6 fim, which were

brought up to the surface of the cell. The negative pressure in the micropipette was constantand varied from — i-o to — 1-5 cm of Brodie's fluid.

We compared the measurements of deep cells in vivo with measurements of pro-trusions pulled from gastrula deep cells in culture (stage 14) (see Table 2). In order toobtain long protrusions we reduced the pressure in the micropipette to a set degreeand then applied it to the cell surface (see also later). It is clear here also that the celldiameter decreases as long protrusions are pulled. In fact, the effect is quite dramatic,as under these conditions we can pull very long protrusions from the cells. Thesemeasurements are, of course, again very approximate, as it is difficult to make accuratemeasurements of the diameter of such spheres and changes in the depth of the cellwere not taken into account. However, it seems clear that cytoplasm must flow intoartificial protrusions, as well as normal ones, as they extend from the cell.

Surface flow during protrusion formation

Does this change in cell shape involve movement of the cell surface, as well ascytoplasmic flow? To answer this question, we marked the surface of deep cells inculture with particles of blood carbon and observed what happened to the particleswhen a protrusion was pulled from the cell. In 3 cases out of 4 the carbon particlesmoved immediately to the mouth of the pipette. This shows that there is surface flowtoward the pipette and the protrusion, when artificial protrusions are produced.

To see if the surface behaves in a similar way when cells actively put out pro-trusions, we observed the movements of carbon particles on cells blebbing in culture.Blebbing is a prelude to lobopodial formation (Trinkaus, 1973) and can be readilywatched under the microscope. If blebs form close to the marks, the marks moveimmediately in the direction of the extending bleb, showing that there is surface flowwhen normal protrusions are produced. It is rather interesting that the marks seem to


move in a sudden surging motion towards the extending bleb, and, then if the blebextends sideways (circus movement) or is resorbed, remain stationary. A bleb wasnever observed to form directly under a carbon particle, so that we do not knowwhether, in such a case, the mark would move backwards toward the cell body as thebleb is resorbed. Carbon particles distant from the bleb did not appear to move at all.So we conclude that when a bleb is formed the membrane is affected only in thevicinity of the bleb.

Stability of protrusions

We have previously noticed that artificial protrusions broke up into globules whenthe length exceeded the circumference (Tickle & Trinkaus, 1973). Normal pro-trusions put out by deep cells in vivo, however, are frequently longer than theircircumference (Table 1). For example cell 2 in Table 1 has a protrusion 27-2/tm inlength and only 15-08 /im in circumference. It seems, therefore, that normal pro-trusions must be structured.

In consequence, it seemed important to try to simulate in vitro the protrusionsformed in vivo. In our previous experiments (Tickle & Trinkaus, 1973), we decreasedthe pressure applied to the cell surface at a steady rate. However, it seems unlikelythat the internal hydrostatic pressure of the cell, which is no doubt in part responsiblefor the thrusting out of protrusions normally (Harris, 1973; DiPasquale, 19756),would be increasing as rapidly as this. Indeed, cells are often observed to form onebleb and retract another at the same time (e.g. cell 1 of Table 1; fig. 4 in Trinkaus,1973). On the contrary, it seems more likely that the internal hydrostatic pressure isnormally a rather constant force, thrusting out protrusions here rather than there,because of local factors, such as a weakening of the cortical cytoplasm. We tried tosimulate this hypothetical situation in vitro by applying negative pressure to the cellsurface at a constant rate. Thus, we reduced the pressure in the micropipette to a setdegree and then applied it to the cell surface. When this was done, we found that wepulled much longer protrusions which were more like those formed in vivo. We mademeasurements on gastrula cells (stage 14) in culture. Pressure was reduced to — i-o to— 1-5 cm of negative pressure of Brodie's fluid. Since the internal diameter of thepipette was 6/im, the circumference of each protrusion is 18/im. Protrusions of15 /im length were pulled from the cells in 30 s (3 measurements) and 30 /im lengthin 10-25 s (4 measurements). Under these conditions, none of these protrusionsbroke up into globules; all were stable for between 1 and 2 min and did not change inlength. After this time, the protrusions grew rapidly, in a matter of seconds, to reachlengths of between 36 and 60 /im. When expelled from the micropipette by applyingpositive pressure, these long protrusions were resorbed in 1-4 min. The short pro-trusions (of about 15 /<m length or less) were resorbed rapidly, usually in about 30 s.Following resorption of the protrusions, the surface of the cell was seen to bleb.

Role of microfilaments

Having established that we could pull protrusions from cells which closely resemblethe protrusions put out by cells normally in vivo by applying negative pressure at a


constant rate, we wanted to see what effect compounds implicated in disturbing thecontractile cortical microfilaments would have on artificial protrusions. The corticalcytoplasm of Fundulus deep cells is rich in microfilaments and long, thin protrusionsappear to contain little else (Hogan & Trinkaus, 1977). When we isolated cells in theabsence of calcium, the cells became thin and flattened on the substratum within afew minutes, producing a fan-like lamella around the edges of the cell. As these cellswere very fragile and tended to burst when touched with a micropipette, it wasdifficult to obtain many measurements of their deformability. In fact, so few measure-ments were obtained that we cannot state whether the cells are indeed more easilydeformed, as their flattened morphology would suggest. It seems that the cells are verysensitive to deprivation of calcium. We also isolated cells in nutrient medium withaugmented calcium. The cells in this medium remain rounded and appear to bleb inthe same way as in normal medium. In the presence of cytochalasin B, the cells didnot bleb. Cells from stage 13^ embryos (early gastrula) were placed in a solution ofcytochalasin B and during 10 min of observation no blebs were initiated. Whennegative pressure was applied to these cells, no protrusions formed in the micro-pipette, rather threads which seemed to be very sticky were pulled off the cells,suggesting a separation of the cell surface from the cortical cytoplasm.

When cytochalasin B was used at this concentration on L cells, nuclei were extrudedfrom some cells but remained attached to the cell body by sticky threads of membraneand cytoplasm (Carter, 1967).

DISCUSSION

Cytoplasmic and surface flow

The change in shape of a Fundulus deep cell during formation of a protrusion fromthe cell surface, whether the protrusion forms normally in vivo or artificially due to theapplication of negative pressure in vitro, is clear evidence that the whole cell is in-volved in protrusion formation and that cytoplasm flows from the cell body into theprotrusion. Protrusion formation cannot be a purely local phenomenon confined to thepart of the cell where the protrusion forms. The considerable cytoplasmic flow thattakes place in these cells suggests a relatively fluid cytoplasm. Consistent with thisthey possess relatively small numbers of microtubules (Hogan & Trinkaus, 1977).The flow of cytoplasm from the cell body into an extending protrusion of its surfacereminds us of the way in which pseudopodia form in amoeboid movement and suggeststhat these vertebrate embryonic cells may be similar to free-living amoebae in theirmanner of movement. However, we have not been able to observe certain details ofthis flow, such as the comparative behaviour of the endoplasm and ectoplasm andsol-gel transformations, which are important in amoeboid movement.

Since there is cytoplasmic flow in these cells and since plasma membranes areknown to be fluid at physiological temperatures (Frye & Edidin, 1970; Singer, 1971),it comes as no surprise that the cell surface of a Fundulus deep cell also flows toward aforming protrusion, as indicated by the movement of carbon particles adhering to thecell surface. This is clear evidence that some of the local increase in cell surface that


occurs as a protrusion forms is due to recruitment of plasma membrane from the cellsurface in the vicinity of the protrusion. It cannot be discounted, however, that localevents might also contribute to the surface of a protrusion, such as assembly of newmembrane (Abercrombie et al. 1970; Harris, 1973). Unfolding of microvilli or folds ofthe cell surface, as suggested for fibroblasts (Wolpert & Gingell, 1969; Follett &Goldman, 1970; Erickson & Trinkaus, 1976) and Amoeba (Czarska & Grebecki,1966), do not seem to be important because in TEM (Hogan & Trinkaus, 1977) andin SEM micrographs (Trinkaus & Erickson, unpublished) the surfaces of deep cellsappear to be quite smooth. Wolpert, Thompson & O'Neill (1964) have shown withmarking experiments that there is also membrane flow in amoebae as pseudopodiaform. So, in this respect, as well as in their possession of cytoplasmic flow, Fundulusdeep cells are similar to Amoeba.

Further evidence to support the idea that surface flow makes an important contri-bution to formation of protrusions could be obtained by examining a situation inwhich surface flow might be restricted; namely, in clumps of cells where the indi-vidual cells are stuck to each other. We believe that there are a number of observationsthat could be explained if surface were not free to flow in areas of contact betweencells. Cells in epithelial cell sheets, which are in contact with and surrounded by othercells do not phagocytose particles dropped on them (Vasiliev et al. 1975). Neuraltube closure involves a shortening in the apical width of the cells and with this theapical surface is thrown into folds and microvilli (Baker & Schroeder, 1967), as ifthe surface could not flow away down the side of the cells because of their circum-ferential contacts. The number of protrusions formed by amphibian gastrula cells inculture is reduced if the cells are in clumps (Holtfreter, 1947). In addition, there isevidence at the molecular level that lipid in the region of membrane proteins is not asfree to move and diffuse as lipid further away (Jost, Griffith, Capaldi & Vanderkoi,1973; Hong & Hubbell, 1972; see discussion in Edidin, 1974). Membrane proteinshave been shown to be concentrated in areas of cell contact (McNutt & Weinstein,1973). In view of these considerations, we would predict that the deformability of acell (the ease with which protrusions can be pulled from its surface) would be de-creased if the cell were adherent to other cells. We have made some such measure-ments but the results were rather variable, perhaps because of varying amounts ofadhesion to other cells. We are not able at present to draw any conclusions.

We showed in a previous study that gastrula cells are more deformable than blastulacells (Tickle & Trinkaus, 1973). Is it possible that gastrula cells are more deformablein part because surface flow takes place more readily in them? This awaits investi-gation. Recently, Johnson & Smith (1976) have shown that amphibian gastrulacells cap fluorescein-labelled concanavalin A, whereas blastula cells do not. Thegreater lateral mobility of bound Con A in gastrula cells may reflect greater intrinsicfluidity of the lipid bilayer. However, this is not the only possible interpretation (seeNicolson, 1974), and, as Edidin & Weiss (1972) have pointed out, although cappingimplies membrane fluidity, it also depends on the locomotory machinery of cells. It isnot surprising, therefore, that mobile gastrula cells form caps whereas sedentaryblastula cells do not. What is somewhat surprising is the result of Roberson, Neri &


Oppenheimer (1975) in a similar study of embryonic sea-urchin cells. They foundthat micromeres of a 32-64 cell stage form caps many hours before they give rise totheir motile descendants, the primary mesenchyme cells.

The flow of cell surface toward a forming protrusion in Fundulus deep cells, ofcourse, contrasts sharply with the situation in moving fibroblasts, epithelial cells, andneurites, where surface flow is away from the protruding leading edge of the cell(Abercrombie et al. 1970; Edidin & Weiss, 1972; Bray, 1973; Harris, 1973; DiPas-quale, 1975 a). We have no explanation for this difference at present. However, it isperhaps relevant to point out that in our study, even though the cells were formingprotrusions of the sort that might be used as locomotory organs, in fact the cells werenot moving. The behaviour of particles on the surface of moving Fundulus deep cellsin vitro has not been studied, because we have not yet succeeded in creating cultureconditions propitious for movement. Nor to our knowledge, has anyone studied thebehaviour of particles on the surfaces of stationary fibroblasts and epithelial cellsduring blebbing or during their initial spreading. However, since the initial expansionof the surface of a fibroblast takes place at the cell margin, with microvilli and blebsdisappearing submarginally as the cell continues to spread (Erickson & Trinkaus,1976), it seems possible that here too, during this initial phase of fibroblast spreading(prior to locomotion), there is some flow of membrane toward the protuberancesforming at the cell margin.

Localization and stability of protrusions

It is clear from the present study that stable protrusions that lengthen rapidly andresemble normal lobopodia can be produced artificially, if a constant value of negativepressure is applied to the cell surface. This suggests that the formation of elongateprotrusions is due normally in part to a constant level of internal hydrostatic pressure.Although this is an interesting suggestion, it does not solve the problem of how aprotrusion forms at one point on the cell surface rather than another. Harris (1973)has proposed that blebbing in tissue culture cells may be due to local weakeningof peripheral resistance to this pressure by functional uncoupling of the cell surfaceand the cortex of the cytoplasm. This appears to be just what happens during blebformation. Although the cortical microfilament layer of a deep cell seems intact acrossthe base of a bleb (Trinkaus & Lentz, 1967), the cortical cytoplasm of the bleb itselfeither lacks microfilaments or has relatively few (Betchaku & Trinkaus, unpublished).A similar situation has also been found in blebs of certain cell lines (L. B. Chen,unpublished; and C. A. Erickson, unpublished), and of gastrula blastomeres ofOryzias latipes in culture (Fujinami, 1976: fig. i), suggesting that this might be awidespread phenomenon.

Although in view of these observations it seems possible that the microfilamentouscortex plays a determining role in the localization of cell protrusions, changes in thecell membrane itself are by no means excluded. If, for example, certain moleculesintercalated mainly into the lipid in the outer half of a particular region of the lipidbilayer, that half of the bilayer would expand at that point and the plasma membranewould evaginate and form a protrusion. We are not aware of examples of this occur-


ring normally. However, the possibility surely exists and Sheetz & Singer (1974)have shown that multiple evaginations of the cell surface appear when erythrocytesare treated with impermeable amphipathic drugs that insert differentially into theouter half of the bilayer.

A long protrusion from a deep cell in vivo usually arises from a bleb (Trinkaus,1973). Frequently, however, these blebs do not grow into long protrusions but eitherare resorbed or, significantly, may spread round the cell in a so-called limnicola orcircus movement (Holtfreter, 1943). Blebs involved in this lateral spreading do notextend to form long protrusions. This suggests that in blastula cells, where limnicolamovements are frequent, localization of protrusions is poor and may in fact be one ofthe reasons why early blastula cells do not form long protrusions.

Another important question concerns the stability of these long protrusions. Justhow are they stabilized ? Their length, stability, and varying form suggest that they arestructured (Rayleigh, 1892; Harvey, 1954). TEM shows long, thin protrusions of deepcells to be filled with a meshwork of microfilaments (Hogan & Trinkaus, 1977), asindeed are the long necks of bottle cells of the amphibian blastopore (Baker, 1965) andof the chick primitive streak (Balinsky & Walther, 1961). Moreover, it has been shownthat the filopodia and microspikes of fibroblasts (Spooner, Yamada & Wessells,1971), epithelial cells (DiPasquale, 19750,6), and neurites (Wessells, Spooner &Luduena, 1973) moving in culture invariably contain an apparently similar meshworkof microfilaments. When these cells are treated with cytochalasin B, these protu-berances retract, suggesting that microfilaments inserting on the plasma membrane(Miranda, Godman & Tanenbaum, 1974) are necessary for maintenance of their form.

Significantly, blebs, which in contrast are short, have an invariant hemisphericform, are unstable and almost always short-lived (Trinkaus, 1973), appear to berelatively structureless. Their cytoplasm is characteristically hyaline and devoid ofeytoplasmic organelles such as mitochondria, microtubules, Golgi and endoplasmicreticulum (Trinkaus & Lentz, 1967), and, as already mentioned, there is a distinctthinning or absence of their microfilamentous cortex. A similar absence of organelles,as well as an absence or thinning of the microfilamentous cortex, has also been foundin the blebs of certain cell lines in culture (L. B. Chen, unpublished; C. A. Erickson,unpublished).

The absence of microtubules in blebs of Fundulus deep cells is consistent with theeffect of treating these cells with colchicine. Blebbing is not inhibited (Trinkaus &Bell, unpublished).

In their early studies of cell deformability, Mitchison & Swann (1954) suggestedprophetically that the eytoplasmic cortex is somehow involved in giving stability tocell form. Since we now know that microfilaments are usually most abundant in thecortical cytoplasm, it seems possible that they have a cytoskeletal function along withpresumed contractile activity and are thus responsible for this cortical stability.Deformability studies on cleaving sea-urchin eggs have provided strong support forthis hypothesis (Wolpert, 1966). As the cleavage furrow forms and its thickenedcircumferential band of cortical microfilaments becomes evident (Schroeder, 1972),its resistance to deformation increases. The effects of calcium-free medium and of


cytochalasin B on the deformability of Fundulus deep cells are also consistent withthis hypothesis and suggest that cortical microfilaments play a cytoskeletal role andgive stability to the form of these cells as well. When calcium is removed from themedium, deep cells become less rigid and short protrusions can be pulled easily, andwith treatment by cytochalasin, the membrane appears to be readily pulled off thecell surface. The absence of calcium presumably inhibits the contractile activity of themicrofilaments, which are present in the cortex of deep cells in abundance (Hogan &Trinkaus, 1977), and thus render the cells more deformable. Cytochalasin may freethe plasma membrane of its attached cortical microfilaments (Miranda et al. 1974)and thus make the cell surface so deformable that application of negative pressureactually tears off pieces of surface membrane.

This research was supported by grants (to J.P.T.) from the NSF (BMS 70-00610) and theNIH (USPHS-HD 07137).

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(Received 15 December 1976)

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