/ . Embryo/, exp. Morph. Vol. 24, 3, pp. 625-640, 1970 6 2 5

Printed in Great Britain

Effect of colcemid on the locomotorybehaviour of fibroblasts

By Ju. M. VASILIEV,11. M. GELFAND, L. V. DOMNINA,O. Y. 1VANOVA, S. G. KOMM AND L. V. OLSHEVSKAJA

From the Institute of Experimental and Clinical Oncologyand the Laboratory of Scientific Cinematography of the

Academy of Medical Sciences of USSR, Laboratory of MathematicalBiology of Moscow State University, Moscow, U.S.S.R.

SUMMARYEffects of metaphase inhibitors (colcemid, colchicine, vinblastine) on mouse and human

embryonic, fibroblast-like cells growing on glass and on an oriented substrate (fish scale)were studied. All three inhibitors caused similar changes in the form of interphase cells andinhibited their directional locomotion. The effects of two inhibitors (colcemid and vin-blastine) were found to be completely reversible. Microcinematographic studies have shownthat the most conspicuous change of locomotory behaviour induced by colcemid was thedisappearance of non-active stable parts of the cell edge; in normal cells only the leadingpart of the edge was actively moving, while in colcemid-treated cells all parts of the edgeeventually became active. Activation of the whole edge made these cells unable to performdirectional translocation.

It is suggested that colcemid and other metaphase inhibitors prevent stabilization of thenon-active state of the cell surface. The possible role of this suggested colcemid-sensitivestabilization mechanism in the normal locomotory behaviour of fibroblasts is discussed.

Electron-microscopic examination has shown that microtubules disappeared from thecytoplasm of colcemid-treated, mouse, fibroblast-like cells. The formation of microtubulesas the possible structural basis of the stabilization of the non-active state of the cell surfaceis discussed.

INTRODUCTION

The main manifestations of the normal locomotory behaviour of culturedfibroblasts and the regulation of this behaviour by cell-cell and cell-substrateinteractions are well known (Abercrombie, 1961, 1965, 1967; Ingram, 1969;Weiss, 1958, 1961, and others). To gain more insight into the possible me-chanisms of these activities it is important to study in detail the effects ofvarious types of inhibitors upon locomotion. Mitotic spindle poisons (colchicine,colcemid, vinblastine) are of special interest in this connection. Besides a charac-teristic action on mitosis, these drugs change the cell form and affect locomotionin a variety of interphase cells such as myoblasts (Godman & Murray, 1953;Bischoff & Holtzer, 1968; Warren, 1968), leucocytes (Malawista & Benesch,

1 Author's address: Institute of Experimental and Clinical Oncology, 6 KaskirskojeShosse, Moscow 478, U.S.S.R.

626 JU. M. VASILIEV AND OTHERS

1967), mast cells (Padawer, 1968), fibroblasts (Vasiliev, Gelfand, Domnina &Rappoport, 1969) and others.

The aim of the experiments described in this paper was to study in detail theeffects of colcemid upon the form and locomotory behaviour of mouse andhuman embryonic, fibroblast-like cells. Results of some experiments with twoother mitotic spindle poisons (colchicine and vinblastine) are also presented.

The results of our experiments give reason to suggest that colcemidselectively affects stabilization of the cell surface, that is, its tendency to remainnon-active. The essential role of this colcemid-sensitive process of surface stabili-zation in the locomotion of normal cells and its possible structural basis arediscussed in the last part of the paper.

MATERIALS AND METHODS

Two types of cells were used: (a) fibroblast-like cells obtained by thetrypsinization of 18- to 20-day-old mouse embryos. The cultures of thesecells were in their first passage, (b) A line of human embryonic, fibroblast-likecells obtained in the Institute of Virus Preparations (Moscow) by the cultiva-tion of cells from the skin of human embryos. These cells were used in their8-2Oth passages. The cells were cultivated in Petri dishes or in penicillin flasks.To study the effect of drugs upon cell migration, wounds were made in 6- to 8-day-old cultures. Cultivation procedures and methods of wounding wereidentical to those described earlier (Vasiliev et al. 1969).

To study the effect of the drugs upon cell orientation we used culturesgrown on the internal surface of fish scales as suggested by Weiss & Taylor(1956). Scales of C. carpio were thoroughly washed, stored in 96 % ethyl alcohol,washed in culture medium and placed at the bottom of culture flasks before thecell suspension was added to the flask. To study the effect of drugs on phago-cytosis, a carmine suspension was added to the medium of 1- to 2-day-oldcultures on glass, simultaneously with colcemid; cultures were examined 6 and24 h later. In various types of experiments living cultures on glass were ex-amined and photographed under a phase-contrast microscope. Cultures grownon glass and on scales were fixed in a mixture of ethyl alcohol and acetic acid(3:1) and stained with Mayer or Jasswoin haematoxylin.

Cultures subjected to electron-microscopic examination were fixed in 5 %glutaraldehyde, then in 2 % osmic acid, and embedded in Araldite. Sectionswere stained with uranyl acetate and Reynolds's lead citrate.

Cultures for time-lapse microcinematography were grown in special chamberswith parallel glass walls. Eagle's culture medium with 10 % bovine serum wasused; the medium in the chambers was changed every 48 h. Cultures werephotographed on 35 mm film with a phase-contrast objective (NA 0-65). In-tervals between frames were 30 or 60 sec; exposure was 1-0-1-5 sec. Developedfilms were examined frame by frame on the projection screen.

Locomotion of fibroblasts 627

RESULTS

General characteristics of the action ofmitoticspindle poisons on fibroblast-like cells

In control culture on glass most cells had a bipolar form; human cells weremore elongated than mouse cells. On fish scales both human and mouse cellsacquired a more elongated form as compared with the same cells on glass (Fig. 2).When colcemid (Ciba, 0-1-0-05/tg/ml) was added to the medium the form ofmost cells changed; long cytoplasmic processes disappeared and the cellsacquired an irregular polygonal form (Figs. 1B, 2B). Often these cells had shortcytoplasmic processes (Fig. 3). These changes of cell form were evident 3-4 hafter the addition of colcemid; in the cultures on scales they developed some-what later (after 10—12 h). As described earlier (Vasiliev et al. 1969) when colce-mid was added to the medium of wounded cultures cell migration into the woundwas stopped a few hours later. Cells blocked in metaphase were often seen incolcemid-treated cultures; later stages of mitosis were absent. A lower concen-tration of colcemid (001 /tg/ml) did not change the cell shape and did notproduce metaphase arrest. Concentrations of colcemid that changed cell formdid not cause any non-specific toxic effects. After 24 h of incubation withcolcemid (01 /*g/ml) the mean number of cells per unit area of culture did notdecrease as compared with cultures before the incubation. The percentage ofcells synthesizing DNA determined autoradiographically in cultures pulse-labelled with [3H]thymidine also was not decreased in these cultures. Colcemid-treated cells phagocytosed particles of carmine as actively as control cells.

If cultures were placed at 4 °C after addition of colcemid, changes of cell formwere not observed in the following 24 h.

Alterations of cell form similar to those caused by colcemid were alsoproduced by two other mitotic spindle poisons: vinblastine sulphate (Richter,Hungary; effective concentrations 005-0005/*g/ml) and colchicine (effectiveconcentrations 0-1-0-01 /tg/ml). Changes produced by all effective concentra-tions of colcemid and vinblastine and by low concentration of colchicine(0-01 /«g/ml) were reversible: if fresh medium was substituted for that containingthe drug, normal cell shape was completely restored 24 h later (Fig. 1C).Normal orientation of cells with regard to the wound edge or to the fibres ofthe scale was also restored. Cells treated with a high concentration of colchicine(01 /fcg/ml) retained their abnormal form 24 h after removal of the drug.

If cultures pre-incubated 24 h with colcemid were then transferred intofresh medium containing puromycin (5-10 /*g/ml) or actinomycin D (0-1 /^g/ml)these inhibitors did not prevent restoration of the normal bipolar cell form.If cultures pre-incubated with colcemid were then transferred into fresh mediumand placed at 4 °C restoration of the normal form was not observed 24 h later.

In a series of experiments the effect of colcemid upon cell attachment to thesubstrate was studied. Colcemid (0-1/tg/ml) was added to culture medium

628 JU. M. YASILIEV AND OTHERS

Fig. 1 Figs. 2 & 3

Fig. 1. Effect of colcemid upon the form of mouse embryo fibroblast-like cells.Phase-contrast optics.(A) Elongated cells in control 6-day-old culture.(B) Changed form of the cells in culture incubated 6 h with colcemid (0-1 /ig/ml).(C) Restoration of normal cell form in culture incubated 24 h with colcemid andthen another 24 h in fresh medium without colcemid.Fig. 2. Effect of colcemid upon the form and orientation of mouse embryo fibro-blast-like cells growing on the surface of the fish scale. Jasswoin haematoxylinstain.

(A) Control culture.(B) Culture incubated 24 h with colcemid (01 /*g/ml).Fig. 3. Mouse fibroblast from culture incubated 24 h with colcemid. Isolatedcell with protrusions of various parts of the cell edge. Phase-contrast optics.

Locomotion offibroblasts 629(Eagle's medium plus 10 % bovine serum) containing a suspension of mouseembryo fibroblasts (3 x 105 cell/ml). Two ml of suspension were placed in eachpenicillin flask containing one 10 x 20 mm coverslip at the bottom. Cultures werekept 6 h at 37 °C, then the coverslips were washed several times in Hanks'solution, fixed and stained. The average number of cells attached to the glassper unit area was counted. The ratio of nuclear overlaps (number of observedoverlaps:number of theoretically expected overlaps) was also counted. Theformula of Abercrombie & Heaysman (1954) modified by Curtis (1961) wasused. The density of attached cells was similar in control and in colcemid-treatedcultures. The ratio of nuclear overlaps was lower in colcemid-treated cultures(014 ± 002 as compared with 0-49 ± 003 in the controls). Thus colcemid had noeffect upon cell attachment to the glass and did not increase cell attachmentto the surface of normal cells. The reduction of nuclear overlaps by colcemidwas possibly related to reduced migratory-activity of the cells, so that colcemidtreated fibroblasts became less able to migrate across the surface of other cells.

Cells of 2-day-old mouse cultures grown for 24 h in the medium containingcolcemid (0-1 /tg/ml) as well as control cultures were subjected to an electron-microscopic examination. The ultrastructure of mouse fibroblasts in controlcultures was similar to that described by previous authors (Movat & Fernando,1962; Cornell, 1969; Goldman & Follett, 1969).

The main differences in the ultrastructure of normal and colcemid-treated cellswere those related to the presence of microtubules in the cytoplasm. Cells in con-trol cultures contained numerous microtubules of various length and about200-250 A in diameter. Microtubules were present in various parts of the cyto-plasm but were especially numerous in wide cytoplasmic processes. Usuallythey were approximately parallel to the lateral surfaces of these processes andended near the anterior ends of the process (Fig. 4 A). In the cytoplasm of thecentral part of the cell body microtubules near the cell surface were usuallyparallel to the long axis of the cell; the orientation of microtubules locatedfarther from the surface was less regular.

Microtubules were not seen in the cytoplasm of fibroblasts treated for 24 hwith colcemid (Fig. 4B). The cytoplasm of these cells contained more free ribo-somes and fewer membrane-bound ribosomes than did that of control cells. Thestructure of other organelles was similar in control and in colcemid-treatedcells.

Microcinematographic studies of cell locomotion

(a) Control cultures. In most experiments 6- to 8-day-old mouse cultureswere filmed; wounds were made 1-2 h before the filming was started. A field ofview containing part of the wound edge was usually selected for filming, so thatmost cells in the field moved approximately in the same direction: from themonolayer into the wound (Fig. 5 A).

Changes accompanying the locomotion of mouse fibroblasts were similar to

630 JU. M. VASILIEV AND OTHERS

Fig. 4. Effect of colcemid upon the microtubules in the cytoplasm of mouseembryo fibroblast-like cells. Electron micrographs.(A) The end of a cytoplasmic process of a cell from the control culture. The cyto-plasm contains numerous microtubules.(B) Peripheral part of the cytoplasm of a cell from a culture incubated 24 h withcolcemid (0-2/*g/ml): microtubules are not seen.

Locomotion of fibroblasts 631those described by previous investigators (Abercrombie, 1961, 1965; Aber-crombie & Ambrose, 1958; Weiss, 1958, 1961, and others). Active and non-active parts of the cell edge could be distinguished in the moving cell CFig. 6).The active leading edge fluctuated backwards and forwards; local protrusionsand withdrawals were repeatedly seen at that part of the edge. The lateral non-

D10/i "

Fig. 5. Trajectories of movements of cell nuclei near the edges of the wounds incontrol (A) and in colcemid-treated (B) mouse embryonic cell cultures. Each lineshows changes of the position of the projection of one nucleus in relation to the fieldof view. This position was determined at intervals of 30 min (points). Directionof movements of each nucleus is shown by the arrow.

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632 JU. M. VASILIEV AND OTHERS

active parts of the edge had a smoother outline and did not fluctuate; theirform was slowly changed in the course of locomotion.

Moving human cells were somewhat different from mouse cells; some of thecytoplasmic processes formed in the anterior part of these cells underwent con-siderable elongation. Sometimes the whole anterior part of the cell underwentelongation and was transformed into a narrow strand of cytoplasm with aflattened leading lamella at its anterior end. Later these long processes oftenunderwent contraction accompanied by the forward translocation of the wholecell body.

Fig. 6. Outline of the edges of a human cell in control culture. Several drawings ofthe same cell were made from the projected film frames. Figures near the arrowsshow the intervals (in min) between frames from which consecutive drawings weremade. Consecutive drawings show changes of the form of the same cell but not ofits position in the field.

When the edges of two cells in human or mouse cultures touched each other,typical manifestations of contact inhibition were observed: immobilization ofthe contacting part of the leading edge, followed by the changes in the directionof locomotion and often by the formation of a relatively firm adhesion betweenthe cells.

(b) Cultures incubated with colcemid. Conditions of filming were similar tothose in control experiments except that colcemid (0-1 /*g/ml) was added to themedium 1 or 2 h before the first frame was taken. Wounds were made 2 h or24 h before the filming began.

During the first 2-8 h, gradual contraction of the long cytoplasmic processeswas observed and the cell form became irregular. Simultaneously the distribu-tion of active and non-active parts of the cell edge gradually changed. Thefluctuations previously observed only in the leading parts of the cell edge gradu-ally spread to all of the free edge, that is to say to all those parts of the edgethat were not in contact with the edges of other cells. Smooth non-active partsof the free edges disappeared (Fig. 7). Those parts of the edges that were contact-ing other cells at this stage remained smooth and non-active. Protrusions that

Locomotion of fibroblasts 633arose and disappeared repeatedly at the free edges did not usually undergo con-siderable elongation. If the protrusion was relatively large, secondary smalloutgrowths were often formed at its edges.

No stable differences in the character of movements of various parts of theedge of isolated cells were observed: certain parts of the edge of isolated cellscould temporarily move more actively than others but these differences werenot substantial and the distribution of more and less active parts often changed.

10//

20

8

Fig. 7. Outline of an isolated human cell from a culture incubated with colcemid.The first drawing was made from the photograph taken after 8 h of incubationwith colcemid. Irregular fluctuations of the whole cell edge are seen.Fig. 8. Outline of the edge of a mouse cell from culture contacting two surfacesof other cells. The first drawing was made from the photograph taken after 12 hof incubation with colcemid.

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634 JU. M. VASILIEV AND OTHERS

At later stages of the action of colcemid (8-12 h and later) the character of theactivity of the free edges did not change. This stage was characterized by gradualactivation of those parts of the edge that were contacting other cells: theseparts of the edge began to move, and their form became irregular (Fig. 8).Usually movements of the two opposite parts of the contacting edges of twocells were activated simultaneously. Those parts of the contacting edges thatwere nearer to the free edges were activated earlier. Activation of contactingedges was often accompanied by their retracting slightly so that a narrow gapwas formed between the cells. Protrusions of activated cell edges moved forwardacross this gap but stopped their forward movement when they touched thesurface of other cells. Movement of cell protrusions across the surface of othercells was very rare. Usually after contact with other cells these protrusionswithdrew, either immediately or after some delay. There were no signs of theformation of firm cell-cell attachments at these contacts; withdrawal ofthe protrusions after contact with the other cell did not leave any strands ofcytoplasm between the two cells. Thus at this stage of incubation with colcemidall parts of the cell edges became active. However, as the movement of thesurface was stopped by contact with other cells, the range of displacementsremained smaller in those parts of the edge that were near to the edges ofother cells.

Activation of the cell edges in colcemid-treated cells was accompanied bygradual cessation of the oriented translocation of these cells into the wound.The position of many nuclei remained unchanged for hours. Those small dis-placements that were observed were of random character (Fig. 5B). Highlyrefractile round cells blocked in mitosis were often seen in filmed colcemid-treated cultures.

In several experiments the restoration of normal cell shape and movementsafter removal of colcemid was photographed. The filming was begun 1 h aftersubstitution of fresh for colcemid-containing medium. The first manifestation ofthe cessation of the colcemid effect was the end of the mitotic block: normalcell division was observed in these cultures even in the first 2 h after removal ofcolcemid. Inactivation of contacting cell edges took place in the following fewhours; it was accompanied by the gradual elongation of the cells. Normalorientation of cells with regard to the wound margin and normal locomotioninto the wound was restored after 15-20 h.

DISCUSSION

Action of mitotic spindle poisons on inter phase fibroblasts

The experiments described above show that several substances which selec-tively affect the mitotic spindle also cause characteristic changes of the form andlocomotory activity of interphase fibroblasts. The minimal concentrations ofthese substances which affected interphase cells were similar to those causing

Locomotion offibroblasts 635metaphase block. The effect of two inhibitors (colcemid and vinblastine) wasfound to be completely reversible.

Colcemid-treated fibroblasts did not migrate into a wound made in a culture.At the same time very active fluctuations of their edges were observed, andthese cells retained the ability to phagocytose particles. Thus cessation of anoriented locomotion was not due to the inhibition of active movements of the

Fig. 9. Curves approximating the outline of a cell with fluctuating edges. Curves areobtained by random changes of the length of the radius in polar co-ordinates. Initiallength of the radius was equal to that of the semicircle. Each 5° the length of radiuswas randomly changed to ± one-tenth of the initial length. Three examples ofcurves obtained by this process are shown.

cell surface. Analysis of microcinematographic data shows that colcemid de-creases the ability of cell edges to retain their stable shape. The leading edge ofnormal fibroblasts fluctuates forwards and backwards. As shown by M. Aber-crombie, J. E. M. Heaysman and S. M. Petrum (personal communication),points a few microns apart on the leading edge perform independent movements.We have not yet made detailed measurements of the displacements of the celledge. Nevertheless our observations make it plausible to assume that all the edge

636 JU. M. VASILIEV AND OTHERS

of colcemid-treated fibroblasts performs localized fluctuations similar to thoseof the leading part of the edge of normal cells. Because of these fluctuations theoutline of the edge of colcemid-treated cells becomes very irregular. The outlineof these cells is crudely approximated by the curves obtained by random varia-tions of the length of radius in polar co-ordinates (Fig. 9). A few other variantsof the curves 'modelling' the outline of cells with fluctuating edges can also beproposed. However, it would be impossible to substantiate the choice betweenthese variants on the basis of the experimental data available at present.

The formation of the active leading edge seems to be an essential part of themechanism of locomotion of fibroblast, although the exact role of this edge isnot clear. The disappearance of the non-active parts of the cell edge in colcemid-treated fibroblasts is probably the cause of the inhibition of directional cellmovements. When all the edge becomes uniformly active, then effective trans-location upon the substrate becomes impossible.

After removal of cultures from colcemid-containing medium, the ability ofthe cell surface to remain non-active is gradually restored. Results of experimentswith puromycin and actinomycin seem to indicate that synthesis of new proteinsand of RNA is not essential for this restoration.

Stabilization of the non-active state of the cell surface

One may suggest that the initial state of the surface of a fibroblast near thecell edge is an active one: any point of this edge continuously changes its positionwith regard to other points. The mechanism of these fluctuations is not clear andwe will not discuss it here. Movements of each part of the edge can be stoppedfor a short time; these halts may occur spontaneously or may be caused by cer-tain factors in the local environment of the cell. These periods of standstill ofa part of the surface are not lasting unless this part is made non-active by somespecial process. The hypothetical process that stabilizes the non-active state ofthe surface can be named 'stabilization'. The results of our experiments are ingood agreement with the suggestion that the effects of the mitotic spindle in-hibitors on interphase fibroblasts can be described as selective inhibition of thisprocess of stabilization.

In each normal cell some part of its edge is stabilized. This partial stabiliza-tion is essential for directional locomotion and also for the determination of theelongated cell form. The elongation of cytoplasmic processes is possibly ac-companied by the stabilization of their lateral edges. In very elongated bipolarcells (human fibroblasts in crowded culture on glass, human and mouse fibro-blasts on fish scales) almost all the cell edge is stabilized. Possibly only theends of the cytoplasmic processes of these cells remain active.

Locomotion of fibroblasts 637

Stabilization of the surface and cell interactionswith the substrate and with other cells

The direction of elongation of fibroblasts growing upon an oriented substrate(e.g. on the fish scale) is determined by the structure of this substrate ('contactguidance' of Weiss, 1963). The nature of this cell-substrate interaction is notknown. It was suggested that orientation depends on the unequal adhesion ofthe cell surface to various structures of the substrate (Weiss & Garber, 1952;Carter, 1965). Fibroblasts growing on the oriented substrate change their formand lose their orientation in colcemid-containing medium. Possibly the inter-action of the cell surface with the substrate is not sufficient for cell orientationunless a non-active state of certain parts of the cell edge is stabilized. One maysuggest that in colcemid-containing medium the guiding effect of the substrateis not absent but is overruled by activation of the whole cell edge.

If this suggestion is correct, colcemid should not affect the selectivity of celladhesion to various substrates. The attachment of fibroblasts to glass is notvisibly affected by colcemid. Experiments with other substrates are now inprogress.

Contact inhibition of movement is another factor that determines localizationof the non-active parts of the cell surface in normal fibroblasts. Contact inhibitionseems to be a complex phenomenon, paralysis of surface movements after con-tact with other cells being one of its main manifestations (Abercrombie, 1967).

According to the hypothesis stated above, paralysis of cell movements aftercontact has two phases: (a) the forward movement of some part of the cellsurface is stopped when this part touches the other cell; (b) then stabilization ofthis halt takes place.

If colcemid acts selectively on the stabilization, then this drug should affectthe second stage of the paralysis but not the first one. Microcinematographicdata are in good agreement with this suggestion. At 8-12 h after addition ofcolcemid to the medium, when the effects of this drug were at their height, onecould see that forward movement of a surface projection was stopped after itscontact with another cell. However, in contrast to control cultures, stablenon-active zones of the edge were not formed.

Thus only the first stage of the paralysis after contact was observed in thesecolcemid-treated cultures. Preservation of the first stage of contact inhibitionof movement may explain also the low ratio of nuclear overlaps in thesecultures.

In summary, it seems reasonable to assume that reactions to external factors(contact with other cells, interaction with the substrate) produce non-stablechanges of spontaneous surface activity, while a colcemid-sensitive intra-cellular process stabilizes the cessation of activity.

In other words, stabilization can be described as the process by which thefibroblast memorizes the effect of external factors changing surface activity.

638 JU. M. VASILIEV AND OTHERS

Any type of memory can function only in association with an opposite processresponsible for 'forgetting'. It is probable that besides a stabilization mechan-ism fibroblasts have also some mechanism that removes the stable state of thesurface. When action of an external factor is discontinued, the stabilized partof the edge can eventually resume active movements. The rate of this dis-appearance of the stable state of the surface depends on cell type: after libera-tion from contact with other cells the lateral surfaces of human fibroblasts areactivated much more slowly than those of mouse cells (Vasiliev et al. 1969).

Possible role of microtubules in the stabilization of the surface

As suggested above, the main effect of colcemid on the locomotory behaviourof fibroblasts can be described as inhibition of surface stabilization. The mainalteration of the electron-microscopic structure of mouse cells induced bycolcemid was disappearance of cytoplasmic microtubules. Selective effects ofcolchicine and colcemid upon the microtubules in the cells of various types hadbeen observed earlier by many authors (Robbins & Gonatas, 1964; Tilney,1965; Behnke & Forer, 1967; Holmes & Choppin, 1968; Tilney & Gibbins,1969, and others). Binding of these drugs to the protein subunits of the micro-tubules (Weisenberg, Borisy & Taylor, 1968) may be the chemical basis of theseeffects.

These facts suggest that the formation of microtubules may be importantfor surface stabilization.

Microtubules in the cytoplasmic processes of normal mouse fibroblasts areusually parallel to the lateral surfaces of these processes. The presence of micro-tubules in cytoplasmic processes has been observed in the cells of many varioustypes (Tilney, 1965; Porter, 1966; Taylor, 1966; Tilney & Gibbins, 1969; Goldman& Follett, 1969). In the course of locomotion of fibroblasts old processes dis-appear and new processes are formed. Therefore it seems probable that dis-assembly of the old microtubules and formation of new structures of this typetake place continuously in moving cells.

Possibly localization and orientation of new microtubules are determined bythe state of cell surface. The general rule may be that microtubules are formedin parallel to those areas of the surface where spontaneous activity is diminished.Once microtubules have been formed under a certain part of the surface, theystabilize the non-active state of this part. At present we have no data suggestingpossible mechanisms for the influence of microtubules upon the state of thesurface. Formation of these structures may change the mechanical deformabilityof the surface, it may polarize movements of fluids and of organelles to variousparts of the surface, etc. The selective effects of colcemid upon the micro-tubules and upon the stabilization of the cell surface are hardly coincidental.However, it would be premature to insist that formation of microtubules is theonly mechanism of stabilization. One cannot exclude participation of somecomponents that have not been revealed by the present electron-microscopic

Locomotion of fibroblasts 639examination. More detailed studies of correlations between the ultra structure ofvarious parts of the cell surface and the locomotory activities of these parts incells of different types are necessary. The validity of suggestions on the possiblerole of stabilization in locomotion and in the determination of cell form does notdepend upon the specific nature of intracellular changes that may be the basis ofthis process.

Stabilization mechanisms similar to those discussed in this paper possibly playa variety of important roles in morphogenesis. Mechanisms of this type may beessential for transformation of the unstable changes of the surface induced byenvironmental factors into more permanent alterations of cell structure.

RESUME

U action de la colcemide sur le comportement locomoteur descellules fibroblastiques

On a etudie l'action d'inhibiteurs de la metaphase (colcemide, colchicine, vinblastine) surdes cellules embryonnaires humaines et de souris de forme fibroblastique, au cours de leurcroissance sur du verre et sur un substrat oriente (ecaille de poisson). Les trois inhibiteurs ontprovoque des changements similaires de la forme des cellules a l'interphase et ont inhibeleur deplacement oriente. On a trouve que les effets de deux inhibiteurs (colcemide et vin-blastine) etaient completement reversibles. Des etudes microcinematographiques ont montreque le changement le plus frappant du comportement locomoteur, induit par la colcemideetait la disparition de parties stables non actives de la bordure cellulaire; dans des cellulesnormales seule la partie principale de la bordure se deplace activement, tandis que dans lescellules traitees par la colcemide toutes les parties du bord cellulaire deviennent en definitiveactives. L'activation de la bordure toute entiere rend ces cellules incapables d'executer undeplacement oriente.

On suggere que la colcemide et d'autres inhibiteurs de la metaphase empechent la stabilisa-tion de l'etat non-actif de la surface cellulaire. Le role eventuel joue par ce mecanisme destabilisation, sensible a la colcemide, dans le comportement locomoteur normal des fibro-blastes est discute.

L'observation au microscope electronique a montre que des microtubules disparaissent ducytoplasme des cellules de souris de forme fibroblastique traitees a la colcemide. La formationde microtubules est discutee en tant que base structurale eventuelle de la stabilisation del'etat non-actif de la surface cellulaire.

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{Manuscript received 20 March 1970)