/. Embryo/, exp. Morph. Vol. 24, 3, pp. 625-640, 1970
625
Printed in Great Britain
Effect of colcemid on the locomotory
behaviour of fibroblasts
By Ju. M. VASILIEV, 1 1. M. GELFAND, L. V. DOMNINA,
O. Y. 1VANOVA, S. G. KOMM AND L. V. OLSHEVSKAJA
From the Institute of Experimental and Clinical Oncology
and the Laboratory of Scientific Cinematography of the
Academy of Medical Sciences of USSR, Laboratory of Mathematical
Biology of Moscow State University, Moscow, U.S.S.R.
SUMMARY
Effects 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 and
inhibited their directional locomotion. The effects of two inhibitors (colcemid and vinblastine) were found to be completely reversible. Microcinematographic studies have shown
that the most conspicuous change of locomotory behaviour induced by colcemid was the
disappearance of non-active stable parts of the cell edge; in normal cells only the leading
part of the edge was actively moving, while in colcemid-treated cells all parts of the edge
eventually became active. Activation of the whole edge made these cells unable to perform
directional translocation.
It is suggested that colcemid and other metaphase inhibitors prevent stabilization of the
non-active state of the cell surface. The possible role of this suggested colcemid-sensitive
stabilization mechanism in the normal locomotory behaviour offibroblastsis discussed.
Electron-microscopic examination has shown that microtubules disappeared from the
cytoplasm of colcemid-treated, mouse, fibroblast-like cells. The formation of microtubules
as the possible structural basis of the stabilization of the non-active state of the cell surface
is discussed.
INTRODUCTION
The main manifestations of the normal locomotory behaviour of cultured
fibroblasts and the regulation of this behaviour by cell-cell and cell-substrate
interactions are well known (Abercrombie, 1961, 1965, 1967; Ingram, 1969;
Weiss, 1958, 1961, and others). To gain more insight into the possible mechanisms of these activities it is important to study in detail the effects of
various types of inhibitors upon locomotion. Mitotic spindle poisons (colchicine,
colcemid, vinblastine) are of special interest in this connection. Besides a characteristic action on mitosis, these drugs change the cell form and affect locomotion
in 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 Kaskirskoje
Shosse, Moscow 478, U.S.S.R.
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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 the
effects of colcemid upon the form and locomotory behaviour of mouse and
human embryonic, fibroblast-like cells. Results of some experiments with two
other mitotic spindle poisons (colchicine and vinblastine) are also presented.
The results of our experiments give reason to suggest that colcemid
selectively affects stabilization of the cell surface, that is, its tendency to remain
non-active. The essential role of this colcemid-sensitive process of surface stabilization in the locomotion of normal cells and its possible structural basis are
discussed in the last part of the paper.
MATERIALS AND METHODS
Two types of cells were used: (a) fibroblast-like cells obtained by the
trypsinization of 18- to 20-day-old mouse embryos. The cultures of these
cells were in their first passage, (b) A line of human embryonic, fibroblast-like
cells obtained in the Institute of Virus Preparations (Moscow) by the cultivation of cells from the skin of human embryos. These cells were used in their
8-2Oth 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 8day-old cultures. Cultivation procedures and methods of wounding were
identical to those described earlier (Vasiliev et al. 1969).
To study the effect of the drugs upon cell orientation we used cultures
grown 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 the
cell suspension was added to the flask. To study the effect of drugs on phagocytosis, a carmine suspension was added to the medium of 1- to 2-day-old
cultures on glass, simultaneously with colcemid; cultures were examined 6 and
24 h later. In various types of experiments living cultures on glass were examined and photographed under a phase-contrast microscope. Cultures grown
on 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. Sections
were stained with uranyl acetate and Reynolds's lead citrate.
Cultures for time-lapse microcinematography were grown in special chambers
with parallel glass walls. Eagle's culture medium with 10 % bovine serum was
used; the medium in the chambers was changed every 48 h. Cultures were
photographed on 35 mm film with a phase-contrast objective (NA 0-65). Intervals between frames were 30 or 60 sec; exposure was 1-0-1-5 sec. Developed
films were examined frame by frame on the projection screen.
Locomotion of
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627
RESULTS
General characteristics of the action ofmitotic
spindle poisons on fibroblast-like cells
In control culture on glass most cells had a bipolar form; human cells were
more elongated than mouse cells. On fish scales both human and mouse cells
acquired 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 of
most cells changed; long cytoplasmic processes disappeared and the cells
acquired an irregular polygonal form (Figs. 1B, 2B). Often these cells had short
cytoplasmic processes (Fig. 3). These changes of cell form were evident 3-4 h
after the addition of colcemid; in the cultures on scales they developed somewhat later (after 10—12 h). As described earlier (Vasiliev et al. 1969) when colcemid was added to the medium of wounded cultures cell migration into the wound
was stopped a few hours later. Cells blocked in metaphase were often seen in
colcemid-treated cultures; later stages of mitosis were absent. A lower concentration of colcemid (001 /tg/ml) did not change the cell shape and did not
produce metaphase arrest. Concentrations of colcemid that changed cell form
did not cause any non-specific toxic effects. After 24 h of incubation with
colcemid (01 /*g/ml) the mean number of cells per unit area of culture did not
decrease as compared with cultures before the incubation. The percentage of
cells synthesizing DNA determined autoradiographically in cultures pulselabelled with [3H]thymidine also was not decreased in these cultures. Colcemidtreated cells phagocytosed particles of carmine as actively as control cells.
If cultures were placed at 4 °C after addition of colcemid, changes of cell form
were not observed in the following 24 h.
Alterations of cell form similar to those caused by colcemid were also
produced by two other mitotic spindle poisons: vinblastine sulphate (Richter,
Hungary; effective concentrations 005-0005/*g/ml) and colchicine (effective
concentrations 0-1-0-01 /tg/ml). Changes produced by all effective concentrations of colcemid and vinblastine and by low concentration of colchicine
(0-01 /«g/ml) were reversible: if fresh medium was substituted for that containing
the 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 of
the 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 into
fresh 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 medium
and 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 the
substrate was studied. Colcemid (0-1/tg/ml) was added to culture medium
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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 and
then another 24 h in fresh medium without colcemid.
Fig. 2. Effect of colcemid upon the form and orientation of mouse embryo fibroblast-like cells growing on the surface of the fish scale. Jasswoin haematoxylin
stain.
(A) Control culture.
(B) Culture incubated 24 h with colcemid (01 /*g/ml).
Fig. 3. Mouse fibroblast from culture incubated 24 h with colcemid. Isolated
cell 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 mouse
embryo fibroblasts (3 x 105 cell/ml). Two ml of suspension were placed in each
penicillin flask containing one 10 x 20 mm coverslip at the bottom. Cultures were
kept 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 glass
per unit area was counted. The ratio of nuclear overlaps (number of observed
overlaps:number of theoretically expected overlaps) was also counted. The
formula of Abercrombie & Heaysman (1954) modified by Curtis (1961) was
used. The density of attached cells was similar in control and in colcemid-treated
cultures. 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 no
effect upon cell attachment to the glass and did not increase cell attachment
to the surface of normal cells. The reduction of nuclear overlaps by colcemid
was possibly related to reduced migratory-activity of the cells, so that colcemid
treated 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 containing
colcemid (0-1 /tg/ml) as well as control cultures were subjected to an electronmicroscopic examination. The ultrastructure of mouse fibroblasts in control
cultures 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 cells
were those related to the presence of microtubules in the cytoplasm. Cells in control cultures contained numerous microtubules of various length and about
200-250 A in diameter. Microtubules were present in various parts of the cytoplasm but were especially numerous in wide cytoplasmic processes. Usually
they were approximately parallel to the lateral surfaces of these processes and
ended near the anterior ends of the process (Fig. 4 A). In the cytoplasm of the
central part of the cell body microtubules near the cell surface were usually
parallel to the long axis of the cell; the orientation of microtubules located
farther from the surface was less regular.
Microtubules were not seen in the cytoplasm of fibroblasts treated for 24 h
with colcemid (Fig. 4B). The cytoplasm of these cells contained more free ribosomes and fewer membrane-bound ribosomes than did that of control cells. The
structure of other organelles was similar in control and in colcemid-treated
cells.
Microcinematographic studies of cell locomotion
(a) Control cultures. In most experiments 6- to 8-day-old mouse cultures
were filmed; wounds were made 1-2 h before the filming was started. A field of
view containing part of the wound edge was usually selected for filming, so that
most cells in the field moved approximately in the same direction: from the
monolayer 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 mouse
embryo fibroblast-like cells. Electron micrographs.
(A) The end of a cytoplasmic process of a cell from the control culture. The cytoplasm contains numerous microtubules.
(B) Peripheral part of the cytoplasm of a cell from a culture incubated 24 h with
colcemid (0-2/*g/ml): microtubules are not seen.
Locomotion of
fibroblasts
631
those described by previous investigators (Abercrombie, 1961, 1965; Abercrombie & Ambrose, 1958; Weiss, 1958, 1961, and others). Active and nonactive parts of the cell edge could be distinguished in the moving cell CFig. 6).
The active leading edge fluctuated backwards and forwards; local protrusions
and withdrawals were repeatedly seen at that part of the edge. The lateral non-
D
10/i
"
Fig. 5. Trajectories of movements of cell nuclei near the edges of the wounds in
control (A) and in colcemid-treated (B) mouse embryonic cell cultures. Each line
shows changes of the position of the projection of one nucleus in relation to the field
of view. This position was determined at intervals of 30 min (points). Direction
of movements of each nucleus is shown by the arrow.
40
E M B 24
632
JU. M. VASILIEV AND OTHERS
active parts of the edge had a smoother outline and did not fluctuate; their
form was slowly changed in the course of locomotion.
Moving human cells were somewhat different from mouse cells; some of the
cytoplasmic processes formed in the anterior part of these cells underwent considerable elongation. Sometimes the whole anterior part of the cell underwent
elongation and was transformed into a narrow strand of cytoplasm with a
flattened leading lamella at its anterior end. Later these long processes often
underwent contraction accompanied by the forward translocation of the whole
cell body.
Fig. 6. Outline of the edges of a human cell in control culture. Several drawings of
the same cell were made from the projected film frames. Figures near the arrows
show the intervals (in min) between frames from which consecutive drawings were
made. Consecutive drawings show changes of the form of the same cell but not of
its 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 of
the contacting part of the leading edge, followed by the changes in the direction
of locomotion and often by the formation of a relatively firm adhesion between
the cells.
(b) Cultures incubated with colcemid. Conditions of filming were similar to
those in control experiments except that colcemid (0-1 /*g/ml) was added to the
medium 1 or 2 h before the first frame was taken. Wounds were made 2 h or
24 h before the filming began.
During the first 2-8 h, gradual contraction of the long cytoplasmic processes
was observed and the cell form became irregular. Simultaneously the distribution of active and non-active parts of the cell edge gradually changed. The
fluctuations previously observed only in the leading parts of the cell edge gradually spread to all of the free edge, that is to say to all those parts of the edge
that were not in contact with the edges of other cells. Smooth non-active parts
of the free edges disappeared (Fig. 7). Those parts of the edges that were contacting other cells at this stage remained smooth and non-active. Protrusions that
Locomotion of
fibroblasts
633
arose and disappeared repeatedly at the free edges did not usually undergo considerable elongation. If the protrusion was relatively large, secondary small
outgrowths were often formed at its edges.
No stable differences in the character of movements of various parts of the
edge of isolated cells were observed: certain parts of the edge of isolated cells
could temporarily move more actively than others but these differences were
not 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 incubation
with colcemid. Irregular fluctuations of the whole cell edge are seen.
Fig. 8. Outline of the edge of a mouse cell from culture contacting two surfaces
of other cells. The first drawing was made from the photograph taken after 12 h
of incubation with colcemid.
40-2
634
JU. M. VASILIEV AND OTHERS
At later stages of the action of colcemid (8-12 h and later) the character of the
activity of the free edges did not change. This stage was characterized by gradual
activation of those parts of the edge that were contacting other cells: these
parts 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 two
cells were activated simultaneously. Those parts of the contacting edges that
were nearer to the free edges were activated earlier. Activation of contacting
edges was often accompanied by their retracting slightly so that a narrow gap
was formed between the cells. Protrusions of activated cell edges moved forward
across this gap but stopped their forward movement when they touched the
surface of other cells. Movement of cell protrusions across the surface of other
cells was very rare. Usually after contact with other cells these protrusions
withdrew, either immediately or after some delay. There were no signs of the
formation of firm cell-cell attachments at these contacts; withdrawal of
the protrusions after contact with the other cell did not leave any strands of
cytoplasm between the two cells. Thus at this stage of incubation with colcemid
all parts of the cell edges became active. However, as the movement of the
surface was stopped by contact with other cells, the range of displacements
remained smaller in those parts of the edge that were near to the edges of
other cells.
Activation of the cell edges in colcemid-treated cells was accompanied by
gradual cessation of the oriented translocation of these cells into the wound.
The position of many nuclei remained unchanged for hours. Those small displacements that were observed were of random character (Fig. 5B). Highly
refractile round cells blocked in mitosis were often seen in filmed colcemidtreated cultures.
In several experiments the restoration of normal cell shape and movements
after removal of colcemid was photographed. The filming was begun 1 h after
substitution of fresh for colcemid-containing medium. The first manifestation of
the cessation of the colcemid effect was the end of the mitotic block: normal
cell division was observed in these cultures even in the first 2 h after removal of
colcemid. Inactivation of contacting cell edges took place in the following few
hours; it was accompanied by the gradual elongation of the cells. Normal
orientation of cells with regard to the wound margin and normal locomotion
into 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 selectively affect the mitotic spindle also cause characteristic changes of the form and
locomotory activity of interphase fibroblasts. The minimal concentrations of
these substances which affected interphase cells were similar to those causing
Locomotion offibroblasts
635
metaphase block. The effect of two inhibitors (colcemid and vinblastine) was
found 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, and
these cells retained the ability to phagocytose particles. Thus cessation of an
oriented 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 are
obtained by random changes of the length of the radius in polar co-ordinates. Initial
length of the radius was equal to that of the semicircle. Each 5° the length of radius
was randomly changed to ± one-tenth of the initial length. Three examples of
curves obtained by this process are shown.
cell surface. Analysis of microcinematographic data shows that colcemid decreases the ability of cell edges to retain their stable shape. The leading edge of
normal fibroblasts fluctuates forwards and backwards. As shown by M. Abercrombie, 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 cell
edge. 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 those
of the leading part of the edge of normal cells. Because of these fluctuations the
outline of the edge of colcemid-treated cells becomes very irregular. The outline
of these cells is crudely approximated by the curves obtained by random variations of the length of radius in polar co-ordinates (Fig. 9). A few other variants
of the curves 'modelling' the outline of cells with fluctuating edges can also be
proposed. However, it would be impossible to substantiate the choice between
these 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 the
mechanism of locomotion of fibroblast, although the exact role of this edge is
not clear. The disappearance of the non-active parts of the cell edge in colcemidtreated fibroblasts is probably the cause of the inhibition of directional cell
movements. When all the edge becomes uniformly active, then effective translocation upon the substrate becomes impossible.
After removal of cultures from colcemid-containing medium, the ability of
the cell surface to remain non-active is gradually restored. Results of experiments
with puromycin and actinomycin seem to indicate that synthesis of new proteins
and 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 the
cell edge is an active one: any point of this edge continuously changes its position
with regard to other points. The mechanism of these fluctuations is not clear and
we will not discuss it here. Movements of each part of the edge can be stopped
for a short time; these halts may occur spontaneously or may be caused by certain factors in the local environment of the cell. These periods of standstill of
a part of the surface are not lasting unless this part is made non-active by some
special process. The hypothetical process that stabilizes the non-active state of
the surface can be named 'stabilization'. The results of our experiments are in
good agreement with the suggestion that the effects of the mitotic spindle inhibitors on interphase fibroblasts can be described as selective inhibition of this
process of stabilization.
In each normal cell some part of its edge is stabilized. This partial stabilization is essential for directional locomotion and also for the determination of the
elongated cell form. The elongation of cytoplasmic processes is possibly accompanied by the stabilization of their lateral edges. In very elongated bipolar
cells (human fibroblasts in crowded culture on glass, human and mouse fibroblasts on fish scales) almost all the cell edge is stabilized. Possibly only the
ends of the cytoplasmic processes of these cells remain active.
Locomotion offibroblasts
637
Stabilization of the surface and cell interactions
with 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 ('contact
guidance' of Weiss, 1963). The nature of this cell-substrate interaction is not
known. It was suggested that orientation depends on the unequal adhesion of
the cell surface to various structures of the substrate (Weiss & Garber, 1952;
Carter, 1965). Fibroblasts growing on the oriented substrate change their form
and lose their orientation in colcemid-containing medium. Possibly the interaction of the cell surface with the substrate is not sufficient for cell orientation
unless a non-active state of certain parts of the cell edge is stabilized. One may
suggest that in colcemid-containing medium the guiding effect of the substrate
is not absent but is overruled by activation of the whole cell edge.
If this suggestion is correct, colcemid should not affect the selectivity of cell
adhesion to various substrates. The attachment of fibroblasts to glass is not
visibly affected by colcemid. Experiments with other substrates are now in
progress.
Contact inhibition of movement is another factor that determines localization
of the non-active parts of the cell surface in normal fibroblasts. Contact inhibition
seems to be a complex phenomenon, paralysis of surface movements after contact with other cells being one of its main manifestations (Abercrombie, 1967).
According to the hypothesis stated above, paralysis of cell movements after
contact has two phases: (a) the forward movement of some part of the cell
surface is stopped when this part touches the other cell; (b) then stabilization of
this halt takes place.
If colcemid acts selectively on the stabilization, then this drug should affect
the second stage of the paralysis but not the first one. Microcinematographic
data are in good agreement with this suggestion. At 8-12 h after addition of
colcemid to the medium, when the effects of this drug were at their height, one
could see that forward movement of a surface projection was stopped after its
contact with another cell. However, in contrast to control cultures, stable
non-active zones of the edge were not formed.
Thus only the first stage of the paralysis after contact was observed in these
colcemid-treated cultures. Preservation of the first stage of contact inhibition
of movement may explain also the low ratio of nuclear overlaps in these
cultures.
In summary, it seems reasonable to assume that reactions to external factors
(contact with other cells, interaction with the substrate) produce non-stable
changes of spontaneous surface activity, while a colcemid-sensitive intracellular process stabilizes the cessation of activity.
In other words, stabilization can be described as the process by which the
fibroblast memorizes the effect of external factors changing surface activity.
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JU. M. VASILIEV AND OTHERS
Any type of memory can function only in association with an opposite process
responsible for 'forgetting'. It is probable that besides a stabilization mechanism fibroblasts have also some mechanism that removes the stable state of the
surface. When action of an external factor is discontinued, the stabilized part
of the edge can eventually resume active movements. The rate of this disappearance of the stable state of the surface depends on cell type: after liberation from contact with other cells the lateral surfaces of human fibroblasts are
activated 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 behaviour
of fibroblasts can be described as inhibition of surface stabilization. The main
alteration of the electron-microscopic structure of mouse cells induced by
colcemid was disappearance of cytoplasmic microtubules. Selective effects of
colchicine and colcemid upon the microtubules in the cells of various types had
been 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 microtubules (Weisenberg, Borisy & Taylor, 1968) may be the chemical basis of these
effects.
These facts suggest that the formation of microtubules may be important
for surface stabilization.
Microtubules in the cytoplasmic processes of normal mouse fibroblasts are
usually parallel to the lateral surfaces of these processes. The presence of microtubules in cytoplasmic processes has been observed in the cells of many various
types (Tilney, 1965; Porter, 1966; Taylor, 1966; Tilney & Gibbins, 1969; Goldman
& Follett, 1969). In the course of locomotion of fibroblasts old processes disappear and new processes are formed. Therefore it seems probable that disassembly of the old microtubules and formation of new structures of this type
take place continuously in moving cells.
Possibly localization and orientation of new microtubules are determined by
the state of cell surface. The general rule may be that microtubules are formed
in parallel to those areas of the surface where spontaneous activity is diminished.
Once microtubules have been formed under a certain part of the surface, they
stabilize the non-active state of this part. At present we have no data suggesting
possible mechanisms for the influence of microtubules upon the state of the
surface. Formation of these structures may change the mechanical deformability
of the surface, it may polarize movements of fluids and of organelles to various
parts of the surface, etc. The selective effects of colcemid upon the microtubules and upon the stabilization of the cell surface are hardly coincidental.
However, it would be premature to insist that formation of microtubules is the
only mechanism of stabilization. One cannot exclude participation of some
components that have not been revealed by the present electron-microscopic
Locomotion of
fibroblasts
639
examination. More detailed studies of correlations between the ultra structure of
various parts of the cell surface and the locomotory activities of these parts in
cells of different types are necessary. The validity of suggestions on the possible
role of stabilization in locomotion and in the determination of cell form does not
depend upon the specific nature of intracellular changes that may be the basis of
this process.
Stabilization mechanisms similar to those discussed in this paper possibly play
a variety of important roles in morphogenesis. Mechanisms of this type may be
essential for transformation of the unstable changes of the surface induced by
environmental factors into more permanent alterations of cell structure.
RESUME
U action de la colcemide sur le comportement locomoteur des
cellules fibroblastiques
On a etudie l'action d'inhibiteurs de la metaphase (colcemide, colchicine, vinblastine) sur
des cellules embryonnaires humaines et de souris de forme fibroblastique, au cours de leur
croissance sur du verre et sur un substrat oriente (ecaille de poisson). Les trois inhibiteurs ont
provoque des changements similaires de la forme des cellules a l'interphase et ont inhibe
leur deplacement oriente. On a trouve que les effets de deux inhibiteurs (colcemide et vinblastine) etaient completement reversibles. Des etudes microcinematographiques ont montre
que le changement le plus frappant du comportement locomoteur, induit par la colcemide
etait la disparition de parties stables non actives de la bordure cellulaire; dans des cellules
normales seule la partie principale de la bordure se deplace activement, tandis que dans les
cellules traitees par la colcemide toutes les parties du bord cellulaire deviennent en definitive
actives. L'activation de la bordure toute entiere rend ces cellules incapables d'executer un
deplacement oriente.
On suggere que la colcemide et d'autres inhibiteurs de la metaphase empechent la stabilisation de l'etat non-actif de la surface cellulaire. Le role eventuel joue par ce mecanisme de
stabilisation, sensible a la colcemide, dans le comportement locomoteur normal des fibroblastes est discute.
L'observation au microscope electronique a montre que des microtubules disparaissent du
cytoplasme des cellules de souris de forme fibroblastique traitees a la colcemide. La formation
de microtubules est discutee en tant que base structurale eventuelle de la stabilisation de
l'etat non-actif de la surface cellulaire.
REFERENCES
M. (1961). The bases of the locomotory behaviour of fibroblasts. Expl Cell
Res., Suppl. 8, pp. 188-198.
ABERCROMBIE, M. (1965). The locomotory behaviour of cells. In Cells and Tissues in Culture.
Methods, Biology and Physiology, vol. i (ed. E. N. Willmer), pp. 177-202. London and
New York: Academic Press.
ABERCROMBIE, M. (1967). Contact inhibition; the phenomenon and its biological implications. Natn. Cancer Inst. Monogr. 26, 249-277.
ABERCROMBIE, M. & AMBROSE, E. J. (1958). Interference microscope studies of cell contacts
in tissue culture. Expl Cell Res. 15, 332-345.
ABERCROMBIE, M. & HEAYSMAN, J. E. M. (1954). Observations of the social behaviour of
cells in tissue culture. II. 'Monolayering' of fibroblasts. Expl Cell Res. 6, 293-306.
BEHNKE, O. & FORER, A. (1967). Evidence for four classes of microtubules in individual cells.
/. Cell Sci. 2, 169-192.
BISCHOFF, R. & HOLTZER, H. (1968). The effect of mitotic inhibitors on myogenesis in vitro.
J. CellBiol.36, 111-127.
ABERCROMBIE,
640
JU. M. VASILIEV AND OTHERS
S. B. (1965). Principles of cell motility; the direction of cell movement and cancer
invasion. Nature, Lond. 208, 1183-1187.
CORNELL, R. (1969). Spontaneous neoplastic transformation in vitro: ultrastructure of transformed cell strains and tumors produced by injection of cell strains. /. Natn. Cancer
Inst. 43, 891-906.
CURTIS, A. S. G. (1961). Control of some cell-contact reactions in tissue culture. /. Natn.
Cancer Inst. 26, 253-268.
GODMAN, G. C. & MURRAY, M. R. (1953). Influence of colchicine on the form of skeletal
muscle in tissue culture. Proc. Soc. exp. Biol. Med. 84, 668-672.
GOLDMAN, R. D. & FOLLETT, E. A. C. (1969). The structure of major cell processes of isolated
BHK 21 fibroblasts. Expl Cell Res. 57, 263-276.
HOLMES, K. V. & CHOPPIN, P. W. (1968). On the role of microtubules in movement and
alignment of nuclei in virus-induced syncytia. /. Cell Biol. 39, 526-543.
INGRAM, V. M. (1969). A side view of moving fibroblasts. Nature, Lond. 222, 641-644.
MALAWISTA, S. E. & BENESCH, K. G. (1967). Human polymorphonuclear leukocytes,
demonstration of microtubules and the effect of colchicine. Science, TV. Y. 156, 521-523.
MOVAT, H. Z. & FERNANDO, N. V. P. (1962). The fine structure of connective tissue. I. The
fibroblast. Expl mol. Path. 1, 509-534.
PADAWER, J. (1968). Paramitotic effects of colchicine: further studies of the mast-cell assay.
Proc. Soc. exp. Biol. Med. 127, 194-199.
PORTER, K. R. (1966). Cytoplasmic microtubules and their function. In Principles of Biomolecular Organization. Ciba Fdn Symp. (ed. G. E. W. Wolstenholme and M. O. Connor),
pp. 308-356. London: Churchill.
ROBBINS, E., & GONATAS, N. K. (1964). Histochemical and ultrastructural studies onHeLa
cell cultures exposed to spindle inhibitors with special reference to the interphase cell.
/. Histochem. Cytochem. 12, 704-711.
TAYLOR, A. C. (1966). Microtubules in the microscopic and cortical cytoplasm of isolated
cells. /. Cell Biol. 28, 155-168.
TILNEY, L. G. (1965). Microtubules in the asymmetric arms of Actinosphaerium and their
response to cold, colchicine and hydrostatic pressure. Anat. Rec. 151, 426 (Abstr.).
TILNEY, L. G. & GIBBINS, J. R. (1969). Microtubules and filaments in the filopodia of the
secondary mesenchyme cells of Arbacia punctata and Echinarachnius parma. J. Cell Sci.
5, 195-210.
VASILIEV, JU. M., GELFAND, I. M.,DOMNINA,L. V. & RAPPOPORT, R. I. (1969). Wound healing
processes in cell cultures. Expl Cell Res. 54, 83-93.
WARREN, R. H. (1968). The effect of colchicine on myogenesis in vivo in Rana pipiens and
Rhodnius prolixus (Hemiptera). /. Cell Biol. 39, 544-555.
"WEISENBERG, R. C , BORISY, G. G. & TAYLOR, E. W. (1968). The colchicine binding protein
of mammalian brain and its relation to microtubules. Biochemistry, N. Y. 7, 4466-4479.
WEISS, P. (1958). Cell contact. Int. Rev. Cytol. 7, 391-423.
WEISS, P. (1961). Guiding principles in cell locomotion and cell aggregation. Expl Cell Res.,
Suppl. 8, pp. 260-281.
WEISS, P. (1963). Cell interactions. Canadian Cancer Conference, vol. 5, pp. 241-275. London,
New York: Academic Press.
WEISS, P. & GARBER, B. (1952). Shape and movement of mesenchyme cells as functions of the
physical structure of the medium, contributions to a quantitative morphology. Proc. natn.
Acad. Sci. U.S.A. 38, 264-280.
WEISS, P. & TAYLOR, A. C. (1956). Fish scales as substratum for uniform orientation of cells
in vitro. Anat. Rec. 124, 381-382.
CARTER,
{Manuscript received 20 March 1970)