Download Locomotion of Fundulus Deep Cells during Gastrulation1

AMER. ZOOL., 21:401-411 (1981)
Locomotion of Fundulus Deep Cells during Gastrulation1
J. P. TRINKAUS
Department of Biology, Yale University, New Haven, Connecticut 06520 and
Marine Biological Laboratory, Woods Hole, Massachusetts 02543
AND
™
C. A.
ERICKSON
Department of Zoology, University of California, Davis, Davis, California 95616 and
Marine Biological Laboratory, Woods Hole, Massachusetts 02543
SYNOPSIS. Mechanism of locomotion of deep cells of Fundulus heteroclitus was studied in
vivo during gastrulation with the aid of time lapse cinemicrography (Nomarski differential
interference contrast optics), scanning electron microscopy of cells known to be moving
at the time of fixation, and cell culture. These are our findings. 1) Deep cells usually move
rapidly, at about 10—15 /i.m/min, regardless of whether they move by blebbing or spreading. Evidence suggests that this high speed is associated with weak adhesion of the trailing
edge: it remains rounded, without large retraction fibers, and it advances continuously
with advance of the leading edge, not sporadically, as it would if it adhered strongly. 2)
In contrast, when stationary cells in close contact separate, they remain connected by
retraction fibers, suggesting strong punctate adhesions. 3) Locomotion by shortening of
a long lobopodium is really a form of spreading movement; the tip of a lobopodium
always spreads. Also, since speed of shortening decreases with continuance, it may depend
primarily on elastic recoil rather than active contraction. 4) Fundulus deep cells appear to
move in two ways: a) protrusion of blebs, followed by much cytoplasmicflow;b) protrusion
of lamellipodia, accompanied by filopodia and frequent cell shortening. 5) Filopodia were
not found except at the leading edge of a spreading lamellipodium and often spread
themselves; perhaps filopodia and lamellipodia are interconvertible. 6) A lamellipodial
margin may form undulations in vivo that move backward like ruffles in vitro. 7) At all
times, whether stationary or moving, the surface of deep cells is smooth, raising unanswered questions concerning the source of surface for their rapid protrusive activity.
movements of deep cells during gastrulaAlthough the surface activity and loco- nonO u r
unanswered questions concern
motion of deep cells of the Fundulus biasmaml
y t h e morphology of the adhesion of
toderm have been studied in situ in considerable detail during blastula and gastrula d e e P c e l l s t o t h e i r normal substrata and to
stages (Trinkaus, 1973), a number of ques- e a c h o t h e r d u n n g movement and details
tions were left unanswered, due primarily o f t h e morphology of locomotor extento the limited use in these studies of No- s l o n s o f t h e leading edge. For example, do
marski differential interference contrast deep cells adhere over a broad area of conoptics (DIC) and to the absence of obser- t a c t o r mainly at highly local points at or
vations with the scanning electron micro- n e a r t h e i r margins, like fibroblasts (Harris,
scope. We have since applied both of these 1 9 7 3 : I z z a r d a n d Lochner, 1980)? Is the
techniques to further study of the loco- surface of a deep cell pulled out to form
motion of these fascinating cells, both in retraction fibers when it moves away from
vivo and in cell culture, and in conse- contacts with other cells? And can the
quence have been able to expand our adhesions of these cells be related to their
knowledge and understanding of the pattern and speed of movement?
In the previous study (Trinkaus, 1973),
deep cells were observed to form blebs, elonp
lik
i
fd
• From the Symposium on Developmental Biology of &* finger-like
extensions
referred
to as
Fishes presented at the Annual Meeting of the Amerlobopodia, broad lamellipodia, and hloican Society of Zoologists, 27-30 December 1979, at podia of varying degrees of thickness. But
Tampa, Florida.
because of the limitations of the in vito sitINTRODUCTION
401
402
J. P. TRINKHAUS AND C. A. ERICKSON
uation (light must pass through an egg 1.8
mm in diameter) and our previous almost
exclusive dependence on bright-field optics, crucial details of the morphology of
these protrusions were lacking. Are blebs
really smooth surfaced? Indeed, are deep
cells in general smooth surfaced, like
spread fibroblasts, or covered with microprotrusions, such as microvilli, like rounded fibroblasts (Erickson and Trinkaus,
1976) and like the yolk syncytial layer of
the Fundulus egg (Betchaku and Trinkaus,
1978)? Or, are they covered with microfolds, like cells of the Fundulus enveloping
layer before they are spread (Betchaku
and Trinkaus, 1978)? What is the structural relationship between lamellipodia
and filopodia? They seem to grade off into
one another in films. SEM is clearly necessary to obtain such details. Also, do the
lamellae of moving deep cells show undulating activity or ruffling as they
spread on the relatively flat substratum of
the yolk syncytial layer (YSL) at the floor
of the segmentation cavity in vivo, as do
fibroblasts when they spread on plane glass
or plastic substrata in vitro? Finally, it was
previously noted that Fundulus deep cells
often move quite rapidly within the blastoderm and that this speed correlates with
the presence of a rounded cell body (Trinkaus, 1973). This implies that the trailing
cell body adheres less firmly to the substratum than the trailing edge of a moving
fibroblast in vitro. If so, the movement of
the trailing edge of a deep cell should be
continuous, responding more or less immediately to the pull of the advancing
leading edge, and not spasmodic like that
of a fibroblast (Chen, 1981). Also, there
should be no morphological evidence of
firm adhesion to the substratum.
The primary purpose of this paper is to
report the results of our efforts to answer
these questions by time-lapse cinemicrography, using DIC optics, by cell culture, and
by scanning electron microscopy.
All details of methods, determinations
of rate of movement and illustrations of
deep cells, both in the living state, as
viewed in films, and in the fixed state, as
viewed in SEM, are presented in a full
publication of the results of this study
which will appear shortly (Trinkaus and
Erickson, in preparation). The present
paper is intended to be no more than an
extended summary of some of the contents
of that paper.
£
MATERIALS AND METHODS
The materials for these studies were
deep blastomeres of early to late gastrula
stages of Fundulus heteroclitus (stages 13 to
18) (Armstrong and Child, 1965). Eggs
were prepared for in vivo studies of living
blastomeres in situ, as described in Trinkaus (1973), and filmed, using DIC.
The Fundulus egg lends itself particularly well to study of moving cells with SEM.
For when a blastoderm is removed from
the underlying yolk syncytial layer (YSL)
during mid to late gastrula stages, a certain
number of deep cells remain stuck to the
YSL just under the embryonic shield. If
such an egg, bereft of its blastoderm, is
placed in a nutrient tissue culture medium,
these cells soon begin to move and filming
has shown that their mode of movement
under these circumstances does not differ
from that in an intact, unoperated egg.
Since their motile activity can be observed
prior to fixation and they can be identified
after fixation, one is able to determine with
certainty that a particular cell observed in
the SEM was moving at the time of fixation
and in which direction, in contrast to most
SEM studies of cells presumed to be moving inside opaque embryos where the motile state of each cell cannot be determined
prior to fixation. In our SEM study, Leibovitz L-15 (GIBCO) was the tissue culture
medium and fixation was in gluteraldehyde (3%), followed by post-osmication.
For observations of cell movement in
culture, deep cells were scooped out of
early gastrula blastoderms (stage 1314-14)
and cultured in Leibovitz L-15 medium at
room temperature, 21O-23°C.
RESULTS AND DISCUSSION
Our new information relates particularly to protrusive activity, mode of movement, pattern of adhesion, and surface
contour of cells utilizing both blebs and la-
LOCOMOTION OF FUNDULUS DEEP CELLS
mellipodia and filopodia for their movement.
Blebbing movement
A As previously observed (Trinkaus, 1973,
i fable 1), cells using blebs as organs of locomotion tend to move quite fast, at about
10 fim/min. It was also observed that these
cells almost always retain a rounded cell
body during this rapid movement. A predominantly rounded cell body suggests
weak adhesions to the substratum, since
there is little or no obvious morphological
indication of adhesion, such as scalloping
of the trailing edge or the formation of
retraction fibers, where the cell surface is
pulled out to a point because of strong
adhesion to its substratum. In the present
study, we were able to film five deep cells
involved in blebbing movement in vivo
with DIC optics for long enough periods
to gain additional information on this matter. The trailing edge of each of these cells
appeared to remain rounded throughout
each period of observation. Although very
fine retraction fibers might well not be resolved in these films, in spite of the use of
DIC optics, large ones, and other gross
distortions of the cell margin, such as scalloping, would be readily detected (see Fig.
6 of Trinkaus, 1973). None of this was
seen. Significantly, in one of the two instances where the trailing edge did not advance during one 20-sec period of observation, it lost its smooth rounded contour
and appeared slightly distorted, as if its
adhesion strengthened momentarily. When
it resumed its smooth contour, it advanced
immediately after this slight hesitation.
If the adhesion of the trailing edge is
actually weak, as suggested by this morphological evidence, we would expect its
advance to be quite continuous, responding more or less immediately to the pull of
the continually advancing leading edge,
and not erratic or spasmodic, as in fibroblasts where the trailing edge adheres
strongly and is relatively immobile for relatively long periods (Chen, 1981). We
therefore plotted the distance advanced by
the trailing edge of each of these cells during succeeding 20-sec intervals during the
403
entire period of observation and from this
calculated the rate in //,m/min for each 20sec interval. From these determinations it
is clear that, except for a few major fluctuations in speed, the advance of the trailing edge of these cells was remarkably constant during the period of observation.
The distance advanced during each 20-sec
interval rarely more than doubled from
one 20-sec interval to the next, or even
from 1 min to the next and usually varied
much less than that. Further, the trailing
edge of each of these cells always advanced
during each 20-sec interval, with only three
exceptions. This is in marked contrast to
fibroblast movement. Incidentally, the
speed of these cells during the period of
observation was quite fast, 9.1 ± 2.4 /u.m/
min to 12.3 ± 4.3 /xm/min, confirming
previous observations. It should also be
emphasized that the rate of advance of the
trailing edge of all 5 cells was approximately the same as the overall rate of advance of the leading edge throughout the
period of observation, again in marked
contrast to fibroblasts moving in vitro. We
say "approximately" because the leading
edge usually advances unevenly over a
broad front and selection of which part of
the margin is leading at any given moment
is bound to be arbitrary to a degree. For
example, hemispheric blebs themselves
form very rapidly at one locus or another
at the leading edge, in 2-10 sec, depending somewhat upon their size, but the
movement of cytoplasm into them and the
extension of the rest of the leading edge
in between blebs takes place much more
slowly—indeed, at about the same rate as
retraction of the trailing edge. Incidentally, in all of these moving cells, blebs were
observed to form exclusively somewhere
on the front or sides of the broad leading
edge, never on the sides of the rear of the
cell or at the trailing edge proper. This
would tend to reinforce a continuation of
movement in a particular direction (Chen,
1979; Trinkaus, 1980; Kolega, 1981), as
indeed actually occurs in these cells, at
least for short periods.
This relatively steady advance of the
trailing edge strongly supports the conclusion reached from the morphological evi-
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J. P. TRINKHAUS AND C. A. ERICKSON
dence—that the trailing edge of these cells
is only weakly adherent. It offers little or
no resistance to the tug of the leading edge
and follows along immediately. There are
small variations in rate that may be due to
variations in the adhesiveness of the trailing edge or in the tension exerted by the
extending leading edge or in the force of
contraction of the leading part of the cell.
But we have no evidence on this matter.
We have no certain information from
SEM concerning the adhesions made by
cells known to be involved in blebbing
movement at the time of fixation. For
some curious reason, we did not succeed
in preserving any cells moving like this on
the exposed YSL. However, we do have
some micrographs of deep cells that were
apparently moving among other deep cells
at the time of fixation and have a bulbous
extension at one end of the cell that has
the size and form of a protrusion at the
leading edge of a cell involved in blebbing
locomotion. These micrographs confirm
the smooth, rounded appearance of the
trailing edge, as viewed in the living state
in films. Moreover, no retraction fibers too
fine to be resolved by the light microscope
were evident.
Good information on the distribution of
adhesions of a rounded cell to the surfaces
of other cells comes from other deep cells
that appeared not to be moving, but whose
locomotor activity was not actually known
with certainty at the time of fixation, and
from certain rounded cells adhering to
other cells on the exposed YSL, which
were known with certainty not to be moving at the time of fixation. These cells are
visibly attached to each other by focal
adhesions, but it is of course not clear
whether these are their sole sites of attachment. From such surface images, we do
not know, for example, whether each cell
is in contact with its substratum submarginally as well. One can only say that no
such contacts were observed in thin sections of deep cells on the YSL (Trinkaus
and Lentz, 1967, Fig. 14); in transmission
electron migrographs the cells seem merely
to be resting on the tips of the microvilli of the YSL. But, then, if there were
such contacts and they were truly focal,
they would be easily missed in thin sections.
Spreading movement
In the first study of Fundulus deep cell
movement (Trinkaus, 1973), three niode-j^
of movement were discerned. These were
called blebbing movement (which we have
just discussed), lobopodial movement, and
lamellipodial movement. The term lobopodial movement described a mode whose
dominant feature is the quick shortening
of a thick elongate finger-like leading extension which pulled the cell body forward
(Triankaus, 1973, Fig. 17). The term lamellipodial movement described a mode
whose dominant feature is the spreading
of the leading edge on the substratum to
form a broad, relatively flattened lamella,
or lamellipodium. With the optical limits
of the previous study, one could not be
certain of the morphology of the tip of the
lobopodia (or phallopodia, as we have
come to call them because of their form).
In some instances, it seemed rounded (e.g.,
Trinkaus, 1973, Fig. 16A), in others slightly spread (e.g., Trinkaus, 1973, Fig. 20).
Moreover, it was observed that as the lobopodium shortened, pulling the cell body
and trailing edge forward rapidly, oftentimes its tip moved forward at the same
time, indicating that the terminal adhesion
of the leading edge was not immobile but
was moving itself. Clearly, we had to have
a closer look at the morphology and activity of such an advancing tip. For if its advance is accompanied by spreading, it
would be more properly termed a lamella
or lamellipodium. If so, this mode of
movement would more properly be considered to be simply an elongate variant of
lamellipodial movement.
Another question left unresolved in the
previous study was the locomotor activity
of filopodia. Although rather thick filopodia were sometimes observed (e.g.,
Trinkaus, 1973, Fig. 21), their role, if any,
in cell movement was not determined. In
particular, their relation to spreading lamellipodia was left unclear; for these two
protrusive activities frequently seemed to
occur together. Moreover, again because
LOCOMOTION OF FUNDULUS DEEP CELLS
of the poor resolution of bright field microscopy and the optical problems posed
by the rather large diameter of the Fundulus egg, we could not be certain whether
very fine filopodia, with the dimensions of
Jtttiose of other embryonic cells such as sea
urchin mesenchyme, were present or not.
For all this, we needed DIC optics and
SEM. We also needed more detail on the
adhesion of the trailing edge of these cells,
as discussed above for cells engaged in
blebbing movement.
Although it was clearly established in the
previous study that some deep cells move
by means of broad spreading lamellae, no
details were observed. Does the exposed
surface of an advancing lamella show ruffling? What is the pattern of adhesion of
the lamella and of the rest of the cell to the
substratum and to other cells. Do they
form retraction fibers upon retraction? A
particularly important morphological
question that has needed attention is the
relation between lamellipodia and filopodia. In the previous study, a lamellipodium
sometimes seemed to divide longitudinally
into filopodia and at other times the leading edge of a lamellipodium seemed to terminate in filar extensions just at the limit
of resolution. Culturing deep cells, to take
advantage of the superior optical conditions of cell culture, and SEM, as well as
DIC in vivo, were used to obtain a more
accurate picture of what is going on.
The superior resolution provided by
DIC optics has enabled us to settle decisively the question concerning the activity
of the leading edge of cells moving by
means of the rapid shortening of a long
phallopodium. In all cases in which the tip
was in focus, it was found to be spread on
the substratum. Moreover, in most cases in
which the extended leading part of the cell
shortened rapidly, thereby quickly pulling
the rounded cell body forward, it itself advanced exactly in a manner previously referred to as lamellipodial movement. Further, in many cells clearly engaged in
"lamellipodial movement" a good deal of
the advance of the trailing edge is also due
to a shortening of the portion of the cell
back of the leading lamellar region. It
405
seems reasonable to conclude, therefore,
that the separate designation "lobopodial
movement" is misleading. It is simply an
extreme variant of what normally occurs
much of the time during so-called "lamellipodial movement."
Although it is usually assumed that rapid shortening of a cell or a part of a cell is
due to active contraction, it could as well
be due to the passive recoil of an elastic
system under tension, or to a combination
of both (Francis and Allen, 1971; Trinkaus, 1973; Izzard, 1974; Chen, 1981). Izzard (1974) showed conclusively that the
shortening of the long filopodia of moving
tunic cells of Botryllus is indeed due to active contraction. On the other hand, however, Chen (1981) has found that the
shortening of the taut, extended tail of a
moving fibroblast upon scission of its attachment to the glass substratum is due to
a mixture of the two processes: passive
elastic recoil in its first rapid phase, and
active contraction in its second slow phase.
It was, therefore, of interest to observe
that in our most clear-cut case of shortening, unaccompanied by simultaneous
advance of the leading lamella, retraction
of the trailing edge occurs very rapidly at
first, and then gradually slows down to
zero. This is exactly what one would expect
of the retraction of an elastic system under
tension. For, as tension is dissipated, there
would be less and less force for retraction
and it would correspondingly slow down
and finally cease when the declining tension equals the force resisting shortening.
It seems therefore, that a large part of the
rapid retraction of elongate deep cells may
depend primarily on elastic recoil rather
than active contraction. (It is, of course,
conceivable that increasing adhesion of the
trailing edge is responsible for the decrease
in rate, but we have no eidence on this.)
We wish now to direct attention to the
leading lamella, or lamellipodium. All observations in vivo are based on study of
deep cells moving on the YSL. This substratum was selected for three reasons. 1)
Since the YSL is relatively planar (compared to the surfaces of other deep cells
or the undersurface of the enveloping layer,
406
J. P. TRINKHAUS AND C. A. ERICKSON
even though it is covered with microvilli
[Betchaku and Trinkaus, 1978]), it was
much easier to keep the cell in focus for a
long enough period to secure a meaningful
sequence. 2) One can fix cells moving on the
YSL directly for viewing with SEM, as previously explained (see above, p. 402). 3) The
nuclei of the YSL provide convenient
markers in the substratum and are particularly valuable in estimating the amount of
drift of the egg when it happened to move
during the observations, so that appropriate corrections could be made in estimating speed. The ventral region of the blastoderm, submarginal to the germ ring, was
selected for filming the movements of living cells in situ because there are fewer
cells there and it was therefore easier to
observe the movement of cells as individuals. The dorsal region under the embryonic shield was selected for observing cells
with SEM for reasons already given (see p.
402). At least in so far as lamellipodial
movement is concerned, we have no reason to believe that cells move differently
on the YSL in the two regions. Cells were
filmed moving both on the ventral YSL in
the intact egg and on the exposed dorsal
YSL in Leibovitz L-15 nutrient medium
after removal of the blastoderm and no differences were detected.
As noted in the previous study (Trinkaus,
1973), the breadth of the leading lamella
varies considerably from cell to cell and during the movement of any one cell, from very
broad to very narrow. In addition, there
seems to be a partial one-way antagonism
between blebbing and spreading. Although the leading edge is often observed
to spread somewhat during blebbing
movement, broad lamellipodia are rarely
observed to bleb.
Undulations or ruffles were rarely seen
on the upper surface of lamellipodia, but,
when observed, these ruffles form at or
near the leading margin of a lamellipodium and then propagate backward a
short distance on the upper surface, as on
fibroblasts in culture. We believe this to be
the first time ruffles have been observed
on cells moving in vivo during normal development. But this is an observation of
doubtful significance, since ruffles are not
known in any instance to be necessary for
movement (Trinkaus, 1976). It is of interest that ruffling has only been observed on
the lamellipodia of deep cells moving on
the YSL, where their upper surface is exposed to the fluid of the segmentation cavity. This reminds one of ruffling cells in
culture, where it always occurs on the upper cell surface which is bathed in the fluid
medium. It must be said, however, that if
deep cells otherwise situated were not observed to ruffle, we would not know
whether that indicated concealment of ruffling by other cells lying on top or genuine
absence of ruffling.
The increased resolution provided by
DIC optics has clearly shown that spreading by deep cells in vivo to form lamellipodia is a more widespread phenomenon
than previously suspected. Among the important features of these lamellipodia that
remain unresolved are the not uncommon
apparent transformation of broad lamellae
into an array of filopodia and the detailed
activity of the extending leading edge of
a lamellipodium. Recourse to cell culture
and SEM have contributed useful information on these matters. We have no guarantee, of course, that the behavior of deep
cells under the artificial conditions of cell
culture is the same as in the developing
blastoderm, nor that their appearance after fixation represents accurately their
morphology at the instant of fixation. And,
indeed, one of the main reasons for studying these cells and other cells in the living
state in their natural surroundings has
been our concern that the plane solid glass
or plastic substratum and the artificial media of cell culture would impose a special
kind of morphology and locomotor behavior on the cell and that fixation would
modify detailed surface contour in some
way. Be that as it may, if a cell's gross morphology and mode of movement in culture
and in SEM does resemble what we see in
the embryo, we have good reason to expect
that the better optical conditions of culture
and the higher magnification of SEM
could contribute reliable detail. Fortunately, we were able to persuade deep cells to
move in culture in a few instances and,
moreover, to move in a manner superfidaily identical to what has been observed
in vivo: a broad, flat lamellipodium spread-
LOCOMOTION OF FUNDULUS DEEP CELLS
ing over the substratum, followed continually and without hesitation by a rounded
cell body with a rounded trailing edge. In
addition, the speed of movement of deep
cells in vitro, 6-8 /xm/min, was comparable
0 o the speed of such cells in vivo (and much
faster, for example, than fibroblasts in vitro). It was, therefore, of much interest to
discover a marked tendency of the broad
lamellipodium of such a cell to form long
ridges or folds aligned in the direction of
cell movement. Since even in culture these
folds appeared at first glance to be "filopodia," they might provide an explanation
for the apparent "transformation" of lamellipodia into "filopodia" that we occasionally see under less favorable optical
conditions in vivo. This evidence against
the division of lamellipodia into filopodia
is corroborated by the fact that we have
never observed filopodia serving as exclusive organs of locomotion in either our
films or in SEM of fixed deep cells. In no
instance in SEM have cells known to be
moving at the time of fixation been observed to be equipped solely with the kind
of thick "filopodia" that we think we see in
vivo in the living blastoderm. On the contrary, lamellipodia in SEM sometimes appear folded in the same way as in culture.
It therefore seems reasonable to conclude
tentatively that Fundulus deep cells do not
move exclusively by means of filopodia in
the fashion of primary mesenchyme cells
of sea urchins (Gustafson and Wolpert,
1961) or tunic cells of tunicates (Izzard,
1974).
This is not to say, however, that filopodia are unimportant in the movement of
deep cells. On the contrary, one of the
striking revelations of SEM is the invariable presence of what appear to be fine filopodia protruding from the advancing
leading edge of each lamellipodium.
These filopodia are too fine to be resolved
by light microscopy in vivo in the blastoderm and demonstrate strikingly the utility of SEM. They are often <0.5 /xm thick
at the base and <0.2 (xm thick at the tip
and along most of their length; also, they
are often branched. They adhere to the
YSL at their tips and seemingly at certain
points subterminally as well. Since they invariably extend beyond the broad expanse
407
of the lamellipodium, they appear to constitute the true leading edge of these cells.
Just how they relate functionally to the lamellar region is unknown. On such a matter, observation of fixed cells with SEM in
and of itself can only provide hints. It is
possible that the leading edge of the lamellipodium spreads forward between its
protruding filopodia, filling in the spaces
and using the filopodia, with their adhesions to the substratum, as anchors stabilizing the advance of the leading edge.
This has been observed in culture for epithelial cells (Buckley and Porter, 1967;
DiPasquale, 1975) and platelets (Allen et
al., 1979). For Fundulus deep cells, however, this is pure speculation. It is also possible that they represent retraction fibers
remaining after previously extended lamellipodia had pulled back just before or
at the time of fixation. We think this to be
unlikely for two reasons. 1) Recently, with
the superior optical conditions of cell culture, Shure and one of us (Shure and
Trinkaus, in preparation) have observed
living deep cells forming exceedingly fine
filopodia. Hence, they are obviously capable of forming such protrusions. 2) The
majority of these filar extensions observed
with SEM on the YSL are not taut and
straight, like retraction fibers under tension, and many are branched.
Possibly, the two forms of protrusive activity are interconvertible. Lamellipodia of
Fundulus deep cells are often so narrow
that it is questionable whether it is semantically more accurate to refer to them
as narrow lamellipodia or as broad filopodia. And, conversely, filopodia appear
to be able to spread on the substratum to
such a degree that they really have become
lamellar. Lamellipodia can become distinctly filar and filopodia distinctly lamellar. Unfortunately, we have not yet been
able to check the validity of this idea in
living deep cells in vivo because of optical
limitations. The discovery of the composite
morphology of the leading portion of
these cells poses a problem in terminology.
How are we to designate the mode of deep
cell movement that we have previously
termed "lamellipodial"? We propose that
it now be called "spreading movement,"
408
J. P. TRINKHAUS AND C. A. ERICKSON
for spreading activity is an invariable, if
not dominant, feature of this mode of
deep cell movement, even though filopodia seem to be almost always present. This
also distinguishes it from blebbing movement, in which spreading is certainly not
a dominant feature, though often evident.
These observations lead to an important
conclusion. Fundulus deep cells appear to
translocate during and after gastrulation
in but two ways: predominantly by spreading, with the participation of filopodia, and
predominantly by blebbing, with rapid and
massive cytoplasmic flow and often a small
amount of spreading. And, even the differences between these two seem in part
quantitative.
Other extensions of the cell surface that
are routinely observed in tissue culture of
many kinds of cells but which were not observed on deep cells in vivo in the previous
study (Trinkaus, 1973) are retraction fibers. Since they mark the points on the
surface of a retracted fibroblast where the
cell is known by interference reflection
microscopy to have been in closest contact
with the substratum (Izzard and Lochner,
1976; 1980) and by microdissection to adhere most strongly (Chen, 1981), it seems
reasonable to assume that the location of
retraction fibers can be taken as a sure sign
of localized strong adhesion, where other
means, such as interference reflexion microscopy (Curtis, 1964), cannot be applied.
Both DIC optics and SEM have revealed
that Fundulus deep cells frequently form
retraction fibers when they pull away from
other deep cells and from the surface of
the YSL. If these retraction fibers do indeed mark the sites of the strongest adhesions between these cells, we can conclude
that Fundulus deep cells in vivo, like fibroblasts in vitro, do not adhere strongly over
a broad area but only at certain delimited
foci.
Significantly, in each case where a retraction fiber is visible in films, the cell surface to which it is attached is pulled out in
a pointed conical form. And, where one is
observed to detach or rupture and disappear, the cell surface in that region rapidly
retracts and rounds off. In one case the
trailing edge of a cell retracted at the incredible rate of 48.0 /xm/min during a 12sec period immediately after disappearance of a large retraction fiber. Another
cell retracted at 7.0 /xm/min during 10 sec
after disappearance of its retraction fiber^
In each instance, the trailing edge appeared rounded within seconds after disappearance of the retraction fibers. Since
observation of an obvious retraction fiber
is associated with a pointed contour of the
trailing edge and disappearance of a retraction fiber with a rounding of the trailing edge, it seems reasonable to conclude
that observation of a rounded trailing edge
indicates either lack of retraction fibers,
i.e., of firm adhesions, or the presence of
very slender ones that are easily broken.
SEM and cell culture confirm this conclusion. Moving cells with rounded cell bodies
fixed and viewed in SEM possess only very
thin retraction fibers, <0.1 fim thick,
which would be undetectable in films of
living cells in vivo, whereas those with a
tapering pointed trailing edge have thick
retraction fibers, which would be readily
visible in films of living cells in vivo. Moving cells with rounded cell bodies viewed
in cell culture, where thin retraction fibers
would be readily detected with DIC, were
not observed to possess retraction fibers at
the trailing edge.
Since cells with rounded trailing edges
either lack retraction fibers or possess only
a few extremely delicate ones, one would
expect that the adhesions of the cell body
of such cells would offer little resistence to
the forward tug of the lamellipodia and
filopodia of the leading edge and should
respond immediately to it. This should
yield two results. 1) The movement of the
cell body should follow right along with
the movement of the leading edge and the
movement of the trailing edge should be
continuous, not sporadic and spasmodic as
in fibroblasts in vitro (Chen, 1981), if
movement of the leading edge is steady. 2)
If lamellipodia and filopodia protrude
rapidly, movement of the whole cell
should be correspondingly rapid, as in
blebbing movement. Indeed, by analogy,
movement of fibroblasts in vitro is much
LOCOMOTION OF FUNDULUS DEEP CELLS
more rapid when not restrained by strong
adhesion of the trailing edge, such as just
after retraction of the trailing edge, when
for a short time it has a rounded contour
(Chen, 1979; Dunn, 1980). To test these
ijwppotheses, the rate of movement of the
rounded trailing edge of seven deep cells
involved in spreading movement in vivo on
the YSL was measured during succeeding
20-sec intervals and the rate/min for each
20-sec interval calculated. The results correspond closely to the predictions. 1) Although the rate of movement of the
rounded trailing edge varied somewhat in
some cells from interval to interval, in
every case its movement was continuous.
The trailing edge followed along quickly
after the leading edge, responding immediately. 2) Because of this immediate response to the tug of the rapidly moving
leading edge, the rate of movement of the
cell was invariably fast, 9.6 ± 1.6 to 16.3
± 2.5 /nm/min, much faster than that of
fibroblasts in vitro, and generally of the
same order of magnitude of deep cells
with a rounded trailing edge moving by
blebbing movement. In some instances, indeed, the rate was as high as 20 /Am/min
for short periods of time, comparable to
that of polymorphonuclear leukocytes in
culture (Zigmond, 1978). We have no explanation for such an augmentation of
rate.
409
and (of course) retraction fibers—is invariably smooth. This smoothness of surface is in marked contrast, incidentally, to
the surface of the enveloping layer and of
the YSL of the same egg during the blastula stage and early epiboly, when they are
covered with folds (EVL) and microvilli
(YSL) (Trinkaus and Lentz, 1967; Betchaku and Trinkaus, 1978). Clearly, Fundulus cells other than deep cells can form
these structures. In one sense, this is a
rather mysterious situation, for there is
substantial evidence that fibroblasts in vitro
(Follett and Goldman, 1970; Erickson and
Trinkaus, 1976) and the YSL of Fundulus
(Betchaku and Trinkaus, 1978) depend on
a ready supply of extra cell surface held in
reserve in many minor protuberances for
the large increases in cell surface that occur as they begin to spread (for a recent
discussion, see Trinkaus, 1980). In the absence of further information, of course, we
can do little more than speculate about
how Fundulus cells obtain the large local
increases in surface required for the very
rapid protrusive activity that accompanies
and is, in fact, the key element in their extraordinarily rapid locomotion. There
must either be massive manufacture of
new surface, massive exocytosis, or massive
movement of surface from one part of the
cell to another. We know nothing about
the possibility of new surface being synthesized and there is no evidence from
TEM
for massive exocytosis (Trinkaus and
Surface contour
Lentz, 1967; Hogan and Trinkaus, 1977)
A curious and at present inexplicable (although it must be admitted that addifeature of Fundulus deep cells during gas- tion of surface by exocytosis might easily
trulation—both those involved in blebbing be missed in ultra-thin sections). There is
movement and in spreading movement— definite evidence, however, that favors
is their constantly smooth surface. Never, massive movement of cell surface. Marks
in any of the numerous scanning electron on the surface of Fundulus deep cells in
micrographs we have taken of deep cells, vitro move toward a forming bleb, whether
within the blastoderm or on the exposed it is artificially induced by application of
YSL, have we ever observed microprotu- negative pressure and/or occurs naturally
berances, such as microvilli, microfolds, or (Tickle and Trinkaus, 1977). And, more
microblebs, on their surface. Nor have impressively, the translocation of fibrothey ever been seen in TEM (Trinkaus and blasts in vitro also seems to involve the
Lentz, 1967; Hogan and Trinkaus, 1977). shifting of surface material, once they are
This statement applies both to stationary completely spread and their surface comcells and cells in active movement. The pletely smooth. Protrusive activity at one
surface of all parts of the cell—rounded locus on the cell surface is accompanied or
cell body, blebs, lamellipodia, filopodia
410
J. P. TRINKHAUS AND C. A. ERICKSON
of cells to glass. A study by interference reflection microscopy. J. Cell Biol. 20:199-215.
DiPasquale, A. 1975. Locomotory activity of epithelial cells in culture. Exp. Cell Res. 94:191-215.
Dunn, G. A. 1980. Mechanisms of fibroblast locomotion. In A. S. G. Curtis and J. D. Pitts (eds.),
Cell adhesion and motility, pp. 409-423. C a m ^
bridge University Press, Cambridge.
Erickson, C. A. and J. P. Trinkaus. 1976. Microvilli
and blebs as sources of reserve surface membrane during cell spreading. Exp. Cell Res.
99:375-384.
Follett, E. A. C. and R. D. Goldman. 1970. The occurrence of microvilli during spreading and
growth of BHK21/C13 fibroblasts. Exp. Cell Res.
59:121-136.
Francis, D. W. and R. D. Allen. 1971. Induced birefringence as evidence of endoplasmic viscoelasticity in Chaos carohnensis. J. Mechanochem.
Cell Motility 1:1-6.
Gustafson, T. and L. Wolpert. 1961. Studies on the
cellular basis of morphogenesis in the sea urchin
ACKNOWLEDGMENTS
embryo. Directed movements of primary mesenchyme cells in normal and vegetalized larvae.
We are indebted to the following for
Exp. Cell Res. 24:64-79.
their aid: W. S. Ramsey and T. Betchaku Harris, A. 1973. Location of cellular adhesions to
(cinemicrography), P. B. Bell, Jr. (cell culsolid substrata. Dev. Biol. 35:97-114.
ture), and A. S. Pooley (scanning electron Hogan.J. C, Jr. and J. P. Trinkaus. 1977. Intercellular junctions, intramembranous particles, and
microscopy). This work was supported by
cytoskeletal elements of deep cells of the Fungrants from the NSF (BMS 70-00610) and
dulus gastrula. J. Embryol. Exp. Morph. 40:125the NIH (USPHS-H3-07137 and CA141.
22451) to J.P.T. and by a fellowship from lzzard, C. S. 1974. Contractile filopodia and in vivo
cell movements in the tunic of the ascidian, Boan NIH Training Grant HP 00032 to
tryllus schlossen. J. Cell Sci. 15:513-535.
C.A.E.
lzzard, C. S. and L. R. Lochner. 1976. Cell-to-substrate contacts in living fibroblasts: An interferREFERENCES
ence reflection study with an evaluation of the
technique. J. Cell Sci. 21:129-159.
Allen, R. D., L. R. Zacharski, S. T. Widirstky, R. Rosenstein, L. M. Zaitlin, and D. R. Burgess. 1979. lzzard, C. S. and L. R. Lochner. 1980. Formation of
cell-to-substrate contacts during fibroblast motilTransformation and motility of human platelets.
ity: An interference-reflexion study. J. Cell Sci.
Details of the shape change and release reaction
42:81-116.
observed by optical and electron microscopy. J.
Cell Biol. 83:126-142.
Kolega, J. 1981. The movement of cell clusters in
vitro: Morphology and directionality. J. Cell Sic.
Armstrong, P. B. and J. S. Child. 1965. Stages in the
(In press).
normal development of Fundulus heteroclitus.
Biol. Bull. 128:143-168.
Tickle, C. and J. P. Trinkaus. 1977. Some clues as
Betchaku, T. and J. P. Trinkaus. 1978. Contact reto the formation of protrusions by Fundulus deep
lations, surface activity, and cortical microfilcells. J. Cell Sci. 26:139-150.
aments of marginal cells of the enveloping layer Trinkaus, J. P. 1973. Surface activity and locomotion
and of the yolk syncytial and yolk cytoplasmic
of Fundulus deep cells during blastula and gaslayers of Fundulus before and during epiboly. J.
trula stages. Dev. Biol. 30:68-103.
Exp. Zool. 206:381-426.
Trinkaus, J. P. 1976. On the mechanism of metaBuckley, I. K. and K. R. Porter. 1967. Cytoplasmic
zoan cell movements. In G. Poste and G. L. Nifibrils in living cultured cells. Protoplasma
colson (eds.), The cell surface in animal development.
64:349-380.
Cell Surface Reviews 1:225-329.
Chen, W.-T. 1979. Induction of spreading during Trinkaus, J. P. 1980. Formation of protrusions of
fibroblast movement. J. Cell Biol. 81:684-691.
the cell surface during tissue cell movement. In
R. O. Hynes and C. F. Fox (eds.), Tumor cell surChen, W.-T. 1981. Mechanism of retraction of the
faces and malignancy. Progress in Clinical and Bitrailing edge during fibroblast movement. J. Cell
ological Research 41:887-906.
Biol. (In press)
Curtis, A. S. G. 1964. The mechanism of adhesion Trinkaus, J. P. and T. L. Lentz. 1967. Surface spe-
preceded by retraction of an approximately equal amount of surface at another locus
(Weiss and Garber, 1952; Chen, 1979;
Dunn, 1980). We have noted in the present study that the protrusive activity of a
moving deep cell tends to be confined to
the general region of the leading edge (see
above p. 403), but as yet have no quantitative information on the matter. And, unfortunately, Fundulus deep cells are seldom flat enough in vivo to lend themselves
to a straightforward planimetric study of
their surface area, as are fibroblasts in vitro
(Chen, 1979). Hence the kind of estimates
of local changes in surface area that are
necessary to provide evidence on this point
do not seem feasible at present.
L O C O M O T I O N O F FUNDULUS
DEEP CELLS
411
cializations of Fundulus cells and their relation to
structure of the medium. Contributions to a
cell movements during gastrulation. J. Cell Biol.
quantitative morphology. Proc. Natl. Acad. Sci.
32:139-153.
U.S.A. 38:264-280.
Weiss, P. and B. Garber. 1952. Shape and movement Zigmond, S. H. 1978. Chemotaxis by polymorphoof mesenchyme cells as functions of the physical
nuclear leukocytes. J. Cell Biol. 77:269-287.