Download Cleavage Furrow Establishment—A Preliminary to Cylindrical

AMER. ZOOL., 13:941-948 (1973).
Cleavage Furrow Establishment—A Preliminary to Cylindrical Shape
Change
RAYMOND RAPPAPORT
Department of Biological Sciences, Union College, Schenectady, New York 12308
and The Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672
SYNOPSIS: The predictable pattern of cell shape changes characterizing animal development must be a consequence of control mechanisms that are at least analogous to
those operating in dividing cells. When cells change shape by an internal mechanism,
it is implied that they also contain systems that will activate and deactivate the
mechanism, localize it within the cell, insure proper timing, and impart the proper
vectoral qualities. Experimental investigations designed to elucidate similar processes
in dividing cells reveal that the physical mechanism that accomplishes cytokinesis is
established at or near the equatorial cell surface by the mitotic apparatus. The process
is completed by late metaphase or early anaphase. In cleaving eggs a pair of asters can
substitute for the intact mitotic apparatus. The nature of the stimulus which apparently passes from the mitotic apparatus to the surface is presently unknown. It moves
toward the surface at about 6 microns per minute and requires about 1 minute to
establish the mechanism. The resulting equatorial contractile activity is initially isotropic but becomes anisolropic at the beginning of visible constriction.
The embryonic cell's ability to alter its
shape by application of intrinsic physical
force at a predictable time and in a predictable manner strongly suggests the existence of control systems which have hardly yet been subjected to experimental
analysis. Activity of this kind implies the
existence of a system for establishing a
force-producing mechanism in a localized
cell region at a time consistent with or dictated by an overall developmental time
table. The mechanism must be activated
and deactivated and some means must exist
for insuring that the force has the proper
vectoral qualities. Investigations concerning the mechanisms of animal cell division
have dealt with similar mechanisms for
about 100 years, and perhaps a brief account of some of the thought and technique
that has evolved in studies of cytokinesis
may prove useful in studies of morphogenesis.
Three long-range goals have historically
determined the direction taken by investigations on cytokineses. The first goal is
elucidation of the nature of the force that
accomplishes division. The second is locaThe original investigations described in this report were supported by giants from the National
Science Foundation.
941
tion of the force-producing mechanism
within the cell, and the third goal is an
understanding of the process that establishes the mechanism. Simple observation
(regardless of elegance or magnification)
has rarely furnished conclusive information on any of these subjects. Observations
of division do not usually permit discrimination between cause and effect and may
not reveal whether a structure or phenomenon present during division is causally related to it. That cell division depends upon
the operation and interaction of many cell
structures and processes has been evident
since the process was first clearly viewed.
Its complex nature means that unequivocal
interpretation of experimental results is
possible only when the cell's basic fabric
is intact and functioning. The constraints
thus placed upon experimental design are
severe. Progress has been accomplished by
stepwise, systematic elimination of hypothetical alternatives rather than by heroic
breakthroughs.
For reasons of experimental convenience,
cleaving eggs and blastomeres are the most
commonly used experimental material for
studies of cytokinesis. However, investigations in which the responses to experimentation of cleaving eggs and adult tis-
942
RAYMOND RAPPAPORT
FIG. 1. (left) Manipulation chamber. E, egg; M,
microscope objective; P, micropipette; W, wall
made of coverslip. (right) Relation between position of mitotic figure and cleavage plane, a-c, Successive stages of cleavage when the spindle is removed: a, before removal of the spindle; b, after
removal; c, furrow appears at a predetermined
position, d-f, Successive stages of cleavage when
the mitotic figure is displaced by the removal of
a part of egg protoplasm: d, before the displacement of the mitotic figure; e, after displacementastral rays of the lower aster are much elongated;
f, furrow appears at a predetermined position. In
both series, cleavage planes are independent of
shifted position of asters. (From Hiramoto, 1956.)
sue cells were compared (Rappaport and
Rappaport, 1968) revealed no fundamental
differences. The outstanding morphological differences between these two kinds of
cells are the relatively greater size of the
asters during the cleavage divisions and the
more irregular form of adult tissue cells.
The location of the cleavage mechanism was determined by experiments in
which regions and organelles were removed, displaced, or specifically disorganized. Persistence of division despite
such localized disruption implies that the
affected area makes no essential contribution to the process. When the mitotic apparatus, comprising the spindle, asters,
and chromosomes, is removed at late metaphase or later, while the sea urchin is still
spherical, division ensues and a normallooking cleavage furrow bisects the egg into a pair of enucleated blastomeres (Fig.
1) (Hiramoto, 1956). Hiramoto (1965) also showed that after anaphase the mitotic
apparatus and immediately surrounding
cytoplasm could be displaced by a large
oil droplet or disrupted by injection of
isotonic sucrose solutions and sea water
without blocking cleavage. In other similar experiments, cleavage of sand dollar
eggs was unaffected when the cytoplasm
in the equatorial plane was vigorously
stirred with a needle (Fig. 2) (Rappaport,
1966). Vertebrate tissue cells behave in
similar fashion, as newt kidney cells continue to divide while the mitotic apparatus and associated cytoplasm are pushed
back and forth through the equatorial
plane (Rappaport and Rappaport, 1968).
These experiments, and others, performed
on cleaving cells indicate that the furrowing process in animal cells is in no way
physically dependent upon the arrangement or presence of any structures in the
subsurface cytoplasm. They have been interpreted as indicating that the physical
mechanism that accomplishes division is
in or attached to the surface.
Although the mitotic apparatus plays no
physical role in furrowing, it appears to
be the structure that determines where the
furrow will appear. One of the earliest
clear demonstrations of this relationship
CLEAVAGE FURROW ESTABLISHMENT
FIG. 2. Cleavage in a sand dollar egg with a moving needle inserted through the cleavage plane.
The needle was swept back and forth during the
period between the photographs. (From Rappaport, 1966.)
was that of Conklin (1917) who produced
abnormally large polar bodies by centrifuging the meiotic apparatus to the center of the egg before metaphase. By manipulating the cell's geometry before the furrow is established, it has been shown that,
in cleaving eggs, the asters of the mitotic
apparatus can substitute for the intact
mitotic apparatus (Rappaport, 1961; Hiramoto, 1971). After a spherical sand dollar
egg is reshaped into a torus by forcing a
glass ball completely through it, the first
cleavage results in a horseshoe-shaped binucleate cell (Fig. 3). As the egg enters
the second cleavage cycle, the two mitotic
apparatuses form in the arms of the horseshoe and extend toward its bend. Two furrows form in the arms of the horseshoe
adjacent to the spindle of the intact mitotic
apparatus and almost simultaneously a
943
third furrow forms in the bend of the
horseshoe between two asters that were
never joined by a spindle. Other experiments have shown that furrows will form
between cytasters (Wilson, 1901) and
sperm asters and between combinations of
amphiasters and sperm asters (Sugiyama,
1951). Although the dispensability of the
spindle has been clearly shown in cleaving
eggs, none of the results thus far achieved
eliminate the possibility that the spindle
may share the asters' capacity for furrow
establishment, even though it may normally remain unused by reason of geometrical
circumstances. No comparable analysis of
the role of asters in furrow establishment
in tissue cells has yet been accomplished,
and the relatively larger spindle in these
cells lies closer to the equatorial surface.
I have speculated elsewhere (Rappaport,
1971) that the important components of
the mitotic apparatus for this function may
be its linear elements. Whether linear elements of the asters or spindle were primarily responsible for furrow establishment could depend upon their relative
sizes and the geometrical relations obtaining in the cell.
Since it is customary to consider the furrowing mechanism as a modified portion
of the cell surface, a brief discussion of
the meaning of the term "surface" in this
context is pertinent. In echinoderm eggs,
the plasma membrane is underlain by a
cytoplasmic region which has for some
years been termed the cortex. The cortex
was demonstrated in echinoderm eggs by
microdissection (Chambers, 1921) and by
centrifugation (Marsland, 1939). Its dimensions were carefully determined by
Hiramoto (1957). It comprises a S-4/i thick
layer that is inseparable from the plasma
membrane and is denser than the cytoplasm lying immediately beneath it. In
dividing cells, the cortex is thickest at the
base of the furrow. Pigment granules may
be held within it and dense strands of cytoplasm may be pulled from its undersurface with needles. Unfortunately no ultrastructural basis for these cortical properties has been clearly demonstrated, and
nearly all studies on the cell cortex have
944
RAYMOND RAPPAPORT
FIG. 3. Cleavage of a torus-shaped cell. Condition
of the mitotic apparatus is shown in line drawings. The position of the spindle is shown by a
double line. Note synchrony with controls. Initial
temperature 19.5 C. Timing begins at fertilization.
Upper left: immediately before furrowing (69
min). Upper right: first cleavage completed, producing a binucleate cell (79 min). Lower left:
second cleavage; two cells have divided from the
free ends of the horseshoe and the binucleate cell,
and the binucleate cell is dividing between the
polar regions of the asters of the second division
(142 min) . Lower right: division completed; each
cell contains one nucleus (144 min) . (From Rappaport, 1961.)
concerned marine invertebrate eggs. However, it appears that the cleavage mechanism is established in what must be considered the cortical region in those cells
where the mechanism has apparently been
observed (Schroeder, 1968; Arnold, 1968,
1969). More information concerning the
structure, behavior, and constitution of the
cortex is highly desirable.
The mitotic apparatus apparently determines the position of the furrow by es-
tablishing the division mechanism in a
localized portion of the cell surface. Any
part of the surface can be changed into
furowing mechanism, and the mitotic apparatus could accomplish its role by altering the constitution of the subsurface cytoplasm. The alteration, or stimulation,
might result from materials added to or
subtracted from the local area, or from
changes that move by propagation rather
than transport (Rappaport, 1965). The in-
CLEAVAGE FURROW ESTABLISHMENT
II
FIG. 4. Cleavage of a sand dollar egg attenuated
by tensile stress. The dark sphere is a glass bead.
(From Rappaport, 1960.)
50
1II 1 + 1
-*
1
teraction between mitotic apparatus and
surface appears to be completed by late
metaphase or early anaphase, for thereafter
the furrow develops and functions after
the two are experimentally isolated from
each other (Hiramoto, 1956). Establishment of the division mechanism in the cell
surface imposes regional functional differentiation which persists for a relatively
brief part of the cell cycle. It is logical to
propose that the part of the surface influenced by the mitotic apparatus is altered
in physical properties and behavior, and
that these alterations precipitate division.
Other parts of the cell surface would, presumably, play no essential physical role
in the process. Although both the polar
(Swann and Mitchison, 1958; Wolpert,
1960) and equatorial (Rappaport, 1965)
surfaces have been suggested as the recipients of mitotic apparatus stimulation,
present evidence supports the latter alternative. No essential geometrical relation
between the mitotic apparatus and the
polar surfaces can be shown to exist. For
instance, cells that are extremely attenuated by attached weights long before the
position of the furrow is determined cleave
S 30-
1
20 -
-r*
10 -
1
1
i
i
i
30
35
40
45
50
Interastrol distance (/x)
FIG. 5. Summary of data from experiments in
which the distance between the asters (interastral
distance) and the distance from a line drawn between the astral centers to the equator (spindleto-surface distance) are varied. When the spindleto-surface distance is 35^ or more and the interastral distance is 35^t or more, furrowing almost
invariably fails. If the spindle-to-surface distance
is reduced to 20^ or less, furrowing occurs in conjunction with a 35fi interastral distance. Plus indicates a furrow formed adjacent to the asters;
minus indicates no furrow formation in that location. (From Rappaport, 1969.)
normally, although the distance from the
mitotic apparatus to the polar surfaces is
greatly increased (Fig. 4) (Rappaport,
I960). On the other hand, relatively small
alterations of the distances between the
asters and the distances from the asters to
the equatorial surface have profound effects on furrow formation (Fig. 5). When
the distance between asters is increased,
furrows do not form. Furrows will form,
however, in surfaces located close to abnormally distant asters (Rappaport, 1969).
The observation that a deficiency arising
from increase in one dimension can be
remedied by decreasing the other dimension lends support to the proposal that
furrows may be established by the joint
946
RAYMOND RAPPAPORT
action of the asters on the equatorial surface.
The stimulation process was not an
early subject of speculation and experimentation, as was the physical nature of the
division mechanism, and investigations
concerning its nature are relatively recent. We have no information concerning
the constitution of the stimulus, and detailed information will probably await a
clearer conception of the physico-chemical basis of the functioning division mechanism. Experimentation has permitted a
rough characterization of some aspects of
the process. In many experiments bearing
on this topic, the cell's shape is altered
early in the mitotic cycle so that the furrow's position is established under unusual
geometrical conditions. Frequently advantage is taken of the fact that in flattened
cells the furrows appear in the margins
and not in the flattened surfaces. In normal and experimentally altered cells the
time and position of the furrow's first appearance is correlated with the position of
the mitotic apparatus. When the apparatus
is centered, as in typical sea urchin eggs,
the furrow appears at the same time at all
points on the equatorial circumference and
it constricts symmetrically. In some eggs
the nucleus is excentric. In these cases, typified by Astriclypeus manni, the furrow appears first at the surface closest to the
mitotic apparatus so that the egg is temporarily heart-shaped (Dan and Dan,
1947). Subsequently, furrowing begins on
the diametrically opposite side and then
the entire equatorial surface engages in
the division process. In cases of extreme
nuclear excentricity, such as is found in
coelenterate eggs, furrows form at the surface closest to the mitotic apparatus, and
the diametrically opposite surface never
actively participates. Furrowing is unilateral, as the furrow cuts through from one
side only. These cleavage patterns are consequences of the geometrical relation between the mitotic apparatus and the surface; for eggs which normally form symmetrical furrows will form unilateral furrows and vice versa when the mitotic apparatus is shifted (Rappaport and Con-
rad, 1963).
The observation that the time of the
furrow's appearance is correlated with distance from the mitotic apparatus permits
estimation of the rate of movement of the
stimulus across the cell. In a flattened cell
with an excentric nucleus, the furrow appears first at the equatorial surface closer
to the mitotic apparatus, and later at the
opposite, more distant, margin. All other
things being equal, the time difference between the appearance of the two furrows
should be proportional to the difference
in distances between the two margins and
the mitotic apparatus. In making the determinations, the distance from the spindle
center to opposite equatorial margins was
measured; a stopwatch was started when
the furrow appeared in the nearer margin
and stopped when it appeared in the more
distant margin. In a series of determinations made on sand dollar eggs with varying degrees of nuclear excentricity the relation between time and distance was linear
(Fig. 6), and the rate was calculated to be
6.3 ± 1.8 microns per min (Rappaport,
1972). This rate is slower than free diffusion (Swann, 1951), and the types of cytoplasmic streaming usually studied (Wolpert, 1965); it approximates the rate of
microtubular growth (Bajer and Mole'Bajer, 1972). These data may be correlated
with the observations that the linear appearance of the astral rays is based primarily upon their microtubular content
(Rebhun and Sander, 1967) and that the
position of the furrow is established at late
metaphase or early anaphase when the astral rays achieve maximum length (Wilson,
1895).
The observation that a very excentric
mitotic apparatus only establishes a furrow in the nearer equatorial margin has
provided a method for estimating the time
necessary for furrow establishment (Rappaport and Ebstein, 1965). After the furrow
develops in the nearer region of a sand dollar egg with an experimentally displaced
nucleus, a furrow can be established in
the more distant margin if it is pushed toward the mitotic apparatus and held there.
Under these circumstances, the total time
CLEAVAGE FURROW ESTABLISHMENT
12
DISTANCE
16
20
(microns)
24
28
FIG. 6. The relation between the difference in
distance between the spindle and the near and
distant equatorial cell margin and the difference
in time between the appearance of the furrow in
the near and distant margins. (From Rappaport,
1972.)
between pushing in the surface and the appearance of an active furrow is 3y£ min,
regardless of the length of time the surface is held close to the mitotic apparatus.
When the mitotic apparatus and surface
are held together for 1 min, the surface
upon release resumes its original contour
beyond the limit of influence of the mitotic
apparatus, and then 2i/2 min after release,
develops a furrow. Exposure of surface to
the mitotic apparatus for less than 1 min
fails to produce a furrow. These results
suggest that a stimulus period of about
1 min irreversibly alters the exposed surface; during the ensuing 2i/2 min latent
period, the molecular reorganization essential for establishment of the functional
division mechanism takes place.
These observations permit the construction of a very approximate time table for
events immediately preceding cytokinesis.
If we use as an example an echinoderm
egg with a 150 micron diameter, and assume that the stimulus originates near the
centrosomes, and we determine that time
0 is the moment of appearance of the active furrow, the stimulus would begin to
move toward the surface at about T =
—12 min. At T =: —S]/2 m i n the stimulus
reaches the surface and a t T = —2i/2 min
the furrow's position is established. At
some time between T = —2\/2 min and
T = 0, equatorial contraction begins.
947
Scott's (1960) study of the movement of
cortical pigment granules revealed that the
initial contraction is isotropic but becomes
anisotropic and circumferential with the appearance of the furrow. His observation
suggests that the cleavage stimulus may
initially impart no vectoral information to
the surface.
We now have a general idea of what
goes on in a cell immediately before and
during division at a very complex level of
organization. Several active cell components have been identified, as has the nature of their participation. It is reasonable
to expect that analogous events occur in
developing cells that change their form by
application of intrinsic forces. A clear understanding of the mechanisms which enable such cells to control the time, location, and orientation of the internally applied stresses will require rarely used and,
perhaps, refreshing experimental approaches to morphogenesis.
FIG. 7. Egg outlines and corresponding pigment
granules patterns during 4 stages of cleavage in
an Arbacia egg. Patterns shrink two dimensionally
in cells marked 13 and 14, but only circumferentially in 15 and 16. (From A. Scott, 1960.)
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RAYMOND RAPPAPORT
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