2239
Journal of Cell Science 107, 2239-2248 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Isolation and characterization of a sea urchin zygote cortex that supports in
vitro contraction and reactivation of furrowing
Gary R. Walker1, Robert Kane2 and David R. Burgess1,*
1246 Crawford Hall, Department of Biological Sciences, University of Pittsburgh,
2Kewalo Marine Laboratory, University of Hawaii, Honolulu, HI 96822, USA
Pittsburgh, PA 15260, USA
*Author for correspondence
SUMMARY
The isolation of the cortex of the sea urchin blastomere by
detergent lysis was explored with the aim of analyzing components important in the structure and function of the
cortical cytoskeleton, and their relationship to such
phenomena as contraction. Buffered EGTA medium supplemented with isotonic glycerol and with magnesium, at a
level close to the reported internal cellular concentration,
yields stable cytoskeletal cortices that retain their spherical
shape. Cortices prepared this way contain actin, myosin,
fascin and spectrin, components normally associated with
the cortical cytoskeleton in a similar distribution to that in
intact zygotes. They retain the organized cortical filamentous structure, including the actin-fascin bundles that form
cores of microvilli. ATP and NaCl caused changes in
cortical shape, described as either contraction or
expansion, respectively. Spectrin, but not myosin, was
partially extracted by NaCl, resulting in expansion of the
cortex that suggests a role for spectrin in maintenance of
cortical structure. ATP (but not ADP nor ATPγS), which
caused the partial removal of myosin and spectrin, led to
the contraction of the cortex, consistent with a role for
myosin in cortical tension. In cortices isolated from
dividing eggs, the zygotes retained their cleavage furrows
and ATP induced continuation of furrow progression. This
preparation appears to be a useful in vitro model for
cytokinesis.
INTRODUCTION
nuclear envelope breakdown and recede during cytokinesis;
however, there is no correlation between peaks of cortical
stiffness or tension and cytokinesis (Hiramoto, 1990). Before
cell division the cortical actin cytoskeleton undergoes structural changes that can result in increased cortical tension (Usui
and Yoneda, 1982; Vacquier, 1981). At the time of division
cortical actin filaments become re-organized (Hamaguchi and
Mabuchi, 1988; Schroeder and Otto, 1988; Yonemura and
Mabuchi, 1987) and actin must interact with myosin for contraction of the cleavage furrow (Mabuchi and Okuno, 1977).
Recent evidence suggests that there is an accumulation of both
actin and myosin into the contractile ring for cytokinesis
(Schroeder and Otto, 1988; Cao and Wang, 1990). However,
since the entire cortex is competent to contract and form
furrows upon stimulation by the astral complex (Rappaport,
1988), it is likely that the cortical actin cytoskeleton is not
locally unique until stimulated to form the contractile ring. In
addition, actin and myosin and their associated proteins relocalize to the cortex after fertilization and during contractile
ring formation (Hamaguchi and Mabuchi, 1988; Schroeder and
Otto, 1988; Bryan and Kane, 1978; Fishkind et al., 1990a,b;
Hosoya and Mabuchi, 1984; Hosoya et al., 1986; Ishidate and
Mabuchi, 1988; Kuramochi et al., 1986; Mabuchi, 1983;
Mabuchi et al., 1985; Mabuchi and Kane, 1987; Schatten et al.,
1986; Wang and Spudich, 1984).
Study of the regulation of cortical actin’s interaction with
Cytokinesis, mediated by the actin/myosin-II-based contractile
ring, is an essential part of mitotic cell division in most cell
types. While in vitro models for mitosis have been developed
in recent years, a reproducibly functional reconstituted model
for cytokinesis has been elusive. This is somewhat surprising
due to the early success by Hoffmann-Berling (1954) in
demonstrating ATP-induced cleavage furrow continuation in
glycerinated models of fibroblasts. Cande (1980) extended this
work on other cultured cells permeabilized with Brij and polyethylene glycol. More recently, Yoshimoto and Hiramoto
(1985) have shown that sea urchin zygotes, saponin-permeabilized at mid-cleavage but still intact, can be induced to
continue cleavage in an ATP-dependent manner. In contrast to
the study of mitotic models facilitated by the ease of isolation
of the mitotic apparatus, these early reconstitutions of cytokinesis occurred in fairly intact cells but not in the isolated contractile ring.
The sea urchin egg has been a favorable cell for investigating cortical activity and cytokinesis, as the cells are large and,
after insemination, very large numbers of zygotes develop synchronously and undergo a series of complex cortically
mediated events associated with fertilization and cleavage.
After fertilization, the cortex of many eggs undergoes cycles
of increased stiffness or tension, which generally peak at
Key words: cytoskeleton, furrowing, contraction, zygote cortex
2240 G. R. Walker, R. Kane and D. R. Burgess
other cytoskeletal proteins during cytokinesis would benefit
from the use of an in vitro system. Such a system would require
the isolation of an intact cortical framework. Ideally, a cortex
preparation should retain not only the native organization but
also functional (i.e. contractile) activity. The intact egg
cleavage furrow has been isolated from newt eggs, partially
characterized and contraction reactivated in vitro; but, unfortunately, the hand microdissection technique appears to
preclude mass isolation for biochemical study (Mabuchi et al.,
1988). The cortex of the sea urchin egg has been mass isolated
by a variety of methods (Vacquier, 1981; Schroeder and Otto,
1988; Spudich and Spudich, 1979; Burgess and Schroeder,
1977; Sakai, 1960; Mabuchi et al., 1980; Begg and Rebhun,
1979; Vacquier and Moy, 1980). Mass isolation of cleavage
furrows from dividing sea urchin eggs has also been accomplished (Schroeder and Otto, 1988; Yonemura et al., 1991).
However, many of these preparations result in cortices tenaciously adherent to substrata precluding contractile properties,
are from the unfertilized cortex, or have lost their contractile
properties. Reports of cleavage in sea urchin blastomeres permeabilized by low concentrations of saponin (Yoshimoto and
Hiramoto, 1985) suggest that detergent-based isolation
methods may be capable of yielding functional cortical preparations from sea urchin blastomeres. Similar detergentinsoluble cytoskeletons derived from Dictyostelium are
capable of contracting in response to ATP (Kuczmarski et al.,
1991). Detergent-induced lysis of sea urchin blastomeres has
also been used for the recent isolation of the contractile ring,
which lacks the ability to contract in vitro (Yonemura et al.,
1991). These detergent-based procedures appear effective in
stabilizing the cell cortex and in retaining its actin-based
cytoskeleton (Spudich and Spudich, 1979; Vacquier and Moy,
1980; Vacquier, 1975; Kane, 1986).
One aim of the studies reported here is to extend previous
work and utilize a detergent-based procedure for the isolation
of stabilized cortices from fertilized and dividing sea urchin
eggs, in order to study the contribution of actin-associated
proteins to the structural and contractile properties of the egg
cortex. The cortices isolated for these studies retain their
microvilli, actin meshwork and actin-associated proteins. As in
intact eggs, changes in cytoskeletal composition of the isolated
cortex are seen in response to egg activation. The association
of myosin and spectrin in the isolated cortex is effected by ATP
and salt, with high salt preferentially removing spectrin, and
inducing cortical expansion and thinning. Treatment of cortices
with ATP, but not ATPγS or ADP, results in a overall ‘contraction’ in interphase cortices and in a continued slow contraction of the contractile ring in cortices from dividing eggs.
MATERIALS AND METHODS
Cortex isolation
The eggs of the Hawaiian sea urchin Tripneustes gratilla, the Florida
urchin Lytechinus variegatus, and the California urchin Strongylocentrotus purpuratus, were used in these experiments. The preparations and properties of isolated cortices were the same in all species,
so references to species are generally omitted, except that much of the
initial characterization was done with eggs from Tripneustes by
Robert Kane. Shedding of gametes was induced by the injection of
0.5 M KCl and eggs were dejellied by brief exposure to pH 5.0 sea
water. Egg volumes were measured after centrifugation of dejellied
eggs at 220 g for 30 seconds. The fertilization membrane and hyaline
layer were removed by transfer of eggs to 1 M glycine or 1 M urea
at 30-40 seconds after sperm addition (Kane, 1973). Transfers of fertilized eggs were done by hand centrifugation, using an approximate
ratio of 1 ml of eggs to 35 ml of demembranation solution. At 5
minutes after sperm addition the glycine or urea was replaced by the
medium to be used for growing the blastomeres. The zygotes were
then washed three times in a 19:1 (v/v) mixture of isotonic NaCl and
KCl containing 5 mM MgSO4 + 0.1 mM Ca2+.
Cortices were isolated by washing the cells once in a 1 M glycerol
solution containing 5 mM MgCl2, buffered at pH 8.3 with 5 mM Tris,
and then transferred to isolation medium for lysis. This medium was
buffered at pH 6.9-7.0 with 20 mM PIPES and contained 1 M
glycerol, 5 mM EGTA, 5 mM MgCl2 and 0.5% Nonidet P-40. Thirty
ml of isolation medium was added to a cell pellet of 1 ml and stirred
gently while the cells lysed; cells were then hand centrifuged, the
supernatant volume was aspirated to 8 ml and the cells were transferred to a loose-fitting Dounce homogenizer. Gentle homogenization
freed the cortices from adhering cytoplasm, usually requiring 25-50
strokes for Tripneustes or 5-8 strokes for the other species. Cortices
were collected by centrifugation at 220-800 g for 1-5 minutes and
washed in 2-3 changes of the same medium without Nonidet.
Morphological analysis
Analysis of the effects of various agents on the cortex morphology
was accomplished by two types of experiments using cortices isolated
from fertilized eggs. One series of experiments involved the use of a
cortex shape-scoring system and was useful in quantifying the effect
of agents on cortex morphology. Cortices were isolated, treated for
30 minutes with buffer alone or buffer containing either 2 mM ADP,
2 mM ATP, 2 mM ATPγS or 250 mM NaCl, and then fixed in 2%
glutaraldehyde. A large population (>100) of cortices were scored, in
a double-blind experiment, on the basis of shape into 4 categories
(type I, smooth and spherical; type II, wrinkled and spherical; type
III, oval or rugby football-shaped; and type IV, fully collapsed and
wrinkled). In some experiments the diameters of cortices were
measured and averaged. Alternatively, cortices were placed on
protamine-coated microscope slides after isolation and directly
viewed during perfusion with 10-20 mM ATP, ADP- or 250 mM
NaCl-containing media. Photomicrographs were taken with Zeiss
Universal or Leitz Dialux microscopes using phase-contrast and epifluorescence optics. Photomicrographs were taken with Technical Pan
2415 film (Kodak) at ASA 200.
The distribution of myosin and spectrin was analyzed by indirect
immunofluorescence using rabbit antibodies to egg myosin
(Yabkowitz and Burgess, 1987) or spectrin (Fishkind et al., 1990a,b),
a generous gift from Dr David Begg. Bound primary antibody was
visualized using FITC-conjugated goat anti-rabbit antibody. All
samples were fixed with 3.7% formaldehyde in isolation buffer for 30
minutes on ice and then blocked with 1% BSA in isolation buffer as
a precaution. Actin was visualized by use of rhodamine-labeled phalloidin. Photomicrographs were taken using T-Max film (Kodak) at
ASA 1600 and were developed in T-Max developer.
Experiments for assessing the quantitative extraction of specific
proteins from the cortices utilized western or dot blots with antibodies
against myosin or spectrin. Cortices isolated as described above were
washed two additional times in isolation buffer without detergent by
centrifugation at 10,000 g for 10 minutes. To determine the proteins
that potentially are extracted from the cortex, resuspended cortices
were incubated in 20 mM ATP, 20 mM ADP, 20 mM ATPγS, 250
mM NaCl or isolation buffer alone for 30 minutes on ice followed by
centrifugation at 10,000 g for 10 minutes. Experiments determining
the effect of ATP concentration on extraction used the following ATP
concentrations: 1.25, 2.5, 5, 10 and 20 mM. Pellets and supernatants
were placed in equal amounts of SDS sample buffer and all samples
were loaded proportionately on SDS-gels or dot blots. Relative
Isolation of a contractile egg cortex 2241
amounts of specific proteins were determined by using 125I-goat antirabbit to probe for the specific antibody of interest bound to the dot
blot or western blot, followed by radioactive detection using an Ambis
radioanalytical imaging system (AMBIS Systems Inc., San Diego,
CA). Protein concentrations were determined by the method of
Minamide and Bamburg (1990).
Electron microscopy and polyacrylamide gel
electrophoresis
Cortices were prepared for electron microscopy by fixation in 2% glutaraldehyde containing 0.2% tannic acid (Begg et al., 1978) and
postfixed in 1% osmium tetroxide in the same medium. Blastomeres
were fixed using the method of Eisenman and Alfert (1982). Cortices
and blastomeres were dehydrated in ethanol and embedded in Araldite
or LR White. Sections were cut on a Reichert or Dupont MT-5000
ultramicrotome, stained with lead citrate and uranyl acetate, and
examined and photographed in a Philips 201 or 301 electron microscope.
Electrophoretic analysis of isolated cortex proteins was accomplished using SDS-polyacrylamide minislab gels according to the procedures of Laemmli et al. (1970) and Matsudaira and Burgess (1978).
Two-dimensional gel electrophoresis involving isoelectric focusing
was performed according to O’Farrell (1975). Western blots were
performed (Towbin et al., 1979) using microslab gels with antibodies
against egg myosin (Yabkowitz and Burgess, 1987) and egg spectrin
(Fishkind, 1987). In addition, antibodies to fascin (Otto et al., 1980),
antibodies to α-tubulin (a generous gift from Dr Charles Walsh, University of Pittsburgh) and antibodies to apical lamina extracellular
matrix proteins (Burke et al., 1991; a generous gift from Dr Robert
Burke, University of Victoria) were used to detect the presence of
these proteins in the isolated cortex.
PIPES buffer, ATP, EGTA, Nonidet P-40 and heavy meromyosin
were obtained from Sigma Chemical Co., St Louis, MO.
RESULTS
Isolated cortex composition and structure
Cortices of blastomeres do not survive detergent lysis in a
buffered glycerol-EGTA solution, but intact cortices can be
recovered (Fig. 1) if the glycerol medium contains 5 mM
magnesium (Kane, 1986), a range reported for the internal egg
concentration (Sui and Shen, 1986.). The lysed cells remained
intact (Fig. 1B) until cortices were freed of cytoplasm by gentle
homogenization and washing, which caused some deformation
from their original shape (Fig. 1C). Washing cortices removed
most of this cytoplasmic debris leaving behind a cortical shell
(Fig. 2A). Cortices obtained by this method display an
elaborate actin filament meshwork immediately under what
sometimes appears to be a prominent hyaline layer, distinct as
an electron-dense lamina, when visualized by electron
microscopy (Fig. 2B). Actin cores derived from the microvilli
were distinctly evident in all cortices. At higher magnifications
(Fig. 2B), the microvillar actin cores displayed a typical
bundled appearance, and decoration of the actin with HMM
revealed arrowheads pointing toward the actin meshwork as in
intact eggs (data not shown).
The isolated cortex was composed of numerous proteins
ranging from low to very high molecular mass as analyzed by
polyacrylamide gel electrophoresis (Fig. 2C). Not surprisingly,
actin was the major protein component of the cortex as
analyzed on one- or two-dimensional PAGE (Figs 2C, 3A).
Immunoblot analysis indicated the presence of other actin-
Fig. 1. The isolation of the detergent-extracted sea urchin egg cortex
cytoskeleton. (A) Phase-contrast micrograph of detergent-lysed eggs.
(B) Detergent-lysed eggs after gentile homogenization (cortex
cytoskeleton). (C) Cortex cytoskeleton after washing in detergentfree buffer, showing some deformation of morphology. The shape of
the isolated cortex is somewhat deformed.
associated cytoskeletal proteins such as fascin, spectrin and
myosin II (Fig. 3A). These components are similar to those
present in TAME-isolated cortices (Spudich and Spudich,
1979; Kane, 1986), as prepared by Vacquier and Moy (1980),
as prepared by shearing eggs adherent to polylysine-coated
dishes (Schroeder and Otto, 1988) or as prepared by detergent
lysis (Yonemura et al., 1991).
F-actin (Fig. 3B), as analyzed using rhodamine-phalloidin,
was distributed throughout most of the cortex in a latticework,
and in certain focal planes the pattern was punctate. At higher
magnifications these punctate spots appeared as fine, short
linear rods that likely corresponded to microvillar cores (Fig.
3B). Occasionally, if removal of fertilization envelopes was not
complete, fertilization envelopes were isolated with the
cortices; however, they were distinguished from cortices by
their lack of staining with rhodamine-phalloidin and by their
2242 G. R. Walker, R. Kane and D. R. Burgess
being much thinner and with much smoother surfaces than
cortices. The distribution of myosin and spectrin by immunofluorescence was as an aggregated, diffuse meshwork (Fig.
3C,D). Myosin appeared as a fine felt-like patterns in some
areas and more punctate in other areas (Fig. 3C). Spectrin
displayed a pattern of distribution similar to that of myosin
(Fig. 3D).
Cortical response to fertilization
The effect of egg activation on the presence of specific proteins
associated with the cytoskeleton was analyzed by immunoblotting. Actin (data not shown) and myosin levels increased dramatically, as has been shown before (see Mabuchi, 1986, for
review; Hamaguchi and Mabuchi, 1988; Begg and Rebhun,
1979; Begg et al., 1978; Mabuchi, 1986). The amount of
myosin associated with the cortex, relative to actin, increased
greater than tenfold after fertilization whereas the amount of
spectrin did not change significantly (Fig. 4). Tubulin, which
is abundant in the unfertilized egg as a soluble pool, was not
associated with the actin cortical cytoskeleton of unfertilized
eggs, but after fertilization tubulin became associated with this
cytoskeletal fraction (Fig. 4). Immunofluorescence detection of
tubulin in cortices of fertilized eggs showed a few microtubules, but extensive networks of microtubules were not
present (data not shown). The presence of an extracellular
matrix protein found in the surface lamina of the fertilized egg,
which is stored in the cortical granules of unfertilized eggs
(Burke et al., 1991), was also examined. This lamina protein
was not associated with the cortical granule-free cortical actin
cytoskeleton of unfertilized eggs but became associated with
the isolated cortical cytoskeleton after fertilization (Fig. 4).
Fig. 2. Isolated cortex cytoskeletal composition. (A) Electron
micrographs reveal that the ultrastructure of the washed isolated cortex
is characterized by a filamentous actin meshwork (A, arrow).
Microvillar cores (B, arrows) are still evident in the actin meshwork of
isolated cortical cytoskeletons. These microvillar cores have a structure
reminiscent of those found in intact eggs. Some cortices retain some of
the hyaline layer (B, arrowheads). Bars: A, 3 µm; B, 0.4 µm. The
isolated cortex contains a variety of peptides (C) with a wide range of
molecular mass and isoelectric pI values. The predominant protein is
actin (as indicated). Molecular mass is indicated at the left, in kDa.
Nucleotide- or salt-induced cortical changes
Cortices vary in shape after washing and centrifugation,
ranging from spherical to crumpled cylindrical (types I-IV, see
Materials and Methods), with the majority falling in types I
and II (Fig. 5). After ATP treatment, a shift in the distribution
of cortices was observed, resulting in significantly fewer class
I (spherical) and more class IV (crumpled) cortices. NaCl
affected cortex shape in the opposite way to that of ATP, in
that significantly more cortices were present as type I
(spherical). These observations were confirmed and extended
in a separate series of experiments by direct measurement of
cortices treated with ATP, ADP, ATPγS or NaCl (Table 1). No
shape changes were noted in buffer alone or in ADP-containing buffer. However, the relative diameters of ATP-treated
cortices were significantly smaller than those of control or
ADP-treated cortices, in all experiments. Both ATPγS and
NaCl treatment resulted in cortices whose diameters appeared
to be greater than those of controls in this experiment.
However, these differences were not statistically significant.
Direct microscopic observation of treated cortices further documented the effects of ATP or NaCl. Cortices from non-dividing
post-fertilized eggs treated with ATP, but not ADP, contracted
(Fig. 6) and became thinner or less phase-dense. In cortices from
interphase cells, this shape change resulted in a global contraction of spherical cortices. After 20 minutes or more, ATP-treated
cortices began to revert to their original shape or expand. On the
other hand, NaCl caused the cortex to expand and, as with ATPtreatment, to become less phase-dense.
Analysis of cortices by SDS-PAGE and western blotting
Isolation of a contractile egg cortex 2243
Fig. 3. Specific actin
cytoskeleton-associated proteins
can be found in these
preparations. (A) Immunoblot
analysis of isolated cortex
cytoskeletal proteins
(Coomassie lane) shows the
presence of several actinassociated proteins, myosin (m),
fascin (f), and spectrin (s).
Microvillar staining by
phalloidin is very evident at
higher magnifications, covering
the surface of the cortex and
appearing as finger-like
projections (B, arrowheads).
Myosin (C) and spectrin (D)
distributions are also somewhat
diffuse in the isolated cortex.
Bars: C, 24 µm; D, 33 µm; B,
8.6 µm.
Fig. 4. Fertilization has an effect
on the composition of the isolated
cortex cytoskeleton (CSK):
Coomassie staining of CSK
proteins shows that the complexity
of protein present increases upon
fertilization (UF, unfertilized;
90'PF, 90 minute post-fertilized).
Approximately equivalent amounts
of actin were loaded for each pair
of unfertilized and 90 minute postfertilized samples. Immunoblot
analysis confirms the fact that
some proteins become associated
with the CSK after fertilization
(tubulin and the extracellular
matrix protein 180 kDa
(ECM180)). Fertilization increases
the association of some proteins,
such as myosin. Spectrin seems to
be present in the cytoskeleton at
similar levels before and after egg
activation. Numbers at the left
indicate molecular mass in kDa.
after incubation in salt or nucleotide revealed a differential
effect on the solubility of cortical cytoskeletal proteins. The
treatment of the isolated cortical cytoskeleton from post-fertilized eggs with either 20 mM ATP or ATPγS is effective in the
removal of actin (not shown), spectrin and myosin (Fig. 7).
Isolation buffer alone or ADP did not remove a significant
amount of actin, myosin or spectrin. Salt (250 mM NaCl) was
also effective in the extraction of some actin (not shown) and
spectrin, but did not lead to extraction of myosin (Fig. 7) from
the isolated cortex. High concentrations of ATP (20 mM)
extracted up to about 70% of spectrin and 50% of myosin. The
concentration dependence of myosin and spectrin extraction by
ATP was analyzed more quantitatively and is summarized in
Fig. 8. At all concentrations ATP extracted proportionately
more spectrin than myosin. More than 50% of the spectrin was
extracted by 250 mM NaCl, whereas myosin was not quantitatively removed by NaCl. A higher salt concentration of 500
mM was required to remove myosin (data not shown).
2244 G. R. Walker, R. Kane and D. R. Burgess
progresses to completion in around 7 minutes in these species,
furrowing in ATP-reacted cortices continued for approximately 10 minutes until furrowing stopped and in some cases
the furrow began to regress.
Table 1. The effect of nucleotides or NaCl on cortex
diameter
Control
Mean
100±15.9
relative
diameter
ADP
ATP *
ATPγS
NaCl
97.4±18.6
82.4±15.4
112.4±14.6
109.3±17.3
Relative diameters of cortices subjected to different conditions are
compared with control cortices. The shortest diameter was used, averaged and
normalized to 100. There was no significant difference between ADP,
ATPγS, NaCl and the control cortices.
*There was a statistically significant difference between diameters of ATPtreated cortices and the control cortices.
Cleavage stage cortices
When cortices were isolated from zygotes undergoing
cleavage, they retained their cleavage furrow (Fig. 9A). In the
region of the contractile ring, circumferentially oriented
filaments were apparent by phase-contrast microscopy (Fig.
9B). Cortices retaining their furrow were best prepared when
zygotes were within their first one-third of the way through
cleavage. Zygotes beyond two-thirds of the way through
cleavage generally did not yield cleavage furrow-containing
cortices, likely due to the cortices breaking in two. Interestingly, when early cleavage stage cortices were treated with
ATP, the cleavage furrow progressed (Fig. 9C). ATP-induced
continuation of cleavage furrow constriction never resulted in
complete cytokinesis of the cortex, but progression continued
to 25-50% of full cleavage from cortices that were from 1030% of full cleavage when isolated. While the normal furrow
% of cortices
40
30
20
10
0
The roles and regulation of specific actin-associated proteins
in the structural integrity and dynamic activity of the egg
cortical cytoskeleton during cytokinesis is not fully understood
(see Conrad and Schroeder, 1990; Mabuchi, 1986; Hepler,
1989; Rappaport, 1986; Salmon, 1986; Satterwhite and
Pollard, 1992, for excellent reviews). The cortex participates
in a variety of cellular functions that require a wide range of
proteins with different properties and activities. The protocol
developed in this study results in cortices that appear by morphology and composition to be similar to the native egg cortex.
Analysis of these cortices suggests that spectrin plays a role in
maintaining the rigid cortical structure of the blastomere and
that myosin is involved in contraction and in maintaining
cortical tension. While these conclusions have been inferred or
demonstrated in other studies of intact cells, the present work
documents these roles in an in vitro model of the egg cortex.
Moreover, this cortex preparation supports partial in vitro reactivation of the contractile ring.
Our work complements and extends the work of others on
the isolated egg cortex. Consistent with our observation that
the entire cortex is competent to undergo ATP-induced con-
AAControl
60
50
DISCUSSION
ATP
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
I
NaCl
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
II
AAAA
AAAA
AAAA
AAAA
AAAA
III
Cortex type
AAAA
AAAA
AAAA
AAAA
IV
Fig. 5. The effect of ATP or NaCl on cortex shape.
(A) Graph depicting the effects of ATP and NaCl on
isolated cortex shape. The shape types are described as
follows: type I cortex is smooth and spherical; type II
is wrinkled and spherical; type III is oval or rugby
football-shaped; and type IV is fully collapsed and
wrinkled. ATP decreases the proportion of cortices
that are round and smooth, leading to a corresponding
increase in collapsed and wrinkled cortices. NaCl acts
to increases the proportion that are smooth and round.
A representative cortex for each classification type is
displayed by the micrograph under the appropriate
data.
Isolation of a contractile egg cortex 2245
traction, several different lines of experimentation show that in
the intact egg the entire cortex is competent to contract. For
instance, manipulations of the astral complex of the mitotic
A
B
C
Fig. 6. Shape changes in the isolated cortical cytoskeleton. Direct
observation shows shape changes experienced by the isolated cortex
during treatment with ATP or NaCl. ATP induces the cortex to
contract in size (B) and NaCl causes a slight enlargement in cortex
profile (C). Perfusion of isolation buffer alone (A) or ADP (data not
shown) do not result in shape changes. Micrographs on the left are
cortices before perfusion and micrographs on the right are the same
cortices 10 minutes after perfusion.
γ
apparatus (Rappaport, 1986), measurements of cortical tension
(Usui and Yoneda, 1982), cortical contractions induced by
calcium ionophore application (Ezzell, 1983), and observations
of rhythmic cortical contractions in fertilized eggs (Yoneda and
Schroeder, 1984), are all consistent with our finding that the
entire isolated cortex undergoes ATP-dependent contractions.
Recently, Rappaport and Rappaport (1993) demonstrated that
premature and multiple furrows can be induced by manipulating the positions of asters in eggs. Detailed analysis of the large
number of proteins present in the present preparation may
provide new members of the family of proteins that are
localized to the contractile ring as was done with another
cortex preparation (Yonemura et al., 1991). The finding that
this present preparation, when isolated early in cytokinesis,
supports continued contraction of the contractile ring is consistent with the reports of Cao and Wang (1990) that the contractile ring uses pre-existing actin filaments for its contraction.
Spectrin is an important actin binding protein present in the
egg membrane skeleton (Fishkind et al., 1987, 1990a,b;
Schatten et al., 1986). Evidence from other systems suggests
that spectrin plays a general role in plasma membrane morphology and mechanical properties (see review by Bennett and
Gilligan, 1993). The spectrin cytoskeleton influences cell
shape by affecting the ‘elasticity’ of the actin network under
the membrane and may play a role in determining the distribution of various membrane proteins. The association of
spectrin with various membrane organelles in the sea urchin
egg (Fishkind et al., 1990a,b) indicates that spectrin plays a
fundamental role in the functional structure of membrane
systems within the sea urchin egg. Spectrin having a role in
stabilizing membrane-cytoskeleton interactions would be consistent with it being present at constant levels in unfertilized
and fertilized egg cortical cytoskeletons.
Cortex shape must be a function of the mechanical properties of the cortical cytoskeleton. It is not surprising that spectrin
would play a role in cortex shape. Low salt treatment selectively removes spectrin, resulting in cortices that become thin
and expand. These conditions, which do not promote contraction or myosin removal, cause changes in the shape of isolated
cortices, characterized as a rounding up or relaxing. The difference in the types of changes caused by ATP versus NaCl
may be due to ATP inducing myosin-based contraction
(discussed below) in addition to weakening the cortex due to
extraction of spectrin. This is consistent with the observation
that some ATP-treated cortices relax after the initial period of
contraction. Interestingly, spectrin was also removed from the
Fig. 7. Proteins extracted from the CSK in
vitro by treatment with ATP, ATPγS or NaCl:
western blot analysis shows that cortices
treated with buffer alone or buffer containing
20 mM ADP retain both myosin (upper panel)
and spectrin (lower panel) in a pelletable form
(P) and very little of either of these proteins
are found in the supernatant (S). Spectrin and
myosin are released into the supernatant (S)
by treatment with 20 mM ATP or ATPγS,
with some retention in the pellet (P). Only
spectrin is released into the supernatant
whereas myosin is retained in the pellet (P)
after NaCl (250 mM) treatment.
2246 G. R. Walker, R. Kane and D. R. Burgess
Fig. 8. Quantification of myosin and spectrin extraction. The ATP
concentration dependency of spectrin and myosin extraction was
determined by averaging results of three experiments. Spectrin and
myosin extracted by ATP were quantified by dot blotting with
radiolabeled secondary antibody. The concentration of ATP for
maximum spectrin extraction (about 10 mM) is less than that
required for maximum myosin extraction.
egg cortex by high levels of ATP in a manner similar to and
at similar concentrations to those described for ATP effects on
spectrin removal from red blood cell ghosts (Sheetz and
Casaly, 1980). Removal of spectrin probably destabilizes the
whole spectrin/actin network leading to a general dissociation
of the cytoskeletal matrix. We cannot rule out the possibility
that other key cytoskeletal proteins may play a structural role
in the cortex and may also be removed by low salt or ATP.
The effects of ATP on extraction of myosin and spectrin
provide further insight into the anchorage of these proteins to
the cortical cytoskeleton. The quantitative results presented in
Fig. 5 suggest that spectrin and myosin are associated with the
cortex via distinct mechanisms, since the concentration dependency and amount of spectrin extracted are different than that
for myosin removal. Low concentrations of ATP, which result
in minimal myosin extraction but some spectrin extraction, are
sufficient to induce contraction of the cortex. The nature of
attachment of myosin to the actin cortex is unknown, although
it may be in a rigor complex with cortical actin, since it is
extractable with ATP but not with ADP. The specific manner
in which myosin is associated with the actin in the cortex may
be critically important in order for actomyosin to function in
Fig. 9. Direct observation of the effect of ATP on the shape of isolated dividing zygote cortices. Cortices isolated at the time of the first
division retain their furrow (A) and display linear structures reminiscent of contractile rings (B, arrows). ATP was perfused across a
microscope slide and phase-contrast micrographs were taken at the times indicated (minutes) after ATP addition. Bars: B, 17 µm; C0', 32 µm.
Isolation of a contractile egg cortex 2247
contraction (Aguado and Kuczmarski, 1993). The fact that
ATP-induced contraction and a thinning of the cortex are consistent with the observation that the contractile ring disassembles as it contracts (Schroeder, 1972). On the other hand, it is
likely that spectrin is attached via its actin binding sites, since
cortical spectrin is readily extracted by low salt, conditions that
do not extract myosin. These findings are in contrast to those
of Schroeder and Otto (1988), who found that very high levels
of salt (600 mM) or ATP did not extract either actin or myosin
from cortices prepared by shearing cytoplasm away from
zygotes adherent to a substratum. However, our study utilized
analysis by PAGE of the supernatants after salt or nucleotide
treatment, whereas the Schroeder and Otto study assayed
extraction by immunofluorescence, which is non-quantitative.
We also note little change in the immunofluorescent distribution of actin, myosin and spectrin with salt or nucleotide
treatment in cortices isolated by our methods (data not shown),
likely because less than one half of the cytoskeletal proteins
are removed by these treatments.
Cortical contractions are likely the result of an actomyosin
contractile event, since they are caused by ATP but not by
either ADP or ATPγS. This is not surprising due to the strong
evidence that myosin II is responsible for contraction of the
contractile ring of not only eggs but all cells (see Satterwhite
and Pollard, 1992, for recent review). ATPγS is a substrate
for kinases, such as myosin light chain kinase, but is not
utilized by the myosin ATPase (Kerrick et al., 1981). Our
results showing that cortices become crumpled throughout
after ATP treatment are consistent with myosin being evenly
distributed throughout the cortex and with myosin playing a
role of generating tension or stiffness, qualities known to be
dynamic and cyclic in the fertilized egg (Hiramoto, 1990;
Usui and Yoneda, 1982). Cortical myosin could also play a
more general role in controlling shape or in providing for
general tension of the cell cortex. Since the entire cortex has
been shown to be able to contract (Rappaport, 1986), it is
likely that actin and myosin provide this tensile force in the
cortex. Interestingly, while it has been recently shown that
myosin II is relocalized to the furrow of dividing Dictyostelium cells (Fukui et al., 1989; Kitanishi-Yumura and
Fukui, 1989), it appears that in eggs myosin is present
throughout the cortex, making the entire cortex competent to
contract. Myosin II’s presence throughout the egg cortex also
is consistent with the finding that myosin II is likely responsible for overall cortical tension and capping of surface
receptors in Dictyostelium (Pasternak et al., 1989). It is then
understandable that in the unfertilized egg cortex, which has
very little tension associated with it, myosin is present in
lower amounts than in the cortex of fertilized eggs that are
capable of exhibiting contractile behavior.
The isolated cortical cytoskeletons used in this study are also
an effective model system with which to study the regulation
of cytoskeletal proteins for such events as cytokinesis. The
finding that egg cortices prepared in solution are capable of
changes in shape suggests that they may hold great promise for
future studies of the functioning of the contractile ring and
myosin II. Moreover, this preparation may be amenable to the
preparation of cortices from other cell types. This preparation
is like that described by Kuczmarski et al. (1991) and Aguado
and Kuczmarski (1993) for cytoskeletal preparations from Dictyostelium whose ATP-induced shape changes have been
monitored by light scattering. This cortical preparation is also
remarkably similar to that of Yonemura et al. (1991), the only
difference being the ability of the present cortical preparation
to contract in vitro. Apart from a slightly higher NP-40 concentration, the major difference between the present cortical
preparation and that of Yonemura is the inclusion of salt and
0.16 M glucose in their preparation versus the presence of 1 M
glycerol in the present preparation. Unfortunately, like the
hand-isolated contractile ring from newt eggs (Mabuchi et al.,
1988), however, the contractile ring of isolated cleavage stage
cortices prepared according to the present protocol was not
able to complete cytokinesis in vitro. Perhaps the lack of
complete furrowing in vitro is due to the lack of asters, which
likely provide continuing stimulation to the advancing furrow
for its maintenance (Rappaport and Rappaport, 1993;
Hiramoto, 1956; Swann and Mitchison, 1953).
This research was supported by National Institute of Health grants
GM14363 (R.E.K.) and GM40086 (D.R.B.). The authors gratefully
acknowledge the technical assistance of Dr Linda K. Kullama
throughout this investigation, and Mr Michael Anzelmo, Mr Joe
Myerson and Mr Brian Feingold for assistance. D.R.B. dedicates this
paper to the memory of Bob Kane (1931-1993).
REFERENCES
Aguado, V. C. and Kuczmarski, E. R. (1993). Contraction of reconstituted
Dictyostelium cytoskeletons: An apparent role for higher order associations
among myosin filaments. Cell Motil. Cytoskel. 26, 103-114.
Begg, D. A., Rodewald, R. and Rebhun, L. I. (1978). The visualization of
actin filament polarity in thin sections. J. Cell Biol. 79, 846-852.
Begg, D. A. and Rebhun, L. I. (1979). pH regulates the polymerization of actin
in the sea urchin egg cortex. J. Cell Biol. 83, 241-248.
Bennett, V. and Gilligan, D. M. (1993). The spectrin-based membrane
skeleton and micron-scale organization of the plasma membrane. Annu. Rev.
Cell Biol. 9, 27-66.
Bryan, J. and Kane, R. E. (1978). Separation and interaction of the major
components of sea urchin actin gel. J. Mol. Biol. 125, 207-224.
Burgess, D. R. and Schroeder, T. E. (1977.). Polarized bundles of actin
filaments within microvilli of fertilized sea urchin eggs. J. Cell Biol. 74,
1031-1037.
Burke, R. D., Myers, R. L., Sexton, T. L. and Jackson, C. (1991). Cell
movements during the initial phase of gastrulation in the sea urchin embryo.
Dev. Biol. 146, 542-557.
Cande, W. Z. (1980). A permeabilized cell model for studying cytokinesis
using mammalian tissue cultured cells. J. Cell Biol. 87, 326-335.
Cao, L.-g. and Wang, Y.-l. (1990). Mechanism of the formation of the
contractile ring in dividing cultured animal cells. II. Cortical movement of
microinjected actin filaments. J. Cell Biol. 111, 1905-1911.
Conrad, G. W. and Schroeder, T. E. (1990). Cytokinesis: Mechanisms of
furrow formation during cell division. Ann. NY Acad. Sci. 582, 1-325.
Eisenman, E. A. and Alfert, M. (1982). A new fixation procedure for
preserving the ultrastructure of marine invertebrate tissues. J. Microsc. 125,
117-120.
Ezzell, R. M. (1983). Phosphorylation-dependent contraction of actomyosin
gels from amphibian eggs. Nature 306, 620-622.
Fishkind, D. J., Bonder, E.M. and Begg, D.A. (1987). Isolation and
characterization of sea urchin egg spectrin. Cell Motil. Cytoskel. 7, 304-314.
Fishkind, D. J., Bonder, E. M. and Begg, D. A. (1990a). Subcellular
localization of sea urchin egg spectrin: evidence for assembly of the
membrane-skeleton on unique classes of vesicles in eggs and embryos. Dev.
Biol. 142, 439-452.
Fishkind, D. J., Bonder, E. M. and Begg, D. A. (1990b). Sea urchin spectrin in
oogenesis and embryogenesis: a multifunctional integrator of membranecytoskeletal interactions. Dev. Biol. 142, 453-464.
Fukui, Y., Lynch, T. J., Brzeska, H. and Korn, E. D. (1989). Myosin I is
located at the leading edges of locomoting Dictyostelium amoebae. Nature
341, 328-331.
2248 G. R. Walker, R. Kane and D. R. Burgess
Hamaguchi, Y. and Mabuchi, I. (1988). Accumulation of fluorescently
labeled actin in the cortical layer in sea urchin eggs after fertilization. Cell
Motil. Cytoskel. 9, 153-163.
Hepler, P. K. (1989). Calcium transients during mitosis: Observations in flux.
J. Cell Biol. 109, 2567-2573.
Hiramoto, Y. (1956). Cell division without mitotic apparatus. Exp. Cell Res.
11, 630-636.
Hiramoto, Y. (1990). Mechanical properties of the cortex before and during
cleavage. In Cytokinesis: Mechanisms of Furrow Formation During Cell
Division (ed. G. W. Conrad and T. E. Schroeder). Ann. New York Acad. Sci.
vol. 582, pp. 22-30.
Hoffmann-Berling, H. (1954). Adenosintriphosphat als Betriebsstoft von
Zellbewegungen. Biochim. Biophys. Acta 14, 182-194.
Hosoya, H. and Mabuchi, I. (1984). A 45,000-mol-wt protein-actin complex
from unfertilized sea urchin egg affects assembly properties of actin. J. Cell
Biol. 99, 994-1001.
Hosoya, H., Mabuchi, I. and Sakai, H. (1986). An 100-kDa Ca2+ sensitive
actin-fragmenting protein from unfertilized sea urchin egg. Eur. J. Biochem.
154, 233-239.
Ishidate, S. and Mabuchi, I. (1988). A novel actin filament-capping protein
from sea urchin eggs: a 20,000 molecular-weight protein-actin complex. J.
Biochem. 104, 72-80.
Kane, R. E. (1973). Hyalin release during normal sea urchin development and
its replacement after removal at fertilization. Exp. Cell Res. 81, 301-311.
Kane, R. E. (1986). TAME stabilizes the cortex and mitotic apparatus of the
sea urchin egg during isolation. Exp. Cell Res. 162, 495-506.
Kerrick, W. G. L., Hoar, P. E., Cassidy, P. S. and Bridenbaugh, R. L.
(1981). Skinned muscle fibers: function significance of phosphorylation and
calcium-activated tension. In Protein Phosphorylation (ed. O. M. Ora and E.
G. Krebs). Cold Spring Harbor Conf. Cell Prolif. vol. 8, pp. 887-900.
Kitanishi-Yumura, T. and Fukui, Y. (1989). Actomyosin organization during
cytokinesis: reversible translocation and differential redistribution in
Dictyostelium. Cell Motil. Cytoskel. 12, 78-89.
Kuczmarski, E. R., Palivos, L., Aguado, C. and Yao, Z. (1991). Stoppedflow measurement of cytoskeletal contraction: Dictyostelium myosin II is
specifically required for contraction of amoeba cytoskeletons. J. Cell Biol.
114, 1191-1199.
Kuramochi, K., Mabuchi, I. and Owaribe, K. (1986). Spectrin from sea
urchin eggs. Biomed. Res. 7, 65-68.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227, 680-685.
Mabuchi, I. and Okuno, M. (1977). The effect of myosin antibody on the
division of starfish blastomeres. J. Cell Biol. 74, 251-263.
Mabuchi, I., Hosoya, H. and Sakai, H. (1980). Actin in the cortical layer of
the sea urchin egg. Changes in its content during and after fertilization.
Biomed. Res. 1, 417-426.
Mabuchi, I. (1983). An actin depolymerizing protein (Depactin) from starfish
oocytes: properties and interaction with actin. J. Cell Biol. 97, 1612-1621.
Mabuchi, I., Hamaguchi, Y., Kobayashi, T., Hosoya, H. and Tsukita, S.
(1985). Alpha-actinin from sea urchin eggs: biochemical properties,
interaction with actin, and distribution in the cell during fertilization and
cleavage. J. Cell Biol. 100, 375-383.
Mabuchi, I. (1986). Biochemical aspects of cytokinesis. Int. Rev. Cytol. 101,
175-213.
Mabuchi, I. and Kane, R. E. (1987). A 250 K-molecular-weight actin binding
protein from actin-based gels formed in sea urchin egg cytoplasmic extract.
J. Biochem. 102, 947-956.
Mabuchi, I., Tsukita, S. and Sawai, T. (1988). Cleavage furrow isolated from
newt eggs: contraction, organization of the actin filaments, and protein
components of the furrow. Proc. Nat. Acad. Sci. USA 85, 5966-5970.
Matsudaira, P. T. and Burgess, D. R. (1978). SDS microslab linear gradient
polyacrylamide gel electrophoresis. Anal. Biochem. 87, 386-396.
Minamide, L. S. and Bamburg, J. R. (1990). A filter paper dye-binding assay
for quantitative determination of protein without interference from reducing
agents or detergents. Anal. Biochem. 190, 66-70.
O’Farrell, P. H. (1975). High resolution two-dimensional electrophoresis of
proteins. J. Biol. Chem. 250, 4007-4021.
Otto, J. J., Kane, R.E. and Bryan, J. (1980.). Redistribution of actin and
fascin in sea urchin eggs after fertilization. Cell Motil. 1, 31-40.
Pasternak, C., Spudich, J.A. and Elson, E.L. (1989.). Capping of surface
receptors and concomitant cortical tension are generated by conventional
myosin. Nature 341, 549-551.
Rappaport, R. (1986). Establishment of the mechanism of cytokinesis. Int.
Rev. Cytol. 101, 245-281.
Rappaport, R. (1988). Surface behavior in artificially constricted sand dollar
eggs and egg fragments. J. Exp. Zool. 246, 253-257.
Rappaport, R. and Rappaport, B. N. (1993). Duration of division-related
events in cleaving sand dollar eggs. Dev. Biol. 158, 265-273.
Sakai, H. (1960). Studies on sulfhydryl groups during cell division of sea
urchin egg. II. Mass isolation of the egg cortex and change in its -SH groups
during cell division. J. Biochem. Biophys. Cytol. 8, 603-607.
Salmon, E. D. (1986). Cytokinesis in animal cells. Curr. Opin. Cell Biol. 1,
541-547.
Satterwhite, L. L. and Pollard, T. D. (1992). Cytokinesis. Curr. Opin. Cell
Biol. 4, 43-52.
Schatten, H., Cheney, R., Balczon, R., Willard, M., Cline, C., Simerly, C.
and Schatten, G. (1986). Localization of fodrin during fertilization and early
development of the sea urchin and mice. Dev. Biol. 118, 457-466.
Schroeder, T. E. (1972). The contractile ring. II. Determining its brief
existence, volumetric changes, and vital role in cleaving Arbacia eggs. J.
Cell Biol. 53, 419-434.
Schroeder, T. E. and Otto, J. J. (1988). Immunofluorescent analysis of actin
and myosin in isolated contractile rings of sea urchin eggs. Zool. Sci. 5, 713725.
Sheetz, M. P. and Casaly, J. (1980). 2,3-Diphosphoglycerate and ATP
dissociate erythrocyte membrane skeletons. J. Biol. Chem. 255, 99559960.
Spudich, A. and Spudich, J. A. (1979). Actin in Triton-treated cortical
preparations of unfertilized and fertilized sea urchin eggs. J. Cell Biol. 82,
212-226.
Sui, A. L. and Shen, S. S. (1986). Intracellular free magnesium concentration
in the sea urchin egg during fertilization. Dev. Biol. 114, 208-213.
Swann, M. M. and Mitchison, J. M. (1953). Cleavage of sea urchin eggs in
colchicine. J. Exp. Biol. 30, 506-513.
Towbin, H., Staehelin, T. and Gordon, J. (1979). Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedure and
some applications. Proc. Nat. Acad. Sci. USA 76, 4350-4354.
Usui, N. and Yoneda, M. (1982). Ultrastructural basis of the tension increase
in sea-urchin eggs prior to cytokinesis. Dev. Growth Differ. 24, 453-465.
Vacquier, V. D. (1975). The isolation of intact cortical granules from sea
urchin eggs: calcium ions trigger granule discharge. Dev. Biol. 43, 62-74.
Vacquier, V. D. and Moy, G. W. (1980). The cytolytic isolation of the cortex
of the sea urchin egg. Dev. Biol. 77, 178-190.
Vacquier, V. D. (1981). Dynamic changes in the egg cortex. Dev. Biol. 84, 126.
Wang, L. and Spudich, J. A. (1984). A45,900-mol-wt protein from
unfertilized sea urchin eggs severs actin filaments in a calcium-dependent
manner and increases the steady state concentration of nonfilamentous actin.
J. Cell Biol. 99, 844-851.
Yabkowitz, R. and Burgess, D. R. (1987). Low ionic strength solubility of
myosin in sea urchin egg extracts is mediated by a myosin-binding protein. J.
Cell Biol. 105, 927-936.
Yoneda, M. and Schroeder, T. E. (1984). Cell cycle timing in colchicinetreated sea urchin eggs: persistent coordination between the nuclear cycles
and the rhythm of cortical stiffness. J. Exp. Zool. 231, 367-378.
Yonemura, S. and Mabuchi, I. (1987). A wave of actin polymerization in the
sea urchin egg cortex. Cell Motil. Cytoskel. 7, 46-53.
Yonemura, S., Mabuchi, I. and Tsukita, S. (1991). Mass isolation of cleavage
furrows from dividing sea urchin eggs. J. Cell Sci. 100, 73-84.
Yoshimoto, Y. and Hiramoto, Y. (1985). Cleavage in a saponin model of the
sea urchin egg. Cell Struct. Funct. 10, 29-36.
(Received 28 February 1994 - Accepted 14 April 1994)