2041
Journal of Cell Science 109, 2041-2051 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
JCS7061
Function of spindle microtubules in directing cortical movement and actin
filament organization in dividing cultured cells
Douglas J. Fishkind1,2,*, John D. Silverman1 and Yu-li Wang1
1Cell Biology Group, Worcester Foundation for
2Department of Biological Sciences, University
Biomedical Research, Shrewsbury, MA 01545, USA
of Notre Dame, 136 Galvin Life Science Center, Notre Dame, IN 46556, USA
*Author for correspondence at address 2
SUMMARY
The mitotic spindle has long been recognized to play an
essential role in determining the position of the cleavage
furrow during cell division, however little is known about
the mechanisms involved in this process. One attractive
hypothesis is that signals from the spindle may function to
induce reorganization of cortical structures and transport
of actin filaments to the equator during cytokinesis. While
an important idea, few experiments have directly tested
this model. In the present study, we have used a variety of
experimental approaches to identify microtubuledependent effects on key cortical events during normal cell
cleavage, including cortical flow, reorientation of actin
filaments, and formation of the contractile apparatus.
Single-particle tracking experiments showed that the
microtubule disrupting drug nocodazole induces an inhibition of the movements of cell surface receptors following
anaphase onset, while the microtubule stabilizing drug
taxol causes profound changes in the overall pattern of
receptor movements. These effects were accompanied by a
related set of changes in the organization of the actin
cytoskeleton. In nocodazole-treated cells, the three-dimensional organization of cortical actin filaments appeared less
ordered than in controls. Measurements with fluorescencedetected linear dichroism indicated a decrease in the
alignment of filaments along the spindle axis. In contrast,
actin filaments in taxol-treated cells showed an increased
alignment along the equator on both the ventral and dorsal
cortical surfaces, mirroring the redistribution pattern of
surface receptors. Together, these experiments show that
spindle microtubules are involved in directing bipolar flow
of surface receptors and reorganization of actin filaments
during cell division, thus acting as a stimulus for positioning cortical cytoskeletal components and organizing the
contractile apparatus of dividing tissue culture cells.
INTRODUCTION
spindle and equatorial cortex (Rappaport, 1986, 1991; Cao and
Wang, 1996). Moreover, when the mitotic spindle is moved by
micromanipulation, the cleavage furrow regresses and then
reinitiates at a new position, directly coincident with the
midzone of the repositioned spindle (Rappaport, 1985).
Beyond the importance of the spindle in signaling the cortex
and determining the position of the cleavage furrow, relatively
little is known about the mechanisms that control this process.
Results from micromanipulation experiments first suggested
that diffusible signals emanating from spindle asters might
function as a stimulus for the induction of cleavage
(Rappaport, 1986, 1991). While this hypothesis can explain a
number of observations with echinoderm embryos, neither the
nature of the signal nor the coupling of the stimulation to the
cortical actin machinery has yet to be elucidated. In addition,
recent observations suggest that the model may not be fully
applicable to all animal cells (Cao and Wang, 1996). A second
idea for spindle induction of furrowing proposes that the
microtubule and actin system in dividing cells may interact
with each other, thus providing a direct mechanism for the
bipolar spindle apparatus to establish or guide cortical
dynamics (Dan, 1948; White and Borisy, 1983; Mabuchi,
Cell division represents the final phase of the cell cycle, when
chromosomes are segregated during mitosis and the cell is partitioned into two daughter cells during cytokinesis. This highly
integrated process involves both the regulated assembly-disassembly of microtubules for chromosome movements (see
Salmon, 1989a; Wadsworth, 1993; McIntosh, 1994, for
reviews), and the dynamic reorganization of cortical actin
filaments for cell cleavage (reviewed by Mabuchi, 1986;
Salmon, 1989b; Conrad and Schroeder, 1990; Satterwhite and
Pollard, 1992; Fukui, 1993; Fishkind and Wang, 1995).
While mitosis and cytokinesis are often treated as distinct
events, classic experiments of Rappaport (1986, 1991),
Hiramoto (1956, 1971), and others (Wilson, 1925; Dan, 1948;
Swann and Mitchison, 1958) have clearly indicated that the
mitotic spindle plays a key role in controlling cytokinesis. For
example, cytokinesis is inhibited by either removal (Hiramoto,
1956, 1971) or disruption of the mitotic spindle during
metaphase and early anaphase (Chambers, 1938; Beams and
Evans, 1940; Swann and Mitchison, 1953; Hamaguchi, 1975),
or by the presence of physical barriers placed between the
Key words: Cell division, Cortical flow, Cytokinesis, Mitosis,
Spindle apparatus
2042 D. J. Fishkind, J. D. Silverman and Y.-l. Wang
1986; Rappaport, 1986, 1991; Bray and White, 1988). While
both ideas are plausible and not mutually exclusive, there is
presently no direct evidence demonstrating a role for microtubules or other spindle structures in regulating cortical
movement or actin filament reorganization in dividing cells.
To determine if microtubules can modulate cortical structure
and cell cleavage, we have designed a series of experiments to
examine cortical dynamics and actin filament organization in
dividing cultured cells following drug-induced disruptions of
spindle microtubules. This study was aided by the recent development of several light microscopy techniques that facilitate
the acquisition of structural information at the molecular level.
Specifically, by utilizing single particle tracking techniques,
we have been able to directly follow the movement of cell
surface receptors driven by the underlying cortex (Wang et al.,
1994). In addition, using fluorescence-detected linear
dichroism and digital optical sectioning microscopy, we can
determine the predominant orientation and three-dimensional
organization of actin filaments in different regions of dividing
cells (Fishkind and Wang, 1993).
Our results show that both cortical movements and the corresponding alignment of actin filaments along the spindle axis
are severely inhibited by the depolymerization of microtubules
during early anaphase. In contrast, stabilization of anaphase
microtubules by taxol induces profound changes in the pattern
of cell surface movements, coincident with a dramatic reorganization of cortical actin filaments on the dorsal cortex.
Together, these results provide direct evidence that spindle
microtubules can regulate the activity and organization of actin
containing structures in dividing cells, and hence serve an
important function in the assembly and contraction of the
cleavage furrow.
MATERIALS AND METHODS
Cell culture
Normal rat kidney (NRK) cells from the well-spread subclone NRK2 (Fishkind and Wang, 1993) were cultured and maintained in plastic
Petri dishes using Kaighn’s modified F12 medium (F12K, JRH Biosciences, Lenexa, KS) supplemented with 10% fetal bovine serum
(JRH Biosciences, Lenexa, KS), 1 mM L-glutamine, 50 µg/ml streptomycin, 50 units/ml penicillin, at 37°C and 5% CO2. Prior to experiments, cells were plated onto alcohol-dipped, flamed glass coverslips
set into 35 mm dishes or secured to microinjection chambers
(McKenna and Wang, 1989), and cultured for 48 to 72 hours. Many
cells in this subclone maintain their association with neighboring cells
during cytokinesis and do not show appreciable degrees of lateral constriction. This facilitates optical analyses of surface receptor
movement and cortical actin organization (Fishkind and Wang, 1993;
Wang et al., 1994).
Treatment of dividing cells with nocodazole and taxol
Pilot experiments were performed to determine the dose required to
disrupt mitotic processes and to cause microtubule disassembly. Cells
in metaphase were carefully monitored until anaphase onset, at which
time the culture medium in the chamber was rapidly exchanged with
medium containing 1-10 µM nocodazole (Sigma Chemical Co., St
Louis, MO) or 1-10 µM taxol (National Cancer Institute, Bethesda,
MD). Media with 1% dimethyl sulfoxide (DMSO; Sigma) or 1-10 µM
baccatin III (an analog of taxol that has no direct effects on microtubules; Manfredi and Horwitz, 1984) were used as controls. Parallel
observations were performed with phase optics to assess the degree
of inhibition of mitosis and with immunofluorescence staining to
determine the extent of microtubule disruption (see below).
Single-particle tracking analysis
Fluorescent beads for single-particle tracking experiments were
prepared according to the method of Wang et al. (1994). Briefly,
unmodified fluorescent carboxylated beads (L-5221, Molecular
Probes, Eugene, OR) were sonicated, washed, and resuspended in
phosphate-buffered saline (PBS) containing 10 mg/ml bovine serum
albumin (BSA; Sigma, St Louis, MO). Cells were labeled by carefully
drawing off the culture medium, briefly rinsing with two exchanges of
warm Hanks’ balanced salts, and applying a dilute, sonicated suspension of beads onto the dish. Following a 2-3 minute incubation, the
dish was rinsed with several changes of culture medium. Cells were
maintained on a warm microscope stage incubator during the labeling
and subsequent observation period (McKenna and Wang, 1989).
Metaphase cells labeled with beads were monitored with phase
optics until anaphase onset, and then imaged with fluorescence optics
every 15 seconds. Cells that divided without extensive lateral constriction during the period of observation were chosen for analysis, in
order to simplify the interpretation of bead movement. In most experiments cells were co-illuminated for simultaneous fluorescence and
phase-contrast observations. To study the effects of microtubule drugs
on cell surface movements, cells were labeled with beads as above
and incubated until shortly after anaphase onset, when directional
bead movement had occurred for 1-1.5 minutes. The medium was
then rapidly replaced with complete F12K containing either 3.3 µM
nocodazole, 5 µM taxol, 1% DMSO, or 1% DMSO with 5 µM
baccatin III, and further imaged for the duration of the experiment.
The drug concentrations of nocodazole and taxol represent the lowest
doses required to provide optimal effects on the spindle microtubules
of NRK-2 cells as determined from the analysis described above.
Analyses of particle movement, including the determination of directionality and speed, were carried out as previously described by Wang
et al. (1994), using a combination of movie loops and frame-to-frame
particle tracking of single beads.
Fixation and staining of dividing cells
Cells were fixed using the glutaraldehyde-based method of Small et
al. (1981) as previously described (Fishkind and Wang, 1993). Autofluorescence and free-aldehydes were quenched with 0.1% NaBH4 for
5 minutes, and additional non-specific binding was blocked for 1 hour
in PBS/1%BSA. Cells were then stained with a 1:200 dilution of antiβ-tubulin monoclonal antibodies (N357, Amersham Life Sciences,
Arlington Heights, IL) for 12-16 hours at 4°C, followed by 3 brief (10
minute) washes in PBS/1%BSA, and incubation with a 1:200 dilution
of fluorescein labeled sheep anti-mouse secondary antibody (Sigma)
for 2 hours at 37°C. After several PBS washes, cells were stained with
200 nM rhodamine phalloidin (Molecular Probes, Eugene, OR)
diluted in PBS for 1 hour, and then rinsed in PBS for an additional
30 minutes before examination. For digital optical sectioning analysis,
coverslips were mounted in the anti-photobleach medium of Clark and
Meyer (1992) prior to examination.
FDLD and digital optical sectioning microscopy
Measurements on actin filament orientation using FDLD were
performed as described by Fishkind and Wang (1993), except that
data were acquired with a cooled CCD camera with an EEV chip of
576×384 pixels (Princeton Instruments, Inc., Trenton, NJ). The image
was focused on or near the ventral cortex. FDLD values were
measured as before by averaging pixel values within a 2.5 µm
diameter spot (5 µm2 area), positioned either at the center of the cell
(i.e. at the equator) or in the subequatorial zone. Cells were analyzed
at various stages of mitosis from metaphase to telophase. Measurements from at least 10 different cells per stage were averaged to obtain
the mean and standard deviation of the FDLD value for a given region.
The three-dimensional organization of spindle microtubules and
Microtubule effects on actin organization 2043
actin filaments in dividing cells were examined using digital optical
sectioning microscopy as previously described (Fishkind and Wang,
1993). Briefly, a through focus series of optical sections (0.28
µm/section) were acquired from fluorescently stained samples, and
out-of-focus fluorescence was removed using computational methods
based on the nearest neighbor algorithm (Castleman, 1979; Agard,
1984; Shaw and Rawlings, 1991) and point spread functions derived
with our optics. Using custom-based software, stereo pairs were
produced from partial or complete stacks of deconvolved sections by
projecting the images at angles +10° and −10° from the optical axis.
Analysis of data sets were performed with a customized, interactive,
motion display program that allows rotation and stereo viewing of
reconstructed three-dimensional images.
RESULTS
Effects of microtubule disrupting drugs on spindle
structure and function
All experiments were performed on the well-spread subcloned
NRK-2 cells to facilitate observations of mitotic and cortical
structures. During mitosis, these cells remain attached to the substratum and neighboring cells, such that the ventral surface shows
little or no cleavage while the dorsal surface cleaves actively
(Fishkind and Wang, 1993). To test the response of NRK-2
spindle microtubules to drug treatments, we applied nocodazole
Fig. 1. Stereo imaging of the threedimensional organization of
spindle microtubules in control and
taxol-treated dividing NRK-2 cells.
Controls and taxol-treated cells
fixed at different stages of mitosis
were optically sectioned and
reconstructed to show the entire
optical stack of deconvolved
sections. In comparison to the
robust astral and interzonal
microtubules in the anaphase
spindle of control cells (a), taxoltreatment resulted in attenuated
spindles composed of compact
radial arrays of short, microtubule
bundles (b). Later in anaphase (c),
spindle microtubules in taxol
treated cells showed slight
elongation, while interzonal
microtubules appeared as tapered
bundles that terminated at the
equatorial midzone (arrowhead).
Unlike controls, taxol-treated cells
lack overlapping microtubules in
the midzone, showing instead a
gap between opposing bundles of
the two half spindles. Bar, 5 µm.
(an agent known to depolymerize microtubules; DeBrabander et
al., 1976) and taxol (a drug known to stabilize microtubules and
affect the assembly kinetics of tubulin; Schiff and Horwitz, 1980;
Wilson and Jordan, 1994) after anaphase onset and evaluated
their effects on microtubule structure and spindle function.
In control NRK-2 cells, the early anaphase spindle is
composed of two robust asters interconnected by a dense interzonal array of microtubules (Fig. 1a). Upon application of
micromolar levels of nocodazole in early anaphase, spindle
microtubules rapidly disassemble within 5 minutes (DeBrabander et al., 1986; see Fig. 4c below), coincident with
immediate cessation of chromosome separation (Mullins and
Snyder, 1981; Fig. 2d-f below). In cells treated with taxol,
three-dimensional images of early anaphase microtubules
showed attenuated spindles consisting of two compact half
spindles of short microtubule bundles emanating from the
poles (Fig. 1b). Cells treated with taxol later in anaphase and
telophase contained elongated microtubules similar to controls,
however the interzonal region showed a number of bundled
microtubules (Fig. 1c). In all taxol-treated cells, microtubules
appear to converge along the equatorial region, with a pronounced gap of staining between opposing fibers (Figs 1b,c,
5a; Amin-Hanjani and Wadsworth, 1991; Snyder and Mullins,
1993). Note, that the perturbation of microtubules in wellspread NRK-2 cells early in anaphase led to either complete
2044 D. J. Fishkind, J. D. Silverman and Y.-l. Wang
inhibition of cytokinesis as shown by the eventual production
of binucleated cells for nocodazole (Mullins and Snyder, 1981;
DeBrabander et al., 1986), or delays in the progression of cell
division as previously observed for taxol (Amin-Hanjani and
Wadsworth, 1991; Snyder and Mullins, 1993).
Effects of nocodazole and taxol on cortical
dynamics
Given the ability to rapidly manipulate spindle microtubules
with nocodazole and taxol, we asked whether these drugs could
alter cortical dynamics in dividing cells. Movements of surface
receptors on living NRK-2 cells were examined with a single
particle tracking assay (Fig. 2), which allows direct observations and measurements of the movement of cell surface
receptors associated with the underlying cortical actin
cytoskeleton (Wang et al., 1994). As shown previously,
surface-bound beads on control cells showed no organized
movement during metaphase, but began directional movement
(0.68±0.34 µm/minute) toward the equator ~1 minute after
anaphase onset (Fig. 2a-c). These movements were most active
over the central region of the spindle where well-aligned
microtubules fill the midzone (Fig. 1a). Later in anaphase and
Fig. 2. Cortical dynamics following spindle disruption by microtubule drugs nocodazole and taxol. Dividing cells were simultaneously imaged
with phase and fluorescence optics to track the movement of surface-bound beads as a function of the stage of cell division. Following
anaphase onset and the initiation of bead movements (a,d,g), cells were treated with 3.3 µM nocodazole or 5 µM taxol to assess their effects on
cortical dynamics. Control cells (a-c) showed the characteristic centripetal movement of beads toward the equator that occurs 1-2 minutes
following anaphase onset and leads to a slight concentration of beads at the equator (c). Nocodazole caused bead movement to change from bidirectional to a random pattern ~1-2 minutes after the treatment (d-f), coincident with the inhibition of poleward movements of chromosomes.
In taxol-treated cells (g-i), beads rapidly cleared from the dorsal surface overlying the spindle region, and became concentrated along lateral
margins of the spindle and the equator. During the next 5-10 minutes a strong, active flow of beads from the lateral margins toward the equator
accompanied the ingression of the cortex. Images shown were recorded at ~1 (a), 3.5 (b), and 6 (c) minutes after anaphase onset for the control
cell, and ~3 (d,g), 6 (e,h), and 10 (f,i) minutes after anaphase onset for the drug treated cells. The arrows denote vectors of the movement of
several beads. Bar, 5 µm.
Microtubule effects on actin organization 2045
Fig. 3. Stereo image of the
three-dimensional
organization of
microtubules (a) and F-actin
(b,c) in a dividing control
NRK-2 cell at late
anaphase/early telophase,
showing the x-y view of the
cell using all optical
sections (a,b), or the x-y
view of just the top (dorsal)
half of the cell (c). Note that
the bottom (ventral) cortex
contains a well-organized
band of filaments aligned
along the equatorial
direction (b), while the
dorsal cortex shows a
number of filaments
organized along the
longitudinal axis in the
subequatorial region (c). In
addition, many cytoplasmic
filaments appear to lie along
the longitudinal axis. Bar,
5 µm.
telophase, a slight converging pattern of movement caused the
concentration of beads toward the center of the cell (Fig. 2c).
In contrast, cells treated with nocodazole 1-2 minutes
following anaphase onset showed a rapid inhibition of directional bead movement, simultaneous with the cessation of
chromosome separation (Fig. 2d-f) and depolymerization of
microtubules. As a result, few beads accumulated at the
equator (Fig. 2f). While taxol treatment caused only a slight
decrease in the rate of directional bead movement (0.48±0.19
µm/minute, n=51 beads from 8 different cells), there were pronounced changes observed in the pattern of particle movements
along the cortex (Fig. 2g-i). Instead of moving more or less
directly toward the equator, beads tended to deplete first from
the region overlying the spindle, and then began active convergence toward the center of the cell (Fig. 2h,i). The initial
zones of depletion correlated well with the characteristic
pattern of spindle microtubules (Fig. 1b), while the subsequent
converging movement in the midzone was directed toward the
focused ends of microtubules at the equator (Fig. 1c).
Three-dimensional organization of actin filaments in
nocodazole and taxol treated cells
To determine if microtubule-dependent effects on particle
movements reflect underlying changes in the structure of the
contractile apparatus, we examined the three-dimensional
organization of actin filaments in glutaraldehyde-fixed cells
using digital optical sectioning microscopy. Stereo pairs of
spindles were also prepared to allow direct comparison of actin
and microtubule structures (Fig. 3). Previous studies of control
cells indicated an essentially isotropic organization of actin
filaments on the dorsal equatorial cortex, and equatorial
alignment of actin filaments on the ventral equatorial cortex
(Fig. 3b; Fishkind and Wang, 1993). In addition, actin
filaments along the dorsal cortex in regions flanking the
equator showed preferential orientation along the long axis of
the cell (Fig. 3c), similar to alignment of interzonal microtubules in the spindle apparatus (Fig. 3a).
In nocodazole treated cells, the general pattern of actin
organization in late anaphase/early telophase cells appeared
strikingly different from that of control cells with a similar
extent of chromosome separation (Figs 3 and 4). The most pronounced change in actin filament organization was an apparent
reduction in the number of actin filaments oriented parallel to
the long axis along the dorsal cortex and spindle region (Fig.
4a,b), an observation later confirmed quantitatively with FDLD
imaging (see Fig. 6, below). On the ventral cortex along the
equator, actin filaments retained an organization similar to that
in control cells, however the band of filaments appeared
2046 D. J. Fishkind, J. D. Silverman and Y.-l. Wang
Fig. 4. Stereo image of the three-dimensional
organization of F-actin in a nocodazole treated dividing
NRK-2 cell, showing the complete x-y view of the cell
(a), or the x-y view of the dorsal half of the cell (b).
Compared to controls, the cell has a more spread out
appearance and an altered pattern of actin organization.
Note, the ventral cortex retains a broader equatorial band
of filaments (a), while the dorsal surface lacks wellordered longitudinal filaments. Anti-tubulin fluorescence
demonstrated that the majority of spindle microtubules
were disassembled in this cell (c). Bar, 5 µm.
broader and less compact (measured below, FDLD analysis).
The rapid loss of anaphase spindle microtubules following
nocodazole treatment was confirmed by anti-tubulin staining
that showed diffuse fluorescence throughout the cell (Fig. 4c).
In taxol-treated cells, the most dramatic change was an
increase in filament alignment along the equator of the dorsal
cortex (Fig. 5). By late anaphase/early telophase, a striking
band of actin filaments formed along the equator in the central
region of the cell (Fig. 5b,c), where surface-bound particles
were previously observed to converge and concentrate (Fig.
2i). These highly aligned equatorial filaments were localized
in the gap between the ends of microtubule bundles emanating
from the two half spindles (Fig. 5a). Many actin filaments
appeared to extend from the outer lateral margins of the
equator and subequatorial region toward the central region of
the ingressing cleavage furrow (Fig. 5b). The pattern of actin
organization parallels that of bead movement in taxol-treated
cells (Fig. 2g-i), consistent with the idea that surface particle
translocations are linked to movement of the underlying
cytoskeleton (Wang et al., 1994), and that both are sensitive to
modifications of spindle microtubules.
Orientation of actin filaments in nocodazole and
taxol treated cells
To measure quantitatively the effect of microtubule drugs on
the organization of actin, we used FDLD microscopy to
determine the preferred orientation of actin filaments (Fishkind
and Wang, 1993). As previously shown, metaphase cells
generally displayed little preferred orientation of actin
filaments except for a slight longitudinal orientation in the
equatorial region (Fig. 6a). Upon anaphase onset, actin organization underwent a progressive increase in equatorial
alignment along the equator, and longitudinal alignment in
subequatorial regions (Fig. 6d,g,j).
Following nocodazole treatment, anaphase cells maintained a
preferred alignment of actin filaments along the equator similar
to that of controls (Fig. 6b,e,h), however, little or no orientation
was detected along the longitudinal direction in the subequato-
Microtubule effects on actin organization 2047
rial region (Fig. 6e, arrows and 6k, bar graph). This result
suggests that disruption of microtubules may have prevented or
reversed the longitudinal alignment of filaments while allowing
equatorial organization to take place. Moreover, as observed
with optical sectioning microscopy (Fig. 4), the equatorial band
of filaments in late anaphase and telophase cells was slightly
wider than that of controls (Fig. 6h, 7.0±2.0 µm versus 4.6±1.0
µm; n=23 and 30 cells, respectively). In taxol-treated cells, the
overall pattern of filament organization as indicated by FDLD
appeared similar to that in control cells (Fig. 6c,f,i). However,
the positive FDLD value along the equator was slightly higher
in taxol-treated cells than in controls during the early stage of
anaphase (Fig. 6f). The apparent shift in the development of
equatorial alignment may be due to the ability of cells to
continue the organization of actin filaments following the
addition of taxol, or possibly to slight differences in the mitotic
staging relative to the separation of chromosomes (Fig. 6l).
DISCUSSION
The mitotic spindle has long been recognized to play a critical
role in establishing the position of the cleavage furrow in dividing
Fig. 5. Stereo image of the
three-dimensional
organization of
microtubules (a) and Factin (b,c) in a taxol treated
dividing NRK-2 cell,
showing the complete x-y
view of the cell (a,b), or
the x-y view of the dorsal
half of the cell (c). The
telophase spindle is
composed of two welldefined half spindles
containing a dense array of
interzonal microtubules
that are focused and
terminate at the equatorial
midzone (a, arrow). The
ingressing furrow overlies
this region (b), and shows a
dramatically altered pattern
of actin characterized by
highly aligned arrays of
parallel filaments lying
along the dorsal cortex (c,
brackets). Many of these
filaments appear to
originate near the lateral
margins of the cell and
curve in toward the equator
where they become
concentrated and aligned.
Longitudinal filaments are
also observed along the
steep ‘walls’ of the furrow
(arrowheads) and within
the deeper cytoplasm
where the spindle resides.
Bar, 5 µm.
cells (Wilson, 1925; Swann and Mitchison, 1958; Rappaport,
1986, 1991). While many hypotheses have been proposed to
explain how the spindle might function in delivering diffusible
and/or mechanical signals to the cortex (Dan, 1948; Wolpert,
1960; White and Borisy, 1983; Rappaport, 1986, 1991; Devore
et al., 1989; Margolis and Andreassen, 1993), little direct experimental evidence has been obtained to elucidate the role of
spindle microtubules in regulating actin filament organization. In
this study, we have utilized a variety of optical approaches to
analyze the potential role of microtubules in controlling cortical
dynamics and actin organization leading to the assembly of the
contractile apparatus for cyto-kinesis. By combining singleparticle tracking, FDLD measurements, and three-dimensional
imaging, together with the use of well-spread NRK-2 cells to
facilitate the detection of structures, we have provided new
evidence that spindle microtubules promote bipolar cortical flow,
enhance actin filament orientation, and help maintain the structural organization of actin during cell division.
Spindle modulation of cortical dynamics and
longitudinal alignment of actin filaments
The initiation of cytokinesis in tissue culture cells is first
2048 D. J. Fishkind, J. D. Silverman and Y.-l. Wang
Fig. 6. Fluorescence detected linear dichroism microscopy of dividing NRK-2 cells following the disruption of spindle microtubules by
nocodazole or taxol. FDLD imaging allows the detection of the preferential orientation in a population of actin filaments. The displayed images
are representative cells in metaphase (a-c), anaphase (d-f), telophase (g-i) showing the global reorganization of actin filaments in control (a,d,g),
nocodazole (b,e,h), and taxol (c,f,i) treated dividing cells. At metaphase, there is no strong preferred orientation of actin filaments (a-c, greenyellow hue), except for slight alignment along the spindle axis near the equator (a-c, bluish striations). During anaphase and telophase, actin
filaments underwent a dramatic reorganization under all conditions, showing alignments along the equator in the furrow region (d-i, yellow-red
band). However, longitudinal alignment was observed only in control and taxol-treated cells (d,f, bluish striations in the subequatorial zone),
appearing completely absent in nocodazole-treated cells (e, arrows). The color bar denotes the relationship between FDLD values and color.
Quantitative analysis of the development of filament alignments during different stages confirms the progressive increase in positive FDLD
values along the equator of control (j), nocodazole (k), and taxol (l) treated cells (solid bars), and clearly demonstrates the lack of axial
alignment in subequatorial regions of nocodazole-treated cells (striped bars). The slight decrease in subequatorial FDLD values in nocodazoletreated cells in telophase (k) reflects the appearance of reforming stress fibers as the cell reenters interphase (see bluish striations in h). M,
Metaphase (n=11/10/11); AO, Anaphase Onset (n=11/13/12); A, Anaphase-A (n=12/5/11); AB, Anaphase-AB (n=15/15/14), B, Anaphase-B
(n=10/10/13); T, Telophase (n=11/16/11). Bar, 10 µm.
detected upon the movement of cortical actin filaments (Cao
and Wang, 1990) and associated membrane receptors (Wang
et al., 1994) toward the equator following anaphase onset. This
process occurs concomitant with well-documented changes in
spindle structures, including the simultaneous shortening of
kinetochore fibers and elongation of astral and interzonal
microtubules (Salmon, 1989a; Wadsworth, 1993; McIntosh,
1994). During the same period of time, there is a dramatic
realignment of actin filaments along the spindle axis in subequatorial regions (Figs 3 and 6), that directly correlates with
cortical movements to the equator (Fig. 2a-c). The inhibition
of both cortical and chromosome movements within 1-2
minutes of the application of nocodazole (Fig. 2d-f) indicates
that cortical flow is highly sensitive to the disassembly of
anaphase microtubules. At the same time, stereo images and
FDLD measurements reveal a failure of actin filaments to align
Microtubule effects on actin organization 2049
along the spindle axis (Figs 4, 6e,k), demonstrating that microtubules play a critical role in orienting actin filaments during
the early phase of cytokinesis.
Further indications of the regulation of cortical movement
by microtubules were obtained with taxol, an agent known to
cause the stabilization of microtubules in vitro (Schiff et al.,
1979; Wilson et al., 1985). When applied to actively dividing
cells, taxol appeared to affect the dynamic properties of spindle
microtubules, leading to changes in their length, degree of
bundling, and three-dimensional organization (Figs 1b,c, 5a;
Amin-Hanjani and Wadsworth, 1991; Snyder and Mullins,
1993; Wilson and Jordan, 1994). Interestingly, these effects
were also accompanied by dramatic changes in the pattern of
cortical movements (Fig. 2g-i), as well as the reorganization of
actin filaments on the dorsal cortex of the cleavage furrow (Fig.
5b,c). The rapid clearance of beads from the region overlying
the early spindle (Figs 1b,c, 2g,h), together with their strong
convergence to the central region of the cell, suggests that taxol
stabilized spindle microtubules possess an enhanced capacity
to alter the path of cortical movements and the organization of
actin filaments. Furthermore, the site of bead convergence
contains a high concentration of converging cortical actin
filaments, consistent with the idea that bead transport is driven
by the movements of the underlying cortex. Most importantly,
the region of convergence is characterized by the apparent termination of numerous interzonal microtubule bundles (Figs
2h,i, 5a), suggesting that molecular signals involved in generating cortical movements are localized along microtubules and
likely concentrate at or near the ends of these bundles.
Spindle effects on equatorial alignment of actin
filaments
The most prominent actin-containing structures in dividing
cells is the band of aligned filaments lying along the equator,
commonly referred to as the contractile ring. While these
filaments have been proposed as the main contractile element
for cytokinesis (Schroeder, 1970, 1973; Sanger and Sanger,
1980; Sanger et al., 1994), recent studies indicate that they are
more abundant in adherent cells than in round cells, and are
concentrated primarily on the bottom, non-cleaving cortex
(Fishkind and Wang, 1993). On the dorsal cortex of adherent
cells, where active cleavage takes place, actin filaments show
a largely isotropic organization, suggesting filament alignments are more likely a response to resistive forces such as
cell-cell and cell-substratum interactions rather than a prerequisite for cell cleavage.
In this study we have observed that alteration or disruption
of spindle microtubules can also cause pronounced effects on
the equatorial alignment of actin filaments. In taxol treated
cells there was a striking increase in tightly focused equatorial
filaments along the dorsal cortex of the furrow (Fig. 5), while
nocodazole-treated cells showed a broader, less well-organized
arrangement of equatorial filaments (Figs 4 and 6). As
discussed above, one explanation for strong filament alignments in taxol-treated cells may relate to increased concentration of signaling or motor molecules at the end of taxol-stabilized microtubules that drive cortical movements to the
equator. Increases in convergent forces at the equator could
then promote increases in actin filament alignment as first
proposed by White and Borisy (1983). Alternatively, enhanced
filament alignment could reflect increases in cortical resistive
forces due to stabilized spindle structures (Mickey and
Howard, 1995; Vallee, 1995). Similar explanations may
account for the broadening of the equatorial actin filament band
in nocodazole-treated cells. Hence, disassembly of microtubules could lead to disruptions in the distribution of cortical
signals controlling bipolar cortical flow or a decrease in
resistive forces (Hamilton and Snyder, 1983) that elicit structural orientation to actin filaments in the cortex.
Based on the dramatic effects of microtubules on cortical
movements and actin filament organization during cell
division, we propose that cytokinesis in tissue culture cells
consists of at least two temporally overlapping phases, with the
initial stage being regulated by spindle microtubules that
control the longitudinal movement of cortical components into
the cleavage furrow, and a second stage involving activation
of an actin-myosin based equatorial contraction that serves to
cleave the cell into two daughter cells. A third and final stage
of cell cleavage in dividing tissue culture cells likely relies on
proper formation of the mid-body involving additional activities such as interzonal microtubule motors (Yen et al., 1991;
Margolis and Andreassen, 1993; Williams et al., 1995), γtubulin mediated microtubule assembly (Julian et al., 1993;
Shu et al., 1995), and eventual disassembly of mid-body components and fusion of the plasma membrane (Mullins and
Biesele, 1977; Rattner, 1992).
Possible role of microtubules in cytokinesis
Although little is known about the nature of microtubulecortical interactions, our observations provide some important
clues. The most obvious role for microtubules is to stimulate,
guide, and drive active movement of surface receptors (Koppel
et al., 1982; McCaig and Robinson, 1982) and cortical components (Cao and Wang, 1990; Yonemura et al., 1993; Wang
et al., 1994) toward the equator during early anaphase, in order
to properly position the assembly of the contractile apparatus.
It is possible that the spindle interacts mechanically with the
cortex through various accessory proteins such as microtubuleassociated proteins, motors, and other proteins that could serve
to link microtubules, intermediate filaments, and actin
filaments to the membrane (Pollard et al., 1984; Goslin et al.,
1989; Sato et al., 1991; Wang et al., 1993; Katsumoto et al.,
1993; Klymkowski, 1995). This idea is supported by recent
findings implicating cortical interactions of the microtubules in
the positioning and orientation of the spindle in yeast (Palmer
et al., 1992; Li et al., 1993; McMillan and Tatchell, 1994) and
dividing nematode embryos (Hyman and White, 1987; Hyman,
1989; Hird and White, 1993). Alternatively, microtubules may
serve as guiding tracks for the delivery of diffusible signals,
signaling organelles, or other organizational cues to the equatorial cortex (Wright et al., 1993; Waterman-Storer et al., 1993;
Martineau et al., 1995). Given the large family of microtubule
motors already implicated in spindle function (Skoufias and
Scholey, 1993; McIntosh, 1994) and numerous signaling
pathways that affect cytokinesis (Fishkind and Wang, 1995),
the identification of molecules involved in transducing signals
should be forthcoming.
While microtubules seem to play an important role in
cortical dynamics in dividing tissue culture cells, their specific
function in cytokinesis of cells in embryos, yeast, fungi, and
slime molds remains less clear. Past ultrastructural work by
Asnes and Schroeder (1979) on sea urchin eggs, showing an
2050 D. J. Fishkind, J. D. Silverman and Y.-l. Wang
apparent low density of microtubules near the equatorial cortex
during anaphase, first cast some doubt on the stimulatory role
of microtubules in cortical events. However, more recent
confocal studies have clearly shown high densities of microtubules extending out to the cortex during mitosis (White et al.,
1987; Henson et al., 1989), reviving the possibility they may
play a specific role in cortical dynamics during cell division in
the sea urchin embryo (Dan, 1948, 1954; McCaig and
Robinson, 1982). Similarly, the extensive array of microtubules lying parallel to advancing furrow canals in the cellularizing embryo may help function in establishing directional
cues for actin-myosin based furrowing (Warn and Warn, 1986;
Katoh and Ishikawa, 1989; Foe et al., 1993; Schejter and
Wieschaus, 1993; Sullivan and Theurkauf, 1995). Future
studies with these and other experimental systems should
continue to provide us with new and important insights into the
complex process of cytokinesis.
We thank Dr Sally Wheatley, the reviewers, and managing editor
for helpful comments on the manuscript, as well as Dr Susan Horwitz
for advice on the use of taxol and baccatin III. Additionally, we thank
the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer
Institute for supplying taxol and baccatin III, and Drs Richard Vallee
and Howard Shpetner for test materials used in pilot studies. This
work was supported by grants from the American Cancer Society PF3758 (D.J.F.), National Institutes of Health GM32476 (Y.L.W.) and
Human Frontier Science Program (Y.L.W.).
REFERENCES
Agard, D. A. (1984). Optical sectioning microscopy: cellular architecture in
three dimensions. Annu. Rev. Biophys. Bioeng. 13, 191-219.
Amin-Hanjani, S. and Wadsworth, P. (1991). Inhibition of spindle
elongation by taxol. Cell Motil. Cytoskel. 20, 136-144.
Asnes, C. F. and Schroeder, T. E. (1979). Ultrastructural evidence against
equatorial stimulation by aster microtubules. Exp. Cell Res. 122, 327-338.
Beams, H. W. and Evans, T. C. (1940). Some effects of colchicine upon the
first cleavage in Arbacia punctulata. Biol. Bull. 79, 188-198.
Bray, D. and White, J. G. (1988). Cortical flow in animal cells. Science 239,
883-888.
Cao, L.-G. and Wang, Y.-L. (1990). Mechanism of the formation of
contractile ring in dividing cultured animal cells. II. Cortical movement of
micro-injected actin filaments. J. Cell Biol. 111, 1905-1911.
Cao, L. and Wang, Y. (1996). Signals from the spindle midzone are required
for the stimulation of cytokinesis in cultured epithelial cells. Mol. Biol. Cell
7, 225-232.
Castleman, K. R. (1979). Digital Image Processing. Prentice-Hall, Engelwood
Cliffs, NJ. pp. 429.
Chambers, R. (1938). Structural and kinetic aspects of cell division. J. Cell
Comp. Physiol. 12, 149-165.
Clark, S. W. and Meyer, D. I. (1992). Centractin is an actin homologue
associated with the centrosome. Nature 359, 246-250.
Conrad, G. W. and Schroeder, T. E. (editors). (1990). Cytokinesis:
Mechanisms of Furrow Formation During Cell Division, vol. 582, Ann. NY
Acad. Sci. 325 pp.
Dan, J. C. (1948). On the mechanism of astral cleavage. Physiol. Zool. 21, 191218.
Dan, K. (1954). The cortical movement in Arbacia punctulata eggs through
cleavage cycle. Embryologia 2, 115-122.
DeBrabander, M. J., Van de Veire, R. M. L., Aerts, F. E. M., Borgers, M.
and Janssen, P. A. J. (1976). The effects of methyl [5-92-thienylcarbonyl)1H-benzimidazol-2-yl]carbamate, (R 17934; NSC 238159), a new synthetic
antitumoral drug interfering with microtubules, on mammalian cells cultured
in vitro. Cancer Res. 36, 905-916.
DeBrabander, M., Geuens, G., Nuydens, R., Willebords, R., Aerts, F.,
DeMey, J. and with the participation of McIntosh, J.R. (1986).
Microtubule dynamics during the cell cycle: The effects of taxol and
nocodazole on the microtubule system of PtK2 cells at different stages of the
mitotic cycle. Int. Rev. Cytol. 101, 215-274.
Devore, J. J., Conrad, G. W. and Rappaport, R. (1989). A model for astral
stimulation of cytokinesis in animal cells. J. Cell Biol. 109, 2225-2232.
Fishkind, D. J. and Wang, Y.-L. (1993). Orientation and three-dimensional
organization of actin filaments in dividing cultured cells. J. Cell Biol. 123,
837-848.
Fishkind, D. J. and Wang, Y.-L. (1995). New horizons for cytokinesis. Curr.
Opin. Cell Biol. 7, 23-31.
Foe, V. E., Odell, G. M. and Edgar, B. A. (1993). Mitosis and morphogenesis
in the Drosophila embryo: point and counterpoint. In The Development of the
Drosophila melanogaster, vol. I (ed. M. Bate and A. Martinez-Arias), pp.
149-300. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York.
Fukui, Y. (1993). Toward a new concept of cell motility: cytoskeletal dynamics
in amoeboid movement and cell division. Int. Rev. Cytol. 144, 85-127.
Goslin, K., Birgbauer, E., Banker, G. and Solomon, F.. (1989). The role of
cytoskeleton in organizing growth cones: a microfilament-associated growth
cone component depends upon microtubules for its localization. J. Cell Biol.
109, 1621-1631.
Hamaguchi, Y. (1975). Microinjection of colchicine into sea urchin eggs. Dev.
Growth Differ. 17, 111-117.
Hamilton, B. T. and Snyder, J.A. (1983). Acceleration of cytokinesis in PtK1
cells treated with microtubule inhibitors. Exp. Cell Res. 144, 345-351.
Henson, J. H., Begg, D. A., Beaulieu, S. M., Fishkind, D. F., Bonder, E. M.,
Terasaki, M., Lebeche, D. and Kaminer, B. (1989). A calsequestrin-like
protein in the endoplasmic reticulum of the sea urchin: localization and
dynamics in the egg and first cell cycle embryo. J. Cell Biol. 109, 149-161.
Hiramoto, Y. (1956). Cell divisions without mitotic apparatus in sea urchin
eggs. Exp. Cell Res. 11, 630-636.
Hiramoto, Y. (1971). Analysis of cleavage stimulus by means of
micromanipulation of sea urchin eggs. Exp. Cell Res. 68, 291-298.
Hird, S. N. and White, J. G. (1993). Cortical and cytoplasmic flow polarity in
early embryonic cells of Caenorhabditis elegans. J. Cell Biol. 121, 13431355.
Hyman, A. A. and White, J. G. (1987). Determination of cell division axes in
the early embryogenesis of Caenorhabditis elegans. J. Cell Biol. 105, 21232135.
Hyman, A. A. (1989). Centrosome movement in the early divisions of
Caenorhabditis elegans: a cortical site determining centrosome position. J.
Cell Biol. 109, 1185-1193.
Julian, M., Tollon, Y., Lajoie-Mazenc, I., Moisand, A., Mazarguil, H.,
Puget, A. and Wright, M. (1993). γ-Tubulin participates in the formation of
the midbody during cytokinesis in mammalian cells. J. Cell Sci. 105, 145156.
Katoh, K. and Ishikawa, H. (1989). The cytoskeletal involvement in
cellularization of the Drosophila melanogaster embryo. Protoplasma 150,
83-95.
Katsumoto, T., Naguro, T., Yan, Y.-L., Iino, A. and Onodera, K. C. (1993).
The spatial distribution of spindle microtubules in anaphase 3Y1 cells. Biol.
Cell 77, 247-253.
Klymkowski, M. W. (1995). Intermediate filaments: new proteins, some
answers, more questions. Curr. Opin. Cell Biol. 7, 46-54.
Koppel, D. E., Oliver, J. M. and Berlin, R. D. (1982). Surface function during
mitosis. III. Quantitative analysis of ligand-receptor movement into the
cleavage furrow: diffusion vs. flow. J. Cell Biol. 93, 950-960.
Li, Y. Y., Yeh, E., Hays, T. and Bloom, K. (1993). Disruption of mitotic
spindle orientation in yeast dynein mutant. Proc. Nat. Acad. Sci. USA 90,
10096-10100.
Mabuchi, I. (1986). Biochemical aspects of cytokinesis. Int. Rev. Cytol. 101,
175-213.
Manfredi, J. J. and Horwitz, S. B. (1984). Taxol: An antimitotic agent with a
new mechanism of action. Pharmac. Ther. 25, 83-125.
Margolis, R. L. and Andreassen, P. R. (1993). The telophase disc: its possible
role in mammalian cell cleavage. BioEssays 15, 201-207.
Martineau, S. N., Andreassen, P. R. and Margolis, R. L. (1995). Delay of
HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of
a postmitotic spindle and telophase disc correlates with synchronous
cleavage recovery. J. Cell Biol. 131, 191-205.
McCaig, C. D. and Robinson, K. R. (1982). The distribution of lectin
receptors on the plasma membrane of the fertilized sea urchin egg during first
and second cleavage. Dev. Biol. 92, 197-202.
McIntosh, J. R. (1994). The role of microtubules in chromosome movement.
Microtubule effects on actin organization 2051
In Microtubules (ed. J. S. Hyams and C. W. Lloyd), pp. 413-434. Wiley-Liss,
New York.
McKenna, N. C. and Wang, Y.-L. 1989. Culturing cells on the microscope
stage. Meth. Cell Biol. 29, 195-205.
McMillan, J. N. and Tatchell, K. (1994). The JNM1 gene in the yeast
Saccharomyces cerevisiae is required for nuclear migration and spindle
orientation during the mitotic cell cycle. J. Cell Biol. 125, 143-158.
Mickey, B. and Howard, J. (1995). Rigidity of microtubules is increased by
stabilizing agents. J. Cell Biol. 130, 909-917.
Mullins, J. M. and Biesele, J. J. (1977). Terminal phase of cytokinesis in D98S cells. J. Cell Biol. 73, 672-684.
Mullins, J. M. and Snyder, J. A. (1981). Anaphase progression and furrow
establishment in nocodazole-arrested PtK1 cells. Chromosoma 83, 493-505.
Palmer, R. E., Sullivan, D. S. and Koshland, D. (1992). Role of astral
microtubules and actin in spindle orientation and migration in the budding
yeast, Saccharomyces cerevisiae. J. Cell Biol. 119, 583-593.
Pollard, T. D., Selden, S. C. and Maupin, P. (1984). Interaction of actin
filaments and microtubules. J. Cell Biol. 99, 33s-37s.
Rappaport, R. (1985). Repeated furrow formation from a single mitotic
apparatus in cylindrical sand dollar eggs. J. Exp. Zool. 234, 167-171.
Rappaport, R. (1986). Establishment of the mechanism of cytokinesis in
animal cells. Int. Rev. Cytol. 105, 245-281.
Rappaport, R. (1991). Cytokinesis. In Comparative Physiology: Oogenesis,
Spermatogenesis, and Reproduction, vol. 10 (ed. R. K. H. Kinne), pp. 1-36.
Karger, Basel.
Rattner, J. B. (1992). Mapping the mammalian intercellular bridge. Cell Motil.
Cytoskel. 23, 231-235.
Salmon, E. D. (1989a). Microtubule dynamics and chromosome movement. In
Mitosis: Molecules and Mechanisms (ed. J. S. Hyams and B. R. Brinkley),
pp. 119-181. Academic Press, London.
Salmon, E. D. (1989b). Cytokinesis in animal cells. Curr. Opin. Cell Biol. 1,
541-547.
Sanger, J. M. and Sanger, J. W. (1980). Banding and polarity of actin
filaments in interphase and cleaving cells. J. Cell Biol. 86, 568-575.
Sanger, J. M., Dome, J. S., Hock, R. S., Mittal, B. and Sanger, J. W. (1994).
Occurrence of fibers and their association with talin in the cleavage furrows
of PtK2 cells. Cell Motil. Cytoskel. 27, 26-40.
Sato, N., Yonemura, S., Obinata, T., Tsukita, Sa. and Tsukita, Sh. (1991).
Radixin, a barbed end-capping actin-modulating protein, is concentrated at
the cleavage furrow during cytokinesis. J. Cell Biol. 113, 321-330.
Satterwhite, L. L. and Pollard, T. D. (1992). Cytokinesis. Curr. Opin. Cell
Biol. 4, 43-52.
Schejter, E. D. and Wieschaus, E. (1993). Functional elements of the
cytoskeleton in the early Drosophila embryo. Annu. Rev. Cell Biol. 9, 67-99.
Schiff, P. B., Fant, J. and Horwitz, S. B. (1979). Promotion of microtubule
assembly in vitro by taxol. Nature 277, 665-667.
Schiff, P. B. and Horwitz, S. B. (1980). Taxol stabilizes microtubules in mouse
fibroblast cells. Proc. Nat. Acad. Sci. USA 77, 1561-1565.
Schroeder, T. E. (1970). The contractile ring: I. Fine structure of dividing
mammalian (HeLa) cells and the effects of cytochalasin B. Z. Zellforsch.
Mikrosk. Anat. 109, 431-449.
Schroeder, T. E. (1973). Actin in dividing cells: Contractile ring filaments bind
heavy meromyosin. Proc. Nat. Acad. Sci. USA 70, 1688-1692.
Shaw, P. J. and Rawlins, D. J. (1991). Three-dimensional fluorescence
microscopy. Prog. Biophys. Mol. Biol. 56, 187-213.
Shu, H.-B., Li, Z., Palacios, M. J., Li, Q. and Joshi, H. C. (1995). A transient
association of γ-tubulin at the midbody is required for the completion of
cytokinesis during the mammalian cell division. J. Cell Sci. 108, 2955-2962.
Skoufias, D. A. and Scholey, J. M. (1993). Cytoplasmic microtubule-based
motor proteins. Curr. Opin. Cell Biol. 5, 95-104
Small, J. V. (1981). Organization of actin in the leading edge of cultured cells;
Influence of osmium tetroxide and dehydration on the ultrastructure of actin
meshworks. J. Cell Biol. 91, 695-705.
Snyder, J. A. and Mullins, J. M. (1993). Analysis of spindle microtubule
organization in untreated and taxol-treated PTK1 cells. Cell Biol. Int. 17,
1075-1084.
Sullivan, W. and Theurkauf, W. E. (1995). The cytoskeleton and
morphogenesis of the early Drosophila embryo. Curr. Opin. Cell Biol. 7, 1822
Swann, M. M. and Mitchison, J. M. (1953). Cleavage of sea-urchin eggs in
colchicine. J. Exp. Biol. 30, 506-514.
Swann, M. M. and Mitchison, J. M. (1958). The mechanism of cleavage in
animal cells. Biol. Rev. 33, 103-135.
Vallee, R. B. (1995). The use of taxol in cell biology. In Taxol, Science and
Applications (ed. M. Suffness), pp. 259-274. CRC Press, Boca Raton, FL.
Wadsworth, P. (1993). Mitosis: spindle assembly and chromosome motion.
Curr. Opin. Cell Biol. 5, 123-128.
Wang, N., Butler, J. P. and Ingber, D. E. (1993). Mechanotransduction across
the cell surface and through the cytoskeleton. Science 260, 1124-1127.
Wang, Y.-L., Silverman, J. D. and Cao, L.-G. (1994). Single particle tracking
of surface receptor movement during cell division. J. Cell Biol. 127, 963-971.
Warn, R. M. and Warn, A. (1986). Microtubule arrays present during the
syncytial and cellular blastoderm stages of the early Drosophila embryo. Exp.
Cell Res. 163, 201-210.
Waterman-Storer, C. M., Sanger, J. W. and Sanger, J. M. (1993). Dynamics
of organelles in the mitotic spindles of living cells: Membranes and
microtubule interactions. Cell Motil. Cytoskel. 26, 19-39.
White, J. G. and Borisy, G. G. (1983). On the mechanism of cytokinesis in
animal cells. J. Theor. Biol. 101, 289-316.
White, J. G., Amos, W. B. and Fordham, M. (1987). An evaluation of
confocal versus conventional imaging of biological structures by
fluorescence microscopy. J. Cell Biol. 105, 41-48.
Williams, B. C., Riedy, M. F., Williams, E. V., Gatti, M. and Goldberg, M.
L.. (1995). The Drosophila kinesin-like protein KLP3A is a midbody
component required for central spindle assembly and initiation of
cytokinesis. J. Cell Biol. 129, 709-723.
Wilson, E. B. (1925). The Cell in Development and Heredity. MacMillan
Publishing, New York. pp. 1,296.
Wilson, L., Miller, H. P., Farrell, K. W., Snyder, K. B., Thompson, W. C.
and Purich, D. L. (1985). Taxol stabilization of microtubules in vitro:
Dynamics of tubulin addition and loss at opposite microtubule ends.
Biochemistry 24, 5254-5262.
Wilson, L. and Jordan, M. A. (1994). Pharmacological probes of microtubule
function. In Microtubules (ed. J. S. Hyams and C. W. Lloyd), pp. 59-83.
Wiley-Liss, New York.
Wolpert, L. (1960). The mechanics and mechanisms of cleavage. Int. Rev.
Cytol. 10, 163-216.
Wright, B. D., Terasaki, M. and Scholey, J. M. (1993). Roles of kinesin and
kinesin-like proteins in sea urchin embryonic cell division: evaluation using
antibody microinjection. J. Cell Biol. 123, 681-689.
Yen, T. J., Compton, D. A., Wise, D., Zinkowski, R. P., Brinkley, B. R.,
Earnshaw, W. C. and Cleveland, D. W. (1991). CENP-E, a novel human
centromere-associated protein required for progression from metaphase to
anaphase. EMBO J. 10, 1245-1254.
Yonemura, S., Nagafuchi, A., Sato, N. and Tsukita, S. (1993). Concentration
of an integral membrane protein, CD43 (leukosialin, sialophorin), in the
cleavage furrow through the interaction of its cytoplasmic domain with actinbased cytoskeletons. J. Cell Biol. 120, 437-449.
(Received 13 November 1995 – Accepted 17 May 1996)