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Current Biology, Vol. 13, R363–R365, April 29, 2003, ©2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0960-9822(03)00278-1
Cell Division: The Place and Time of
Cytokinesis
Cayetano Gonzalez
Recent studies have provided evidence that, during
cytokinesis, activation of the Pbl–Rho1 pathway by a
protein complex located at the spindle midzone, and
inhibition of this pathway by two mitotic cyclins, may
be major contributing factors controlling the place
and timing of the cleavage furrow.
Successful cell division requires tight control of the
timing and positioning of the cytokinesis furrow, so
that it starts only after the two sets of chromosomes
are fully segregated and proceeds precisely between
them. Furrowing involves the formation of an
actomyosin network, a process regulated by the small
G protein Rho1 [1]. Rho1 activation is regulated by the
balance between the guanine nucleotide exchange
factors (GEFs) and the GTPase-activating proteins
(GAPs): a GEF activates Rho1 by catalyzing the uptake
of GTP, while a GAP stimulates the GTPase activity of
Rho1 leading to its inactivation. In Drosophila the
Rho1-GEF is Pebble (Pbl) a protein known to be
essential for cytokinesis [2,3]. Two recent studies [4,5]
have now shown that activation of Pbl by a protein
complex localised at the site of furrowing, and
regulation of the Pbl-dependent cytokinesis pathway
by the central cell-cycle machinery, may control the
place and time of cytokinesis.
The first key observations made by Somers and
Saint [4] were that RacGAP50C, a Rho family GAP [6],
interacts with Pbl in the yeast two-hybrid assay and
that the two proteins co-immunoprecipitate from
Drosophila embryonic tissues. Moreover, they found
that downregulation of RacGAP50C by RNA interference (RNAi) has a similar effect to mutation of Pbl: both
perturbations impair cytokinesis, leading to a binucleate cell phenotype. The functional relevance of this
was supported by the demonstration that RacGAP50C
RNAi and a dominant-negative pbl mutation interact
synergistically, and by the conservation of these components during animal evolution — humans have Pbl
and RacGAP50C orthologs, known respectively as
ECT2 and MgcRacGAP [4,7,8].
In the nematode Caenorhabditis elegans, the
RacGAP50C ortholog Cyk-4 had previously been
linked to another key cytokinesis protein, the kinesinlike motor protein Zen-4, the fly ortholog of which is
known as Pavarotti (Pav). These two proteins are part
of the multimeric complex known as centralspindlin,
which has microtubule-bundling activity in vitro and is
required for the bundling of microtubules that occurs
in the central region of a dividing cell as mitosis
European Molecular Biology Laboratory, Cell Biology and
Biophysics Programme, Meyerhofstrasse 1, 69117
Heidelberg, Germany. E-mail: [email protected]
Dispatch
proceeds [9,10]. In Drosophila too, cycle 16 cells in
pav mutants have a reduced level of microtubule
bundling and show no cortical furrowing [11]. The new
evidence reported by Somers and Saint [4] shows that
the interaction between RacGAP50C and Pav is
conserved in Drosophila. The mammalian orthologs of
Cyk-4 and Zen-4 also localize to the central spindle
and are essential for cytokinesis [12,13].
The distribution of RacGAP50C throughout mitosis
is highly dynamic. As shown by immunofluorescence:
in prophase RacGAP50C is localized to the cytoplasm;
during metaphase it is found over the mitotic spindle;
just prior to the onset of furrow formation it is concentrated in dots over the central spindle; and during the
earliest stages of furrowing it is found in a more concentrated midzone band. In late anaphase and early
telophase, RacGAP50C localises to the overlapping
central spindle microtubules and to distinct cortical
microtubules. At late telophase, the complex is found
on the midbody, and it moves to the nucleus during
interphase. These results are consistent with earlier
observations on RacGAP50C orthologs [9,11,12], but
the details of the distribution of RacGAP50C during
late anaphase and early telophase are new and very
revealing. Somers and Saint [4] found that, from the
onset of cytokinesis to the end of furrowing, most
RacGAP50C–Pav complexes form a punctuate ring
that can be seen to precisely abut the intracellular
face of the cortical ring which is formed by proteins
including Peanut, Myosin II, Diaphanous and, very
importantly, Pbl itself.
The identification of this double-ring structure at
the cytokinetic furrow, composed of both actomyosin-associated contractile components, including
Pbl, and microtubule-associated RacGAP50C–Pav
complexes, led Somers and Saint [4] to propose a
model in which the Pav–RacGAP50C–Pbl complex
positions the contractile ring and coordinates cytoskeletal remodelling during cytokinesis (Figure 1). The
model is based on the microtubule-mediated arrival
of the RacGAP50C–Pav complex at the equatorial
ring, where it mediates microtubule bundling. Localisation of RacGAP50C at this point would enable its
interaction with cortical Pbl, which is proposed to
activate its RhoGEF activity. The resulting local activation of Rho1 would coordinate F-actin and microtubule remodelling, thereby enabling cytokinesis.
The affinity of the RacGAP50C–Pav complex for
microtubules and the plus-end-directed nature of the
Pav kinesin-like motor protein, together with the
observed distribution of microtubules, appear sufficient to account for localization of the complex to an
equatorial cortical ring. Indeed, the localization of
RacGAP50C–Pav complexes to the microtubule
midzone is independent of its interaction with Pbl, but
dependent on Pav [4]. Moreover, the synergistic effect
of simultaneous loss of RacGAP50C and Pbl function
is consistent with the suggested role of RacGAP50C
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R364
CyclinB–Cdk1
Plasma membrane
CyclinB3–Cdk1
–
Pav
RacGAP50C
Active Rho1
+
Microtubules
• Passenger proteins release
• Astral microtubule growth
• Spindle elongation
• Midbody formation
Pav
RacGAP50C
Pbl
Pav
RacGAP50C
Pbl
+
Pav
RacGAP50C
FURROW
Diaphanous
Citron kinase
Drok
Myosin II
–
Cytokinesis
Current Biology
as activator of Pbl. The model is also consistent with
the known role of microtubules in positioning the
contractile ring [14,15].
The controlled accumulation of RacGAP50C at the
critical place, and consequent activation of Pbl, thus
seems to be essential for specifying the site of furrowing. What about the timing? Furrow initiation is
known to follow degradation of cyclins B and B3 [16],
suggesting that activity of either or both of these two
cyclins opposes furrow initiation and that their degradation might contribute to the timing of cytokinesis.
Indeed, cyclin B is known to have an indirect inhibitory
effect on cytokinesis because it blocks events, such
as the transition to anaphase B or the dissociation of
chromosome passenger proteins from centromeres
[17,18], thought to be required for the onset of cytokinesis. Stabilization of Drosophila cyclin B3, unlike that
of cyclin B, does not block cytokinesis [17], suggesting a clear distinction between the abilities of these
two B-type cyclins to inhibit cytokinesis.
Evidence reported recently in Current Biology by
Echard and O’Farrell [5], however, shows that cyclin B
and cyclin B3 can both directly inhibit cytokinesis.
These authors carried out a detailed study of the
timing of cytokinesis in cells in which either cyclin B
and cyclin B3 levels were reduced by mutation, or
cyclin B3 levels maintained by the expression of
stable forms of the protein [5]. They found that loss of
either cyclin B or cyclin B3 advanced cytokinesis
furrow initiation, while artificially maintained cyclin B3
levels delayed the onset of furrow formation. Despite
the timing differences, in each case cytokinesis proceeded to completion. In addition to the delay, furrow
ingression was slowed in cells expressing stable
cyclin B3. An increased proportion of anaphase cells
in the process of cytokinesis was observed in cyclin B
and cyclin B3 mutant embryos, suggesting that furrow
ingression is also slowed in these cells, perhaps as a
secondary consequence of premature initiation.
Figure 1. A model for the role of
Pav–RacGAP50C–Pbl complex, cyclin B
and cyclin B3 in the regulation of
cytokinesis.
The place of furrow formation during
cytokinesis is specified by the movement
of RacGAP50C to the spindle midzone
(arrows) through the activity of its binding
partner, the plus-end-directed molecular
motor, Pav. There, RacGAP50C associates with the cortical RhoGEF Pbl, resulting in the local activation of Rho1 that
promotes furrowing. The onset of furrow
formation depends on the time of destruction of cyclin B and cyclin B3, most likely
in a complex with Cdk1. By blocking
mitotic exit events (left box) that are
thought to be prerequisites for cytokinesis,
cyclin B has an indirect inhibitory effect on
cytokinesis. Cyclin B and cyclin B3 also
have a direct inhibitory effect upon the
Pbl-dependent pathway of cytokinesis
activation. Any of the proteins that act with
Pbl to control cytokinesis (right box) could
be the target, but Pbl itself is a strong candidate. (Adapted from [4,5].)
Several lines of evidence suggest that at least one of
the mechanisms by which cyclins B and B3 affect the
cytokinesis regulatory network may involve the Pbl
pathway [5]. The first is the similar effects reducing the
Pbl level or expressing stable cyclin B3 have on the
timing of cytokinesis, delaying and slowing furrow formation during mitotic cycle 14. The second is that the
two conditions interact synergistically, so that furrows
are almost absent when stable cyclin B3 is expressed
in pbl mutant embryos. This strongly suggests that
cyclin B3 either inhibits a residual activity of the Pbl
pathway or suppresses a parallel pathway that acts in
conjunction with Pbl. Finally, additional support for the
view that cyclin B and cyclin B3 are inhibitors of the
Pbl-mediated cytokinesis activation pathway is provided by the suppressor effect of cyclin B and cyclin
B3 mutations on the cytokinesis-defective phenotype
caused by the expression of a dominant-negative
version of Pbl.
Together, these observations imply there is a
regulatory connection between cyclins B/B3 and the
Pbl pathway (Figure 1), though they do not define how
directly they are connected. Interestingly, the expression of stable cyclin B or B3 suppresses the normal rise
in Pbl in anaphase, suggesting that cyclin–Cdk regulation of Pbl accumulation may be a major factor controlling the onset of cytokinesis. Moreover, stable cyclin B3
slows spindle maturation, neither the peripheral microtubules that contact the cortex at the midzone nor the
midbody developing fully in its presence. It has been
suggested that delivery of a cleavage stimulus to the
cortex determines the timing of cytokinesis [19]. It will
be interesting to know what effect degradation of cyclin
B3 may have on the dynamics of RacGAP50C localisation during mitosis.
References
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