Download cleeks o` cytokinesis: microtubule sticks and contractile hoops in cell

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Biochemical Society Transactions (2008) Volume 36, part 3
Girds ‘n’ cleeks o’ cytokinesis: microtubule sticks
and contractile hoops in cell division
David M. Glover1 , Luisa Capalbo, Pier Paolo D’Avino, Melanie K. Gatt, Matthew S. Savoian and Tetsuya Takeda
Cancer Research U.K. Cell Cycle Genetics Research Group, University of Cambridge, Department of Genetics, Downing Street, Cambridge CB2 3EH, U.K.
Abstract
Microtubules maintain an intimate relationship with the rings of anillin, septins and actomyosin filaments
throughout cytokinesis. In Drosophila, peripheral microtubules emanating from the spindle poles contact the
equatorial cell cortex to deliver the signal that initiates formation of the cytokinetic furrow. Mutations that
affect microtubule stability lead to ectopic furrowing because peripheral microtubules contact inappropriate
cortical sites. The PAV-KLP (Pavarotti-kinesin-like protein)/RacGAP50C (where GAP is GTPase-activating
protein) centralspindlin complex moves towards the plus ends of microtubules to reach the cell equator.
When RacGAP50C is tethered to the cell membrane, furrowing initiates at multiple non-equatorial sites,
indicating that mis-localization of this single molecule is sufficient to promote furrowing. Furrow formation
and ingression requires RhoA activation by the RhoGEF (guanine-nucleotide-exchange factor) Pebble, which
interacts with RacGAP50C. RacGAP50C also binds anillin, which associates with actin, myosin and septins.
Thus RacGAP50C plays a pivotal role during furrow formation by activating RhoA and linking the peripheral
microtubules with the nascent rings through its interaction with anillin.
Spindle microtubules and cleavage rings
The title for this short paper reflects the Edinburgh venue
of the meeting that catalysed its writing and the realization
that the Scots tongue, a variant of Old English, has acquired
status as a distinct language on the website of the Scottish
Assembly. The Scots ‘girds ‘n’ cleeks’ corresponds to the
English ‘hoops and sticks’, universal children’s toys that
provide an analogy for the multiple hoops of anillin,
actomyosin and septins and the microtubules that organize
them for the process of cell division. We have previously
reviewed how these interactions orchestrate the process of
cytokinesis and refer the reader to that article for a more indepth discussion [1]. The importance of spindle microtubules
in directing the position of cytokinesis was clear from the
early experiments of Rappaport [2]. Since that time, two
apparently opposing models have emerged to account for
their role: one proposing that astral microtubules inhibit
furrow formation close to the spindle poles and the other
suggesting that a sub-population of microtubules actually
deliver a signal to the equatorial cortex. Most probably,
elements of both models are relevant and these mechanisms
are used to varying extents in different organisms or cell types.
In this short article we focus on a series of experiments from
our own laboratory that support the predominance of the
second model in Drosophila cells, and we apologize in advance
both for not giving a wider viewpoint and to scientists whose
work we have not been able to cite because of space restraints.
Key words: anillin, central-spindle microtubule, centralspindlin, cytokinesis, Rac GTPaseactivating protein (RacGAP), Rho guanine-nucleotide-exchange factor (RhoGEF).
Abbreviations used: GAP, GTPase-activating protein; GEF, guanine-nucleotide-exchange factor;
GFP, green fluorescent protein; KLP, kinesin-like protein; PAV-KLP, Pavarotti-KLP; RNAi, RNA
interference.
1
To whom correspondence should be addressed (email [email protected]).
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Authors Journal compilation Unashamedly, we will also focus upon one particular cell
type in Drosophila, the primary spermatocyte. This is our
favourite cell because of the advantages it offers for studying
cytokinesis.
Cytokinesis in Drosophila spermatocytes
One difficulty in using genetic approaches to study cell
division is the fact that many proteins that have a particular
role in prometaphase are reutilized for different ends during
cytokinesis. Thus mutants for such proteins tend to show
metaphase arrest in mitosis due to the spindle assembly
checkpoint. In male meiosis, however, the absence of a strong
spindle assembly checkpoint enables such mutant cells to
proceed through anaphase and telophase and to attempt
cytokinesis. Mutations in asp, for example, cause somatic
cells to arrest at metaphase with highly abnormal spindle
poles, whereas asp mutant spermatocytes proceed beyond this
stage and show pronounced defects in the organization of the
central spindle microtubules that are necessary for cytokinesis
[3,4]. Added to this peculiarity of cell cycle control is the fact
that the primary spermatocyte is one of the largest cell types in
Drosophila and is amenable to short-term culture and direct
observation of meiosis by time-lapse microscopy.
Such observations on living spermatocytes expressing
tubulin tagged with GFP (green fluorescent protein) show
quite clearly that the initiation of furrow formation takes
place when a set of peripheral microtubules extend from the
centrosomes to ‘touch’ the cell cortex at the cell equator
(Figure 1A) [5]. The equatorial rings that form at this
time, first of anillin and subsequently of actin and septins,
retain their contact with and progressively constrict the
central spindle microtubules and will finally divide the cell
Biochem. Soc. Trans. (2008) 36, 400–404; doi:10.1042/BST0360400
Mechanics and Control of Cytokinesis
Figure 1 Furrowing is initiated in Drosophila spermatocytes
when peripheral microtubules emanating from the spindle poles
touch the cell cortex
(A) Selected frames from a time-lapse sequence of a single cell
shown at low (−1 min) and higher (subsequent panels) magnification
of the boxed region. After anaphase onset, peripheral microtubules
(arrowheads) approach the cortex, which they probe (2 min) until
cortical contact is made (4 min), at which time they become bundled
(7 min). Shortly thereafter, the cleavage furrow initiates and ingresses,
thereby coalescing peripheral and interior microtubules (12 and 17 min).
The fluorescence signal for tubulin–GFP is inverted. Time is relative
in two. Failure of the final stages of constriction of
the central spindle microtubules are seen in mutants
of the gene vibrator, previously found to encode the
Drosophila phosphatidylinositol transfer protein; central
spindle formation occurs correctly in such mutants, but
furrow ingression occurs more slowly and the apparent
connection between the constricted cytoskeleton and the
plasma membrane is finally lost and the furrow undergoes
elastic regression [6]. This probably reflects the multiple
functions of phosphoinositol lipids in regulating both actin
dynamics and membrane synthesis.
to anaphase onset. Scale bars, 10 μm. (B) Loss of orbit preferentially
prevents formation of interior but not peripheral microtubules. Peripheral
microtubules still form and probe the cytoplasm (8 min). A more robust
set of peripheral microtubules form (arrowheads and arrows) and contact
the cortex, immediately before furrow formation on the right and left
sides of the cell (23 min). Furrow ingression arrests on the right but
continues on the left side. This furrow advances before also then
ultimately regressing (35 min). Times are relative to anaphase onset.
Scale bar, 10 μm. (C) Abnormally long astral microtubules in klp67A
mutants collect in the upper portion of the cell, where they initiate an
abnormally positioned unilateral cleavage furrow that advances but fails
to fully cleave the cell (12–23 min; arrows). Time is relative to anaphase
onset. Scale bar, 10 μm.
Furrow initiation requires microtubules to
contact the cell cortex
We have studied how the site of the initiation of furrowing
depends upon the contact made by microtubules with the
cortex in mutants in which microtubule stability is altered.
One such mutant is in the gene orbit/mast that encodes
the counterpart of the mammalian protein, CLASP. At
metaphase, microtubule subunits flux along the kinetochore
fibres; tubulin subunits are incorporated at the kinetochore
while being simultaneously lost from microtubule minus
ends at the centrosome. The Orbit protein is required for
tubulin subunit addition, a function that is balanced by
the microtubule-depolymerizing properties of the kinesintype depolymerase KLP (kinesin-like protein) 10A [7]. Thus
down-regulation of both Orbit and KLP10A rescues the
monopolar spindle phenotype shown by down-regulation of
either protein alone. During cytokinesis, Orbit relocalizes
specifically to an interior population of central spindle
microtubules. Consistently, this subset of microtubules are
unstable in orbit mutants. However, the peripheral astral
microtubules still grow out from the centrosomes to touch the
cortex and initiate furrow formation, but in the absence of
the interior central spindle, the furrow is unstable and
cytokinesis fails (Figure 1B) [5]. Thus the peripheral
microtubules are required to initiate the furrow and the
interior central spindle microtubules for its maintenance.
Another microtubule-associated protein whose function
changes in late M-phase is the microtubule-destabilizing
KLP67A protein. In prometaphase and anaphase, KLP67A is
required to facilitate chromosome biorientation and anaphase
movement, whereas in cytokinesis it acquires a microtubule
stabilizing function [7,8]. In KLP67A mutant spermatocytes,
anaphase B spindles elongate with normal kinetics but then
bend towards the cortex. Both peripheral and interior spindle
microtubules then form diminished bundles of abnormally
positioned central spindle microtubules. Importantly, furrows initiate at the sites of central spindle bundles but can
be unilateral or non-equatorially positioned (Figure 1C).
Ectopic furrows can also be stimulated by the interior central
spindle but in general are observed only to form after this
structure buckles to contact the cortex. The central spindles
become increasing unstable over time, actin and anillin fail
to form homogenous bands and cytokinesis fails. Thus the
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Biochemical Society Transactions (2008) Volume 36, part 3
signal to furrow can be delivered wherever the peripheral
microtubules contact the cortex but it can also be provided by
the interior central spindle microtubules under these aberrant
circumstances. Collectively this body of results from wildtype and mutant cells indicate that microtubules do deliver
the signal that initiates furrowing in Drosophila.
RacGAP50C provides the central platform
of the furrow-initiation signalling
machinery
What then is this signal and can we see its association
with microtubules? A likely candidate is the centralspindlin
complex. At the core of this conserved complex are a
motor component, known as PAV-KLP (Pavarotti-KLP) in
Drosophila, ZEN-4 in Caenorhabditis elegans and MKLP1 in mammals, and a GAP (GTPase activating protein),
RacGAP50C or Tum in flies, CYK-4 in nematodes, and
MgcRacGAP in mammals. The motor protein displays plusend directed activity [9–12] and so is potentially able to deliver
a signal to the cell equator. Furthermore, our own studies
of GFP-tagged PAV-KLP in embryonic cells showed it to
become concentrated at the equator of the cell prior to cytokinesis [13]. The GAP component had been reported to
down-regulate the in vitro activity of Rac and Cdc42 GTPases
more efficiently than Rho [14–16] and so did not seem like
the ideal candidate for the signalling molecule. RhoA seemed
more likely because its inactivation prevents cytokinesis in
most systems [17] and its active form accumulates at the
future cleavage site in a microtubule-dependent manner [18].
Studies in Drosophila and mammals indicated that RhoA
activation at the cleavage furrow requires the RhoGEF
(guanine-nucleotide-exchange factor) Pebble-ECT2 [19–21].
The finding by Saint and Somers [22] that Drosophila Pebble
could complex to RacGAP50C led them to propose that the
onset of cytokinesis could be triggered by the microtubulemediated delivery of the centralspindlin complex to the cortex
where its RacGAP component would activate the RhoGEF
Pebble and, in turn, RhoA. The finding that MgcRacGAP
interacts with ECT2 in mammals suggests that this proposed
mechanism has been conserved during evolution [23–26].
Could the centralspindlin complex then indeed be a
platform that permits Pebble/Ect2 to activate RhoA and
initiate cytokinesis? To begin to address this question
we were led to ask what happens to microtubules and
the initiation of furrowing following the inactivation of
either the PAV-KLP or the RacGAP50C components of
centralspindlin. We found that down-regulation of either
component by RNAi (RNA interference) in cultured cells
prevented the onset of furrowing in spite of the continued
growth of astral microtubules to touch the cell cortex at its
equator [27]. Thus the centralspindlin complex seemed to be
essential for the astral microtubules to transmit their signal.
This interpretation was reinforced by our direct time-lapse
observations of the dynamics of the centralspindlin complex
in both mitotically dividing cells in culture and in primary
spermatocytes. In both cell types, we were able to observe
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Authors Journal compilation Figure 2 PAV-KLP tracks to the plus ends of peripheral
microtubles before the onset of furrowing
(A) A time-lapse series showing PAV–GFP dynamics in Drosophila
primary spermatocytes. The corresponding DIC (differential interference
contrast) images in the top panels show furrow ingression (black arrows).
PAV–GFP fluorescence intensifies at the cell equator as cells progress into
anaphase (lower panels). (B) Magnified areas from the indicated boxed
regions in (A) show PAV-KLP becoming concentrated at the plus ends
of peripheral microtubules contacting the cell cortex. Time is shown in
minutes relative to anaphase onset. Scale bars, 10 μm.
PAV-KLP tagged with GFP translocate to the plus ends of
the astral microtubules as they touched the cell cortex to
determine the cleavage plane (Figure 2). If PAV-KLP was
just providing the motor function to take RacGAP50C to
the cortex, we argued that a membrane-tethered version of
RacGAP50C ought to provoke ectopic furrowing. To test
this, we fused an integral plasma membrane component, the
human T-cell receptor CD8, with the full coding region of
RacGAP50C and a GFP tag was inserted between the two
genes to follow its distribution (Figure 3A). When we induced
the expression of this construct in cultured Drosophila cells,
we observed the formation of multiple ectopic furrows
all over the cell’s cortex (Figure 3B) [27]. These furrows
were able to form in the absence of PAV-KLP and they
recruited both anillin and septins. We strongly suspect that
RacGAP50C induces these ectopic furrows because it serves
as a means of recruiting and/or activating the RhoGEF
Pebble. Consistently no equatorial or ectopic furrowing
activity was observed in the CD8–GFP–RacGAP50C cells
after RNAi depletion of Pebble.
We cannot rule out in the above experiments that the
membrane-tethered version of RacGAP50C can also recruit
other factors that co-operate with it to induce ectopic
furrows. Indeed recent results from both our laboratory
[28], and Robert Saint and co-workers [29], indicate that
anillin, one of the first molecules to be recruited to the
furrow in the form of a ring, also physically interacts
directly with RacGAP50C. As anillin also interacts with actin,
myosin and septins, this provides the basis for a physical
Mechanics and Control of Cytokinesis
Figure 3 Mislocalization of RacGAP50C causes ectopic furrowing
(A) Schematic diagram of the inducible CD8–GFP and CD8–GFP–
RacGAP50C constructs that were stably expressed in Drosophila cell
lines. Mt, metallothionein. (B) Following induction of the two constructs
from the metallothionein promoter, cells were examined by immunostaining. The arrows indicate the ectopic furrows, whereas the arrowhead
indicates the equatorial furrow. Scale bar, 10 μm.
ring and the relative roles of phosphatidylinositol lipids in
regulating addition of new membrane and behaviour of the
filamentous actin. Microtubules continue to be central to
the whole process, not least by providing the transport
network that cycles vesicles to and from the constricting
furrow. We are beginning to have some insight into these
processes through studies in a variety of model systems.
The future challenge is to integrate this knowledge into an
understanding of what is one the most fundamental processes
in cell biology.
The work in our laboratory on cytokinesis has been supported by
grants from Cancer Research U.K. and the BBSRC (Biotechnology
and Biological Sciences Research Council).
References
connection between girds and cleeks. Thus RacGAP50C
indeed looks as though it plays a pivotal role in coordinating the process of furrow formation. It appears
to be transported to the plus ends of astral microtubules
that grow out towards the equatorial cortex in a process
that relies upon its ability to form a complex with the
PAV-KLP motor protein or its counterparts in other
organisms. Its ability to induce the formation of ectopic
furrows depends upon the RhoGEF Pebble/Ect2, with
which it interacts directly. This RhoGEF then activates Rho
at the cortex to initiate the furrowing process. RacGAP50C
also captures anillin at the cortex and thus provides the
possibility of recruiting all of the ring components. Rho
itself has also recently been reported to interact directly
with anillin [30], thus raising the possibility that Rho and
RacGAP50C provide mutual reinforcement in establishing
the ring of anillin that serves to organize actin and septins.
Future challenges
The mechanics of the gird ‘n’ cleek are quite different from
those of cytokinesis. Whereas the gird requires a simple
stroke from the cleek to set it in motion, the contractile
apparatus of the cell is mechanically far more complex.
We are on the verge of understanding the events that initiate
formation of the cleavage furrow and its associated contractile
rings. Akin to the gird ‘n’ cleek, this requires that contact
is first established between peripheral microtubules and the
nascent contractile ring. There then begins a process likely
to involve multiple cycles of incremental ring constriction in
which the central spindle microtubules play an essential role.
What the roles of the active versus inactive forms of various
GTPases might be in co-ordinating these events remains to be
clarified. We also have much to learn about the mechanisms
that maintain membrane association with the contractile
1 D’Avino, P.P., Savoian, M.S. and Glover, D.M. (2005) Cleavage furrow
formation and ingression during animal cytokinesis: a microtubule
legacy. J. Cell Sci. 118, 1549–1558
2 Rappaport, R. (1961) Experiments concerning the cleavage stimulus in
sand dollar eggs. J. Exp. Zool. 148, 81–89
3 Riparbelli, M.G., Callaini, G., Glover, D.M. and do Carmo Avides, M.
(2002) A requirement for the Abnormal Spindle protein to organise
microtubules of the central spindle for cytokinesis in Drosophila.
J. Cell Sci. 115, 913–922
4 Wakefield, J.G., Bonaccorsi, S. and Gatti, M. (2001) The Drosophila
protein Asp is involved in microtubule organization during spindle
formation and cytokinesis. J. Cell Biol. 153, 637–648
5 Inoue, Y.H., Savoian, M.S., Suzuki, T., Máthé, E., Yamamoto, M.-T. and
Glover, D.M. (2004) Mutations in orbit/mast reveal that the central
spindle is comprised of two microtubule populations, those that initiate
cleavage and those that propagate furrow ingression. J. Cell Biol. 166,
49–60
6 Gatt, M.K. and Glover, D.M. (2006) The Drosophila phosphatidylinositol
transfer protein encoded by vibrator is essential to maintain
cleavage-furrow ingression in cytokinesis. J. Cell Sci. 119, 2225–2235
7 Savoian, M.S., Gatt, M.K., Riparbelli, M.G., Callaini, G. and Glover, D.M.
(2004) Drosophila Klp67A is required for proper chromosome
congression and segregation during meiosis I. J. Cell Sci. 117, 3669–3677
8 Gatt, M.K., Savoian, M.S., Riparbelli, M.G., Massarelli, C., Callaini, G. and
Glover, D.M. (2005) Klp67A destabilises pre-anaphase microtubules but
subsequently is required to stabilise the central spindle. J. Cell Sci. 118,
2671–2682
9 Adams, R.R., Tavares, A.A.M., Salzberg, A., Bellen, H.J. and Glover, D.M.
(1998) Genes Dev. 12, 1483–1494
10 Nislow, C., Lombillo, V.A., Kuriyama, R. and McIntosh, J.R. (1992)
A plus-end-directed motor enzyme that moves antiparallel microtubules
in vitro localizes to the interzone of mitotic spindles. Nature 359,
543–547
11 Powers, J., Bossinger, O., Rose, D., Strome, S. and Saxton, W. (1998)
A nematode kinesin required for cleavage furrow advancement.
Curr. Biol. 8, 1133–1136
12 Raich, W.B., Moran, A.N., Rothman, J.H. and Hardin, J. (1998) Cytokinesis
and midzone microtubule organization in Caenorhabditis elegans require
the kinesin-like protein ZEN-4. Mol. Biol. Cell 9, 2037–2049
13 Minestrini, G., Harley, A.S. and Glover, D.M. (2003) Localization of
Pavarotti-KLP in living Drosophila embryos suggests roles in reorganizing
the cortical cytoskeleton during the mitotic cycle. Mol. Biol. Cell 14,
4028–4038
14 Hirose, K., Kawashima, T., Iwamoto, I., Nosaka, T. and Kitamura, T.
(2001) MgcRacGAP is involved in cytokinesis through associating with
mitotic spindle and midbody. J. Biol. Chem. 276, 5821–5828
15 Jantsch-Plunger, V., Gonczy, P., Romano, A., Schnabel, H., Hamill, D.,
Schnabel, R., Hyman, A.A. and Glotzer, M. (2000) CYK-4: A Rho family
GTPase activating protein (GAP) required for central spindle formation
and cytokinesis. J. Cell Biol. 149, 1391–1404
16 Somers, W.G. and Saint, R. (2003) A RhoGEF and Rho family
GTPase-activating protein complex links the contractile ring to cortical
microtubules at the onset of cytokinesis. Dev. Cell 4, 29–39
C The
C 2008 Biochemical Society
Authors Journal compilation 403
404
Biochemical Society Transactions (2008) Volume 36, part 3
17 Piekny, A., Werner, M. and Glotzer, M. (2005) Cytokinesis: welcome to
the Rho zone. Trends Cell Biol. 15, 651–658
18 Bement, W.M., Benink, H.A. and von Dassow, G. (2005)
A microtubule-dependent zone of active RhoA during cleavage plane
specification. J. Cell Biol. 170, 91–101
19 Kimura, K., Tsuji, T., Takada, Y., Miki, T. and Narumiya, S. (2000)
Accumulation of GTP-bound RhoA during cytokinesis and a critical
role of ECT2 in this accumulation. J. Biol. Chem. 275, 17233–17236
20 Prokopenko, S.N., Brumby, A., O’Keefe, L., Prior, L., He, Y., Saint, R. and
Bellen, H.J. (1999) A putative exchange factor for Rho1 GTPase is
required for initiation of cytokinesis in Drosophila. Genes Dev. 13,
2301–2314
21 Tatsumoto, T., Xie, X., Blumenthal, R., Okamoto, I. and Miki, T. (1999)
Human ECT2 is an exchange factor for Rho GTPases, phosphorylated
in G2 /M phases, and involved in cytokinesis. J. Cell Biol. 147, 921–928
22 Saint, R. and Somers, W.G. (2003) Animal cell division: a fellowship of
the double ring? J. Cell Sci. 116, 4277–4281
23 Kamijo, K., Ohara, N., Abe, M., Uchimura, T., Hosoya, H., Lee, J.S. and
Miki, T. (2006) Dissecting the role of Rho-mediated signaling in
contractile ring formation. Mol. Biol. Cell 17, 43–55
24 Nishimura, Y. and Yonemura, S. (2006) Centralspindlin regulates ECT2
and RhoA accumulation at the equatorial cortex during cytokinesis.
J. Cell Sci. 119, 104–114
C The
C 2008 Biochemical Society
Authors Journal compilation 25 Yuce, O., Piekny, A. and Glotzer, M. (2005) An ECT2-centralspindlin
complex regulates the localization and function of RhoA. J. Cell Biol. 170,
571–582
26 Zhao, W.M. and Fang, G. (2005) MgcRacGAP controls the assembly of
the contractile ring and the initiation of cytokinesis. Proc. Natl. Acad.
Sci. U.S.A. 102, 13158–13163
27 D’Avino, P.P., Savoian, M.S., Capalbo, L. and Glover, D.M. (2006)
RacGAP50C is sufficient to signal cleavage furrow formation during
cytokinesis. J. Cell Sci. 119, 4402–4408
28 D’Avino, P.P., Takeda, T., Capalbo, L., Zhang, W., Lilley, K.S., Laue, E.D.
and Glover, D.M. (2008) Interaction between anillin and RacGAP50C
connects the actomyosin contractile ring with spindle microtubules at
the cell division site. J. Cell Sci. 121, 1151–1158
29 Gregory, S.L., Ebrahimi, S., Milverton, J., Jones, W.M., Bejsovec, A. and
Saint, R. (2008) Cell division requires a direct link between
microtubule-bound RacGAP and anillin in the contractile ring. Curr. Biol.
18, 25–29
30 Piekny, A.J. and Glotzer, M. (2008) Anillin is scaffold protein that links
RhoA, actin, and myosin during cytokinesis. Curr. Biol. 18, 30–36
Received 23 January 2008
doi:10.1042/BST0360400