Centre for the Molecular Genetics of Development, Research School of Biological Sciences, Australian National University, Canberra ACT 0200, Australia
Author for correspondence (e-mail: robert.saint{at}anu.edu.au)
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Summary |
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Key words: Cytokinesis, Rho GTPase, PBL/Ect2 RhoGEF, Contractile ring, PAV-KLP/MKLP1, Central spindle, Microtubules
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Introduction |
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Appropriate partitioning is achieved by positioning the plane of cleavage between the recently separated chromosomes during anaphase. The regulation of this positioning, as well as many other aspects of cytokinesis, has been the subject of speculation since the late 19th century (Rappaport, 1971). More than a century later, we still have no clear molecular understanding of many of the vital steps involved, including the mechanism that positions the plane of cleavage. Here, we discuss recent studies of regulators of the Rho family of GTPases that have provided a long-sought-after, if speculative, molecular model for the positioning of the contractile ring in animal cells.
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Cytokinesis: more than a pinch of actomyosin |
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Rho GTPases and actin regulation |
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It is now well established that Rho-family GTPases induce changes in actin organization in response to intracellular and extracellular signals. These small G proteins act through a range of effectors, such as the kinases Pak (p21-activated kinase) (reviewed by Bokoch, 2003) and ROCK (Rho kinase) (reviewed by Riento and Ridley, 2003
), and actin-modulating proteins such as the Diaphanous-related formin-homology proteins (reviewed by Evangelista et al., 2003
) and WASp family proteins (reviewed by Badour et al., 2003
). During cytokinesis, Rho GTPase appears to be acting through some of these effectors. The Diaphanous formin-homology protein (Tatsumoto et al., 1999
) and members of the Rho-dependent kinases (Kosako et al., 2000
; Madaule et al., 1998
) have been implicated as targets of activated Rho.
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The role of the mitotic apparatus |
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Although astral microtubules appear to be sufficient to induce furrowing in some cases, microtubule arrangements in cells of different sizes and structures can vary considerably during anaphase. One common feature of anaphase cells is the formation of bundled microtubules in the midzone between the separated chromosomes. These midzone microtubules are collectively referred to as the central spindle. In some cells, such as Drosophila melanogaster spermatocytes, the central spindle contacts the cell cortex (Cenci et al., 1994). A role for the midzone microtubules in generating the cytokinetic stimulus has been suggested previously (Wheatley and Wang, 1996
; Bonaccorsi et al., 1998
; Gatti et al., 2000
; Bucciarelli et al., 2003
). Wheatley and Wang showed that in normal rat kidney (NRK) cells with multiple spindles, furrowing is induced when the midzone microtubules lie close to the cortex and is independent of the position of the spindle poles or astral microtubules. Bonaccorsi et al., have shown that D. melanogaster asterless mutant male meiotic cells, which lack astral microtubules but develop normal midzone microtubules, are capable of undergoing cytokinesis. These and other studies have led Gatti and his colleagues to conclude that formation of the central spindle and the contractile ring apparatus are interdependent (Cao and Wang, 1996
; Gatti et al., 2000
; Giansanti et al., 1998
; Somma et al., 2002
). As noted above, furrowing does not require formation of a complete ring and a local association between microtubules and the cortex is enough to induce local furrowing (Wheatley and Wang, 1996
).
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Connecting the contractile ring to microtubules |
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Three genes in particular have been important to these new developments: those encoding the D. melanogaster Rho GTP exchange factor (GEF); Pebble (PBL) (Hime and Saint, 1992; Lehner, 1992
; Prokopenko et al., 1999
); the plus-end-directed kinesin-like motor proteins known as ZEN-4 in C. elegans (Raich et al., 1998
) and Pavarotti (PAV-KLP) in D. melanogaster (Adams et al., 1998
); and the Rho-family GTPase-activating protein (GAP) known as CYK-4 in C. elegans (Jantsch-Plunger et al., 2000
) and RacGAP50C in D. melanogaster (Somers and Saint, 2003
). Each of these has orthologs in mammals. The mouse ortholog of pbl, Ect2, was originally identified as a proto-oncogene and then shown to be required for cytokinesis (Tatsumoto et al., 1999
). MgcRacGAP, the mammalian ortholog of CYK-4/RacGAP50C, and the CHO1/MKLP1 vertebrate orthologs of ZEN-4/PAV-KLP are also required for cytokinesis (Chen et al., 2002
; Kuriyama et al., 2002
). Thus, all three encoded proteins are evolutionarily conserved cytokinesis factors.
pbl appears to be required for the earliest steps in cytokinesis, because embryonic D. melanogaster cells lacking PBL fail to show evidence of contractile ring function (Lehner, 1992; Prokopenko et al., 1998; Somma et al., 2002
). Genetic interactions and yeast two-hybrid studies indicated that Rho1/RhoA is the target of PBL RhoGEF activity (O'Keefe et al., 2001
; Prokopenko et al., 1999
). PBL localizes to the contractile ring during cytokinesis (Prokopenko et al., 1999
), where it appears to activate Rho locally and trigger the cascade of events that results in contractile ring function.
An important recent development is the discovery that CYK-4 and ZEN-4 associate to form a complex, known as centralspindlin, that can bundle microtubules (Mishima et al., 2002). Microtubule bundling is a feature of the anaphase/telophase microtubule network and appears to play an important role in generating the midzone microtubule network. The centralspindlin complex also appears to be a conserved feature of animal cytokinesis. The human and D. melanogaster CYK-4 and ZEN-4 orthologs form a complex (Mishima et al., 2002
; Somers and Saint, 2003
), and embryonic cells lacking the Drosophila zen-4 ortholog, pav, exhibit defects in microtubule bundling (Adams et al., 1998
).
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A double ring to bind them? |
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The link between the PBL RhoGEF and the centralspindlin complexes suggested a model for the positioning of the contractile ring (Somers and Saint, 2003). This `double ring' model postulates that the kinesin-like protein component (PAV-KLP/ZEN-4//CHO1/MKLP1) moves centralspindlin complexes to the interdigitated or juxtaposed plus ends of microtubules located at the midzone, where they form a microtubule-associated ring underneath the future cleavage site (Fig. 3). The interaction between the RacGAP50C/CYK-4/MgcRacGAP and the PBL/Ect2 is proposed to result in the positioning of the cortical RhoGEF at this site. This, in turn, would result in formation of a ring of activated Rho and, consequently, organization of the actomyosin contractile ring (Fig. 3).
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This model accounts for conflicting observations (discussed above) concerning the roles of the astral microtubules and the midzone/central spindle microtubules. The arrangement and type of microtubule network may, in fact, not be important except in being able to deliver the centralspindlin complex to the midzone cortex. The way this is achieved may depend on the cell type. In large cells, such as those involved in the initial divisions of the sea urchin embryo, where the central spindle is far from the cortex, astral microtubules may play this role. Conversely, in the case of D. melanogaster spermatocytes or NRK cells, it may be the central spindle that delivers the centralspindlin complexes to create the cortical double ring with PBL and induce furrowing.
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A general mechanism for animal cytokinesis? |
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Other evidence, however, is inconsistent with this model. Some of the inconsistencies appear to reflect differences in cytoskeletal organization within different types of cells. For example, whereas studies described above revealed correlations between midzone or astral microtubule concentrations and furrow positioning (Wheatley and Wang, 1996; Giansanti et al., 1998
), a recent report shows that C. elegans furrowing occurs at a local minimum in the microtubule concentration (Dechant and Glotzer, 2003
). In addition, the phenotypes of the cyk-4 and zen-4 mutants suggest that they are required only after the onset of cytokinesis, when the advancing furrow reaches the central spindle (Raich et al., 1998
; Jantsch-Plunger et al., 2000
). The recent observation that Aurora-B-mediated phosphorylation converts MgcRacGAP (the mammalian CYK-4 ortholog) into a RhoGAP (Minoshima et al., 2003
) supports the proposal that this factor inactivates Rho as the advancing furrow meets the midbody (Jantsch-Plunger et al., 2000
).
Other conflicting evidence has come from studies of mammalian cultured cells. In contrast to the behaviour of RacGAP50C RNAi-treated D. melanogaster tissue culture cells (Somers and Saint, 2003), mammalian cells subjected to RNAi directed against CHO1 exhibit later cytokinetic phenotypes (Matuliene and Kurimaya, 2002
). In addition, dominant-negative constructs of both Ect2 and CHO1 also block cytokinesis after the onset of furrowing. These studies should be treated with some caution, however, because it is possible that residual levels of the proteins remain at sufficient levels to induce initial furrowing. Indeed, Echard and O'Farrell (Echard and O'Farrell, 2003
) have observed that the persistence of maternal PBL correlates with incomplete furrowing in cycle 14 pbl mutant cells. Alternatively, it is possible that the PBL-RACGAP50C-PAV-KLP double-ring is not involved in the initial cytokinetic events, but positioned in preparation for a later role at the time of midbody formation, or that it plays a role in relaxing the contacts between epithelial cells so that they can round up for mitosis.
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Conclusion and perspectives: upstream and downstream of Rho activation |
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Even if the double ring model turns out to be true, it explains only a small fraction of the events required for successful cytokinesis. For example, it does not explain how microtubules are established so that the plus ends are juxtaposed or interdigitated for the centralspindlin complexes to accumulate and create the plane of division. It also does not explain how the double ring mechanism becomes active during anaphase B. Activation may involve regulators of late mitotic processes, such as polo-like kinase (Carmena et al., 1998), the aurora kinases (Giet and Glover, 2001
; Schumacher et al., 1998
; Severson et al., 2000
) and INCENP (Kaitna et al., 2000
; Wheatley et al., 2001
), or other factors associated with the centralspindlin-PBL complex, such as the CeCDC-14 phosphatase, which is required for ZEN-4 localization (Gruneberg et al., 2002
).
We also know little about the molecular events that occur between the activation of Rho and the formation and activation of the actomyosin contractile ring. In D. melanogaster, Anillin accumulates before the appearance of the actomyosin network (Field and Alberts, 1995) and in X. laevis, myosin appears before F-actin (Noguchi and Mabuchi, 2001
). Septins are also recruited to the ring, perhaps by anillin-actin complexes (Kinoshita et al., 2002
). The process of furrowing must involve an exquisite molecular motor that allows the membrane-attached ring to contract.
Although many questions associated with animal cytokinesis remain, it is nonetheless clear that the combined experimental resources of key model organisms such as C. elegans, X. laevis and D. melanogaster will provide the answers.
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Footnotes |
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