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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 Dispatch 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 1. O’Keefe, L., Somers, W.G., Harley, A. and Saint, R. (2001). The Pebble GTP exchange factor and the control of cytokinesis. Cell Struct. Funct. 26, 619–626. Current Biology R365 2. Lehner, C.F. (1992). The pebble gene is required for cytokinesis in Drosophila. J. Cell Sci. 103, 1021–1030. 3. 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. 4. 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. 5. Echard, A. and O’Farrell, P.H. (2003). The degradation of two mitotic cyclins contributes to the timing of cytokinesis. Curr. Biol. 13, 373–383. 6. Sotillos, S. and Campuzano, S. (2000). DRacGAP, a novel Drosophila gene, inhibits EGFR/Ras signalling in the developing imaginal wing disc. Development 127, 5427–5438. 7. 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. 8. Touré, A., Dorseui, O., Morin, L., Jegou, B., Reibel, L. and Gacon, G. (1998). MgcRacGAP, a new human GTPase-activating protein for Rac and Cdc42 similar to Drosophila rotundRacGAP gene product, is expressed in male germ cells. J. Biol. Chem. 273, 6019–6023. 9. Mishima, M., Kaitna, S. and Glotzer, M. (2002). Central spindle assembly and cytokinesis require a kinesin-like protein/RhoGAP complex with microtubule bundling activity. Dev. Cell 2, 41–54. 10. 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. 11. Adams, R.R., Tavares, A.A., Salzberg, A., Bellen, H.J. and Glover, D.M. (1998). pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev. 12, 1483–1494. 12. 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. 13. Kuriyama, R., Gustus, C., Terada, Y., Uetake,Y. and Matuliene, J. (2002). CHO1, a mammalian kinesin-like protein, interacts with Factin and is involved in the terminal phase of cytokinesis. J. Cell Biol. 156, 783–790. 14. Gatti, M. Giansanti, M.G. and Bonaccorsi, S. (2000). Relationships between the central spindle and the contractile ring during cytokinesis in animal cells. Microsc. Res. Tech. 49, 202–208. 15. Wang, Y.L. (2001). The mechanism of cytokinesis, reconsideration and reconciliation. Cell Struct. Funct. 26, 633–638. 16. Sigrist, S., Jacobs, H., Stratmann, R. and Lehner, C.F. (1995). Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3. EMBO J. 14, 4827–4838. 17. Parry, D.H. and O’Farrell, P.H. (2001). The schedule of destruction of three mitotic cyclins can dictate the timing of events during exit from mitosis. Curr. Biol. 11, 671–683. 18. Murata-Hori, M. and Tatsuka, M., Wang, Y.L. (2002). Probing the dynamics and functions of Aurora B kinase in living cells during mitosis and cytokinesis. Mol. Biol. Cell 13, 1099–1108. 19. Shuster, C.B. and Burgess, D.R. (1999). Parameters that specify the timing of cytokinesis. J. Cell Biol. 146, 981–992.