A microtubule-dependent zone of active RhoA during cleavage plane specification

Abstract

Cytokinesis in animal cells results from the assembly and constriction of a circumferential array of actin filaments and myosin-2. Microtubules of the mitotic apparatus determine the position at which the cytokinetic actomyosin array forms, but the molecular mechanisms by which they do so remain unknown. The small GTPase RhoA has previously been implicated in cytokinesis. Using four-dimensional microscopy and a probe for active RhoA, we show that active RhoA concentrates in a precisely bounded zone before cytokinesis and is independent of actin assembly. Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis. Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement. We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.

Rho GTPase activity zones and transient contractile arrays

“…(18) Moreover, Rho concentrates at the equator prior to cytokinesis in cultured mammalian cells. (19)(20)(21)(22)(23) Rho activity zones are typically spatially confined to regions with widths on the order of microns, while the linear dimension of the cells in which they form ranges from hundreds of microns (Xenopus oocytes, echinoderm zygotes) to tens of microns (echinoderm blastomeres, cultured mammalian cells).…”

“…(19)(20)(21)(22)(23) Rho activity zones are typically spatially confined to regions with widths on the order of microns, while the linear dimension of the cells in which they form ranges from hundreds of microns (Xenopus oocytes, echinoderm zygotes) to tens of microns (echinoderm blastomeres, cultured mammalian cells). Rho itself was shown to be required for cytokinesis long ago, (1) but these findings, and the demonstration that active Rho dynamically senses microtubule perturbation, (18) imply that Rho zones are part of the long-sought link between microtubule geometry and stimulation of cytokinetic furrowing. It remains to be seen whether Cdc42 and/or Rac activity zones also participate in cytokinesis, but this is consistent with several findings.…”

“…(18) If Rho-GTP experiences a diffusion coefficient similar to myristoylated yellow fluorescent protein (~2 μm 2 /sec;) (39) and has an intrinsic hydrolysis rate on the order of one per 5-50 minutes, only a shallow, transient concentration gradient could be sustained, even with an infinitesimally narrow source. Furthermore, as the source continued to emit active Rho, the domain would simply fill up; the steady state, which would be reached after an hour or so, would have a high concentration of active Rho everywhere, but elevated by 20-30% near the source (Fig.…”

“…(3) During urchin embryonic cytokinesis, Rho zones of approximately normal intensity and breadth appear in cells treated shortly before furrowing with either cytochalasin or latrunculin, implying that this zone is not strictly dependent on F-actin for formation. However, cytochalasin-or latrunculintreated cells exhibit active Rho on dynamic, tubular PM extensions that are pulled into the cytoplasm, apparently on astral microtubules, (18) suggesting that at least some aspects of Rho-GTP's normal distribution is perturbed.…”

“…Second, the cytokinetic Rho zone disappears within 1-2 minutes when the spindle that it is supposed to bisect is physically moved to a new location. (18) Meanwhile, a new Rho zone appears just as rapidly as the old one disappears. Third, photobleaching experiments in budding yeast (45) and Candida albicans (46) indicate that Cdc42 at the bud site is turned over within ~5 seconds; photobleaching experiments in echinoderm embryos likewise indicate rapid turnover within cytokinetic Rho zones (10-30 seconds; unpublished results).…”