Centralspindlin regulates ECT2 and RhoA accumulation at the equatorial cortex during cytokinesis

Abstract

During determination of the cell division plane, an actomyosin contractile ring is induced at the equatorial cell cortex by signals from the mitotic apparatus and contracts to cause cleavage furrow progression. Although the small GTPase RhoA is known to regulate the progression, probably by controlling actin filament assembly and enhancing actomyosin interaction, any involvement of RhoA in division plane determination is unknown. In this study, using a trichloroacetic acid (TCA) fixation protocol we recently developed, we show that RhoA accumulates at the equatorial cortex before furrow initiation and continues to concentrate at the cleavage furrow during cytokinesis. We also demonstrate that both Rho activity and microtubule organization are required for RhoA localization and proper furrowing. Selective disruption of microtubule organization revealed that both astral and central spindle microtubules can recruit RhoA at the equatorial cortex. We find that centralspindlin and ECT2 are required for RhoA localization and furrowing. Centralspindlin is localized both to central spindle microtubules and at the tips of astral microtubules near the equatorial cortex and recruits ECT2. Positional information for division plane determination from microtubules is transmitted to the cell cortex to organize actin cytoskeleton through a mechanism involving these proteins.

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). 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.…”

“…(34)(35)(36) Ect2 associates with MgcRacGAP, (20)(21)(22)(23)37) a component of the centralspindlin complex. (38) The other centralspindlin component, MKLP1, is a plus enddirected microtubule motor protein.…”

“…Centralspindlin bundles microtubules in the spindle midzone, (38) and its components have also been localized to astral microtubules in the region near the equator prior to furrowing. (23,34) In addition, binding of Ect2 to MgcRacGAP may promote Ect2 GEF activity. (20) Disruption of MKLP, MgcRacGAP, or Ect2 prevents both cytokinesis and concentration of Rho at the cell equator.…”

“…(20) Disruption of MKLP, MgcRacGAP, or Ect2 prevents both cytokinesis and concentration of Rho at the cell equator. (20)(21)(22)(23) Thus, it is thought that the centralspindlin complex translocates along microtubules to the cell equator, where it interacts with Ect2. (37) This interaction would concentrate Ect2 and stimulate its GEF activity, resulting in local Rho activation at the equator.…”

Novel Functions of Ect2 in Polar Lamellipodia Formation and Polarity Maintenance during “Contractile Ring-Independent” Cytokinesis in Adherent Cells

“…The Drosophila and Caenorhabditis elegans orthologues of Ect2, Pebble and LET-21, respectively, have both been shown to be required for contractile ring formation and cytokinesis (Prokopenko et al, 1999;Morita et al, 2005). Although Ect2 appears to act as a GEF with all three Rho-type small GTPases in vitro, recent studies suggest that RhoA is the primary downstream target of Ect2 in vivo (Kimura et al, 2000;Yuce et al, 2005;Kamijo et al, 2006;Nishimura and Yonemura, 2006;Birkenfeld et al, 2007).Interestingly, under appropriate conditions in some cell types, cytokinesis proceeds fairly normally without contractile ring activity. For example, when adhering to a substrate, myosin II-null cells of the cellular slime mold Dictyostelium discoideum are able to divide by making use of traction forces, which move the daughter cells away from one another (Neujahr et al, 1997;Zang et al, 1997;Nagasaki et al, 2002).…”