An Anillin-Ect2 Complex Stabilizes Central Spindle Microtubules at the Cortex during Cytokinesis

Paul Frenette,Michael Loloyan,Mena Kinal,Eric Haines,Paknoosh Pakarian,Alisa Piekny,Claude Prigent

doi:10.1371/journal.pone.0034888

Abstract

Cytokinesis occurs due to the RhoA-dependent ingression of an actomyosin ring. During anaphase, the Rho GEF (guanine nucleotide exchange factor) Ect2 is recruited to the central spindle via its interaction with MgcRacGAP/Cyk-4, and activates RhoA in the central plane of the cell. Ect2 also localizes to the cortex, where it has access to RhoA. The N-terminus of Ect2 binds to Cyk-4, and the C-terminus contains conserved DH (Dbl homologous) and PH (Pleckstrin Homology) domains with GEF activity. The PH domain is required for Ect2's cortical localization, but its molecular function is not known. In cultured human cells, we found that the PH domain interacts with anillin, a contractile ring protein that scaffolds actin and myosin and interacts with RhoA. The anillin-Ect2 interaction may require Ect2's association with lipids, since a novel mutation in the PH domain, which disrupts phospholipid association, weakens their interaction. An anillin-RacGAP50C (homologue of Cyk-4) complex was previously described in Drosophila, which may crosslink the central spindle to the cortex to stabilize the position of the contractile ring. Our data supports an analogous function for the anillin-Ect2 complex in human cells and one hypothesis is that this complex has functionally replaced the Drosophila anillin-RacGAP50C complex. Complexes between central spindle proteins and cortical proteins could regulate the position of the contractile ring by stabilizing microtubule-cortical interactions at the division plane to ensure the generation of active RhoA in a discrete zone.

Highlights

Cytokinesis describes the division of a cell into two genetically identical daughter cells and occurs due to the RhoA-mediated ingression of an actomyosin ring
Our results suggest that their interaction may physically link central spindle microtubules with the contractile ring, a function that was previously described for the anillinRacGAP50C complex in Drosophila
The anillin homology domain (AHD) from Drosophila anillin directly binds to RacGAP50C in vitro [33], yet Hela cells depleted of endogenous Cyk-4 had no effect on anillin’s interaction with Ect2 (Figure 1A)

Summary

Introduction

Cytokinesis describes the division of a cell into two genetically identical daughter cells and occurs due to the RhoA-mediated ingression of an actomyosin ring. The mitotic spindle determines the division plane during anaphase and is comprised of astral microtubules that emanate to the poles of the cell, and central spindle microtubules (including centrally positioned astral microtubules) that reach the equatorial cortex. While central spindle microtubules stimulate contractile ring formation in the center of the cell, astral microtubules inhibit the localization of contractile ring components at the poles of the cell [1,2,3]. The molecular components of the astral pathway have not been identified in many cell types, yet they may dominantly determine the division plane in large cells where the central spindle is positioned far from the cortex. The central spindle stimulates contractile ring formation by leading to the activation of RhoA by the GEF, Ect2 [7].

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Results

Conclusion