RhoA and the Rho guanine nucleotide exchange factor ECT-2 are involved in both polarization and cytokinesis. During cytokinesis, interactions of ECT-2 with the Rho GTPase-activating protein CYK-4 promote RhoA activation. A novel protein, NOP-1, acts in parallel with CYK-4 to promote RhoA activation during polarization and cytokinesis.

SUMMARY In metazoans, cytokinesis is triggered by activation of the GTPase RhoA at the equatorial plasma membrane. ECT-2, the guanine nucleotide exchange factor (GEF) required for RhoA activation, is activated by the centralspindlin complex that concentrates on spindle midzone microtubules. However, these microtubules and the plasma membrane are not generally in apposition, and thus the mechanism by which RhoA is activated at the cell equator remains unknown. Here we report that a regulated pool of membrane-bound, oligomeric centralspindlin stimulates RhoA activation. The membrane-binding C1 domain of CYK-4, a centralspindlin component, promotes furrow initiation in C. elegans embryos and human cells. Membrane localization of centralspindlin oligomers is globally inhibited by PAR-5/14-3-3. This activity is antagonized by the chromosome passenger complex (CPC), resulting in RhoA activation at the nascent cleavage site. Therefore, CPC-directed centralspindlin oligomerization during anaphase induces contractile ring assembly at the membrane.

Cytokinesis in animal cells is mediated by a cortical actomyosinbased contractile ring. The GTPase RhoA is a critical regulator of this process as it activates both non-muscle myosin and a nucleator of actin filaments [1]. The site at which active RhoA and its effectors accumulate is controlled by the microtubule-based spindle during anaphase [2]. ECT-2, the guanine nucleotide exchange factor (GEF) that activates RhoA during cytokinesis is regulated by phosphorylation and subcellular localization [3–5]. ECT2 localization depends on interactions with CYK-4/MgcRacGAP, a Rho GTPase activating protein (GAP) domain containing protein [5, 6]. Here, we show that, contrary to expectations, the Rho GTPase activating protein (GAP) domain of CYK-4 promotes activation of RhoA during cytokinesis. Furthermore, we show that the primary phenotype caused by mutations in the GAP domain of CYK-4 is not caused by ectopic activation of CED-10/Rac1 and ARX-2/Arp2. However, inhibition of CED-10/Rac1 and ARX-2/Arp2 facilitates ingression of weak cleavage furrows. These results demonstrate that a GAP domain can contribute to activation of a small GTPase. Furthermore, cleavage furrow ingression is sensitive to the balance of contractile forces and cortical tension.

Shiga-toxin-producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocytosed by intestinal epithelial cells and transported retrogradely through the secretory pathway. It is ultimately translocated to the cytosol where it inhibits protein translation. We found that Shiga toxin transport through the secretory pathway was dependent on the cytoskeleton. Recent studies reveal that Shiga toxin activates signaling pathways that affect microtubule reassembly and dynein-dependent motility. We propose that Shiga toxin alters cytoskeletal dynamics in a way that facilitates its transport through the secretory pathway. We have now found that Rho GTPases regulate the endocytosis and retrograde motility of Shiga toxin. The expression of RhoA mutants inhibited endocytosis of Shiga toxin. Constitutively active Cdc42 or knockdown of the Cdc42-specific GAP, ARHGAP21, inhibited the transport of Shiga toxin to the juxtanuclear Golgi apparatus. The ability of Shiga toxin to stimulate microtubule-based transferrin transport also required Cdc42 and ARHGAP21 function. Shiga toxin addition greatly decreases the levels of active Cdc42-GTP in an ARHGAP21-dependent manner. We conclude that ARHGAP21 and Cdc42-based signaling regulates the dynein-dependent retrograde transport of Shiga toxin to the Golgi apparatus. INTRODUCTIONEnteritis caused by Shigella dysenteria and pathogenic strains of Escherichia coli is a global health threat. These bacteria secrete Shiga toxin that enters intestinal epithelial cells and kills them by blocking translation. In some cases, the toxin escapes the gut and targets the kidney and vascular endothelium resulting in hemolytic-uremic syndrome (Sandvig and van Deurs, 2000; O'Loughlin and RobinsBrowne, 2001;Proulx et al., 2001;Desch and Motto, 2007). Treatment options for Escherichia coli infection and hemolyticuremic syndrome are limited in part because of an incomplete understanding of the molecular mechanisms underlying Shiga toxin's trafficking within cells.Shiga toxin reaches the cytosol by using retrograde transport through the secretory pathway (Sandvig and van Deurs, 2002;Johannes and Popoff, 2008). Shiga toxin is a heteromultimeric protein containing one A subunit and five B subunits. The A subunit is an N-glycosidase that inhibits protein translation, whereas the Shiga toxin B subunits (STxBs) mediate intracellular targeting. STxB binds to the cell surface via a glycolipid receptor, globotriaosyl ceramide (Gb3). Entry is mediated by clathrin-dependent or -independent endocytosis (Lingwood, 1993;Sandvig and van Deurs, 2000). It is transported from early endosomes to the Golgi complex before undergoing COPI-independent retrograde transport to the endoplasmic reticulum (Mallard et al., 1998;Girod et al., 1999;Falguieres et al., 2001;Luna et al., 2002;Lauvrak et al., 2004;McKenzie et al., 2009). The A subunit exits the endoplasmic reticulum into the cytosol where it cleaves the rRNA (Obrig et al., 1985).Shiga toxin usurps several components of the constitutive trafficking machinery t...

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