Trends in Cell Biology
Cytokinesis seriesCytokinesis: welcome to the Rho zone
Introduction
Members of the Rho family of GTPases regulate a wide variety of biological processes. Many, although not all, of these processes involve regulation of the actin cytoskeleton and the contractility of myosin II. Cytokinesis – the generation of two separated daughter cells from a single progenitor – requires the spatial and temporal control of actin and myosin, and various members of the Rho GTPase family control this process in essentially all eukaryotic organisms. This review discusses the current literature that reveals the molecular mechanisms through which Rho GTPases orchestrate cytokinesis. We focus on recent insights into the mechanism of RhoA localization and activation as well as the function of RhoA effector proteins.
Rho family proteins are ∼20-kDa GTPases that simply consist of a characteristic GTPase domain followed by ∼30 amino acids, the very last four of which are subjected to a series of posttranslational modifications resulting in loss of the three terminal residues and prenylation of the C-terminal cysteine residue. This lipid group contributes to interactions with regulatory components and membrane compartments such as the plasma membrane, endosomes and Golgi. The Rho family is distinguished from other classes of small GTPases by a helix that is inserted between the fifth β-strand and the fourth α-helix [1]. Although members of the Rho family are clearly distinct from other members of the Ras-related GTPase superfamily, there is significant divergence within the subfamily (22 members in humans). Many members of the Rho family can be further subdivided on the basis of sequence into three major subsets, known as Rho, Rac and Cdc42 (Figure 1).
Rho family proteins bind to both GTP or GDP with high affinity and have a slow intrinsic GTPase activity (t1/2 ∼5–10 min [2]). The balance between the GTP- and GDP-bound states is regulated by specific guanine nucleotide dissociation inhibitors (GDIs), GDP–GTP exchange factors (GEFs) that promote GTP binding, and GTPase-activating proteins (GAPs) that promote GTP hydrolysis. GEF or GAP domains are commonly found in the eukaryotic genome (102 and 105 proteins in humans, respectively; smart.embl-heidelberg.de), and many proteins that contain GEF or GAP domains contain additional protein- or lipid-interaction domains that might enable them to respond to changes in cell physiology. GEFs and GAPs are not generally selective for a single GTPase in vitro, allowing for immense regulatory complexity. However, in vivo some processes, including cytokinesis, rely on a single GEF, indicating that there is not a lot of functional redundancy among the many proteins containing GEF domains. When Rho proteins bind to GTP, a conformational change occurs that increases their affinity for a specific set of proteins called effectors. Active Rho binding regulates the activity of the effectors and allows Rho proteins to regulate cellular physiology.
Multiple Rho GTPases are implicated in cytokinesis in various eukaryotes (Box 1), but, in animal cells, RhoA is the central player, and its role will be the focus of this review. Cells depleted of RhoA by RNAi, or cells injected with a Clostridial enzyme, C3 transferase, or C3-treated RhoA, show no cortical contractility or furrow formation in most animal cell types studied 3, 4, 5. Vertebrates have two additional proteins highly related to RhoA (RhoB and RhoC). All three proteins are subject to inactivation by the C3 enzyme. Deletion of the gene encoding RhoB (the most divergent of the three) is not lethal, but defects in vascular development occur, although mitotic defects were not reported [6]. Deletion of the gene encoding RhoC reveals that it too is not essential for cell proliferation – rather, RhoC regulates migration of specific cell types, in particular, tumor cells [7]. The possible role of redundancy among RhoA, B and C in development has not yet been addressed. However, the cytokinesis phenotypes induced by depletion of RhoA imply that it is essential for cytokinesis [8].
Section snippets
RhoA effectors
Activated RhoA orchestrates cytokinesis by binding and regulating specific protein effectors that profoundly affect the actomyosin contractile network (Figure 2). The key RhoA effectors involved in cytokinesis are members of the formin family, plus Rho-dependent kinase (ROCK, also known as ROK) and Citron kinase (Citron K). Briefly, formin promotes the polymerization of actin necessary for formation of the contractile ring. ROCK regulates myosin, which is a crucial component of the contractile
RhoA localization
Although there is compelling evidence that active RhoA is required for furrow formation, ingression and stabilization, this requirement does not necessarily imply that RhoA function is spatially restricted. However, this is an appealing possibility, and several independent means have been developed that allow activated RhoA localization during cytokinesis to be assessed 8, 41 (Box 2). These results indicate that, indeed, active RhoA concentrates at the site of furrow formation. Importantly,
Spatiotemporal regulation of RhoA
The ability to detect active RhoA during cytokinesis has allowed a preliminary dissection of the mechanism that generates the zone of active RhoA that drives furrow formation. The RhoGEF ECT2/Pebble is a crucial upstream regulator that is required for cytokinesis in all metazoans analyzed to date (see [46] for review), and, accordingly, it is required for the localization of active RhoA to the equatorial cortex.
The activity of ECT2 appears to be subject to both spatial and temporal regulation
Inactivation of RhoA
Like many other GTPases, RhoA is believed to act as a molecular switch, and the RhoGEF ECT2 switches RhoA on and allows it to engage its effectors and drive furrow formation. At late stages of division, cells become less contractile 55, 56 and the signal from probes that detect active RhoA is attenuated 8, 41, indicating that RhoA is inactivated. What are the mechanisms responsible for switching RhoA off? This question is not yet fully answered. Two mechanisms could cooperate to inactivate
Concluding remarks
There is a plethora of evidence that RhoA and its regulators and effectors play essential roles in cytokinesis in many different types of cells. However, there might be situations in which alternative pathways can substitute for the canonical pathway that is dependent on the actomyosin-based contractile ring. In Dictyostelium, for example, myosin is essential for division in non-adherent cells [64], but cells lacking myosin can divide if they are attached to a substrate [65]. Likewise, yeast
Acknowledgements
We thank of W. Bement (University of Wisconsin, USA) and G. Dassow (University of Washington, USA) for the Xenopus image in Figure 3.
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