Rho Family GTPase-activating Protein Research Articles

ARHGAP4 promotes leukemogenesis in acute myeloid leukemia by inhibiting DRAM1 signaling.

Rho GTPase-activating protein 4 (ARHGAP4) is an important Rho family GTPase-activating protein that is strongly associated with the onset and progression of some tumors. We found that ARHGAP4 mRNA and protein are overexpressed in human acute myeloid leukemia (AML) patients and are associated with a poor prognosis. ARHGAP4 knockdown significantly impairs viability and colony formation capacity and induces apoptosis in AML cells. Further results demonstrate that ARHGAP4 deletion impairs AML progression in vivo. Interestingly, DRAM1 signaling is significantly activated in AML cells with ARHGAP4 knockdown. Our results also indicated that ARHGAP4 might function in AML cells by binding with p53 to inhibit DRAM1. Moreover, knockdown of DRAM1 rescues the defects of ARHGAP4 in AML cells. This newly described role of the ARHGAP4/DRAM1 axis in regulating AML progression may have important therapeutic implications.

Expression Analyses of Rich2/Arhgap44, a Rho Family GTPase-Activating Protein, during Mouse Brain Development

Rho family small GTPases, such as Rho, Rac, and Cdc42, play essential roles during brain development, by regulating cellular signaling and actin cytoskeletal reorganization. Rich2/Arhgap44, a Rac- and Cdc42-specific GTPase-activating protein, has been reported to be a key regulator for dendritic spine morphology and synaptic function. Given the essential roles of Rac and Cdc42 in brain development, Rich2 is supposed to take part in brain development. However, not only the molecular mechanism involved but also the expression profile of Rich2 during neurodevelopment has not yet been elucidated. In this study, we carried out expression analyses of Rich2 by focusing on mouse brain development. In immunoblotting, Rich2 exhibited a tissue-dependent expression profile in the young adult mouse, and the expression was increased during brain development. In immunohistochemical analyses, Rich2 was observed in the cytoplasm of cortical neurons at postnatal day (P) 0 and then came to be enriched in the nucleus with moderate distribution in neuropils at P7. Later at P30, a complex immunostaining pattern of Rich2 was observed; Rich2 was distributed in the nucleus, cytoplasm, and neuropils in many cortical neurons, whereas other neurons frequently displayed little expression. In the hippocampus at P7, Rich2 was distributed mainly in the cytoplasm of excitatory neurons in the cornu ammonis regions, while it was moderately detected in the nucleus in the dentate granule cells. Notably, Rich2 was distributed in excitatory synapses of the cornu ammonis 1 region at P30. Biochemical fractionation analyses also detected Rich2 in the postsynaptic density. Taken together, Rich2 is found to be expressed in the central nervous system in a developmental stage-dependent manner and may be involved in synapse formation/maintenance in cortical neurons.

Antibodies Against Macrophage CD44 Protect Platelets from FcγR-Mediated Phagocytosis: A Potential Mechanism for CD44 Antibody Amelioration of Murine Immune Thrombocytopenia

CD44 is a transmembrane protein that is widely expressed on leukocytes and other cell types. CD44 is the major receptor for hyaluronan, and CD44-hyaluronan binding plays an important role in regulating immune cell migration, inflammation, and wound repair. Interestingly, targeting CD44 with monoclonal antibodies (anti-CD44) has demonstrated therapeutic efficacy in numerous murine autoimmune disease models including passive antibody-mediated immune thrombocytopenia (ITP), where it is effective at a three log-fold-lower dose compared to intravenous immunoglobulin (IVIg). However, the anti-inflammatory mechanisms behind anti-CD44 remain largely unsolved. In passive antibody-mediated ITP, anti-platelet antibody is administered to induce thrombocytopenia by FcγR-dependent and independent mechanisms, and macrophage depletion as well as FcγR blocking antibodies protect platelets from FcγR-dependent removal. The efficacy of anti-CD44 in this model suggest to us that anti-CD44 may be interfering with platelet removal by phagocytes. Here we show that anti-CD44 inhibits macrophage FcγR-mediated phagocytosis and protects platelets from macrophage uptake in vitro. Anti-CD44 inhibited RAW 264.7 macrophage phagocytosis of antibody-opsonized platelets in a dose-dependent manner, up to near complete (>90%) inhibition as compared to isotype controls. Multiple anti-CD44 clones tested were capable of inhibiting macrophage uptake of platelets opsonized with different anti-platelet antibodies. The binding of antibody-opsonized platelets to anti-CD44-treated macrophages was not blocked, suggesting that inhibition of phagocytosis occurs at a post-FcγR binding step. Because CD44 can regulate actin polymerization by recruiting the Rho-family GTPase-activating proteins Tiam1 and Vav2, we asked whether anti-CD44 may be interfering with actin polymerization required for platelet uptake. Live-cell microscopy of RAW 264.7 macrophages transfected with LifeAct (plasmid encoding a fluorescent non-cytotoxic actin binding protein) revealed that anti-CD44 did not affect actin dynamics and filopodia formation in the absence of antibody-opsonized platelets. However, anti-CD44-treated macrophages failed to initiate phagocytic cup formation upon antibody-opsonized-platelet binding, suggesting that inhibition is upstream of actin polymerization initiated by FcγRs. Finally, the ability of anti-CD44 to inhibit platelet uptake also showed complete concordance with a blocking antibody against FcγRIIb/III, implicating that anti-CD44 may be mediating inhibition of the FcγRIII pathway. In conclusion, our data suggests that antibodies against CD44 protect platelets from macrophage phagocytosis in vitro by inhibition of FcγRs, and that inhibition occurs at a step post-binding but before phagocytic cup formation. New therapies for ITP are needed to replace current donor-derived therapeutics and irreversible splenectomy. This research helps to shed light on how anti-CD44 functions to normalize platelet counts in murine passive ITP, and may pave the way for future novel therapeutics to replace IVIg in the treatment of ITP. DisclosuresLazarus:CSL pharmaceuticals: Research Funding; St.Michael's Hosp/The Canadian Blood Services: Other: Patents on monoclonal antibodies as IVIg and anti-D replacements; Rigel pharmaceuticals: Research Funding; Momenta pharmaceuticals: Honoraria.

The neglected terminators: Rho family GAPs in neutrophils.

GTPase-activating proteins (GAPs) accelerate the rate of hydrolysis of GTP bound to small GTPases, thereby limiting the prevalence and concentration of the active, GTP-bound form of these proteins. The large number of potential GAPs acting on members of the Rho family of small GTPases raises the question of specificity or redundancy. In this review, we summarize experimental data obtained on the role of Rho family GAPs in neutrophils, highlight cases where more than one GAP is involved in a physiological function and show examples that GAPs can be involved not only in termination but also in initiation of cellular processes. We demonstrate that the expression-level regulation of GAPs may also occur in short-living cells such as neutrophils. Finally, we provide insight into the existence and structure of molecular complexes in which Rho family GAPs are involved. GAPs play more complex and varied roles than being simple terminators of cellular processes.

CYK-4 functions independently of its centralspindlin partner ZEN-4 to cellularize oocytes in germline syncytia.

Throughout metazoans, germ cells undergo incomplete cytokinesis to form syncytia connected by intercellular bridges. Gamete formation ultimately requires bridge closure, yet how bridges are reactivated to close is not known. The most conserved bridge component is centralspindlin, a complex of the Rho family GTPase-activating protein (GAP) CYK-4/MgcRacGAP and the microtubule motor ZEN-4/kinesin-6. Here, we show that oocyte production by the syncytial Caenorhabditis elegans germline requires CYK-4 but not ZEN-4, which contrasts with cytokinesis, where both are essential. Longitudinal imaging after conditional inactivation revealed that CYK-4 activity is important for oocyte cellularization, but not for the cytokinesis-like events that generate syncytial compartments. CYK-4's lipid-binding C1 domain and the GTPase-binding interface of its GAP domain were both required to target CYK-4 to intercellular bridges and to cellularize oocytes. These results suggest that the conserved C1-GAP region of CYK-4 constitutes a targeting module required for closure of intercellular bridges in germline syncytia.

Sestd1 Encodes a Developmentally Dynamic Synapse Protein That Complexes With BCR Rac1-GAP to Regulate Forebrain Dendrite, Spine and Synapse Formation.

SEC14 and Spectrin domain-1 (Sestd1) is a synapse protein that exhibits a striking shift from the presynaptic to postsynaptic space as neurons mature postnatally in the mouse hippocampus. Hippocampal pyramidal neurons from mice with global genetic deletion of Sestd1 have reduced dendrite arbors, spines, and excitatory synapses. Electrophysiologically this correlates with cell-autonomous reductions in both AMPA- and NMDA-excitatory postsynaptic currents in individual hippocampal neurons from which Sestd1 has been deleted in vivo. These neurodevelopmental and functional deficits are associated with increased activation of the Rho family GTPases Rac1 and RhoA. Co-immunoprecipitation and mass spectrometry reveal that the Breakpoint Cluster Region protein, a Rho GTPase activating protein (GAP), forms complexes with Sestd1 in brain tissue. This complements earlier findings that Sestd1 can also partner with other Rho family GAPs and guanine nucleotide exchange factors. Our findings demonstrate that Sestd1 is a developmentally dynamic synaptic regulator of Rho GTPases that contributes to dendrite and excitatory synapse formation within differentiating pyramidal neurons of the forebrain.

A GAP that Divides.

Cytokinesis in metazoan cells is mediated by an actomyosin-based contractile ring that assembles in response to activation of the small GTPase RhoA. The guanine nucleotide exchange factor that activates RhoA during cytokinesis, ECT-2, is highly regulated. In most metazoan cells, with the notable exception of the early Caenorhabditis elegans embryo, RhoA activation and furrow ingression require the centralspindlin complex. This exception is due to the existence of a parallel pathway for RhoA activation in C. elegans. Centralspindlin contains CYK-4 which contains a predicted Rho family GTPase-activating protein (GAP) domain. The function of this domain has been the subject of considerable debate. Some publications suggest that the GAP domain promotes RhoA activation (for example, Zhang and Glotzer, 2015; Loria, Longhini and Glotzer, 2012), whereas others suggest that it functions to inactivate the GTPase Rac1 (for example, Zhuravlev et al., 2017). Here, we review the mechanisms underlying RhoA activation during cytokinesis, primarily focusing on data in C. elegans. We highlight the importance of considering the parallel pathway for RhoA activation and detailed analyses of cyk-4 mutant phenotypes when evaluating the role of the GAP domain of CYK-4.

Multiparametric Analysis of Cell Shape Demonstrates that β-PIX Directly Couples YAP Activation to Extracellular Matrix Adhesion

SummaryMechanical signals from the extracellular matrix (ECM) and cellular geometry regulate the nuclear translocation of transcriptional regulators such as Yes-associated protein (YAP). Elucidating how physical signals control the activity of mechanosensitive proteins poses a technical challenge, because perturbations that affect cell shape may also affect protein localization indirectly. Here, we present an approach that mitigates confounding effects of cell-shape changes, allowing us to identify direct regulators of YAP localization. This method uses single-cell image analysis and statistical models that exploit the naturally occurring heterogeneity of cellular populations. Through systematic depletion of all human kinases, Rho family GTPases, GEFs, and GTPase activating proteins (GAPs), together with targeted chemical perturbations, we found that β-PIX, a Rac1/Ccd42 GEF, and PAK2, a Rac1/Cdc42 effector, drive both YAP activation and cell-ECM adhesion turnover during cell spreading. Our observations suggest that coupling YAP to adhesion dynamics acts as a mechano-timer, allowing cells to rapidly tune gene expression in response to physical signals.

Preparation of centralspindlin as an active heterotetramer of kinesin and GTPase activating protein subunits for in vitro structural and functional assays.

Centralspindlin is a crucial regulator of animal cytokinesis, consisting of MKLP1 kinesin-6 and CYK4 Rho-family GTPase activating protein (RhoGAP). As a microtubule-bundling protein, it plays a crucial role in the formation of the central spindle. Through distinct accumulation to the antiparallel microtubule overlaps at the central spindle and the midbody, it recruits various downstream factors to the site of cell division as well as anchors the plasma membrane to maintain the narrow intercellular channels between the daughter cells until their final separation (abscission). A unique and functionally important feature of centralspindlin as a kinesin-containing protein complex is that the nonmotor component, CYK4, is not a passive cargo of the MKLP1 motor, but an integrated component of a microtubule-organizing machinery. Thus, for in vitro structural and functional assays, it is pivotal to prepare active stoichiometric complexes of the two components. Discussed here are two complimentary approaches, (1) reconstitution of the complex in bacterial extracts (in extract reconstitution) and (2) purification of a native complex from a mammalian cell line using a localization and affinity purification (LAP) tag.

The RhoGAP activity of CYK-4/MgcRacGAP functions non-canonically by promoting RhoA activation during cytokinesis.

Cytokinesis requires activation of the GTPase RhoA. ECT-2, the exchange factor responsible for RhoA activation, is regulated to ensure spatiotemporal control of contractile ring assembly. Centralspindlin, composed of the Rho family GTPase-activating protein (RhoGAP) MgcRacGAP/CYK-4 and the kinesin MKLP1/ZEN-4, is known to activate ECT-2, but the underlying mechanism is not understood. We report that ECT-2-mediated RhoA activation depends on the ability of CYK-4 to localize to the plasma membrane, bind RhoA, and promote GTP hydrolysis by RhoA. Defects resulting from loss of CYK-4 RhoGAP activity can be rescued by activating mutations in ECT-2 or depletion of RGA-3/4, which functions as a conventional RhoGAP for RhoA. Consistent with CYK-4 RhoGAP activity contributing to GEF activation, the catalytic domains of CYK-4 and ECT-2 directly interact. Thus, counterintuitively, CYK-4 RhoGAP activity promotes RhoA activation. We propose that the most active form of the cytokinetic RhoGEF involves complex formation between ECT-2, centralspindlin and RhoA.

Glucocorticoid receptor DNA binding factor 1 expression and osteosarcoma prognosis.

Glucocorticoid receptor DNA binding factor 1 (GRF-1) is an important Rho family GTPase-activating protein, and the dysregulation of GRF-1 expression maybe involved in tumor progression. However, the role of GRF-1 expression in the osteosarcoma prognosis has been well less elaborated. Here, we conducted a hospital-based case study, including 247 patients with pathologically confirmed osteosarcoma to evaluate the associations between GRF-1 expression and osteosarcoma prognosis. We found that high GRF-1 expression was correlated with poor outcome of osteosarcoma compared with low GRF-1 expression (the median recurrence-free survival times, 11months vs 56months; the median overall survival times, 18months vs 53months). Like tumor stage, the GRF-1 expression was an independent prognostic factor influencing the survival of osteosarcoma [hazard ratio values (95% confidence interval) were 5.39 (3.54-8.20) for recurrence-free survival (RFS) and 6.58 (4.44-9.74) for overall survival (OS), respectively]. Furthermore, the high expression of GRF-1 was significantly associated with larger tumor size, tumor dedifferentiation, and increasing metastasis risk. Functionally, the knockdown of GRF-1 expression inhibited tumor cells proliferation and induced cell apoptosis. These results indicate for the first time that GRF-1 expression may modify osteosarcoma prognosis and may be a potential tumor therapeutic target.

Comprehensive molecular characterization of gastric adenocarcinoma.

Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein–Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also known as PD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies.Supplementary informationThe online version of this article (doi:10.1038/nature13480) contains supplementary material, which is available to authorized users.

BDNF Promotes Axon Branching of Retinal Ganglion Cells via miRNA-132 and p250GAP

A crucial step in the development of the vertebrate visual system is the branching of retinal ganglion cell (RGC) axons within their target, the superior colliculus/tectum. A major player in this process is the neurotrophin brain-derived neurotrophic factor (BDNF). However, the molecular basis for the signaling pathways mediating BDNF action is less well understood. As BDNF exerts some of its functions by controlling the expression of microRNAs (miRNAs), we investigated whether miRNAs are also involved in BDNF-mediated retinal axon branching. Here, we demonstrate that the expression pattern of miRNA-132 in the retina is consistent with its involvement in this process, and that BDNF induces the upregulation of miRNA-132 in retinal cultures. Furthermore, in vitro gain-of-function and loss-of-function approaches in retinal cultures reveal that miRNA-132 mediates axon branching downstream of BDNF. A known target of miRNA-132 is the Rho family GTPase-activating protein, p250GAP. We find that p250GAP is expressed in RGC axons and mediates the effects of miRNA-132 in BDNF-induced branching. BDNF treatment or overexpression of miRNA-132 leads to a reduction in p250GAP protein levels in retinal cultures, whereas the overexpression of p250GAP abolishes BDNF-induced branching. Finally, we used a loss-of-function approach to show that miRNA-132 affects the maturation of RGC termination zones in the mouse superior colliculus in vivo, while their topographic targeting remains intact. Together, our data indicate that BDNF promotes RGC axon branching during retinocollicular/tectal map formation via upregulation of miRNA-132, which in turn downregulates p250GAP.

Polarity in Migrating Neurons Is Related to a Mechanism Analogous to Cytokinesis

Migrating neurons are bipolar, with a leading process and a trailing process [1]. The proximal region of the leading process displays a concentration of F-actin that contributes to the advance of the soma and the centrosome [2-7]. Here, we show that kinesin-6, a microtubule-based motor protein best known for its role in cytokinesis, also concentrates in this region. Depletion of kinesin-6 results in multipolar neurons that either are stationary or continuously change their direction of movement. In such neurons, F-actin no longer concentrates in a single process. During cytokinesis, kinesin-6 forms a complex with a Rho-family GTPase-activating protein called MgcRacGAP to signal to the actin cytoskeleton so that cortical movements are concentrated in the cleavage furrow [8-13]. During neuronal migration, MgcRacGap also concentrates in the proximal region of the leading process, and inhibition of its activity results in a phenotype similar to kinesin-6 depletion. We conclude that neuronal migration utilizes a cytoskeletal pathway analogous to cytokinesis, with kinesin-6 signaling through MgcRacGap to the actin cytoskeleton to constrain process number and restrict protrusive activity to a single leading process, thus resulting in a bipolar neuron able to move in a directed fashion.

Targeting of the RhoGEF Ect2 to the Equatorial Membrane Controls Cleavage Furrow Formation during Cytokinesis

In animal cells, formation of the cytokinetic furrow requires activation of the GTPase RhoA by the guanine nucleotide exchange factor Ect2. How Ect2, which is associated with the spindle midzone, controls RhoA activity at the equatorial cortex during anaphase is not understood. Here, we show that Ect2 concentrates at the equatorial membrane during cytokinesis in live cells. Ect2 membrane association requires a pleckstrin homology domain and a polybasic cluster that bind to phosphoinositide lipids. Both guanine nucleotide exchange function and membrane targeting of Ect2 are essential for RhoA activation and cleavage furrow formation in human cells. Membrane localization of Ect2 is spatially confined to the equator by centralspindlin, Ect2's spindle midzone anchor complex, and is temporally coordinated with chromosome segregation through the activation state of CDK1. We propose that targeting of Ect2 to the equatorial membrane represents a key step in the delivery of the cytokinetic signal to the cortex.

P70 S6 kinase in the control of actin cytoskeleton dynamics and directed migration of ovarian cancer cells

Ovarian cancer is highly metastatic with a poor prognosis. The serine/threonine kinase, p70 S6 kinase (p70(S6K)), which is a downstream effector of phosphatidylinositol 3-kinase/Akt pathway, is frequently activated in ovarian cancer. Here, we show that p70(S6K) is a critical regulator of the actin cytoskeleton in the acquisition of the metastatic phenotype. This regulation is through two important activities: p70(S6K) acts as an actin filament cross-linking protein and as a Rho family GTPase-activating protein. Ectopic expression of constitutively active p70(S6K) in ovarian cancer cells induced a marked reorganization of the actin cytoskeleton and promoted directional cell migration. Using cosedimentation and differential sedimentation assays, p70(S6K) was found to directly bind to and cross-link actin filaments. Immunofluorescence studies showed p70(S6K) colocalized with cytochalasin D-sensitive actin at the leading edge of motile cells. The p70(S6K) did not affect the kinetics of spontaneous actin polymerization, but could stabilize actin filaments by the inhibition of cofilin-induced actin depolymerization. In addition, we showed that p70(S6K) stimulated the rapid activation of both Rac1 and Cdc42, and their downstream effector p21-activated kinase (PAK1), but not RhoA. Depletion of p70(S6K) expression or inhibition of its activity resulted in significant inhibition of actin cytoskeleton reorganization and reduced migration, with a concomitant reduction in Rac1, Cdc42 and PAK1 activation, confirming that the effect was p70(S6K) specific. Similarly, the actin cytoskeleton reorganization/migratory phenotype could be reversed by expression of dominant negative Rac1 and Cdc42, or inhibition of PAK1. These results reveal a new direction for understanding the oncogenic roles of p70(S6K) in tumor progression.

Clustering of Centralspindlin Is Essential for Its Accumulation to the Central Spindle and the Midbody

Cytokinesis in animal cells requires the central spindle and midbody, which contain prominent microtubule bundles. Centralspindlin, a heterotetrameric complex consisting of kinesin-6 and RhoGAP (Rho-family GTPase-activating protein) subunits, is essential for the formation of these structures. Centralspindlin becomes precisely localized to the central spindle, where it promotes the equatorial recruitment of important cytokinetic regulators. These include ECT2, the activator of the small GTPase RhoA, which controls cleavage furrow formation and ingression. Centralspindlin's own RhoGAP domain also contributes to furrow ingression. Finally, centralspindlin facilitates recruitment of the chromosome passenger complex and factors that control abscission. Despite the importance of localized accumulation of centralspindlin, the mechanism by which this motor protein complex suddenly concentrates to the center of interpolar microtubule bundles during anaphase is unclear. Here, we show that centralspindlin travels along central spindle microtubules as higher-order clusters. Clustering of centralspindlin is critical for microtubule bundling and motility along microtubules in vitro and for midbody formation in vivo. These data support a positive feedback loop of centralspindlin clustering and microtubule organization that may underlie its distinctive localization during cytokinesis.

Cytokinesis: GAP Gap

The central spindle regulates cleavage furrow formation and cytokinesis and is composed of antiparallel microtubules bundled by microtubule-associated proteins and kinesin motors. One key protein in the central spindle is a Rho family GTPase-activating protein, CYK-4/MgcRacGAP, whose target is the subject of two new studies that arrive at divergent models.

The PAR-6 Polarity Protein Regulates Dendritic Spine Morphogenesis through p190 RhoGAP and the Rho GTPase

The majority of excitatory synaptic transmission in the brain occurs at dendritic spines, which are actin-rich protrusions on the dendrites. The asymmetric nature of these structures suggests that proteins regulating cell polarity might be involved in their formation. Indeed, the polarity protein PAR-3 is required for normal spine morphogenesis. However, this function is independent of association with atypical protein kinase C (aPKC) and PAR-6. Here we show that PAR-6 together with aPKC plays a distinct but essential role in spine morphogenesis. Knockdown of PAR-6 inhibits spine morphogenesis, whereas overexpression of PAR-6 increases spine density, and these effects are mediated by aPKC. Using a FRET biosensor, we further show that p190 RhoGAP and RhoA act downstream of the PAR-6/aPKC complex. These results define a role for PAR-6 and aPKC in dendritic spine biogenesis and maintenance, and reveal an unexpected link between the PAR-6/aPKC complex and RhoA activity.

Cooperative Assembly of CYK-4/MgcRacGAP and ZEN-4/MKLP1 to Form the Centralspindlin Complex

Cytokinesis in metazoan cells requires a set of antiparallel microtubules that become bundled upon anaphase onset to form a structure known as the central spindle. Bundling of these microtubules requires a protein complex, centralspindlin, that consists of the CYK-4/MgcRacGAP Rho-family GTPase-activating protein and the ZEN-4/MKLP1 kinesin-6 motor protein. Centralspindlin, but not its individual subunits, is sufficient to bundle microtubules in vitro. Here, we present a biochemical and genetic dissection of centralspindlin. We show that each of the two subunits of centralspindlin dimerize via a parallel coiled coil. The two homodimers assemble into a high-affinity heterotetrameric complex by virtue of two low-affinity interactions. Conditional mutations in the regions that mediate complex assembly can be readily suppressed by numerous second site mutations in the interacting regions. This unexpected plasticity explains the lack of primary sequence conservation of the regions critical for this essential protein-protein interaction.