The regulation of mDia1 by autoinhibition and its release by Rho*GTP.
ABSTRACT: Formins induce the nucleation and polymerisation of unbranched actin filaments via the formin-homology domains 1 and 2. Diaphanous-related formins (Drfs) are regulated by a RhoGTPase-binding domain situated in the amino-terminal (N-terminal) region and a carboxy-terminal Diaphanous-autoregulatory domain (DAD), whose interaction stabilises an autoinhibited inactive conformation. Binding of active Rho releases DAD and activates the catalytic activity of mDia. Here, we report on the interaction of DAD with the regulatory N-terminus of mDia1 (mDia(N)) and its release by Rho*GTP. We have defined the elements required for tight binding and solved the three-dimensional structure of a complex between an mDia(N) construct and DAD by X-ray crystallography. The core DAD region is an alpha-helical peptide, which binds in the most highly conserved region of mDia(N) using mainly hydrophobic interactions. The structure suggests a two-step mechanism for release of autoinhibition whereby Rho*GTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that Rho*GTP accelerates the dissociation of DAD from the mDia(N)*DAD complex.
Project description:Diaphanous-related formins are eukaryotic actin nucleation factors regulated by an autoinhibitory interaction between the N-terminal RhoGTPase-binding domain (mDiaN) and the C-terminal Diaphanous-autoregulatory domain (DAD). Although the activation of formins by Rho proteins is well characterized, its inactivation is only marginally understood. Recently, liprin-α3 was shown to interact with mDia1. Overexpression of liprin-α3 resulted in a reduction of the cellular actin filament content. The molecular mechanisms of how liprin-α3 exerts this effect and counteracts mDia1 activation by RhoA are unknown. Here, we functionally and structurally define a minimal liprin-α3 core region, sufficient to recapitulate the liprin-α3 determined mDia1-respective cellular functions. We show that liprin-α3 alters the interaction kinetics and thermodynamics of mDiaN with RhoA·GTP and DAD. RhoA displaces liprin-α3 allosterically, whereas DAD competes with liprin-α3 for a highly overlapping binding site on mDiaN. Liprin-α3 regulates actin polymerization by lowering the regulatory potency of RhoA and DAD on mDiaN. We present a model of a mechanistically unexplored and new aspect of mDiaN regulation by liprin-α3.
Project description:Formin proteins direct the nucleation and assembly of linear actin filaments in a variety of cellular processes using their conserved formin homology 2 (FH2) domain. Diaphanous-related formins (DRFs) are effectors of Rho-family GTPases, and in the absence of Rho activation they are maintained in an inactive state by intramolecular interactions between their regulatory N-terminal region and a C-terminal segment referred to as the DAD domain. Although structures are available for the isolated DAD segment in complex with the interacting region in the N-terminus, it remains unclear how this leads to inhibition of actin assembly by the FH2 domain. Here we describe the crystal structure of the N-terminal regulatory region of formin mDia1 in complex with a C-terminal fragment containing both the FH2 and DAD domains. In the crystal structure and in solution, these fragments form a tetrameric complex composed of two interlocking N+C dimers. Formation of the tetramer is likely a consequence of the particular N-terminal construct employed, as we show that a nearly full-length mDia1 protein is dimeric, as are other autoinhibited N+C complexes containing longer N-terminal fragments. The structure provides the first view of the intact C-terminus of a DRF, revealing the relationship of the DAD to the FH2 domain. Delineation of alternative dimeric N+C interactions within the tetramer provides two general models for autoinhibition in intact formins. In both models, engagement of the DAD by the N-terminus is incompatible with actin filament formation on the FH2, and in one model the actin binding surfaces of the FH2 domain are directly blocked by the N-terminus.
Project description:Formins are key regulators of actin nucleation and polymerization. They contain formin homology 1 (FH1) and 2 (FH2) domains as the catalytic machinery for the formation of linear actin cables. A subclass of formins constitutes the Diaphanous-related formins, members of which are regulated by the binding of a small GTP-binding protein of the Rho subfamily. Binding of these molecular switch proteins to the regulatory N-terminal mDia(N), including the GTPase-binding domain, leads to the release of auto-inhibition. From the three mDia isoforms, mDia1 is activated only by Rho (RhoA, -B, and -C), in contrast to mDia2 and -3, which is also activated by Rac and Cdc42. Little is known about the determinants of specificity. Here we report on the interactions of RhoA, Rac1, and Cdc42 with mDia1 and an mDia1 mutant (mDia(N)-Thr-Ser-His (TSH)), which based on structural information should mimic mDia2 and -3. Specificity is analyzed by biochemical studies and a structural analysis of a complex between Cdc42.Gpp(NH)p and mDia(N)-TSH. A triple NNN motif in mDia1 (amino acids 164-166), corresponding to the TSH motif in mDia2/3 (amino acids 183-185 and 190-192), and the epitope interacting with the Rho insert helix are essential for high affinity binding. The triple N motif of mDia1 allows tight interaction with Rho because of the presence of Phe-106, whereas the corresponding His-104 in Rac and Cdc42 forms a complementary interface with the TSH motif in mDia2/3. We also show that the F106H and H104F mutations drastically alter the affinities and thermodynamics of mDia interactions.
Project description:Formins assemble non-branched actin filaments and modulate microtubule dynamics during cell migration and cell division. At the end of mitosis formins contribute to the generation of actin filaments that form the contractile ring. Rho small GTP-binding proteins activate mammalian diaphanous-related (mDia) formins by directly binding and disrupting an intramolecular autoinhibitory mechanism. Although the Rho-regulated activation mechanism is well characterized, little is known about how formins are switched off. Here we reveal a novel mechanism of formin regulation during cytokinesis based on the following observations; 1) mDia2 is degraded at the end of mitosis, 2) mDia2 is targeted for disposal by post-translational ubiquitin modification, 3) forced expression of activated mDia2 yields binucleate cells due to failed cytokinesis, and 4) the cytokinesis block is dependent upon mDia2-mediated actin assembly as versions of mDia2 incapable of nucleating actin but that still stabilize microtubules have no effect on cytokinesis. We propose that the tight control of mDia2 expression and ubiquitin-mediated degradation is essential for the completion of cell division. Because of the many roles for formins in cell morphology, we discuss the relevance of mDia protein turnover in other processes where ubiquitin-mediated proteolysis is an essential component.
Project description:Inverted formin 2 (INF2) encodes a member of the diaphanous subfamily of formin proteins. Mutations in INF2 cause human kidney disease characterized by focal and segmental glomerulosclerosis. Disease-causing mutations occur only in the diaphanous inhibitory domain (DID), suggesting specific roles for this domain in the pathogenesis of disease. In a yeast two-hybrid screen, we identified the diaphanous autoregulatory domains (DADs) of the mammalian diaphanous-related formins (mDias) mDia1, mDia2, and mDia 3 as INF2_DID-interacting partners. The mDias are Rho family effectors that regulate actin dynamics. We confirmed in vitro INF2_DID/mDia_DAD binding by biochemical assays, confirmed the in vivo interaction of these protein domains by coimmunoprecipitation, and observed colocalization of INF2 and mDias in glomerular podocytes. We investigated the influence of this INF2_DID/mDia_DAD interaction on mDia mediated actin polymerization and on serum response factor (SRF) activation. We find that the interaction of INF2_DID with mDia_DAD inhibited mDia-mediated, Rho-activated actin polymerization, as well as SRF-responsive gene transcriptional changes. Similar assays using the disease-causing E184K and R218Q mutations in INF2_DID showed a decreased effect on SRF activation and gene transcription. The binding of INF2_DID to mDia_DAD may serve as a negative regulatory mechanism for mDias' function in actin-dependent cell processes. The effects of disease-causing INF2 mutations suggest an important role for this protein and its interaction with other formins in modulating glomerular podocyte phenotype and function.
Project description:Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.
Project description:Entamoeba histolytica requires a dynamic actin cytoskeleton for intestinal and systemic pathogenicity. Diaphanous-related formins represent an important family of actin regulators that are activated by Rho GTPases. The E. histolytica genome encodes a large family of Rho GTPases and three diaphanous-related formins, of which EhFormin1 is known to regulate mitosis and cytokinesis in trophozoites. We demonstrate that EhFormin1 modulates actin polymerization through its formin homology 2 domain. Despite a highly divergent diaphanous autoinhibitory domain, EhFormin1 is autoinhibited by an N- and C-terminal intramolecular interaction but activated upon binding of EhRho1 to the N-terminal domain tandem. A crystal structure of the EhRho1·GTP?S-EhFormin1 complex illustrates an EhFormin1 conformation that diverges from mammalian mDia1 and lacks a secondary interaction with a Rho insert helix. The structural model also highlights residues required for specific recognition of the EhRho1 GTPase and suggests that the molecular mechanisms of EhFormin1 autoinhibition and activation differ from those of mammalian homologues.
Project description:The mammalian Diaphanous-related (mDia) formins are cytoskeletal regulators that assemble and, in some cases, bundle filamentous actin (F-actin), as well as stabilize microtubules. The development of small molecule antagonists and agonists that interrogate mDia formin function has allowed us to investigate the roles of formins in disease states. A small molecule inhibitor of FH2 domain (SMIFH2) inhibits mDia-dependent actin dynamics and abrogates tumor cell migration and cell division in vitro and ex vivo tissue explants. mDia formin activation with small molecule intramimics IMM01/02 and mDia2-DAD peptides inhibited glioblastoma motility and invasion in vitro and ex vivo rat brain slices. However, SMIFH2, IMMs, and mDia2 DAD efficacy in vivo remains largely unexplored and potential toxicity across a range of developmental phenotypes has not been thoroughly characterized. In this study, we performed an in vivo screen of early life-stage toxicity in Danio rerio zebrafish embryos 2 days post-fertilization (dpf) in response to SMIFH2, IMM01/02, and mDia2 DAD. SMIFH2 at concentrations ?5-10 ?M induced significant defects in developing zebrafish, including shorter body lengths, tail curvature and defective tail cellularity, craniofacial malformations, pericardial edema, absent and/or compromised vasculature function and flow, depressed heart rates and increased mortality. Conversely, IMM and mDia2 DAD peptides were minimally toxic at concentrations up to 10-20 and 50 ?M, respectively. SMIFH2's therapeutic potential may therefore be limited by its substantial in vivo toxicity at functional concentrations. mDia formin agonism with IMMs and mDia2 DADs may therefore be a more effective and less toxic anti-invasive therapeutic approach.
Project description:Formin proteins utilize a conserved formin homology 2 (FH2) domain to nucleate new actin filaments. In mammalian diaphanous-related formins (DRFs) the FH2 domain is inhibited through an unknown mechanism by intramolecular binding of the diaphanous autoinhibitory domain (DAD) and the diaphanous inhibitory domain (DID).Here we report the crystal structure of a complex between DID and FH2-DAD fragments of the mammalian DRF, mDia1 (mammalian diaphanous 1 also called Drf1 or p140mDia). The structure shows a tetrameric configuration (4 FH2 + 4 DID) in which the actin-binding sites on the FH2 domain are sterically occluded. However biochemical data suggest the full-length mDia1 is a dimer in solution (2 FH2 + 2 DID). Based on the crystal structure, we have generated possible dimer models and found that architectures of all of these models are incompatible with binding to actin filament but not to actin monomer. Furthermore, we show that the minimal functional monomeric unit in the FH2 domain, termed the bridge element, can be inhibited by isolated monomeric DID. NMR data on the bridge-DID system revealed that at least one of the two actin-binding sites on the bridge element is accessible to actin monomer in the inhibited state.Our findings suggest that autoinhibition in the native DRF dimer involves steric hindrance with the actin filament. Although the structure of a full-length DRF would be required for clarification of the presented models, our work here provides the first structural insights into the mechanism of the DRF autoinhibition.
Project description:Mutations in inverted formin 2 INF2 are a common cause of familial FSGS. INF2 interacts with diaphanous-related formins (mDia) and antagonizes mDia-mediated actin polymerization in response to active Rho signaling, suggesting that dysregulation of these pathways may mediate the development of INF2-related FSGS. However, the precise mechanisms by which INF2 regulates actin-dependent podocyte behavior remain largely unknown. Here, we investigated the possible role of INF2 in both lamellipodia-associated actin dynamics and actin-dependent slit diaphragm (SD) protein trafficking by manipulating the expression of INF2 and the activity of Rho/mDia signaling in cultured podocytes. Activation of mDia in the absence of INF2 led to defective formation of lamellipodia and abnormal SD trafficking. Effects of mutations disrupting the INF2-mDia interaction suggested the specificity of the mDia-antagonizing effect of INF2 in maintaining the lamellipodium. Furthermore, we found that SD trafficking requires INF2 interaction with lipid raft components. In summary, INF2 regulates lamellipodial actin dynamics and the trafficking of slit diaphragm proteins by opposing Rho/mDia-mediated actin polymerization. Thus, in podocytes, INF2 appears to be an important modulator of actin-dependent behaviors that are under the control of Rho/mDia signaling.