Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2

Mammalian erythroid cells undergo enucleation, an asymmetric cell division involving extrusion of a pycnotic nucleus enveloped by the plasma membrane. The mechanisms that power and regulate the enucleation process have remained obscure. Here, we show that deregulation of Rac GTPase during a late stage of erythropoiesis completely blocks enucleation of cultured mouse fetal erythroblasts without affecting their proliferation or differentiation. Formation of the contractile actin ring (CAR) on the plasma membrane of enucleating erythroblasts was disrupted by inhibition of Rac GTPases. Furthermore, we demonstrate that mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments, is also required for enucleation of mouse fetal erythroblasts. We show that Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner and that downregulation of mDia2, but not mDia1, by small interfering RNA (siRNA) during the late stages of erythropoiesis blocked both CAR formation and erythroblast enucleation. Additionally, overexpression of a constitutively active mutant of mDia2 rescued the enucleation defects induced by the inhibition of Rac GTPases. These results reveal important roles for Rac GTPases and their effector mDia2 in enucleation of mammalian erythroblasts.     

MAP1A light chain-2 interacts with GTP-RhoB to control epidermal growth factor (EGF)-dependent EGF receptor signaling

Rho GTPases have been implicated in the control of several cellular functions, including regulation of the actin cytoskeleton, cell proliferation, and oncogenesis. Unlike RhoA and RhoC, RhoB localizes in part to endosomes and controls endocytic trafficking. Using a yeast two-hybrid screen and a glutathione S-transferase pulldown assay, we identified LC2, the light chain of the microtubule-associated protein MAP1A, as a novel binding partner for RhoB. GTP binding and the 18-amino acid C-terminal hypervariable domain of RhoB are critical for its binding to MAP1A/LC2. Coimmunoprecipitation and immunofluorescence experiments showed that this interaction occurs in U87 cells. Down-regulation of MAP1A/LC2 expression decreased epidermal growth factor (EGF) receptor expression and modified the signaling response to EGF treatment. We concluded that MAP1A/LC2 is critical for RhoB function in EGF-induced EGF receptor regulation. Because MAP1A/LC2 is thought to function as an adaptor between microtubules and other molecules, we postulate that the RhoB and MAP1A/LC2 interactions facilitate endocytic vesicle trafficking and regulate the trafficking of signaling molecules.     

The human formin FHOD1 contains a bipartite structure of FH3 and GTPase-binding domains required for activation

Formins induce the nucleation and polymerization of unbranched actin filaments. They share three homology domains required for profilin binding, actin polymerization, and regulation. Diaphanous-related formins (DRFs) are activated by GTPases of the Rho/Rac family, whose interaction with the N-terminal formin domain is thought to displace a C-terminal Diaphanous-autoregulatory domain (DAD). We have determined the structure of the N-terminal domains of FHOD1 consisting of a GTPase-binding domain (GBD) and the DAD-recognition domain FH3. In contrast to the formin mDia1, the FHOD1-GBD reveals a ubiquitin superfold as found similarly in c-Raf1 or PI3 kinase. This GBD is recruited by Rac and Ras GTPases in cells and plays an essential role for FHOD1-mediated actin remodeling. The FHOD1-FH3 domain is composed of five armadillo repeats, similarly to other formins. Mutation of one residue in the predicted DAD-interaction surface efficiently activates FHOD1 in cells. These results demonstrate that DRFs have evolved different molecular solutions to govern their autoregulation and GTPase specificity.     

Rho GTPases in animal cell mitosis

The Rho GTPases have been thought to influence cell morphogenesis through remodeling of the actin cytoskeleton. Consistently, downstream targets such as the mDia family of formins and the WASP family proteins induce actin nucleation and polymerization, and another set of downstream effectors, the ROCK family protein kinases, are involved in regulation of actomyosin contractility. However, evidence has now accumulated that Rho GTPases also regulate local dynamics of microtubules. The mDia family proteins, for example, function downstream of Rho to stabilize and align microtubules in interphase cells. Concomitantly, the role of Rho GTPases in animal cell division, once thought to be limited to cytokinesis, has now been shown to extend to mitosis. Recent work indicates that they may function during both spindle orientation and chromosome congression. However, their involvement is cell-type-specific, raising arguments for and against a mitotic role for Rho GTPases.     

A Highlights from MBoC Selection: Rho and Anillin-dependent Control of mDia2 Localization and Function in Cytokinesis

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.     

The formins: active scaffolds that remodel the cytoskeleton

Evolutionarily conserved in eukaryotes, formin homology (FH) proteins, or formins, exert their effects on the actin and microtubule (MT) networks during meiosis, mitosis, the maintenance of cell polarity, vesicular trafficking, signaling to the nucleus and embryonic development. Once thought to be only molecular scaffolds that indirectly affected cellular functions through the binding of other proteins, recent in vitro studies have illustrated that they can function as actin nucleators in the formation of new filaments. The connection between formins and MTs is less well understood. In yeast, the MT effects appear to be dependent on the ability of formins to generate polarized actin cables whereas, in mammalian cells, formin signals that cause MT stabilization and polarization might be more direct. A subclass of formins, the Diaphanous-related formins (Drfs), can act as effectors for Rho small GTPases, yet it is not clear what GTPase binding contributes to formin function.     

The Rho family GTPase Rif induces filopodia through mDia2

Eukaryotic cells produce a variety of specialized actin-rich surface protrusions. These include filopodia-thin, highly dynamic projections that help cells to sense their external environment. Filopodia consist of parallel filaments of actin, bundled by actin crosslinking proteins. The filaments are oriented with their rapidly growing "barbed" ends at the protruding tip and their slowly growing "pointed" ends at the base. Extension occurs by polymerization at the tip and is controlled by regulation of filament capping. The Rho GTPase Cdc42 is a key mediator of filopodia formation, which it regulates through binding CRIB domain-containing effectors. Cdc42 binds and activates the WASP proteins, which in turn activate the actin-nucleating complex Arp2/3. It also binds and activates IRSp53, which recruits the Ena/WASP family protein Mena to the filopodial tip and protects elongating actin filaments from capping. Previously, we identified another Rho family GTPase, Rif, as a potent stimulator of filopodial protrusion through a mechanism that does not require Cdc42. Here we characterize the differences between filopodia induced by these two small GTPases and show that the Rif effector in this pathway is the Diaphanous-related formin mDia2. Thus, Rif and Cdc42 represent two distinct routes to the induction of filopodia-producing structures with both shared and unique properties.     

Differential interactions of the formins INF2, mDia1, and mDia2 with microtubules

A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (K(d) < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements.     

The basic region of the diaphanous-autoregulatory domain (DAD) is required for autoregulatory interactions with the diaphanous-related formin inhibitory domain

Mammalian diaphanous-related (mDia) formins act as Rho GTPase effectors during cytoskeletal remodeling. Rho binding to mDia amino-terminal GTPase-binding domains (GBDs) causes the adjacent Dia-inhibitory domain (DID) to release the carboxyl-terminal Dia-autoregulatory (DAD) domain that flanks the formin homology-2 (FH2) domain. The release of DAD allows the FH2 domain to then nucleate and elongate nonbranched actin filaments. DAD, initially discovered as a region of homology shared between a phylogenetically divergent set of formin proteins, is comprised of a core motif, MDXLLXL, and an adjacent region is comprised of numerous basic residues, typically RRKR in the mDia family. Here, we show that these specific amino acids within the basic region of DAD contribute to the binding of DID and therefore the maintenance of the mDia autoregulatory mechanism. In addition, expression of full-length versions of mDia2 containing amino acid substitutions in either the DAD core or basic regions causes profound changes in the F-actin architecture, including the formation of filopodia-like structures that rapidly elongate from the cell edge. These studies further refine our understanding of the molecular contribution of DAD to mDia control and the role of mDia2 in the assembly of membrane protrusions.     

A novel strategy for specifically down-regulating individual Rho GTPase activity in tumor cells

The Rho family GTPases RhoA, RhoB, and RhoC regulate the actin cytoskeleton, cell movement, and cell growth. Unlike Ras, up-regulation or overexpression of these GDP/GTP binding molecular switches, but not activating point mutations, has been associated with human cancer. Although they share over 85% sequence identity, RhoA, RhoB, and RhoC appear to play distinct roles in cell transformation and metastasis. In NIH 3T3 cells, RhoA or RhoB overexpression causes transformation whereas RhoC increases the cell migration rate. To specifically target RhoA, RhoB, or RhoC function, we have generated a set of chimeric molecules by fusing the RhoGAP domain of p190, a GTPase-activating protein that accelerates the intrinsic GTPase activity of all three Rho GTPases, with the C-terminal hypervariable sequences of RhoA, RhoB, or RhoC. The p190-Rho chimeras were active as GTPase-activating proteins toward RhoA in vitro, co-localized with the respective active Rho proteins, and specifically down-regulated Rho protein activities in cells depending on which Rho GTPase sequences were included in the chimeras. In particular, the p190-RhoA-C chimera specifically inhibited RhoA-induced transformation whereas p190-RhoC-C specifically reversed the migration phenotype induced by the active RhoC. In human mammary epithelial-RhoC breast cancer cells, p190-RhoC-C, but not p190-RhoA-C or p190-RhoB-C, reversed the anchorage-independent growth and invasion phenotypes caused by RhoC overexpression. In the highly metastatic A375-M human melanoma cells, p190-RhoC-C specifically reversed migration, and invasion phenotypes attributed to RhoC up-regulation. Thus, we have developed a novel strategy utilizing RhoGAP-Rho chimeras to specifically down-regulate individual Rho activity and demonstrate that this approach may be applied to multiple human tumor cells to reverse the growth and/or invasion phenotypes associated with disregulation of a distinct subtype of Rho GTPase.     

Unique structural and nucleotide exchange features of the Rho1 GTPase of Entamoeba histolytica

The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.     

Functional characterization of Rho GTPases in Aspergillus niger uncovers conserved and diverged roles of Rho proteins within filamentous fungi

Rho GTPases are signalling molecules regulating morphology and multiple cellular functions including metabolism and vesicular trafficking. To understand the connection between polarized growth and secretion in the industrial model organism Aspergillus niger, we investigated the function of all Rho family members in this organism. We identified six Rho GTPases in its genome and used loss-of-function studies to dissect their functions. While RhoA is crucial for polarity establishment and viability, RhoB and RhoD ensure cell wall integrity and septum formation respectively. RhoC seems to be dispensable for A. niger. RacA governs polarity maintenance via controlling actin but not microtubule dynamics, which is consistent with its localization at the hyphal apex. Both deletion and dominant activation of RacA (Rac(G18V)) provoke an actin localization defect and thereby loss of polarized tip extension. Simultaneous deletion of RacA and CftA (Cdc42) is lethal; however, conditional overexpression of RacA in this strain can substitute for CftA, indicating that both proteins concertedly control actin dynamics. We finally identified NoxR as a RacA-specific effector, which however, is not important for apical dominance as reported for A. nidulans but for asexual development. Overall, the data show that individual Rho GTPases contribute differently to growth and morphogenesis within filamentous fungi.     

The mouse Formin mDia1 is a potent actin nucleation factor regulated by autoinhibition

Formin proteins are widely expressed in eukaryotes and play essential roles in assembling specific cellular actin-based structures. Formins are defined by a Formin Homology 2 (FH2) domain, as well as a proline-rich FH1 domain that binds the actin monomer binding protein, profilin, and other ligands. Constructs including FH2 of budding yeast Bni1 or fission yeast Cdc12 formins nucleate actin filaments in vitro. In this study, we demonstrate that FH2-containing constructs of murine mDia1 (also called p140 mDia or Drf1) are much more potent actin nucleators than the yeast formins. FH1 is necessary for nucleation when actin monomers are profilin bound. mDia1 is a member of the Diaphanous formin subfamily (Dia), whose members contain an N-terminal Rho GTPase binding domain (GBD) and a C-terminal Diaphanous autoinhibitory domain (DAD, ). Based on cellular and in vitro binding studies, an autoinhibitory model for Dia formin regulation proposes that GBD binding to DAD inhibits Dia-induced actin remodeling, whereas Rho binding activates by releasing GBD from DAD. Supporting this model, our results show that an N-terminal mDia1 construct strongly inhibits actin nucleation by the C terminus. RhoA partially relieves inhibition but does so when bound to either GDP or GTP analogs. Both N- and C-terminal mDia1 constructs appear to be multimeric.     

RhoD localization and function is dependent on its GTP/GDP-bound state and unique N-terminal motif

The atypical Rho GTPase RhoD has previously been shown to have a major impact on the organization and function of the actin filament system. However, when first discovered, RhoD was found to regulate endosome trafficking and dynamics and we therefore sought to investigate this regulation in more detail. We found that exogenously expressed RhoD in human fibroblasts localized to vesicles and the plasma membrane and that the active GTP-bound conformation was required for the plasma membrane localization but not for vesicle localization. In contrast to the GTPase deficient atypical Rho GTPases, which have a stalled GTPase activity, RhoD has an elevated intrinsic GDP/GTP exchange activity, rendering the protein constitutively active. Importantly, RhoD can still hydrolyze GTP and we found that an intact GTPase activity was required for efficient fusion of RhoD-positive vesicles. RhoD has a unique N-terminal extension of 14 amino acid residues, which is not present in the classical Rho GTPases RhoA, Cdc42 and Rac1. Deletion of this N-terminal motif often lead to clustering of RhoD positive vesicles, which were found accumulated at the peripheral membrane border. In addition, the number of vesicles per cell was increased manifold, suggesting that the N-terminal motif has an important regulatory role in vesicle dynamics.     

Biochemical characterization of the Rho GTPase-regulated actin assembly by diaphanous-related formins, mDia1 and Daam1, in platelets

The diaphanous-related formins are actin nucleating and elongating factors. They are kept in an inactive state by an intramolecular interaction between the diaphanous inhibitory domain (DID) and the diaphanous-autoregulatory domain (DAD). It is considered that the dissociation of this autoinhibitory interaction upon binding of GTP-bound Rho to the GTPase binding domain next to DID induces exposure of the FH1-FH2 domains, which assemble actin filaments. Here, we isolated two diaphanous-related formins, mDia1 and Daam1, in platelet extracts by GTP-RhoA affinity column chromatography. We characterized them by a novel assay, where beads coated with the FH1-FH2-DAD domains of either mDia1 or Daam1 were incubated with platelet cytosol, and the assembled actin filaments were observed after staining with rhodamine-phalloidin. Both formins generated fluorescent filamentous structures on the beads. Quantification of the fluorescence intensity of the beads revealed that the initial velocity in the presence of mDia1 was more than 10 times faster than in the presence of Daam1. The actin assembly activities of both FH1-FH2-DADs were inhibited by adding cognate DID domains. GTP-RhoA, -RhoB, and -RhoC, but not GTP-Rac1 or -Cdc42, bound to both mDia1 and Daam1 and efficiently neutralized the inhibition by the DID domains. The association between RhoA and Daam1 was induced by thrombin stimulation in platelets, and RhoA-bound endogenous formins induced actin assembly, which was inhibited by the DID domains of Daam1 and mDia1. Thus, mDia1 and Daam1 are platelet actin assembly factors having distinct efficiencies, and they are directly regulated by Rho GTPases.     

A New Look at Rho GTPases in the Cell Cycle: Their Role in Kinetochore-Microtubule Attachment

Rho GTPases including Rho, Rac and Cdc42 are involved in cell morphogenesis by inducing specific types of actin cytoskeleton and alignment and stabilization of microtubules. Previous studies suggest that they also regulate cell cycle progression; Rho, Rac and Cdc42 regulate the G1-S progression and Rho controls cytokinesis. However, a role of Rho GTPases in nuclear division has not been definitely shown. We have recently found that Cdc42 and its downstream effector mDia3 are involved in bi-orientation and stabilization of spindle microtubules attachment to kinetochores and regulate chromosome alignment and segregation. Here, we discuss how this is coordinated with other events in mitosis, particularly, with the action of Rho in cytokinesis and how attachment of microtubules to kinetochores is achieved and stabilized. We also discuss redundancy of Cdc42 and Cdc42-related GTPase(s) and potential mechanisms of chromosome instability in cancer     

LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics

The small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. We show that in PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho kinase (ROCK)-LIM kinase (LIMK)-cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways.