Rho Subfamily Of GTPases Research Articles

Functional integrity of the contractile actin cortex is safeguarded by multiple Diaphanous-related formins

The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA - /E -/H - and racE - mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.

Mouse Macrophages Completely Lacking Rho Subfamily GTPases (RhoA, RhoB, and RhoC) Have Severe Lamellipodial Retraction Defects, but Robust Chemotactic Navigation and Altered Motility

RhoA is thought to be essential for coordination of the membrane protrusions and retractions required for immune cell motility and directed migration. Whether the subfamily of Rho (Ras homolog) GTPases (RhoA, RhoB, and RhoC) is actually required for the directed migration of primary cells is difficult to predict. Macrophages isolated from myeloid-restricted RhoA/RhoB (conditional) double knock-out (dKO) mice did not express RhoC and were essentially "pan-Rho"-deficient. Using real-time chemotaxis assays, we found that retraction of the trailing edge was dissociated from the advance of the cell body in dKO cells, which developed extremely elongated tails. Surprisingly, velocity (of the cell body) was increased, whereas chemotactic efficiency was preserved, when compared with WT macrophages. Randomly migrating RhoA/RhoB dKO macrophages exhibited multiple small protrusions and developed large "branches" due to impaired lamellipodial retraction. A mouse model of peritonitis indicated that monocyte/macrophage recruitment was, surprisingly, more rapid in RhoA/RhoB dKO mice than in WT mice. In comparison with dKO cells, the phenotypes of single RhoA- or RhoB-deficient macrophages were mild due to mutual compensation. Furthermore, genetic deletion of RhoB partially reversed the motility defect of macrophages lacking the RhoGAP (Rho GTPase-activating protein) myosin IXb (Myo9b). In conclusion, the Rho subfamily is not required for "front end" functions (motility and chemotaxis), although both RhoA and RhoB are involved in pulling up the "back end" and resorbing lamellipodial membrane protrusions. Macrophages lacking Rho proteins migrate faster in vitro, which, in the case of the peritoneum, translates to more rapid in vivo monocyte/macrophage recruitment.

S-23 : Innate immune sensing of bacteria and bacterial virulence by the cytosolic inflammasome complex

Inflammasomes are large cytoplasmic complexes that mediate caspase-1 activation, cytokine maturation and macrophage inflammatory death. We demonstrate that the NAIP family of NOD-like proteins (NLRs) are cytosolic receptors for various bacterial products and activate caspase-1 through another NLR protein NLRC4. In mice, NAIP5 directly recognizes flagellin while NAIP1 and NAIP2 are receptors for the needle and rod subunit of bacterial toxin-injecting type III secretion system (T3SS), respectively. Human NAIP (hNAIP) is the ortholog of mouse NAIP1 and also recognizes the T3SS needle protein derived from a wide range of bacterial pathogens including Salmonella typhimurium, enterohaemorrhagic Escherichia coli, Shigella flexneri and Burkholderia spp. Ligation of the NAIPs by the corresponding ligands promotes their physical association with NLRC4, resulting in caspase-1 activation and anti-bacteria defense. We further discover that Pyrin, another candidate pattern recognition receptor encoded by the familial Mediterranean fever disease gene MEFV, forms an inflammasome complex in response to bacteria or bacterial toxin-induced inactivating modifications of host Rho proteins. These include glucosylation by Clostridium difficile cytotoxin TcdB, adenylylation by FIC-domain bacterial effectors, ADP-ribosylation by Clostridium botulinum C3 toxin as well as deamidation by Burkholderia cenocepacia, which all occur in the switch I region in Rho-subfamily GTPases. Loss of the Pyrin inflammasome causes elevated intra-macrophage growth of B. cenocepacia and diminished lung inflammation in mice. The two inflammasome pathways not only play an important role in restricting bacterial infections but also serve as a mechanism for distinguishing pathogenic bacteria from non-pathogenic ones such as commensals.

Abstract 2640: HEF1 is an important regulator of mesenchymal and amoeboid invasion in breast cancer.

Abstract In previous studies, the focal adhesion-associated scaffolding protein HEF1, also known as NEDD9, has been implicated as a possible dynamic switch between the mesenchymal and amoeboid migration methods of melanoma cancer cells. Management of this switch provides a possible mechanism of control that could potentially open up new strategies for treatments of metastatic cancers. We have sought to investigate whether HEF1’s involvement in cell migration could also be applied to breast cancer. To monitor the switch between the two methods of migration, we decided to monitor activation of the Rac1 and RhoA proteins of the Rho subfamily of GTPases, which are renowned for their roles in actin cytoskeletal dynamics and have been linked to cancer cell invasion. High levels of active Rac1 result in an elongated cell morphology accompanied by mesenchymal cell movement, whereas high active RhoA levels result in a rounded cell morphology accompanied by amoeboid cell movement. Using a series of GST-tag pulldown experiments, we pulled and quantified amounts of active Rac1 and RhoA from an MDA-MB-231LN human breast cancer cell line grown in both 2D and 3D conditions under a HEF1 shRNA knockdown. Our HEF1-reduced cell line shows a decrease in active Rac1 and an increase in active RhoA compared to a control cell line with normal HEF1 levels. With this data, we aim to further substantiate HEF1 as a relevant key player in the balance between mesenchymal and amoeboid dominance in cancer cells. Citation Format: Brandon Jones, Elena Pugacheva. HEF1 is an important regulator of mesenchymal and amoeboid invasion in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2640. doi:10.1158/1538-7445.AM2013-2640

Rho proteins are negative regulators of TLR2, TLR3, and TLR4 signaling in astrocytes

The family of Toll-like receptors (TLRs) expressed by innate immune cells recognizes a spectrum of microbial components as well as molecules released from injured tissues. TLR ligation activates intracellular signaling cascades that culminate in the up-regulation of proinflammatory genes. We have recently demonstrated that the up-regulation of inflammatory cytokines mediated by TLR4 in astrocytes is negatively controlled by the monomeric GTPases of Rho subfamily. The present study was undertaken to examine further the involvement of Rho proteins in the inflammatory response of astrocytes elicited by the ligation of three TLRs that use divergent signaling pathways. Astrocyte cultures established from newborn rat brains were challenged with ligands of TLR2, TLR3, and TLR4. The expression of genes encoding interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNFalpha), interferon-beta (IFNbeta), and inducible nitric oxide synthase (NOS2) was up-regulated 24 hr after the challenge as determined by real-time RT-PCR. Pretreatment of the cells with toxin B, which specifically inactivates Rho proteins, enhanced the up-regulation of gene expression. The extent of this enhancement was both receptor and gene dependent. The enhancing effect of Rho protein inactivation was also evident at the protein level of IL-6 and NOS2 as revealed by ELISA and immunoblot analyses, respectively. These results suggest that Rho proteins control TLR-mediated up-regulation of inflammatory genes in astrocytes by interfering with multiple events along the signaling pathways.

Transcriptomal profiling of the cellular transformation induced by Rho subfamily GTPases

We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.

Mechanism of Redox-mediated Guanine Nucleotide Exchange on Redox-active Rho GTPases

Rho GTPases regulate multiple cellular processes including actin cytoskeletal rearrangements, transcriptional regulation, and oxidant production. The studies described herein demonstrate that small molecule redox agents, in addition to protein regulatory factors, can regulate the activity of redox-active Rho GTPases. A novel (GXXXXGK(S/T)C) motif, conserved in a number of Rho GTPases, appears critical for redox-mediated guanine nucleotide dissociation in vitro. A detailed molecular mechanism for redox regulation of GXXXXGK(S/T)C motif-containing Rho GTPases is proposed.

Biochemical Clustering of Monomeric GTPases of the Ras Superfamily

To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence. Here we developed a novel analytical platform allowing a systematic comparison of protein families based on their biochemical properties. This approach was validated on the Rho subfamily of GTPases. We used two high throughput methods, referred to as AlphaScreen and FlashPlate, to measure nucleotide binding capacity, exchange, and hydrolysis activities of small monomeric GTPases. These two technologies have the characteristics to be very sensitive and to allow homogenous and high throughput assays. To analyze and integrate the data obtained, we developed an algorithm that allows the classification of GTPases according to their enzymatic activities. Integration and hierarchical clustering of these results revealed unexpected features of the small Rho GTPases when compared with primary sequence-based trees. Hence we propose a novel phylobiochemical classification of the Ras superfamily of GTPases.

Separate Signals in Blood Cell Development

Guanosine triphosphatases (GTPases) of the Ras superfamily regulate broad cellular functions, including activity of the cytoskeleton, apoptosis, gene transcription, and intracellular trafficking. The Rho subfamily GTPases, Rac-1 and Rac-2, have been implicated in several of these activities within hematopoietic stem cell and leukocytes. Using conditional deletion of the Rac-1 and Rac-2 genes, Gu et al. determined that although both proteins are required to regulate actin assembly, Rac-1 specifically controls stem cell proliferation with Rac-2 governing cellular migration and production of the superoxide burst in neutrophils. In B cells, Walmsley et al. observed vital cooperation between the two GTPases, with deletion of both alleles resulting in a block in B cell development. This finding corresponded with a signaling defect and failure to express the receptor for the Baff protein, a critical regulator of B cell function and development. Y. Gu, M.-D. Filippi, J. A. Cancelas, J. E. Siefring, E. P. Williams, A. C. Jasti, C. E. Harris, A. W. Lee, R. Prabhakar, S. J. Atkinson, D. J. Kwiatkowski, D. A. Williams, Hematopoietic cell regulation by Rac1 and Rac2 guanosine triphosphatases. Science 302 , 445-449 (2003). [Abstract] [Full Text] M. J. Walmsley, S. K. T. Ooi, L. F. Reynolds, S. Harless Smith, S. Ruf, A. Mathiot, L. Vanes, D. A. Williams, M. P. Cancro, V. L. J. Tybulewicz, Critical roles for Rac1 and Rac2 GTPases in B cell development and signaling. Science 302 , 459-462 (2003). [Abstract] [Full Text]

Phospholipases stimulate secretion in RBL mast cells.

Roles for glycerophospholipids in exocytosis have been proposed, but remain controversial. Phospholipases are stimulated following the activation of the high-affinity receptor for immunoglobulin E (IgE) in mast cells. To study the biochemical sequelae that lead to degranulation, broken cell systems were employed. We demonstrate that the addition of three distinct types of exogenous phospholipases (i.e., bcPLC, scPLD, and tfPLA(2)), all of which hydrolyze phosphatidylcholine (PC), trigger degranulation in permeabilized RBL-2H3 cells, a mucosal mast cell line. Production of bioactive lipids by these phospholipases promotes release of granule contents through the plasma membrane and acts downstream of PKC, PIP(2), and Rho subfamily GTPases in regulated secretion. These exogenous phospholipase-induced degranulation pathways circumvent specific factors activated following stimulation of the IgE receptor as well as in ATP- and GTP-dependent intracellular pathways. Taken together, these results suggest that regulated secretion may be achieved in vitro in the absence of cytosolic factors via phospholipase activation and that products of PC hydrolysis can promote exocytosis in mast cells.

Multiple roles of the Dcdc42 GTPase during wing development in Drosophila melanogaster.

The Rho sub-family of GTPases, comprising Rho, Rac and Cdc42. regulates many biological processes, including morphogenesis, cell polarity, migration, the cell cycle and gene expression. It is important to develop genetic approaches to allow the dissection, in vivo, of the mechanisms of GTPase regulation and signal transmission, and their biological consequences. In this regard, wing development in Drosophila melanogaster is an excellent model system. To investigate the functions of the Drosophila Cdc42 GTPase (Dcdc42), we generated phenotypes during wing development, by expression of the dominant-negative N17 and L89 mutants of Dcdc42. We have identified roles for Dcdc42 in wing growth, and in cell fate choice during the development of the wing veins and the peripheral nervous system. Reduction of Dcdc42 signalling following over-expression of Dcdc42N17 resulted in a broader but more diffuse domain characterised by wing-margin sensory bristles. This was correlated with a broadened stripe of wingless expression along the dorsal-ventral boundary of third-instar wing imaginal discs. Together with genetic interactions with loss- and gain-of-function Notch alleles, these data support a role for wild-type Dcdc42 as a negative regulator of Notch signalling.

The guanine nucleotide exchange factor trio mediates axonal development in the Drosophila embryo.

Recent analysis of Rho subfamily GTPases in Drosophila revealed roles for Rac and Cdc42 during axonogenesis. Here, we describe the identification and characterization of the Drosophila counterpart of Trio, a guanine nucleotide exchange factor (GEF) that associates with the receptor phosphatase LAR and regulates GTPase activation in vertebrate cells. Mutants deficient in trio activity display defects in both central and peripheral axon pathways reminiscent of phenotypes observed in embryos deficient in small GTPase function. Double mutant analysis shows that trio interacts with Rac in a dose-sensitive manner but not with Rho. Moreover, reduction of trio activity potentiates the phenotype of mutations in the LAR homolog Dlar, suggesting that these proteins collaborate in orchestrating the cytoskeletal events that underlie normal axonogenesis.

Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2.

The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 and kinase-dead LIMK2 (LIMK2/KD) were respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylated cofilin and induced formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation was stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induced stress fibers and filopodia, whereas both Rho- induced stress fibers and Cdc42-induced filopodia were abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac did not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization.

Impact of amino acids 22-27 of Rho-subfamily GTPases on glucosylation by the large clostridial cytotoxins TcsL-1522, TcdB-1470 and TcdB-8864.

Here we report data describing some principles of the interaction between small GTP-binding proteins and large Clostridial cytotoxins (LCTs). Our investigation was based on the differential glucosylation of Rac1 versus RhoA by LCTs TcsL-1522, TcdB-1470 and TcdB-8864. Chimeric RhoA/Rac1 proteins and GTPases mutated at defined regions or single amino acids were used as substrates. Starting with chimeric Rac/Rho proteins we demonstrated that proteins containing the N-terminal 73 amino acids of Rac1 (but not those of RhoA) were efficiently glucosylated. Within this stretch, three regions differ significantly in Rac1 and RhoA. Regions containing amino acids 41-45 and 50-54 had no effect on toxin induced glucosylation, whereas amino acids 22-27 had a drastic impact on the potential of all three toxins to covalently modify the GTPases. Point mutations K25T of RhoA (numbering according to Rac1) and K27A of Cdc42 significantly increased glucosylation by the cytotoxins; introduction of lysines at the equivalent positions of Rac1 hindered modification. Our experiments demonstrate the influence of this charged residue on GTPase-LCT interactions. Amino acids 22-27 are part of the transition between the alpha1-helix to the switch I region of small GTP-binding proteins; both are known structures for specificity determination of the interactions with physiologic partners. Comparing these structures with data from our investigation we suggest that TcsL-1522, TcdB-1470 and TcdB-8864 mimic aspects of the physiologic interactions of small GTP-binding proteins.

Effects of Clostridium difficile toxin B on activation of rat peritoneal mast cells

Clostridium difficile toxin B that inactivates Rho subfamily proteins by glucosylation, inhibited dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) and phorbol 12-myristate 13-acetate (PMA)-induced mast cell activation by 80 to 90% in a concentration- and time dependent manner with a delay of about 30 min. Activation of mast cells by compound 48/80 and calcium ionophore A23187 was maximally inhibited by about 50%. Inhibition by toxin B was observed with suspended, attached and permeabilised mast cells. C3 ADP-ribosyltransferase, which selectively inactivates RhoA,B,C subtype proteins inhibited antigen, compound 48/80, PMA, A23187 and GTP[S]-induced degranulation of permeabilised mast cells C3-induced inhibition of stimulated histamine release was smaller than that observed with toxin B and both inhibitory effects were not additive. These findings suggest the involvement of Rho subtype GTPases and additionally, of other members of the Rho subfamily GTPases in activation of rat peritoneal mast cells.

The role of the cytoskeleton in the morphological changes occurring during neuronal differentiation

Abstract Development of neurons from neuroblasts involves the extension of cytoplasmic processes (neurites) that mature into axons and dendrites in response to distinct extracellular signals. Changes in the cytoskeleton are crucial for neurite outgrowth and maturation. The initial sprouting of neurites requires rearrangements of actin microfilaments which are possibly under the control of proteins belonging to the Rho subfamily of GTPases. Little is known about the extracellular stimuli which regulate the activity of these GTPases in developing neurons. Likewise, the molecular mechanisms by which actin microfilament dynamics and organization are modified after the activation of Rho subfamily GTPases are not yet clear. The consolidation and maturation of growing neurites that become axons and dendrites requires the participation of other cytoskeletal elements, particularly microtubules. The expression and phosphorylation of specific microtubule-associated proteins may be important for axon and dendrite development. There is some information about the protein kinases and phosphatases that act on microtubule-associated proteins. However, much less is known about the signal transduction pathways that regulated microtubule-associated protein expression and phosphorylation in response to distinct extracellular signals.

Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis.

The Rho subfamily of GTPases has been shown to regulate cellular morphology. We report the discovery of a new member of the Rho family, named RhoL, which is equally similar to Rac, Rho, and Cdc42. Expression of a dominant-negative RhoL transgene in the Drosophila ovary caused nurse cells to collapse and fuse together. Mutant forms of Cdc42 mimicked this effect. Expression of constitutively active RhoL led to nurse cell subcortical actin breakdown and disruption of nurse cell-follicle cell contacts, followed by germ cell apoptosis. In contrast, Rac activity was specifically required for migration of a subset of follicle cells called border cells. All three activities were necessary for normal transfer of nurse cell cytoplasm to the oocyte. These results suggest that Rho protein activities have cell type-specific effects on morphogenesis.

Selective activation of the JNK signaling cascadeand c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs

The Rho subfamily of GTPases is involved in control of cell morphology in mammals and yeast. The mammalian Rac and Cdc42 proteins control formation of lamellipodia and filopodia, respectively. These proteins also activate MAP kinase (MAPK) cascades that regulate gene expression. Constitutively activated forms of Rac and Cdc42Hs are efficient activators of a cascade leading to JNK and p38/Mpk2 activation. RhoA did not exhibit this activity, and none of the proteins activated the ERK subgroup of MAPKs. JNK, but not ERK, activation was also observed in response to Dbl, an oncoprotein that acts as a nucleotide exchange factor for Cdc42Hs. Results with dominant interfering alleles place Rac1 as an intermediate between Ha-Ras and MEKK in the signaling cascade leading from growth factor receptors and v-Src to JNK activation. JNK and p38 activation are likely to contribute to the biological effects of Rac, Cdc42Hs, and Dbl on cell growth and proliferation.