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Bacterial toxin fatty-acylates lysine residues A toxin produced by the bacterium that causes cholera has a catalytic activity that contributes to its effects on the cytoskeleton of host cells. Zhou et al. determined the protein structure of the Rho guanosine triphosphatase (GTPase)–inactivation domain of the toxin from Vibrio cholerae and found it to be similar to that of a human fatty acyltransferase. Indeed, the toxin peptide could catalyze fatty acylation of lysine residues of Rho-family GTPases, which regulate the actin cytoskeleton. Such covalent modification of lysine residues in mammalian proteins had been noted before, but the enzymes responsible were not known. Science , this issue p. 528

Biological systems are highly sensitive to changes in their environment. Indeed, the molecular basis of the environmental stress response suggests that the specialized stress responses share more commonalities than previously believed. Here, we used the nematode C. elegans to gain insight into the role of Rho signaling during two common environmental challenges, oxidative and thermal stress. In response to heat shock (HS), wild type (N2) worms demonstrated reduced viability which was rescued by genetic suppression of CDC42 and RHO-1. Visualization of F-actin by phalloidin-rhodamine underscored a strict correlation between the levels of F-actin following GTPase suppression and survival. Additionally, genetic ablation of OSG-1, a Guanine Nucleotide Exchange Factor (GEF) previously implicated in oxidative stress, was associated with constitutively lower levels of F-actin and increased mortality. However, upon an oxidative insult F-actin stability decreased in N2 worms, a rescue of this affect was observed in OSG-1 null worms, consistent with the resistance exhibited by these worms to oxidative stress (OS). Together these data suggest that during conditions of thermal or oxidative stress Rho signaling promotes vulnerability by altering actin dynamics. Thus, the stability of the actin cytoskeleton, in part through a conserved mechanism mediated by Rho signaling, is a crucial factor for the cell's survival to environmental challenges.

Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed. Here we investigated the signaling pathways involved in TNT formation by macrophages using multiple imaging techniques including super-resolution microscopy (3D-SIM) and live-cell imaging including the use of FRET-based Rho GTPase biosensors. We found that formation of TNTs required the activity and differential localization of Cdc42 and Rac1. The downstream Rho GTPase effectors mediating actin polymerization through Arp2/3 nucleation, Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous 2 (WAVE2) proteins are also important, and both pathways act together during TNT biogenesis. Finally, TNT function as measured by transfer of cellular material between cells was reduced following depletion of a single factor demonstrating the importance of these factors in TNTs. Given that the characterization of TNT formation is still unclear in the field; this study provides new insights and would enhance the understanding of TNT formation towards investigating new markers.

DOCK6 is a RAC1/CDC42 guanine nucleotide exchange factor, however, little is known about its function and sub-cellular localization. DOCK6 regulates the balance between RAC1 and RHOA activity during cell adhesion and is important for CDC42-dependent mitotic chromosome alignment. Surprisingly, a cell intrinsic adaptation mechanism compensates for errors in these DOCK6 functions that arise as a consequence of prolonged DOCK6 depletion or complete removal in DOCK6 knockout cells. Down-regulation of the ubiquitin-like modifier ISG15 accounts for this adaptation. Strikingly, although most other DOCK family proteins are deployed on the plasma membrane, here we show that DOCK6 localizes to the endoplasmic reticulum (ER) in dependence of its DHR-1 domain. ER localization of DOCK6 opens up new insights into its functions.

Melanoma is a highly lethal cutaneous tumor, killing affected patients through development of multiple poorly immunogenic metastases. Suboptimal activation of immune system by melanoma cells is often due to molecular modifications occurring during tumor progression that prevent efficient recognition of melanoma cells by immune effectors. Statins are HMG-CoA reductase inhibitors, which block the mevalonate synthesis pathway, used by millions of people as hypocholesterolemic agents in cardiovascular and cerebrovascular diseases. They are also known to inhibit Rho GTPase activation and Rho dependent signaling pathways. Rho GTPases are regarded as molecular switches that regulate a wide spectrum of cellular functions and their dysfunction has been characterized in various oncogenic process notably in melanoma progression. Moreover, these molecules can modulate the immune response. Since 10 years we have demonstrated that Statins and other Rho GTPases inhibitors are critical regulators of molecules involved in adaptive and innate anti-melanoma immune response. In this review we summarize our major observations demonstrating that these pharmacological agents stimulate melanoma immunogenicity and suggest a potential use of these molecules to promote anti-melanoma immune response.

Rho GTPases regulate cytoskeletal and cell adhesion dynamics and thereby coordinate a wide range of cellular processes, including cell migration, cell polarity and cell cycle progression. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation to regulate their ability to activate effector proteins and to elicit cellular responses. However, it has become apparent that Rho GTPases are regulated by post-translational modifications and the formation of specific protein complexes, in addition to GTP-GDP cycling. The canonical regulators of Rho GTPases - guanine nucleotide exchange factors, GTPase-activating proteins and guanine nucleotide dissociation inhibitors - are regulated similarly, creating a complex network of interactions to determine the precise spatiotemporal activation of Rho GTPases.

Summary During tooth development, ameloblasts differentiate from inner enamel epithelial cells to enamel-forming cells by modulating the signal pathways mediating epithelial–mesenchymal interaction and a cell-autonomous gene network. The differentiation process of epithelial cells is characterized by marked changes in their morphology and polarity, accompanied by dynamic cytoskeletal reorganization and changes in cell–cell and cell–matrix adhesion over time. Functional ameloblasts are tall, columnar, polarized cells that synthesize and secrete enamel-specific proteins. After deposition of the full thickness of enamel matrix, ameloblasts become smaller and regulate enamel maturation. Recent significant advances in the fields of molecular biology and genetics have improved our understanding of the regulatory mechanism of the ameloblast cell life cycle, mediated by the Rho family of small GTPases. They act as intracellular molecular switch that transduce signals from extracellular stimuli to the actin cytoskeleton and the nucleus. In our review, we summarize studies that provide current evidence for Rho GTPases and their involvement in ameloblast differentiation. In addition to the Rho GTPases themselves, their downstream effectors and upstream regulators have also been implicated in ameloblast differentiation.

The Rho GTPases are simple enzymes with complex roles in regulating cell morphology, gene transcription, cell cycle progression, apoptosis and tumour progression. The picture has been further complicated by the steady rise in the number of known Rho GTPases as well as in the number of known regulators and target proteins of these GTPases. Recent implications of Rho effectors in human disease, however, might give important clues to how specificity is achieved in cell signalling pathways employing Rho GTPases.

Background Fibroblast-like synoviocytes (FLS) play critical roles on joint inflammation and destruction of cartilage and bone in rheumatoid arthritis (RA) due to their aggressive behavior including increased migration. The Rho family of small GTPases are the master regulators of actin cytoskeleton remodeling which leads to pseudopodia formation and migration. Peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) β is a transcriptional regulator which plays important roles on regulating multiple signaling pathways. Our previous study revealed that elevated PGC-1β in RA-FLS promoted their migration and invasion. However, the underlying mechanism remains elusive. Objectives To investigate the role of PGC-1β on regulating pseudopodia formation of RA-FLS and its underlying mechanism. Methods Synovial tissues were obtained from six patients with active RA and FLS were isolated and cultured in vitro. RA-FLS were transfected with a lentivirus vector for PGC-1β knockdown or over-expression, and transfected with same lentivirus vectors marked Lv-sh-GFP or Lv-GFP as negative controls. The change of cytoskeleton was stained with fluorescent phalloidin to visualize polymerized actin in lentivirus transfected RA-FLS. The mRNA expression of RhoA, Rac1, and Cdc42 was measured by qPCR. Their activity was measured using a Rac1, RhoA or Cdc42 Pulldown & G-Lisa Activation Assay Kit. Results 1) PGC-1β knockdown inhibited lamellipodia and filopodia formations of RA-FLS compared with Lv-sh-GFP transfection group (cells with lamellipodia: 24% ± 7% vs. 40% ± 5%, P=0.035; cells with filopodia: 34% ± 9% vs. 49% ± 4%, P=0.041), while PGC-1β over-expression promoted lamellipodia and filopodia formations of RA-FLS compared with Lv-GFP transfection group (cells with lamellipodia: 50% ± 4% vs. 34% ± 6%, P=0.040; cells with filopodia: 67% ± 7% vs. 52% ± 6%, P=0.045, Figure 1A). 2) PGC-1β knockdown or over-expression did not affect the mRNA expression of Rac1, RhoA or Cdc42 (all P>0.05, Figure 1B). However, PGC-1β knockdown suppressed the activity of Rac1, RhoA and Cdc42 (Pulldown assays: 65%∼78% reduction; G-LISA assays: 28%∼53% reduction, all P Conclusion Our findings revealed that elevated PGC-1β in RA-FLS promoted pseudopodia formation by activating Rho family proteins which imply a novel target on regulating RA-FLS migration. Acknowledgement This work was supported by National Natural Science Foundation of China (81671612 and 81801606), Guangdong Natural Science Foundation (2017A030313576, 2017A030310236 and 2018A030313541) and Guangdong Medical Scientific Research Foundation (A2017109). Disclosure of Interests None declared

E-cadherin is an adherens junction protein that forms intercellular contacts in epithelial cells. Downregulation of E-cadherin is frequently observed in epithelial tumors and it is a hallmark of epithelial–mesenchymal transition (EMT). However, recent findings suggest that E-cadherin plays a more complex role in certain types of cancers. Previous studies investigating the role of E-cadherin mainly used gene-knockdown systems; therefore, we used the CRISPR/Cas9n system to develop E-cadherin-knockout (EcadKO) ovarian cancer RMG-1 cell to clarify the role of E-cadherin in RMG-1 cells. EcadKO RMG-1 cells demonstrated a complete loss of the adherens junctions and failed to form cell clusters. Cell–extracellular matrix (ECM) interactions were increased in EcadKO RMG-1 cells. Upregulation of integrin beta1 and downregulation of collagen 4 were confirmed. EcadKO RMG-1 cells showed decreased β-catenin levels and decreased expression of its transcriptional target cyclin D1. Surprisingly, a marked decrease in the migratory ability of EcadKO RMG-1 cells was observed and the cellular response to Rho GTPase inhibitors was diminished. Thus, we demonstrated that E-cadherin in RMG-1 cells is indispensable for β-catenin expression and β-catenin mediated transcription and Rho GTPase-regulated directionally persistent cell migration., Highlights • E-cadherin loss diminished the formation of cell clusters in RMG-1 cells. • E-cadherin loss depleted β-catenin expression in RMG-1 cells. • E-cadherin loss markedly decreased cell migration and response to RhoGTPase inhibitors during cell migration in RMG-1 cells.

The animal Ras/Rho-related GTPas gene family is represented by a sole family of ROP proteins in plants. Plant ROPs presumably interact with cell surface receptors in response to specific stimuli including abiotic, biotic, and hormonal elicitors to mediate diverse signaling responses. There are both similarities and at the same time differences at the regulatory level in plant versus non-plant Rho GTPases. ROPs are apparently capable of interacting with multiple effector molecules affecting several cellular biochemical responses such as actin dynamics, protein degradation, and interaction with the environment. ROPs are thus regarded as multifunctional proteins that integrate several cellular signals and coordinate diverse pathways to regulate growth, development, and stress responses.

Rho GTPases, which control processes such as cell proliferation and cytoskeleton remodeling, are often hyperexpressed in tumors. Several members, such as RhoA/B/C, must be isoprenylated to interact with their effectors. Statins, by inhibiting the synthesis of prenyl groups, may affect RhoA/B/C activity and represent a promising tool in anticancer therapy.

SUMMARY Burkholderia cenocepacia is an opportunistic bacterial pathogen that causes severe pulmonary infections in cystic fibrosis and chronic granulomatous disease patients. B. cenocepacia can survive inside infected macrophages within the B. cenocepacia-containing vacuole (BcCV) and to elicit a severe inflammatory response. By inactivating the host macrophage Rho GTPases, the bacterial effector TecA causes depolymerization of the cortical actin cytoskeleton. In this study, we find that B. cenocepacia induces the formation of large cytosolic F-actin clusters in infected macrophages. Cluster formation requires the nucleation-promoting factor WASH, the Arp2/3 complex, and TecA. Inactivation of Rho GTPases by bacterial toxins is necessary and sufficient to induce the formation of the cytosolic actin clusters. By hijacking WASH and Arp2/3 activity, B. cenocepacia disrupts interactions with the endolysosomal system, thereby delaying the maturation of the BcCV., In Brief Despite causing profound inhibition of host cell Rho GTPases, Burkholderia cenocepacia induces F-actin polymerization near endomembranes, particularly around phagosomes. Walpole et al. show that WASH, an Arp2/3 activator, is required for this de novo F-actin polymerization. The F-actin clusters formed around phagosomes delay their maturation, preventing their fusion with lysosomes., Graphical Abstract

In addition to direct activation of caspase-1 and induction of apoptosis by SipB, invasive Salmonella stimulates multiple signaling pathways that are key regulators of host cell survival. Nevertheless, little is known about the relative contributions of these pathways to Salmonella-mediated death of macrophages. We studied human monocytic U937 cells and found that apoptosis was induced by invading wild-type Salmonella typhimurium but not by phagocytosed, serum-opsonized, noninvasive Salmonella mutants. Pretreating U937 cells with inhibitors of tyrosine kinases or phosphatidylinositol-3 kinase (PI-3K) completely blocked phagocytosis of opsonized Salmonella mutants but did not affect invasion by wild-type Salmonella or the apoptosis caused by invasion. However, pretreatment with GGTI-298, a geranylgeranyltransferase-1 inhibitor that prevents prenylation of Cdc42 and Rac1, suppressed Salmonella-induced apoptosis by ∼70%. Transduction of Tat fusion constructs containing dominant-negative Cdc42 or Rac1 significantly inhibited Salmonella-induced cell death, indicating that the cytotoxicity of Salmonella requires activation of Cdc42 and Rac. In contrast to phagocytosis of opsonized bacteria, invasion by S. typhimurium stimulated Cdc42 and Rac1, regardless of the activities of tyrosine- or PI-3K. Moreover, Salmonella infection activated Akt protein in a tyrosine-kinase or PI-3K-dependent manner, and a reduced expression of Akt by antisense transfection rendered the cells more sensitive to apoptosis induced by opsonized Salmonella. These results indicate that direct activation of Cdc42 and Rac1 by invasive Salmonella is a prerequisite of Salmonella-mediated death of U937 cells, whereas the simultaneous activation of Akt by tyrosine kinase and PI-3K during receptor-mediated phagocytosis protects cells from apoptosis.

We previously demonstrated that exogenous expression of a truncated form of the tight junction protein ZO-3 affected junctional complex assembly and function. Current results indicate that this ZO-3 construct influences actin cytoskeleton dynamics more globally. We show that expression of the amino-terminal half of ZO-3 (NZO-3) in Madin-Darby canine kidney cells results in a decreased number of stress fibers and focal adhesions and causes an increased rate of cell migration in a wound healing assay. We also demonstrate that RhoA activity is reduced in NZO-3–expressing cells. We determined that ZO-3 interacts with p120 catenin and AF-6, proteins localized to the junctional complex and implicated in signaling pathways important for cytoskeleton regulation and cell motility. We also provide evidence that NZO-3 interacts directly with the C terminus of ZO-3, and we propose a model where altered interactions between ZO-3 and p120 catenin in NZO-3–expressing cells affect RhoA GTPase activity. This study reveals a potential link between ZO-3 and RhoA-related signaling events.

Although vesicular trafficking is essential for a large variety of cellular processes, the regulation of vesicular trafficking is still poorly understood. Members of the Rho family of small GTPases have recently emerged as important control elements of many stages of vesicular trafficking, providing new insight into the regulation of these events. We will discuss the diverse roles played by Rho proteins in membrane trafficking and focus on the biological implications of these functions.

The members of the Rho subfamily of small GTPases are key regulators of the actin cytoskeleton. However, recent studies have provided evidence for multiple additional roles for these signalling proteins in controlling endocytic traffic. Here we review our current understanding of Rho GTPase action within the endocytic pathway and examine the potential points of convergence with the more established, actin-based functions of these signalling proteins.

Schwann cells (SCs) are a highly plastic cell type capable of undergoing phenotypic changes following injury or disease. SCs are able to upregulate genes associated with nerve regeneration and ultimately achieve functional recovery. During the regeneration process, the extracellular matrix (ECM) and cell morphology play a cooperative, critical role in regulating SCs, and therefore highly impact nerve regeneration outcomes. However, the roles of the ECM and mechanotransduction relating to SC phenotype are largely unknown. Here, we describe the role that matrix stiffness and cell morphology play in SC phenotype specification via known mechanotransducers YAP/TAZ and RhoA. Using engineered microenvironments to precisely control ECM stiffness, cell shape, and cell spreading, we show that ECM stiffness and SC spreading downregulated SC regenerative associated proteins by the activation of RhoA and YAP/TAZ. Additionally, cell elongation promoted a distinct SC regenerative capacity by the upregulation of Rac1/MKK7/JNK, both necessary for the ECM and morphology changes found during nerve regeneration. These results confirm the role of ECM signaling in peripheral nerve regeneration as well as provide insight to the design of future biomaterials and cellular therapies for peripheral nerve regeneration.

Megakaryocytes differentiate from hematopoietic stem cells in the bone marrow. The transition of megakaryocytes to platelets is a complex process. Thereby, megakaryocytes extend proplatelets into sinusoidal blood vessels, where the proplatelets undergo fission to release platelets. Defects in platelet production can lead to a low platelet count (thrombocytopenia) with increased bleeding risk. Rho GTPases comprise a family of small signaling G proteins that have been shown to be master regulators of the cytoskeleton controlling many aspects of intracellular processes. The generation of Pf4-Cre transgenic mice was a major breakthrough that enabled studies in megakaryocyte-/platelet-specific knockout mouse lines and provided new insights into the central regulatory role of Rho GTPases in megakaryocyte maturation and platelet production. In this review, we will summarize major findings on the role of Rho GTPases in megakaryocyte biology with a focus on mouse lines in which knockout strategies have been applied to study the function of the best-characterized members Rac1, Cdc42 and RhoA and their downstream effector proteins.

Platelets are a critical cell for prevention of bleeding. Part of the response to the formation of the thrombus is the activation of the actin cytoskeleton, with an inability to effectively activate the cytoskeleton linked to thrombus formation defects and instability. The control of this process is linked to activation of the Rho GTPases, Cdc42, Rac1, and RhoA, although additional small GTPases such as Rif and Rap have been shown to play roles in platelet function.Here we describe the methodology to accurately understand how Rho GTPases are activated in platelets. Due to the technical limitations of working with platelets, such as their lack of ability to be transfected, the majority of work has been carried out either using inhibitors of Rho GTPases or within knockout mouse models. Studies can be conducted both in suspension samples and in spread platelets. In suspension the platelets will undergo a shape change response, but will not be able to spread. In spread platelets it is possible to examine the effects of the matrix environment, such as concentration, type, and stiffness on Rho GTPase function within platelet activation and platelet spreading.

N-cadherin, a member of the Ca2+-dependent cell–cell adhesion molecule family, plays an essential role in skeletal muscle cell differentiation. We show that inhibition of N-cadherin–dependent adhesion impairs the upregulation of the two cyclin-dependent kinase inhibitors p21 and p27, the expression of the muscle-specific genes myogenin and troponin T, and C2C12 myoblast fusion. To determine the nature of N-cadherin–mediated signals involved in myogenesis, we investigated whether N-cadherin–dependent adhesion regulates the activity of Rac1, Cdc42Hs, and RhoA. N-cadherin–dependent adhesion decreases Rac1 and Cdc42Hs activity, and as a consequence, c-jun NH2-terminal kinase (JNK) MAPK activity but not that of the p38 MAPK pathway. On the other hand, N-cadherin–mediated adhesion increases RhoA activity and activates three skeletal muscle-specific promoters. Furthermore, RhoA activity is required for β-catenin accumulation at cell–cell contact sites. We propose that cell–cell contacts formed via N-cadherin trigger signaling events that promote the commitment to myogenesis through the positive regulation of RhoA and negative regulation of Rac1, Cdc42Hs, and JNK activities.

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that β-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit the expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of β-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases.

Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) is a key process of peritoneal fibrosis. Rapamycin has been previously shown to inhibit EMT of PMCs and prevent peritoneal fibrosis. In this study, we investigated the undefined molecular mechanisms by which rapamycin inhibits EMT of PMCs. To define the protective effect of rapamycin, we initially used a rat PD model which was daily infused with 20 mL of 4.25% high glucose (HG) dialysis solution for 6 weeks to induce fibrosis. The HG rats showed decreased ultrafiltration volume and obvious fibroproliferative response, with markedly increased peritoneal thickness and higher expression of α-smooth muscle actin (α-SMA) and transforming growth factor-β1. Rapamycin significantly ameliorated those pathological changes. Next, we treated rat PMCs with HG to induce EMT and/or rapamycin for indicated time. Rapamycin significantly inhibited HG-induced EMT, which manifests as increased expression of α-SMA, fibronectin, and collagen I, decreased expression of E-cadherin, and increased mobility. HG increased the phosphorylation of PI3K, Akt, and mTOR. Importantly, rapamycin inhibits the RhoA, Rac1, and Cdc42 activated by HG. Moreover, rapamycin repaired the pattern of F-actin distribution induced by HG, reducing the formation of stress fiber, focal adhesion, lamellipodia, and filopodia. Thus, rapamycin shows an obvious protective effect on HG-induced EMT, by inhibiting the activation of Rho GTPases (RhoA, Rac1, and Cdc42).

Rho GTPases are molecular switches that modulate multiple intracellular signaling processes by means of various effector proteins. As a result, Rho GTPase activities are tightly spatiotemporally regulated in order to ensure homeostasis within the cell. Though the roles of Rho GTPases during neural development have been well documented, their participation during neurodegeneration has been far less characterized. Herein we discuss our current knowledge of the role and function of Rho GTPases and regulators during neurodegeneration, and highlight their potential as targets for therapeutic intervention in common neurodegenerative disorders.

Significance: Oxidation and reduction events are critical to physiological and pathological processes and are highly regulated. Herein, we present evidence for the role of Ras and Rho GTPases in controlling these events and the unique underlying mechanisms. Evidence for redox regulation of Ras GTPases that contain a redox-sensitive cysteine (X) in the conserved NKXD motif is presented, and a growing consensus supports regulation by a thiyl radical-mediated oxidation mechanism. We also discuss the debate within the literature regarding whether 2e− oxidation mechanisms also regulate Ras GTPase activity. Recent Advances: We examine the increasing in vitro and cell-based data supporting oxidant-mediated activation of Rho GTPases that contain a redox-sensitive cysteine at the end of the conserved phosphoryl-binding loop (p-loop) motif (GXXXXG[S/T]C). While this motif is distinct from Ras, these data suggest a similar 1e− oxidation-mediated activation mechanism. Critical Issues: We also review the data showing that the unique p-loop placement of the redox-sensitive cysteine in Rho GTPases supports activation by 2e− cysteine oxidation. Finally, we examine the role that Ras and Rho GTPases play in controlling key oxidant-regulating enzymes in the cell, and we speculate on a feedback mechanism. Future Directions: Given that these GTPases and redox-regulating enzymes are involved in multiple physiological and pathological processes, we discuss future experiments that may clarify the interplay between them. Antioxid. Redox Signal. 18, 250–258.

Ras and Rho family GTPases control a wide variety of cellular processes, and the signaling downstream of these GTPases is influenced by their subcellular localization when activated. Since only a minority of total cellular GTPases is active, observation of the total subcellular distribution of GTPases does not reveal where active GTPases are localized. In this chapter, we describe the use of effector recruitment assays to monitor the subcellular localization of active Ras and Rho family GTPases. The recruitment assay relies on preferential binding of downstream effectors to active GTPases versus inactive GTPases. Tagging the GTPase-binding-domain (GBD) of a downstream effector with a fluorescent protein produces a probe that is recruited to compartments where GTPases are active. We describe an example of a recruitment assay using the GBD of PAK1 to monitor Rac1 activity and explain how the assay can be expanded to determine the subcellular localization of activation of other GTPases.

Rho GTPases are master regulators of actomyosin structure and dynamics and play pivotal roles in a variety of cellular processes including cell morphology, gene transcription, cell cycle progression, and cell adhesion. Because aberrant Rho GTPase signaling activities are widely associated with human cancer, key components of Rho GTPase signaling pathways have attracted increasing interest as potential therapeutic targets. Similar to Ras, Rho GTPases themselves were, until recently, deemed "undruggable" because of structure-function considerations. Several approaches to interfere with Rho GTPase signaling have been explored and show promise as new ways for tackling cancer cells.This review focuses on the recent progress in targeting the signaling activities of three prototypical Rho GTPases, that is, RhoA, Rac1, and Cdc42. The authors describe the involvement of these Rho GTPases, their key regulators and effectors in cancer. Furthermore, the authors discuss the current approaches for rationally targeting aberrant Rho GTPases along their signaling cascades, upstream and downstream of Rho GTPases, and posttranslational modifications at a molecular level.To date, while no clinically effective drugs targeting Rho GTPase signaling for cancer treatment are available, tool compounds and lead drugs that pharmacologically inhibit Rho GTPase pathways have shown promise. Small-molecule inhibitors targeting Rho GTPase signaling may add new treatment options for future precision cancer therapy, particularly in combination with other anti-cancer agents.

Cell movement is essential during embryogenesis to establish tissue patterns and to drive morphogenetic pathways and in the adult for tissue repair and to direct cells to sites of infection. Animal cells move by crawling and the driving force is derived primarily from the coordinated assembly and disassembly of actin filaments. The small GTPases, Rho, Rac, and Cdc42, regulate the organization of actin filaments and we have analyzed their contributions to the movement of primary embryo fibroblasts in an in vitro wound healing assay. Rac is essential for the protrusion of lamellipodia and for forward movement. Cdc42 is required to maintain cell polarity, which includes the localization of lamellipodial activity to the leading edge and the reorientation of the Golgi apparatus in the direction of movement. Rho is required to maintain cell adhesion during movement, but stress fibers and focal adhesions are not required. Finally, Ras regulates focal adhesion and stress fiber turnover and this is essential for cell movement. We conclude that the signal transduction pathways controlled by the four small GTPases, Rho, Rac, Cdc42, and Ras, cooperate to promote cell movement.

Ultraviolet light crossing the ozone layer in the atmospheric barrier affects all forms of living beings on earth. In eukaryotic cells, the nucleotide excision repair (NER) pathway protects the DNA by removing cyclobutane pyrimidine dimers (CPDs) and 6-4-photoproduct (6-4-PP) lesions caused by ultraviolet (UV) light, allowing cells to proliferate. On the other hand, adhesion and invasion processes, primarily regulated by the typical Rho GTPases Rho, Rac, and Cdc42, are also affected by UV radiation effects. Studies focused on determining whether or not these GTPases might affect the NER pathway in different cell models are enlightening and should start with classical experimental methodologies. In this chapter we describe two methods (host cell reactivation assay, or HCR, and slot-blots for CPDs and 6-4-PPs) to assess the direct or indirect involvement of these three GTPases on the NER pathway.

The Rho GTPases (Rho, Rac, and Cdc42) regulate assembly of the actin cytoskeleton in many cells, including leukocytes. Recent work in identifying the protein targets of these GTPases is providing greater insight into the mechanisms used by cells to control cytoskeletal dynamics for a variety of purposes.

The last decades have witnessed an exponential increase in our knowledge of Rho GTPase signaling network which further highlighted the cross talk between these proteins and the complexity of their signaling pathways. In this review, we summarize the upstream and downstream players from Rho GTPases that are mainly involved in actin polymerization leading to cell motility and potentially playing a role in cancer cell metastasis.

Bacterial pathogens developed several strategies to overcome defense systems of eukaryotic hosts. Within the infection process, they need to attach to and cross through epithelial layers, escape from the innate and adaptive immune response, and find a physiological niche to survive. One target to modulate the host-pathogen interaction in order to deceit pathogen resistance is the actin cytoskeleton and its regulators: the family of Rho GTPases. Some bacterial toxins catalyze a covalent modification of Rho GTPases to keep these molecular switches in a constitutive active or inactive state. This leads to rearrangement of the actin cytoskeleton. Toxin-treated cells show typical morphological changes depending on substrate specificity and action of the toxins. In this chapter, we discuss the classes of bacterial toxins based on their mode of action, their recombinant expression (specifically CNF1), intoxication and subsequent morphological changes of the actin cytoskeleton, and cell shape.

IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis., Federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health; Intramural Research Program of National Institutes of Health; Frederick National Laboratory; Center for Cancer Research; Intramural Research Program of the National Institutes of Health Clinical Center; Scientific and Technological Research Council of Turkey (TÜBİTAK)

The ability of cells to adhere and to exert contractile forces governs their capacity to move within an organism. The cytoskeletal regulators of the Rho GTPase proteins are involved in control of the contractile forces of cells. To elucidate the basis of cell migration, we analyzed contractile forces and nanoscale adhesion-related particles in single cells expressing constitutively active variants of Rho GTPases by using traction-force microscopy and ultra-high-resolution stimulated emission depletion microscopy, respectively. RhoAV14 induced large increases in the contractile forces of single cells, with Rac1L61 and RhoDV26 having more moderate effects. The RhoAV14- and RhoDV26-induced forces showed similar spatial distributions and were accompanied by reduced or unaltered cell spreading. In contrast, the Rac1L61-induced force had different, scattered, force distributions that were linked to increased cell spreading. All three of these Rho GTPase activities caused a loss of thick stress fibers and focal adhesions and a more homogenous distribution of nanoscale adhesion-related particles over the ventral surface of the cells. Interestingly, only RhoAV14 increased the density of these particles. Our data suggest a Rac1-specific mode for cells to generate contractile forces. Importantly, increased density and a more homogenous distribution of these small adhesion-related particles promote cellular contractile forces.

Rho GTPases, including RhoA, Rac1 and Cdc42, are key regulators of cell migration in animals. In a recent issue of Nature, two papers present delicate studies on precise roles and spatiotemporal coordination of Rho GTPases in live cells, using either a photoactivatable Rac1 or specific biosensors, together with computational multiplexing method. In addition to implications in cell migration, analytical tools and methods used in these works can also be extended into other live cell studies on complex cellular/molecular events.

Astrocytes modulate and support neuronal and synapse function via numerous mechanisms that often rely on diffusion of signalling molecules, ions or metabolites through extracellular space. As a consequence, the spatial arrangement and the distance between astrocyte processes and neuronal structures are of functional importance. Likewise, changes of astrocyte structure will affect the ability of astrocytes to interact with neurons. In contrast to neurons, where rapid morphology changes are critically involved in many aspects of physiological brain function, a role of astrocyte restructuring in brain physiology is only beginning to emerge. In neurons, small GTPases of the Rho family are powerful initiators and modulators of structural changes. Less is known about the functional significance of these signalling molecules in astrocytes. Here, we review recent experimental evidence for the role of RhoA, Cdc42 and Rac1 in controlling dynamic astrocyte morphology as well as experimental tools and analytical approaches for studying astrocyte morphology changes.

The actin cytoskeleton is essential for the biology of osteoclasts, in particular during bone resorption. As key regulators of actin dynamics, the small GTPases of the Rho family are very important in the control of osteoclast activity. The study of Rho GTPase signaling pathways is essential to uncover the mechanisms of bone resorption and can have interesting applications for the treatment of osteolytic diseases. In this chapter, we describe various techniques to obtain primary osteoclasts from murine bone marrow cells, to measure Rho GTPase activation levels, to monitor bone resorption activity of osteoclasts and to introduce the expression of proteins of interest using a retroviral approach. We illustrate the different methods with experimental examples of the effect of Rac1 activation by the exchange factor Dock5 on bone resorption by osteoclasts.

Rho GTPases contribute to multiple cellular processes that could affect cancer progression, including cytoskeletal dynamics, cell cycle progression, transcriptional regulation, cell survival and vesicle trafficking. In vitro several Rho GTPases have oncogenic activity and/or can promote cancer cell invasion, and this correlates with increased expression and activity in a variety of cancers. Conversely, other family members appear to act as tumour suppressors and are deleted, mutated or downregulated in some cancers. Genetic models are starting to provide new information on how Rho GTPases affect cancer development and progression. Here, we discuss how Rho GTPases could contribute to different steps of cancer progression, including proliferation, survival, invasion and metastasis.

© 2008 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0). The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3β homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases. Deutsche Forschungsgemeinschaft

Podocyte injury is a key event in proteinuric kidney disease and eventually glomerular scarring. While adrenomedullin (AM), a potent vasodilatory peptide, has been reported to confer renoprotection in several experimental models of kidney diseases, its effect on injured podocytes and the related mechanism is still largely unknown. Employing Western blotting analysis, immunoprecipitation and immunofluorescence, we investigated the effects of AM on the expressions of podocyte cytoskeletal proteins and Rho-family small GTPases (Rho GTPases) in puromycin aminonucleoside (PAN)-induced podocyte injury, both in cultured podocytes and in PAN nephrosis rats. Urinary protein excretion and the morphologic changes of kidney in PAN nephrosis rats were evaluated. Glutathione-S-transferase pull-down assay was applied for Rho GTPases activity. PAN induced massive albuminuria and morphologic injury, which were significantly mitigated by AM administration. AM significantly antagonized not only the PAN-decreased expressions of synaptopodin, nephrin, CD2AP and podocin, but also the PAN-disrupted interactions between synaptopodin–RhoA, nephrin–CD2AP, and CD2AP–Rac1–cortactin. These effects of AM in cultured podocytes were mostly significantly blocked by H89, a PKA inhibitor. AM dramatically upregulated the PAN-induced Rho GTPases protein expressions and their activities. PAN increased the expressions of endogenous AM and its receptor RAMP2 which was furthermore upregulated by AM administration. AM alleviated podocyte injury induced by PAN both in cell culture and in PAN nephrosis. The beneficial effects of AM on podocytes can be attributable to direct modulation of podocyte cytoskeletal proteins and Rho GTPases, mainly via a PKA-dependent pathway.

Purpose of review This review summarizes our current understanding of the roles of Rho GTPases in early erythropoiesis, downstream of cytokine signaling, and in terminal erythroblast maturation and enucleation, as master regulators of the cytoskeleton and cytokinesis. Recent findings Similarities of structural and signaling requirements of erythroblast enucleation with the cytokinesis process have been confirmed and expanded in the last year, suggesting that enucleation is a form of asymmetric cell division. Myosin, the classic actin partner in cytokinesis, was shown to play an essential role in enucleation. Studies with multispectral high-speed cell imaging in flow demonstrated a sequential process requiring establishment of polarity through a unipolar microtubule spindle in orthochromatic erythroblasts, followed by Rac-directed formation of a contractile actomyosin ring and coalescence of lipid rafts between reticulocyte and pyrenocyte, steps which reiterate the choreography of cytokinesis. mDia2, a Rho effector known to play a role in enucleation, was also found essential for erythroblast cytokinesis as its deficiency in mice caused failure of primitive erythropoiesis and embryonic death. Summary Further elucidation of the role of Rho GTPases in the erythroid lineage development may reveal potential targets for improving red blood cell production in vivo and in vitro.

The Rho GTPases are essential regulators of basic cellular processes, including cell migration, cell contraction and cell division. Most studies still involve just the three canonical members, RhoA, Rac1 and Cdc42, although the Rho GTPases comprise at least 20 members. The aim of this review is to highlight some of the recent advances in our knowledge regarding the less-studied Rho members, with the focus on RhoD and Rif. The phenotypic alterations to cell behaviour that are triggered by RhoD and Rif suggest that they have unique impacts on cytoskeletal dynamics that distinguish them from the well-studied members of the Rho GTPases. In addition, RhoD has a role in the regulation of intracellular transport of vesicles. Taken together, the available data indicate that RhoD and Rif have functions as master regulators in the integration of cytoskeletal reorganisation and membrane trafficking.

Rho-family GTPases are the major regulators of the actin and microtubule cytoskeleton. Regulators of the GTPases include GEFs and GAPs, which control activation and deactivation, respectively, of this family of molecular switches. This study explores the role of the GEF Tiam2 in breast cancer cell proliferation and invasion, which was initially discovered using a siRNA screen in MDA-MB-231 breast cancer cells. Protein expression studies are performed using a panel of cell lines, revealing that Tiam2 expression is upregulated in Ras mutant cells. Additionally, Tiam2 depletion causes MDA-MB-231 cells to form fewer colonies in soft agar colony formation assays. Work continues on this project to determine how Tiam2 contributes to breast cancer proliferation and invasion using both cell culture and mouse models.