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Rho GTPases are a family of small G proteins that regulate a wide array of cellular processes related to their key roles controlling the cytoskeleton. Cancer is a multistep disease caused by the accumulation of genetic mutations and epigenetic alterations, from the initial stages of cancer development when cells in normal tissues undergo transformation, to the acquisition of invasive and metastatic traits, responsible for a large number of cancer related deaths. In this review, we discuss the role of Rho GTPase signaling in cancer in every step of disease progression. Rho GTPases contribute to tumor initiation and progression, by regulating proliferation and apoptosis, but also metabolism, senescence, and cancer cell stemness. Rho GTPases play a major role in cell migration and in the metastatic process. They are also involved in interactions with the tumor microenvironment and regulate inflammation, contributing to cancer progression. After years of intensive research, we highlight the importance of relevant models in the Rho GTPase field, and we reflect on the therapeutic opportunities arising for cancer patients., The work was supported by Cancer Research UK (CRUK) C33043/A24478 (to V.S.-M., E.C.-M., O.M., and L.K.); Barts Charity (to V.S.M., R.S., and S.L.G.); The Harry J. Lloyd Charitable Trust (to J.L.O. and V.S.-M); and Comunidad de Madrid (Atraccion de Talento-2019-T1/BMD-13642) (to J.L.O.).

Membrane binding and unbinding dynamics play a crucial role in the biological activity of several non-integral membrane proteins, which have to be recruited to the membrane in order to perform their functions. By localizing to the membrane, these proteins are able to induce downstream signal amplification in their respective signaling pathways. Here we present a 3D computational approach using reaction-diffusion equations to investigate the relation between membrane localization of Focal Adhesion Kinase (FAK), Ras homolog family member A (RhoA) and signal amplification of the YAP/TAZ signaling pathway. Our results show that the theoretical scenarios, in which FAK is membrane-bound, yield robust and amplified YAP/TAZ nuclear translocation signals. Moreover, we predict that the amount of YAP/TAZ nuclear translocation increases with cell spreading, confirming the experimental findings in the literature. In summary, our in silico predictions show that when the cell membrane interaction area with the underlying substrate increases, for example through cell spreading, this leads to more encounters between membrane-bound signaling partners and downstream signal amplification. Since membrane activation is a motif common to many signaling pathways, this study has important implications for understanding the design principles of signaling networks.

Pathological bone loss diseases (osteolysis, Paget’s diseases) are commonly caused by the excessive differentiation and activity of osteoclasts. The Rho GTPases family members Rac1/2 (Rac1 and Rac2) have been reported for their special role in exerting multiple cellular functions during osteoclastic differentiation, which includes the most prominent function on dynamic actin cytoskeleton rearranging. Besides that, the increasing studies demonstrated that the regulating effects of Rac1/2 on the osteoclastic cytoskeletal organization are through the GEFs member Dock5. Although the amount of relevant studies on this topic is still limited, several excellent studies have been reported that extensively explored the molecular mechanisms involved in Rac1/2 and Dock5 during the osteoclastogenesis regulation, as well as their role as the therapeutic target in bone loss diseases. Herein, in this review, we aim to focus on recent advances studies for extensively understanding the role of Rho GTPases Rac1/2 and Dock5 in osteoclastogenesis, as well as their role as a potential therapeutic target in regulating osteoclastogenesis.

Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) byiEscherichia coli/i, a commensal of the digestive tract and a source of urinary tract pathogens. Bioinformatics analyses of a large collection ofiE. coli/igenomes from EnteroBase, enriched in clinical isolates of worldwide origins, suggest the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene,icnf1/i, is preferentially distributed in four common sequence types (ST) encompassing the pandemiciE. coli/iMDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment, with known enhanced capacities to colonize the gastrointestinal tract. Statistical projections based on this dataset point to a global expansion oficnf1/i-positive multidrug-resistant ST131 strains from subcladeiH/i30Rx/C2, accounting for a rising prevalence oficnf1/i-positive strains in ST131. Despite the absence of phylogeographical signals,icnf1/i-positive isolates segregated into clusters in the ST131-iH/i30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the sameicnf1/iallele. The suggested dominant expansion oficnf1/i-positive strains in ST131-iH/i30Rx/C2 led us to uncover the competitive advantage conferred byicnf1/ifor gut colonization to the clinical strain EC131GY ST131-iH/i30Rx/C2 versusicnf1/i-deleted isogenic strain. Complementation experiments showed that colon tissue invasion was compromised in the absence of deamidase activity on Rho GTPases by CNF1. Hence, gut colonization factor function oficnf1/iwas confirmed for another clinical strain ST131-iH/i30Rx/C2. In addition, functional analysis of theicnf1/i-positive clinical strain EC131GY ST131-iH/i30Rx/C2 and aicnf1/i-deleted isogenic strain showed no detectable impact of the CNF1 gene on bacterial fitness and inflammation during the acute phase of bladder monoinfection. Together these data argue for an absence of role of CNF1 in virulence during UTI, while enhancing gut colonization capacities of ST131-iH/i30Rx/C2 and suggested expansion oficnf1/i-positive MDR isolates in subclade ST131-iH/i30Rx/C2.

Dedicator of cytokinesis 10 (Dock10) is a guanine nucleotide exchange factor for Cdc42 and Rac1 that regulates the JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) signaling cascades. In this study, we characterized the roles of Dock10 in the myocardium. In vitro: we ablated Dock10 in neonatal mouse floxed Dock10 cardiomyocytes (NMCMs) and cardiofibroblasts (NMCFs) by transduction with an adenovirus expressing Cre-recombinase. In vivo, we studied mice in which the Dock10 gene was constitutively and globally deleted (Dock10 KO) and mice with cardiac myocyte-specific Dock10 KO (Dock10 CKO) at baseline and in response to two weeks of Angiotensin II (Ang II) infusion. In vitro, Dock10 ablation differentially inhibited the α-adrenergic stimulation of p38 and JNK in NMCM and NMCF, respectively. In vivo, the stimulation of both signaling pathways was markedly attenuated in the heart. The Dock10 KO mice had normal body weight and cardiac size. However, echocardiography revealed mildly reduced systolic function, and IonOptix recordings demonstrated reduced contractility and elevated diastolic calcium levels in isolated cardiomyocytes. Remarkably, Dock10 KO, but not Dock10 CKO, exaggerated the pathological response to Ang II infusion. These data suggest that Dock10 regulates cardiac stress-related signaling. Although Dock10 can regulate MAPK signaling in both cardiomyocytes and cardiofibroblasts, the inhibition of pathological cardiac remodeling is not apparently due to the Dock10 signaling in the cardiomyocyte.

Background Evidence indicates that Gα12, Gα13, and its downstream effectors, RhoA and Rac1, regulate neuronal morphology affected by stress. This study was aimed at investigating whether repeated stress influences the expression of proteins related to the Gα12/13 intracellular signaling pathway in selected brain regions sensitive to the effects of stress. Furthermore, the therapeutic impact of β(1)adrenergic receptors (β1AR) blockade was assessed. Methods Restraint stress (RS) model in mice (2 h/14 days) was used to assess prolonged stress effects on the mRNA expression of Gα12, Gα13, RhoA, Rac1 in the prefrontal cortex (PFC), hippocampus (HIP) and amygdala (AMY). In a separate study, applying RS model in rats (3–4 h/1 day or 14 days), we evaluated stress effects on the expression of Gα12, Gα11, Gαq, RhoA, RhoB, RhoC, Rac1/2/3 in the HIP. Betaxolol (BET), a selective β1AR antagonist, was introduced (5 mg/kg/p.o./8–14 days) in the rat RS model to assess the role of β1AR in stress effects. RT-qPCR and Western Blot were used for mRNA and protein assessments, respectively. Results Chronic RS decreased mRNA expression of Gα12 and increased mRNA for Rac1 in the PFC of mice. In the mice AMY, decreased mRNA expression of Gα12, Gα13 and RhoA was observed. Fourteen days of RS exposure increased RhoA protein level in the rats’ HIP in the manner dependent on β1AR activity. Conclusions Together, these results suggest that repeated RS affects the expression of genes and proteins known to be engaged in neural plasticity, providing potential targets for further studies aimed at unraveling the molecular mechanisms of stress-related neuropsychiatric diseases.

The Rho family of small GTPases (Rho GTPases) are the master regulators of the actin cytoskeleton and consist of 22 members. Previous studies implicated dysregulation of Rho GTPases in podocytes in the pathogenesis of proteinuric glomerular diseases. Rho GTPases are primarily regulated by the three families of proteins; guanine nucleotide exchange factors (GEFs; 82 members), GTPase-activating proteins (GAPs; 69 members), and GDP dissociation inhibitors (GDIs; 3 members). Since the regulatory proteins far outnumber their substrate Rho GTPases and act in concert in a cell/context-dependent manner, the upstream regulatory mechanism directing Rho GTPases in podocytes is largely unknown. In this review, we summarize recent advances in the understanding of the role of Rho GTPase regulatory proteins in podocytes, including the known mutations of these proteins that cause proteinuria in humans. We also provide critical appraisal of the in vivo and in vitro studies and identify the knowledge gap in the field that will require further studies.

Given the key role of cell migration in cancer metastasis, there is a critical need for in vitro models that better capture the complexities of in vivo cancer cell microenvironments. Using both two-dimensional (2D) and three-dimensional (3D) culture models, recent research has demonstrated the role of both matrix and ligand densities in cell migration. Here, we leveraged our previously developed 2.5D sandwich culture platform to foster a greater understanding of the adhesion-dependent migration of glioblastoma cells with a stiffness gradient. Using this model, we demonstrated the differential role of stiffness gradients in migration in the presence and absence of adhesion moieties. Furthermore, we observed a positive correlation between the density of cell adhesion moieties and migration, and a diminished role of stiffness gradients at higher densities of adhesion moieties. These results, i.e., the reduced impact of stiffness gradients on adhesion-dependent migration relative to adhesion-independent migration, were confirmed using inhibitors of both mechanotransduction and cell adhesion. Taken together, our work demonstrates the utility of sandwich culture platforms that present stiffness gradients to study both adhesion-dependent and -independent cell migration and to help expand the existing portfolio of in vitro models of cancer metastasis.

The inability to simultaneously observe all of the important Rho GTPases (Cdc42, Rac1, and RhoA) has prevented us from obtaining evidence of their coordinated regulation during cell deformation. Here, we propose Motion-Triggered Average (MTA), an algorithm that converts individually observed GTPases into pseudo-simultaneous observations. Using the time series obtained by MTA and mathematical model, we succeeded for the first time in decoding the cell edge velocity from the three GTPase activities to provide clear numerical evidence for coordinated cell edge regulation by the three GTPases. We found that the characteristics of the obtained activities were consistent with those of previous studies, and that GTPase activities and their derivatives were involved in edge regulation. Our approach provides an effective strategy for using single-molecule observations to elucidate problems hampered by the lack of simultaneous observations.

Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding β1 and β3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal β1/β3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal β1/β3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis.

Nematode-trapping (NT) fungi can form unique infection structures (traps) to capture and kill free-living nematodes and, thus, can play a potential role in the biocontrol of nematodes. Arthrobotrys oligospora is a representative species of NT fungi. Here, we performed a time course transcriptome sequencing (RNA-seq) analysis of transcriptomes to understand the global gene expression levels of A. oligospora during trap formation and predation. We identified 5,752 unique differentially expressed genes, among which the rac gene was significantly upregulated. Alternative splicing events occurred in 2,012 genes, including the rac and rho2 gene. Furthermore, we characterized three Rho GTPases (Rho2, Rac, and Cdc42) in A. oligospora using gene disruption and multiphenotypic analysis. Our analyses showed that AoRac and AoCdc42 play an important role in mycelium growth, lipid accumulation, DNA damage, sporulation, trap formation, pathogenicity, and stress response in A. oligospora. AoCdc42 and AoRac specifically interacted with components of the Nox complex, thus regulating the production of reactive oxygen species. Moreover, the transcript levels of several genes associated with protein kinase A, mitogen-activated protein kinase, and p21-activated kinase were also altered in the mutants, suggesting that Rho GTPases might function upstream from these kinases. This study highlights the important role of Rho GTPases in A. oligospora and provides insights into the regulatory mechanisms of signaling pathways in the trap morphogenesis and lifestyle transition of NT fungi. IMPORTANCE Nematode-trapping (NT) fungi are widely distributed in terrestrial and aquatic ecosystems. Their broad adaptability and flexible lifestyles make them ideal agents for controlling pathogenic nematodes. Arthrobotrys oligospora is a model species employed for understanding the interaction between fungi and nematodes. Here, we revealed that alternative splicing events play a crucial role in the trap development and lifestyle transition in A. oligospora. Furthermore, Rho GTPases exert differential effects on the growth, development, and pathogenicity of A. oligospora. In particular, AoRac is required for sporulation and trap morphogenesis. In addition, our analysis showed that Rho GTPases regulate the production of reactive oxygen species and function upstream from several kinases. Collectively, these results expand our understanding of gene expression and alternative splicing events in A. oligospora and the important roles of Rho GTPases in NT fungi, thereby providing a foundation for exploring their potential application in the biocontrol of pathogenic nematodes.

Chronic stress can increase the risk of developing a substance use disorder in vulnerable individuals. Numerous models have been developed to probe the underlying neurobiological mechanisms, however most prior work has been restricted to male rodents, conducted only in rats, or introduces physical injury that can complicate opioid studies. Here we sought to establish how chronic psychosocial stress influences fentanyl consumption in male and female C57BL/6 mice. We used chronic social defeat stress (CSDS), or the modified vicarious chronic witness defeat stress (CWDS), and used social interaction to stratify mice as stress-susceptible or resilient. We then subjected mice to a 15 day fentanyl drinking paradigm in the home cage that consisted of alternating forced and choice periods with increasing fentanyl concentrations. Male mice susceptible to either CWDS or CSDS consumed more fentanyl relative to unstressed mice, and exhibited increased preference for fentanyl. CWDS-susceptible female mice did not differ from unstressed mice during the forced periods, but showed increased preference for fentanyl. We also found decreased expression of nucleus accumbens Rho GTPases in male, but not female mice following stress and fentanyl drinking. We also compare fentanyl drinking behavior in mice that had free access to plain water throughout. Our results indicate that stress-sensitized fentanyl consumption is dependent on both sex and behavioral outcomes to stress.

A growing body of evidence implicates requisite roles for GTP and its binding proteins (Rho GTPases) in the cascade of events leading to physiological insulin secretion from the islet beta cell. Interestingly, chronic exposure of these cells to hyperglycaemic conditions appears to result in sustained activation of specific Rho GTPases (e.g. Rac1) leading to significant alterations in cellular functions including defects in mitochondrial function and nuclear collapse culminating in beta cell demise. One of the objectives of this review is to highlight our current understanding of the regulatory roles of GTP and Rho GTPases in normal islet function (e.g. proliferation and insulin secretion) as well potential defects in these signalling molecules and metabolic pathways that could contribute islet beta cell dysfunction and loss of functional beta cell mass leading to the onset of diabetes. Potential knowledge gaps in this field and possible avenues for future research are also highlighted. Abbreviations: ARNO: ADP-ribosylation factor nucleotide binding site opener; CML: carboxyl methylation; Epac: exchange protein directly activated by cAMP; ER stress: endoplasmic reticulum stress; FTase: farnesyltransferase; GAP: GTPase activating protein; GDI: GDP dissociation inhibitor; GEF: guanine nucleotide exchange factor; GGTase: geranylgeranyltransferase; GGpp: geranylgeranylpyrophosphate; GGPPS: geranylgeranyl pyrophosphate synthase; GSIS: glucose-stimulated insulin secretion; HGPRTase: hypoxanthine-guanine phosphoribosyltransferase; IMPDH: inosine monophosphate dehydrogenase; α-KIC: α-ketoisocaproic acid; MPA: mycophenolic acid; MVA: mevalonic acid; NDPK: nucleoside diphosphate kinase; NMPK: nucleoside monophosphate kinase; Nox2: phagocyte-like NADPH oxidase; PAK-I: p21-activated kinase-I; β-PIX: β-Pak-interacting exchange factor; PRMT: protein arginine methyltransferase; Rac1: ras-related C3 botulinum toxin substrate 1; Tiam1: T-cell lymphoma invasion and metastasis-inducing protein 1; Trx-1: thioredoxin-1; Vav2: vav guanine nucleotide exchange factor 2

Despite treatment advances, radioresistance and metastasis markedly impair the benefits of radiotherapy to patients with malignancies. Functioning as molecular switches, Rho guanosine triphosphatases (GTPases) have well-recognized roles in regulating various downstream signaling pathways in a wide range of cancers. In recent years, accumulating evidence indicates the involvement of Rho GTPases in cancer radiotherapeutic efficacy and metastasis, as well as radiation-induced metastasis. The functions of Rho GTPases in radiotherapeutic efficacy are divergent and context-dependent; thereby, a comprehensive integration of their roles and correlated mechanisms is urgently needed. This review integrates current evidence supporting the roles of Rho GTPases in mediating radiotherapeutic efficacy and the underlying mechanisms. In addition, their correlations with metastasis and radiation-induced metastasis are discussed. Under the prudent application of Rho GTPase inhibitors based on critical evaluations of biological contexts, targeting Rho GTPases can be a promising strategy in overcoming radioresistance and simultaneously reducing the metastatic potential of tumor cells.

Objective: This study evaluated the involvement of Rho GTPases proteins in the regulation of cytodifferentiation of the SCC-4 human oral squamous cell carcinoma cell line. Methods: Cytokeratin and vimentin immunofluorescence and F-actin staining, assays were performed with control cells and Clostridium difficile 1, 2 and 4 μg/mL Toxin A (Rho GTPases inhibitor) treated SCC-4 cells on three-dimensional MatrigelTM for 24 h. Samples were analyzed by using confocal laser microscopy. Significances were p

Cancer progression and metastasis are processes that involve significant cellular changes. Many of these changes include alterations in the activity of the Rho GTPase family of proteins. Rho GTPases are signaling proteins that function as molecular switches and are involved in the regulation of most major cellular processes. Cancer development is often associated with abnormalities in Rho GTPase signaling. Rho GTPase signaling is regulated by two families of proteins, guanine nucleotide-exchange factors (RhoGEFs) and GTPase activating proteins (RhoGAPs), that function upstream of the Rho proteins to regulate their activation and inactivation, respectively. While initial work has focused on the role of RhoGEFs in cancer, the RhoGAP family members are rapidly being established as key regulators of cancer development and progression. The aim of this review is to summarize our advances in understanding the role of RhoGAPs in cancer and to discuss their significance in the development of therapeutics.

IQ motif-containing GTPase-activating proteins (IQGAPs) modulate a wide range of cellular processes by acting as scaffolds and driving protein components into distinct signaling networks. Their functional states have been proposed to be controlled by members of the RHO family of GTPases, among other regulators. In this study, we show that IQGAP1 and IQGAP2 can associate with CDC42 and RAC1-like proteins but not with RIF, RHOD, or RHO-like proteins, including RHOA. This seems to be based on the distribution of charged surface residues, which varies significantly among RHO GTPases despite their high sequence homology. Although effector proteins bind first to the highly flexible switch regions of RHO GTPases, additional contacts outside are required for effector activation. Sequence alignment and structural, mutational, and competitive biochemical analyses revealed that RHO GTPases possess paralog-specific residues outside the two highly conserved switch regions that essentially determine the selectivity of RHO GTPase binding to IQGAPs. Amino acid substitution of these specific residues in RHOA to the corresponding residues in RAC1 resulted in RHOA association with IQGAP1. Thus, electrostatics most likely plays a decisive role in these interactions.

Memory impairments are associated with many brain disorders such as autism, Alzheimer’s disease, and depression. Forming memories involves modifications of synaptic transmission and spine morphology. The Rho family small GTPases are key regulators of synaptic plasticity by affecting various downstream molecules to remodel the actin cytoskeleton. In this paper, we will review recent studies on the roles of Rho proteins in the regulation of hippocampal long-term potentiation (LTP) and long-term depression (LTD), the most extensively studied forms of synaptic plasticity widely regarded as cellular mechanisms for learning and memory. We will also discuss the involvement of Rho signaling in spine morphology, the structural basis of synaptic plasticity and memory formation. Finally, we will review the association between brain disorders and abnormalities of Rho function. It is expected that studying Rho signaling at the synapse will contribute to the understanding of how memory is formed and disrupted in diseases.

Enhanced invasiveness is one of the defining biological traits of glioblastoma cells, which exhibit an infiltrative nature that severely hinders surgical resection. Among the molecular lesions responsible for GBM aggressiveness, aberrant receptor tyrosine kinase (RTK) signalling is well-characterised. Enhanced RTK signalling directly impacts a myriad of cellular pathways and downstream effectors, which include the Rho GTPase family, key regulators of actin cytoskeletal dynamics. Here, we have analysed the functional crosstalk between oncogenic signals emanating from RTKs and Rho GTPases and focused on the specific contribution of Rnd3 to the invasive phenotype of GBM in this context. We found that RTK inhibition with a panel of RTK inhibitors decreased cell motility and cell invasion and promoted dramatic actin cytoskeleton reorganisation through activation of the RhoA/Rho-associated protein kinase 1 (ROCK) axis. RTK inhibition also significantly decreased Rnd3 expression levels. Consistently, shRNA-mediated Rnd3 silencing revealed that Rnd3 depletion promoted substantial changes in the actin cytoskeleton and reduced cell motility and invasion capacity, recapitulating the effects observed upon RTK inhibition. Our results indicate that Rnd3 is a crucial mediator of RTK oncogenic signalling involved in actin cytoskeletal reorganisation, which contributes to determining the invasive phenotype of GBM cells.

Simple Summary Whilst mutations in genes encoding RHO GTPase proteins are rare in different type of cancers, altered expression of several RHO GTPases has been reported in a variety of human malignancies. As key regulators of gene expression, lncRNAs coordinate a wide range of molecular processes, including post-translational regulation through miRNA sponging. The purpose of the present study was to address the current state of knowledge about the lncRNAs involved in the regulation of the expression of the RHO GTPases including RHOA, RHOB, RHOC, RAC1, and CDC42, with a specific focus on the regulatory mechanism of lncRNAs as the molecular sponges of miRNAs. Considering the critical roles of lncRNAs in malignancies, lncRNA-based therapeutics are representing promising approaches in cancer treatment through novel technologies. In this regard, well-characterized examples of lncRNAs associated with tumorigenicity present experimental frameworks for future studies in this rapidly evolving field. Abstract RHO GTPases are critical signal transducers that regulate cell adhesion, polarity, and migration through multiple signaling pathways. While all these cellular processes are crucial for the maintenance of normal cell homeostasis, disturbances in RHO GTPase-associated signaling pathways contribute to different human diseases, including many malignancies. Several members of the RHO GTPase family are frequently upregulated in human tumors. Abnormal gene regulation confirms the pivotal role of lncRNAs as critical gene regulators, and thus, they could potentially act as oncogenes or tumor suppressors. lncRNAs most likely act as sponges for miRNAs, which are known to be dysregulated in various cancers. In this regard, the significant role of miRNAs targeting RHO GTPases supports the view that the aberrant expression of lncRNAs may reciprocally change the intensity of RHO GTPase-associated signaling pathways. In this review article, we summarize recent advances in lncRNA research, with a specific focus on their sponge effects on RHO GTPase-targeting miRNAs to crucially mediate gene expression in different cancer cell types and tissues. We will focus in particular on five members of the RHO GTPase family, including RHOA, RHOB, RHOC, RAC1, and CDC42, to illustrate the role of lncRNAs in cancer progression. A deeper understanding of the widespread dysregulation of lncRNAs is of fundamental importance for confirmation of their contribution to RHO GTPase-dependent carcinogenesis.

Cytokinesis is required to cleave the daughter cells at the end of mitosis and relies on the spatiotemporal control of RhoA GTPase. Cytokinesis failure can lead to changes in cell fate or aneuploidy, which can be detrimental during development and/or can lead to cancer. However, our knowledge of the pathways that regulate RhoA during cytokinesis is limited, and the role of other Rho family GTPases is not clear. This is largely because the study of Rho GTPases presents unique challenges using traditional cell biological and biochemical methods, and they have pleiotropic functions making genetic studies difficult to interpret. The recent generation of optogenetic tools and biosensors that control and detect active Rho has overcome some of these challenges and is helping to elucidate the role of RhoA in cytokinesis. However, improvements are needed to reveal the role of other Rho GTPases in cytokinesis, and to identify the molecular mechanisms that control Rho activity. This review examines some of the outstanding questions in cytokinesis, and explores tools for the imaging and control of Rho GTPases.

Small Ras superfamily GTPases are highly conserved regulatory factors of fungal cell wall biosynthesis and morphogenesis. Previous experiments have shown that the Rho4-like protein of the maize anthracnose fungus Colletotrichum graminicola, formerly erroneously annotated as a Rho1 protein, physically interacts with the β-1,3-glucan synthase Gls1 (Lange et al., 2014; Curr. Genet. 60:343–350). Here, we show that Rho4 is required for β-1,3-glucan synthesis. Accordingly, Δrho4 strains formed distorted vegetative hyphae with swellings, and exhibited strongly reduced rates of hyphal growth and defects in asexual sporulation. Moreover, on host cuticles, conidia of Δrho4 strains formed long hyphae with hyphopodia, rather than short germ tubes with appressoria. Hyphopodia of Δrho4 strains exhibited penetration defects and often germinated laterally, indicative of cell wall weaknesses. In planta differentiated infection hyphae of Δrho4 strains were fringy, and anthracnose disease symptoms caused by these strains on intact and wounded maize leaf segments were significantly weaker than those caused by the WT strain. A retarded disease symptom development was confirmed by qPCR analyses. Collectively, we identified the Ras GTPase Rho4 as a new virulence factor of C. graminicola.

Cdc42 is a member of the Rho family of small GTPases and a master regulator of the actin cytoskeleton, controlling cell motility, polarity and cell cycle progression. This small G protein and its regulators have been the subject of many years of fruitful investigation and the advent of functional genomics and proteomics has opened up new avenues of exploration including how it functions at specific locations in the cell. This has coincided with the introduction of new structural techniques with the ability to study small GTPases in the context of the membrane. The role of Cdc42 in cancer is well established but the molecular details of its action are still being uncovered. Here we review alterations found to Cdc42 itself and to key components of the signal transduction pathways it controls in cancer. Given the challenges encountered with targeting small G proteins directly therapeutically, it is arguably the regulators of Cdc42 and the effector signalling pathways downstream of the small G protein which will be the most tractable targets for therapeutic intervention. These will require interrogation in order to fully understand the global signalling contribution of Cdc42, unlock the potential for mapping new signalling axes and ultimately produce inhibitors of Cdc42 driven signalling.

The Rho family of GTPases represents highly conserved molecular switches involved in a plethora of physiological processes. Fission yeast Schizosaccharomyces pombe has become a fundamental model organism to study the functions of Rho GTPases over the past few decades. In recent years, another fission yeast species, Schizosaccharomyces japonicus, has come into focus offering insight into evolutionary changes within the genus. Both fission yeasts contain only six Rho-type GTPases that are spatiotemporally controlled by multiple guanine–nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and whose intricate regulation in response to external cues is starting to be uncovered. In the present review, we will outline and discuss the current knowledge and recent advances on how the fission yeasts Rho family GTPases regulate essential physiological processes such as morphogenesis and polarity, cellular integrity, cytokinesis and cellular differentiation.

A lesser known but crucially important downstream effect of Rho family GTPases is the regulation of gene expression. This major role is mediated via the cytoskeleton, the organization of which dictates the nucleocytoplasmic shuttling of a set of transcription factors. Central among these is myocardin-related transcription factor (MRTF), which upon actin polymerization translocates to the nucleus and binds to its cognate partner, serum response factor (SRF). The MRTF/SRF complex then drives a large cohort of genes involved in cytoskeleton remodeling, contractility, extracellular matrix organization and many other processes. Accordingly, MRTF, activated by a variety of mechanical and chemical stimuli, affects a plethora of functions with physiological and pathological relevance. These include cell motility, development, metabolism and thus metastasis formation, inflammatory responses and—predominantly-organ fibrosis. The aim of this review is twofold: to provide an up-to-date summary about the basic biology and regulation of this versatile transcriptional coactivator; and to highlight its principal involvement in the pathobiology of kidney disease. Acting through both direct transcriptional and epigenetic mechanisms, MRTF plays a key (yet not fully appreciated) role in the induction of a profibrotic epithelial phenotype (PEP) as well as in fibroblast-myofibroblast transition, prime pathomechanisms in chronic kidney disease and renal fibrosis.

Chemical and optogenetic methods for post-translationally controlling protein function have enabled new discoveries and the engineering of synthetic cellular functions. However, most of these methods only confer single-input, single-output control. To increase the diversity of post-translational behaviors that can be programmed we built a system based on a single protein receiver that can integrate multiple drug inputs, including approved therapeutics. Our system translates drug inputs into diverse outputs with engineered reader proteins that provide variable dimerization states of the receiver protein. We show that our single receiver protein architecture can be used to program diverse cellular responses, including graded and proportional dual-output control of transcription and mammalian cell signaling. We apply our tools to titrate the competing activities of the Rac and Rho GTPases to control cell morphology. Our receiver protein and suite of reader proteins provides researchers with a versatile toolset to post-translationally program mammalian cellular processes and to engineer cell therapies.

Metastasis is responsible for as many as 90% of cancer-associated deaths in patients. The metastatic process is a result of tumor cell migration and invasion associated with morphological changes and increased expression of several genes involved in cell migration. We have already shown that monocyte chemotactic protein-1-induced protein-1 (MCPIP1), a negative regulator of inflammatory processes, influences cell morphology, prevents the epithelial to mesenchymal transition program, and regulates metastasis in clear cell renal cell carcinoma (ccRCC). However, the mechanism by which MCPIP1 influences cell migratory potential is unknown. In this study, we investigated how MCPIP1 affects ccRCC cell migration. We showed that MCPIP1 prevents morphological transformation and drastically reduces the migration of ccRCC cells. MCPIP1 decreases the levels of Rho GTPases and reduces the phosphorylation of FAK at Tyr-397 and Tyr-861 and Src at Tyr-418. The loss of MCPIP1 RNase activity results in actin remodeling, an increase in the levels of Rho proteins and the phosphorylation of FAK on Tyr-397, which leads to Tyr-418 Src phosphorylation and an increase in migration activity. Moreover, we observed increased expression of IL-1β in ccRCC cells and tumors lacking MCPIP1 RNase activity. Additionally, microarray analysis of tissues from patients with ccRCC revealed changes in the expression of several genes correlated with migration as tumor progression occurred. This study indicates an important role of MCPIP1 as a regulator of migratory potential in ccRCC.

Leakage from blood vessels into tissues is governed by mechanisms that control endothelial barrier function and vascular leakage to maintain homeostasis. Dysregulated endothelial permeability contributes to many conditions and can influence disease morbidity and treatment. Diverse approaches used to study endothelial permeability have yielded a wealth of valuable insights. Yet, ongoing questions, technical challenges, and unresolved controversies relating to the mechanisms and relative contributions of barrier regulation, transendothelial sieving, and transport of fluid, solutes, and particulates complicate interpretations in the context of vascular physiology and pathophysiology. Here, we describe recent in vivo findings and other advances in understanding endothelial barrier function with the goal of identifying and reconciling controversies over cellular and molecular processes that regulate the vascular barrier in health and disease.

Förster resonance energy transfer (FRET) biosensors are popular and useful for directly observing cellular signaling pathways in living cells. Until recently, multiplex imaging of genetically encoded FRET biosensors to simultaneously monitor several protein activities in one cell was limited due to a lack of spectrally compatible FRET pair of fluorescent proteins. With the recent development of miRFP series of near-infrared (NIR) fluorescent proteins, we are now able to extend the spectrum of FRET biosensors beyond blue-green-yellow into NIR. These new NIR FRET biosensors enable direct multiplex imaging together with commonly used cyan-yellow FRET biosensors. We describe herein a method to produce cell lines harboring two compatible FRET biosensors. We will then discuss how to directly multiplex-image these FRET biosensors in living cells. The approaches described herein are generally applicable to any combinations of genetically encoded, ratiometric FRET biosensors utilizing the cyan-yellow and NIR fluorescence.

Cells and tissues are continuously exposed to both chemical and physical stimuli and dy-namically adapt and respond to this variety of external cues to ensure cellular homeostasis, regulated development and tissue‐specific differentiation. Alterations of these pathways promote disease progression—a prominent example being cancer. Rho GTPases are key regulators of the remodeling of cytoskeleton and cell membranes and their coordination and integration with different biological processes, including cell polarization and motility, as well as other signaling networks such as growth signaling and proliferation. Apart from the control of GTP–GDP cycling, Rho GTPase activity is spatially and temporally regulated by post‐translation modifications (PTMs) and their assembly onto specific protein complexes, which determine their controlled activity at distinct cellular compartments. Although Rho GTPases were traditionally conceived as targeted from the cytosol to the plasma membrane to exert their activity, recent research demonstrates that active pools of different Rho GTPases also localize to endomembranes and the nucleus. In this review, we discuss how PTM‐driven modulation of Rho GTPases provides a versatile mechanism for their com-partmentalization and functional regulation. Understanding how the subcellular sorting of active small GTPase pools occurs and what its functional significance is could reveal novel therapeutic opportunities. Spanish Ministerio de Ciencia e Innovación (CSD2009-0016, SAF2014-51876-R, SAF2017-83130-R, BFU2016-81912-REDC, and IGP-SO-MINSEV1512-07-2016); Fundació La Marató de TV3 (674/C/2013 and 201936-30-31); Worldwide Cancer Research Foundation (# 15-0404); Asociación Española Contra el Cáncer (PROYE20089DELP), Fundación Obra social La Caixa (AtheroConvergence, HR20-00075) and Fondo Europeo de Desarrollo Regional and Centro Nacional de Investigaciones Cardiovasculares Carlos III is supported by the Ministerio de Ciencia e Innovación and the Pro CNIC

Rho proteins operate as key regulators of the cytoskeleton, cell morphology and trafficking. Acting as molecular switches, the function of Rho GTPases is determined by guanosine triphosphate (GTP)/guanosine diphosphate (GDP) exchange and their lipidation via prenylation, allowing their binding to cellular membranes and the interaction with downstream effector proteins in close proximity to the membrane. A plethora of in vitro studies demonstrate the indispensable function of Rho proteins for cytoskeleton dynamics within different cell types. However, only in the last decades we have got access to genetically modified mouse models to decipher the intricate regulation between members of the Rho family within specific cell types in the complex in vivo situation. Translationally, alterations of the expression and/or function of Rho GTPases have been associated with several pathological conditions, such as inflammation and cancer. In the context of the GI tract, the continuous crosstalk between the host and the intestinal microbiota requires a tight regulation of the complex interaction between cellular components within the intestinal tissue. Recent studies demonstrate that Rho GTPases play important roles for the maintenance of tissue homeostasis in the gut. We will summarize the current knowledge on Rho protein function within individual cell types in the intestinal mucosa in vivo, with special focus on intestinal epithelial cells and T cells.

Summary: In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) can both promote and suppress tumor progression. Here, we show that the Rho effector protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 is associated with reduced PSC proliferation, contractility, and alpha-smooth muscle actin (α-SMA) stress fibers. In spheroid co-cultures with PDAC cells, loss of PKN2 prevents PSC invasion but, counter-intuitively, promotes invasive cancer cell outgrowth. PKN2 deletion induces a myofibroblast to inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo. Further, deletion of PKN2 in the pancreatic stroma induces more locally invasive, orthotopic pancreatic tumors. Finally, we demonstrate that a PKN2KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast function can limit important stromal tumor-suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.

Cleft lip and/or palate are a split in the lip, the palate or both. This results from the inability of lip buds and palatal shelves to properly migrate and assemble during embryogenesis. By extracting primary cells from a cleft patient, we aimed at offering a better understanding of the signaling mechanisms and interacting molecules involved in the lip and palate formation and fusion. With Rho GTPases being indirectly associated with cleft occurrence, we investigated the role of the latter in both. First, whole exome sequencing was conducted in a patient with cleft lip and palate. Primary fibroblastic cells originating from the upper right gingiva region were extracted and distinct cellular populations from two individuals were obtained: a control with no cleft phenotype and a patient with a cleft lip and palate. The genetic data showed three candidate variables in ARHGEF18, EPDR1, and CUL7. Next, the molecular data showed no significant change in proliferation rates between healthy patient cells and CL/P patient cells. However, CL/P patient cells showed decreased migration, increased adhesion and presented with a more elongated phenotype. Additionally, RhoA activity was upregulated in these cells, whereas Cdc42 activity was downregulated, resulting in loss of polarity. Our results are suggestive of a possible correlation between a dysregulation of Rho GTPases and the observed phenotype of cleft lip and palate patient cells. This insight into the intramolecular aspect of this disorder helps link the genetic defect with the observed phenotype and offers a possible mechanism by which CL/P occurs.

Epac1 (exchange protein activated by cAMP) stabilizes the endothelial barrier, but detailed studies are limited by the side effects of pharmacological Epac1 modulators and transient transfections. Here, we compare the key properties of barriers between endothelial cells derived from wild-type (WT) and Epac1-knockout (KO) mice myocardium. We found that KO cell layers, unlike WT layers, had low and cAMP-insensitive trans-endothelial resistance (TER). They also had fragmented VE-cadherin staining despite having augmented cAMP levels and increased protein expression of Rap1, Rac1, RhoA, and VE-cadherin. The simultaneous direct activation of Rac1 and RhoA by CN04 compensated Epac1 loss, since TER was increased. In KO-cells, inhibition of Rac1 activity had no additional effect on TER, suggesting that other mechanisms compensate the inhibition of the Rac1 function to preserve barrier properties. In summary, Epac1 is crucial for baseline and cAMP-mediated barrier stabilization through mechanisms that are at least partially independent of Rac1.

Neurological and neuropsychiatric disorders are mediated by several pathophysiological mechanisms, including developmental and degenerative abnormalities caused primarily by disturbances in cell migration, structural plasticity of the synapse, and blood-vessel barrier function. In this context, critical pathways involved in the pathogenesis of these diseases are related to structural, scaffolding, and enzymatic activity-bearing proteins, which participate in Ca2+- and Ras Homologs (Rho) GTPases-mediated signaling. Rho GTPases are GDP/GTP binding proteins that regulate the cytoskeletal structure, cellular protrusion, and migration. These proteins cycle between GTP-bound (active) and GDP-bound (inactive) states due to their intrinsic GTPase activity and their dynamic regulation by GEFs, GAPs, and GDIs. One of the most important upstream inputs that modulate Rho GTPases activity is Ca2+ signaling, positioning ion channels as pivotal molecular entities for Rho GTPases regulation. Multiple non-selective cationic channels belonging to the Transient Receptor Potential (TRP) family participate in cytoskeletal-dependent processes through Ca2+-mediated modulation of Rho GTPases. Moreover, these ion channels have a role in several neuropathological events such as neuronal cell death, brain tumor progression and strokes. Although Rho GTPases-dependent pathways have been extensively studied, how they converge with TRP channels in the development or progression of neuropathologies is poorly understood. Herein, we review recent evidence and insights that link TRP channels activity to downstream Rho GTPase signaling or modulation. Moreover, using the TRIP database, we establish associations between possible mediators of Rho GTPase signaling with TRP ion channels. As such, we propose mechanisms that might explain the TRP-dependent modulation of Rho GTPases as possible pathways participating in the emergence or maintenance of neuropathological conditions.

In cell migration, polarization is the process by which a stationary cell breaks symmetry and initiates motion. Although a lot is known about the mechanisms involved in cell polarization, the role played by myosin contraction remains unclear. In addition, cell polarization by mechanical impact has received little attention. Here, we study the influence of myosin activity on cell polarization and the initiation of motion induced by mechanical cues using a computational model for keratocytes. The model accounts for cell deformation, the dynamics of myosin and the signaling protein RhoA (a member of the Rho GTPases family), as well as the forces acting on the actomyosin network. Our results show that the attainment of a steady polarized state depends on the strength of myosin down- or up-regulation and that myosin upregulation favors cell polarization. Our results also confirm the existence of a threshold level for cell polarization, which is determined by the level of polarization of the Rho GTPases at the time the external stimuli vanish. In all, this paper shows that capturing the interactions between the signaling proteins (Rho GTPases for keratocytes) and the compounds of the motile machinery in a moving cell is crucial to study cell polarization.

Typical Rho GTPases include the enzymes RhoA, Rac1, and Cdc42 that act as molecular switches to regulate essential cellular processes in eukaryotic cells such as actomyosin dynamics, cell cycle, adhesion, death and differentiation. Recently, it has been shown that different conditions modulate the activity of these enzymes, but their functions still need to be better understood. Here we examine the interplay between RhoA and the NER (Nucleotide Excision Repair) pathway in human cells exposed to UVA, UVB or UVC radiation. The results show high levels and accumulation of UV-induced DNA lesions (strand breaks and cyclobutane pyrimidine dimers, CPDs) in different cells with RhoA loss of function (LoF), either by stable overexpression of negative dominant RhoA (RhoA-N19 mutant), by inhibition with C3 toxin or by transient silencing with siRNA. Cells under RhoA LoF showed reduced levels of γH2AX, p-Chk1 (Ser345) and p-p53 (Ser15) that reflected causally in their accumulation in G1/S phases, in low survival rates and in reduced cell proliferation, also in accordance with the energy of applied UV light. Even NER-deficient cells (XPA, XPC) or DNA translesion synthesis (TLS)-deficient cells (XPV) showed substantial hypersensitivity to UV effects when previously submitted to RhoA LoF. In contrast, analyses of apoptosis, necrosis, autophagy and senescence revealed that all cells displaying normal levels of active RhoA (RhoA-GTP) are more resistant to UV-promoted cell death. This work reaffirms the role of RhoA protein signaling in protecting cells from damage caused by UV radiation and demonstrates relevant communicating mechanisms between actin cytoskeleton and genomic stability.

Migrating cells need to coordinate distinct leading and trailing edge dynamics but the underlying mechanisms are unclear. Here, we combine experiments and mathematical modeling to elaborate the minimal autonomous biochemical machinery necessary and sufficient for this dynamic coordination and cell movement. RhoA activates Rac1 via DIA and inhibits Rac1 via ROCK, while Rac1 inhibits RhoA through PAK. Our data suggest that in motile, polarized cells, RhoA–ROCK interactions prevail at the rear whereas RhoA-DIA interactions dominate at the front where Rac1/Rho oscillations drive protrusions and retractions. At the rear, high RhoA and low Rac1 activities are maintained until a wave of oscillatory GTPase activities from the cell front reaches the rear, inducing transient GTPase oscillations and RhoA activity spikes. After the rear retracts, the initial GTPase pattern resumes. Our findings show how periodic, propagating GTPase waves coordinate distinct GTPase patterns at the leading and trailing edge dynamics in moving cells.

Epithelial cells form highly organized polarized sheets with characteristic cell morphologies and tissue architecture. Cell–cell adhesion and intercellular communication are prerequisites of such cohesive sheets of cells, and cell connectivity is mediated through several junctional assemblies, namely desmosomes, adherens, tight and gap junctions. These cell–cell junctions form signalling hubs that not only mediate cell–cell adhesion but impact on multiple aspects of cell behaviour, helping to coordinate epithelial cell shape, polarity and function. This review will focus on the tight and adherens junctions, constituents of the apical junctional complex, and aims to provide a comprehensive overview of the complex signalling that underlies junction assembly, integrity and plasticity.

Genetically encoded optogenetic tools are increasingly popular and useful for perturbing signaling pathways with high spatial and temporal resolution in living cells. Here, we show basic procedures employed to implement optogenetics of Rho GTPases in a macrophage cell line. Methods described here are generally applicable to other genetically encoded optogenetic tools utilizing the blue-green spectrum of light for activation, designed for specific proteins and enzymatic targets important for immune cell functions.

Glioblastoma is a highly aggressive brain tumor that is characterized by its unparalleled invasiveness. Invasive glioblastoma cells not only escape surgery and focal therapies but also are more resistant to current radio- and chemo-therapeutic approaches. Thus, any curative therapy for this deadly disease likely should include treatment strategies that interfere with glioblastoma invasiveness. Understanding glioblastoma invasion mechanisms is therefore critical. We discuss the strengths and weaknesses of various glioblastoma invasion models and conclude that robust experimental evidence has been obtained for a pro-invasive role of Ephrin receptors, Rho GTPases, and casein kinase 2 (CK2). Extensive interplay occurs between these proteins, suggesting the existence of a glioblastoma invasion signaling network that comprises several targets for therapy.

The RhoGTPases are characterized as membrane-associated molecular switches that cycle between active, GTP-bound and inactive, GDP-bound states. However, 90–95% of RhoGTPases are maintained in a soluble form by RhoGDI, which is generally viewed as a passive shuttle for inactive RhoGTPases. Our current understanding of RhoGTPase:RhoGDI dynamics has been limited by two experimental challenges: direct visualization of the RhoGTPases in vivo and reconstitution of the cycle in vitro. We developed methods to directly image vertebrate RhoGTPases in vivo or on lipid bilayers in vitro. Using these methods, we identified pools of active and inactive RhoGTPase associated with the membrane, found that RhoGDI can extract both inactive and active RhoGTPases, and found that extraction of active RhoGTPase contributes to their spatial regulation around cell wounds. These results indicate that RhoGDI directly contributes to the spatiotemporal patterning of RhoGTPases by removing active RhoGTPases from the plasma membrane., eLife digest Organisms rely on many signaling molecules to control how their cells grow, divide and heal. For example, when the cell membrane is damaged, two signaling proteins, Rho and Cdc42, are recruited to wounds and activated to promote repair. Active Rho and active Cdc42 form two concentric rings at the membrane to direct the closure of the wound. Rho and Cdc42 belong to the RhoGTPase family, a group of proteins that act as molecular switches and alternate between active and inactive forms. At the level of the cell, RhoGTPases are only active in the tiny patches of the membrane where they bind. However, individual proteins hop on and off membranes in a matter of seconds, only staying bound for short periods. This mechanism is controlled by a regulatory protein known as RhoGDI, and it allows RhoGTPases to form precise patterns of activity at membranes – such as the rings that surround a wound site. However, it was not known exactly how RhoGDI regulates the activity of RhoGTPases over space and time, partly because it is difficult to study these proteins in the laboratory. To fill this knowledge gap, Golding, Visco et al. developed new fluorescent probes to track Rho and Cdc42 in wounded cells from frogs and on artificial membranes. The experiments showed that pools of inactive Cdc42 accumulated on membranes, alongside the active form of the protein. RhoGDI removed both active and inactive RhoGTPases from artificial and frog cell membranes. In fact, removing active Rho and Cdc42 proteins from the cell membrane was necessary to form the spatial patterns of RhoGTPase activity observed in wounded frog cells. The findings of Golding, Visco et al. help to understand how RhoGDI proteins regulate RhoGTPases and provide new tools to further study these proteins. In humans, mutations in either RhoGDI or Cdc42 are responsible for severe conditions such as Nephrotic Syndrome Type 8 or Takenouchi-Kosaki syndrome. In the future, this work may aid the development of treatments and cures for these conditions.

Sphingosine‐1‐phosphate (S1P) is a crucial regulator in vascular inflammation. Our recent study found that under pathophysiological concentration in active anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV), S1P participated in MPO‐ANCA‐positive IgG‐induced glomerular endothelial cell (GEnC) activation via a S1P receptor (S1PR)‐dependent way. However, the downstream signalling pathways are not fully clear yet. In this study, we demonstrated that Rho guanosine triphosphatases (GTPases) signalling pathways, RhoA and Rac1 in particular, were implicated in MPO‐ANCA‐positive IgG‐mediated GEnCs activation enhanced by pathophysiological concentration of S1P in AAV. These results provide mechanistic insights into vascular barrier dysfunction in AAV, which may facilitate the development of effective therapies.

Despite therapeutic advances, resistance to chemotherapy remains a major challenge to patients with malignancies. Rho GTPases are essential for the development and progression of various diseases including cancer, and a vast number of studies have linked Rho GTPases to chemoresistance. Therefore, understanding the underlying mechanisms can expound the effects of Rho GTPases towards chemotherapeutic agents, and targeting Rho GTPases is a promising strategy to downregulate the chemo-protective pathways and overcome chemoresistance. Importantly, exceptions in certain biological conditions and interactions among the members of Rho GTPases should be noted. In this review, we focus on the role of Rho GTPases, particularly Rac1, in regulating chemoresistance and provide an overview of their related mechanisms and available inhibitors, which may offer novel options for future targeted cancer therapy.

Objective— Pulmonary artery smooth muscle cells (PASMCs) from neprilysin (NEP) null mice exhibit a synthetic phenotype and increased activation of Rho GTPases compared with their wild-type counterparts. Although Rho GTPases are known to promote a contractile SMC phenotype, we hypothesize that their sustained activity decreases SM-protein expression in these cells. Approach and Results— PASMCs isolated from wild-type and NEP −/− mice were used to assess levels of SM-proteins (SM-actin, SM-myosin, SM22, and calponin) by Western blotting, and were lower in NEP −/− PASMCs compared with wild-type. Rac and Rho (ras homology family member) levels and activity were higher in NEP −/− PASMCs, and ShRNA to Rac and Rho restored SM-protein, and attenuated the enhanced migration and proliferation of NEP −/− PASMCs. SM-gene repressors, p-Elk-1, and Klf4 (Kruppel lung factor 4), were higher in NEP −/− PASMCs and decreased by shRNA to Rac and Rho. Costimulation of wild-type PASMCs with PDGF (platelet-derived growth factor) and the NEP substrate, ET-1 (endothelin-1), increased Rac and Rho activity, and decreased SM-protein levels mimicking the NEP knock-out phenotype. Activation of Rac and Rho and downstream effectors was observed in lung tissue from NEP −/− mice and humans with chronic obstructive pulmonary disease. Conclusions— Sustained Rho activation in NEP −/− PASMCs is associated with a decrease in SM-protein levels and increased migration and proliferation. Inactivation of RhoGDI (Rho guanine dissociation inhibitor) and RhoGAP (Rho GTPase activating protein) by phosphorylation may contribute to prolonged activation of Rho in NEP −/− PASMCs. Rho GTPases may thus have a role in integration of signals between vasopeptides and growth factor receptors and could influence pathways that suppress SM-proteins to promote a synthetic phenotype.

SummaryThe cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic reorganizations during a variety of essential biological processes including cell adhesion, cell migration, and cell division1,2. During cell division and cell locomotion, for example, waves of filamentous-actin (F-actin) assembly and disassembly develop in the cell cortex in a process termed “cortical excitability”3–7. In developing frog and starfish embryos, cortical excitability is generated through coupled positive and negative feedback, with rapid activation of Rho-mediated F-actin assembly followed in space and time by F-actin-dependent inhibition of Rho8,9. These feedback loops are proposed to serve as a mechanism for amplification of active Rho signaling at the cell equator to support furrowing during cytokinesis, while also maintaining flexibility for rapid error correction in response to movement of the mitotic spindle during chromosome segregation10. In this paper, we develop an artificial cortex based onXenopusegg extract and supported lipid bilayers (SLBs), to investigate cortical Rho and F-actin dynamics11. This reconstituted system spontaneously develops two distinct dynamic patterns: singular excitable Rho and F-actin waves and non-traveling oscillatory Rho and F-actin patches. Both types of dynamic patterns have properties and dependencies similar to the cortical excitability previously characterizedin vivo9. These findings directly support the longstanding speculation that the cell cortex is a self-organizing structure and present a novel approach for investigating mechanisms of Rho-GTPase-mediated cortical dynamics.HighlightsAn artificial cell cortex comprisingXenopusegg extract on a supported lipid bilayer self-organizes into complex, dynamic patterns of active Rho and F-actinWe identified two types of reconstituted cortical dynamics – excitable waves and coherent oscillationsReconstituted waves and oscillations require Rho activity and F-actin polymerization

Gravity plays an important role in normal tissue maintenance. The ability of stem cells to repair tissue loss in space through regeneration and differentiation remains largely unknown. To investigate the impact of microgravity on blood vessel formation from pluripotent stem cells, we employed the embryoid body (EB) model for vasculogenesis and simulated microgravity by clinorotation. We first differentiated mouse embryonic stem cells into cystic EBs containing two germ layers and then analyzed vessel formation under clinorotation. We observed that endothelial cell differentiation was slightly reduced under clinorotation, whereas vascular branch morphogenesis was markedly enhanced. EB-derived endothelial cells migrated faster, displayed multiple cellular processes, and had higher Cdc42 and Rac1 activity when subjected to clinorotation. Genetic analysis and rescue experiments demonstrated that Cdc42 but not Rac1 is required for microgravity-induced vascular branch morphogenesis. Furthermore, affinity pull-down assay and mass spectrometry identified Rap1GDS1 to be a Cdc42 guanine nucleotide exchange factor, which was upregulated by clinorotation. shRNA-mediated knockdown of Rap1GDS1 selectively suppressed Cdc42 activation and inhibited both baseline and microgravity-induced vasculogenesis. This was rescued by ectopic expression of constitutively active Cdc42. Taken together, these results support the notion that simulated microgravity activates Cdc42 via Rap1GDS1 to promote vascular branch morphogenesis.

Rho GTPases such as Rho, Rac and Cdc42 are important regulators of the cortical cytoskeleton in processes including cell division, locomotion and repair. In these processes, Rho GTPases assume characteristic patterns wherein the active GTPases occupy mutually exclusive “zones” in the cell cortex. During cell wound repair, for example, a Rho zone encircles the wound edge and is in turn encircled by a Cdc42 zone. Here we evaluated the contributions of crosstalk between Rho and Cdc42 to the patterning of their respective zones in wounded Xenopus oocytes using experimental manipulations in combination with mathematical modeling. The results show that the position of the Cdc42 zone relative the Rho zone and relative to the wound edge is controlled by the level of Rho activity. In contrast, the outer boundary of the Rho zone is limited by the level of Cdc42 activity. Models based on positive feedback within zones and negative feedback from Rho to the GEF-GAP Abr to Cdc42 capture some, but not all, of the observed behaviors. We conclude that GTPase zone positioning is controlled at the level of Rho activity and we speculate that the Cdc42 zone or something associated with it limits the spread of Rho activity. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Summary Ras GTPases in complex with Guanosine triphosphate (GTP) or GTP analog exhibit dynamic equilibrium between two interconvertible conformations—an inactive state 1 and an active state 2. Unlike Ras, it remains unclear if the GTP-bound form of Rho GTPases also exhibits multiple conformational states. Here, we describe a protocol for structural and biochemical analyses of RhoA GTPase. This protocol can be adapted for the characterization of other Rho GTPases. For details on the use and execution of this protocol, please refer to Lin et al. (2021)., Graphical abstract, Highlights • Purification of wild-type and mutant RhoA in both GDP- and GTP analog-bound forms • Structural analyses of RhoA using both X-ray crystallography and NMR spectrometry • Biochemical assays of RhoA for effector binding and nucleotide exchange, Ras GTPases in complex with GTP or GTP analog exhibit dynamic equilibrium between two interconvertible conformations—an inactive state 1 and an active state 2. Unlike Ras, it remains unclear if the GTP-bound form of Rho GTPases also exhibits multiple conformational states. Here, we describe a protocol for structural and biochemical analyses of RhoA GTPase. This protocol can be adapted for the characterization of other Rho GTPases.

Metastasis is a process by which cancer cells escape from the location of the primary tumor invading normal tissues at distant organs. Chromosomal instability (CIN) is a hallmark of human cancer, associated with metastasis and therapeutic resistance. The centrosome plays a major role in organizing the microtubule cytoskeleton in animal cells regulating cellular architecture and cell division. Loss of centrosome integrity activates the p38-p53-p21 pathway, which results in cell-cycle arrest or senescence and acts as a cell-cycle checkpoint pathway. Structural and numerical centrosome abnormalities can lead to aneuploidy and CIN. New findings derived from studies on cancer and rare genetic disorders suggest that centrosome dysfunction alters the cellular microenvironment through Rho GTPases, p38, and JNK (c-Jun N-terminal Kinase)-dependent signaling in a way that is favorable for pro-invasive secretory phenotypes and aneuploidy tolerance. We here review recent data on how centrosomes act as complex molecular platforms for Rho GTPases and p38 MAPK (Mitogen activated kinase) signaling at the crossroads of CIN, cytoskeleton remodeling, and immune evasion via both cell-autonomous and non-autonomous mechanisms.