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Ras homology (RHO) GTPases are signalling proteins that have crucial roles in triggering multiple immune functions. Through their interactions with a broad range of effectors and kinases, they regulate cytoskeletal dynamics, cell polarity and the trafficking and proliferation of immune cells. The activity and localization of RHO GTPases are highly controlled by classical families of regulators that share consensus motifs. In this Review, we describe the recent discovery of atypical modulators and partners of RHO GTPases, which bring an additional layer of regulation and plasticity to the control of RHO GTPase activities in the immune system. Furthermore, the development of large-scale genetic screening has now enabled researchers to identify dysregulation of RHO GTPase signalling pathways as a cause of many immune system-related diseases. We discuss the mutations that have been identified in RHO GTPases and their signalling circuits in patients with rare diseases. The discoveries of new RHO GTPase partners and genetic mutations in RHO GTPase signalling hubs have uncovered unsuspected layers of crosstalk with other signalling pathways and may provide novel therapeutic opportunities for patients affected by complex immune or broader syndromes.

BACKGROUND: Previous research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking. METHODS: We conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped in vivo, podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting Srgap1. To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; in situ proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated SRGAP1 knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics. RESULTS: We demonstrated SRGAP1 localization to podocyte foot processes in vivo and to cellular protrusions in vitro. Srgap1(fl/fl)*Six2Cre but not Srgap1(fl/fl)*hNPHS2Cre knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of Srgap1 by hNPHS2Cre resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore, SRGAP1-knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery in vitro. Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS. CONCLUSIONS: SRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.

The Rho GTPase family accounts for as many as 20 members. Among them, the archetypes RhoA, Rac1, and Cdc42 have been the most well-characterized. Like all members of the small GTPases superfamily, Rho proteins act as molecular switches to control cellular processes by cycling between active, GTP-bound and inactive, GDP-bound states. The 14-3-3 family proteins comprise seven isoforms. They exist as dimers (homo- or hetero-dimer) in cells. They function by binding to Ser/Thr phosphorylated intracellular proteins, which alters the conformation, activity, and subcellular localization of their binding partners. Both 14-3-3 proteins and Rho GTPases regulate cell cytoskeleton remodeling and cell migration, which suggests a possible interaction between the signaling pathways regulated by these two groups of proteins. Indeed, more and more emerging evidence indicates the mutual regulation of these two signaling pathways. There have been many documented reviews of 14-3-3 protein and Rac1 separately, but there is no review regarding the interaction and mutual regulation of these two groups of proteins. Thus, in this article we thoroughly review all the reported interactions between the signaling pathways regulated by 14-3-3 proteins and Rho GTPases (mostly Rac1).

Rho family GTPases are signaling molecules that orchestrate cytoskeletal dynamics in a variety of cellular processes. Because they effect localized changes to the cytoskeleton only in their active (GTP-bound) conformation, the ability to monitor the active state of Rho GTPases in space and time is critical for understanding their function. Here, we summarize popular tools used for live imaging of active Rho GTPases, outlining advantages and drawbacks of these approaches. Additionally, we highlight key features of the Xenopus laevis embryo that make it well-suited for epithelial cell biology and discuss how application of Rho activity reporters in the Xenopus laevis embryo led to the discovery of a novel phenomenon, junctional Rho flares.

The Ras-related C3 botulinum toxin substrate 1 (Rac1) belongs to the family of Ras-homologous small GTPases. It is well characterized as a membrane-bound signal transducing molecule that is involved in the regulation of cell motility and adhesion as well as cell cycle progression, mitosis, cell death and gene expression. Rac1 also adjusts cellular responses to genotoxic stress by regulating the activity of stress kinases, including c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinases as well as related transcription factors. Apart from being found on the inner side of the outer cell membrane and in the cytosol, Rac1 has also been detected inside the nucleus. Different lines of evidence indicate that genotoxin-induced DNA damage is able to activate nuclear Rac1. The exact mechanisms involved and the biological consequences, however, are unclear. The data available so far indicate that Rac1 might integrate DNA damage independent and DNA damage dependent cellular stress responses following genotoxin treatment, thereby coordinating mechanisms of the DNA damage response (DDR) that are related to DNA repair, survival and cell death.

Phagocytosis is defined as a cellular uptake pathway for particles of greater than 0.5 μm in diameter. Particle clearance by phagocytosis is of critical importance for tissue health and homeostasis. The ultimate goal of anti-pathogen phagocytosis is to destroy engulfed bacteria or fungi and to stimulate cell-cell signaling that mount an efficient immune defense. In contrast, clearance phagocytosis of apoptotic cells and cell debris is anti-inflammatory. High capacity clearance phagocytosis pathways are available to professional phagocytes of the immune system and the retina. Additionally, a low capacity, so-called bystander phagocytic pathway is available to most other cell types. Different phagocytic pathways are stimulated by particle ligation of distinct surface receptors but all forms of phagocytosis require F-actin recruitment beneath tethered particles and F-actin re-arrangement promoting engulfment, which are controlled by Rho family GTPases. The specificity of Rho GTPase activity during the different forms of phagocytosis by mammalian cells is the subject of this review.

SUMMARYMore than half of human tumors exhibit aberrantly dysregulated phosphoinositide signaling, yet how this is controlled remains not fully understood. While somatic mutations of PI3K, PTEN and Ras account for many cases of the hyperactivated lipid signals, other mechanisms for these dysfunctions in cancer are also being discovered. We report here that TNFAIP8 interacts with PtdIns(4,5)P2and PtdIns(3,4,5)P3and is likely to be hijacked by cancer cells to facilitate directional migration during malignant transformation. TNFAIP8 maintains the quiescent cellular state by sequestering inactive Rho GTPases in the cytosolic pool, which can be set free upon chemoattractant activation at the leading edge. Consequently, loss of TNFAIP8 results in severe defects of chemotaxis and adhesion. Thus, TNFAIP8, whose expression can be induced by inflammatory cytokines such as TNFαfrom tumor microenvironment, represents a molecular bridge from inflammation to cancer by linking NF-κB pathway to phosphoinositide signaling. Our study on the conserved hydrophobic cavity structure will also advisein silicodrug screening and development of new TNFAIP8-based strategies to combat malignant human diseases.

Rho GDP dissociation inhibitors (RhoGDIs) play important roles in various cellular processes, including cell migration, adhesion, and proliferation, by regulating the functions of the Rho GTPase family. Dissociation of Rho GTPases from RhoGDIs is necessary for their spatiotemporal activation and is dynamically regulated by several mechanisms, such as phosphorylation, sumoylation, and protein interaction. The expression of RhoGDIs has changed in many human cancers and become associated with the malignant phenotype, including migration, invasion, metastasis, and resistance to anticancer agents. Here, we review how RhoGDIs control the function of Rho GTPases by regulating their spatiotemporal activity and describe the regulatory mechanisms of the dissociation of Rho GTPases from RhoGDIs. We also discuss the role of RhoGDIs in cancer progression and their potential uses for therapeutic intervention.

Rho GTPases are a family of small GTPases, which play an important role in the regulation of the actin cytoskeleton. Not surprisingly, Rho GTPases are crucial for cell migration and therefore highly important for cancer cell invasion and the formation of metastases. In addition, Rho GTPases are involved in growth and survival of tumor cells, in the interaction of tumor cells with their environment, and they are vital for the cancer supporting functions of the tumor stroma. Recent research has significantly improved our understanding of the regulation of Rho GTPase activity, the specificity of Rho GTPases, and their function in tumor stem cells and tumor stroma. This review summarizes these novel findings and tries to define challenging questions for future research.

The ubiquitin ligase RNF8 is known to induce epithelial-to-mesenchymal (EMT) transition and metastasis in triple-negative breast cancer (TNBC). Besides EMT, Rho GTPases have been shown as key regulators in metastasis. In this study, we investigated the role of RNF8 in regulating Rho GTPases and cell motility. We find that RNF8 knockdown in TNBC cells attenuates the protein and mRNA levels of Ras homolog family member A (RHOA) and cell division cycle 42 (CDC42). We show that the formation of filopodia, focal adhesions, and the association of focal adhesions to stress fibers is impaired upon RNF8 knockdown. Cell migration is significantly inhibited by RNF8 knockdown. Our study suggests a potential novel role for RNF8 in mediating cell migration in TNBC through regulation of the Rho GTPases RHOA and CDC42.

Recently, Rho GTPases substrates include Rac (Rac1 and Rac2) and Cdc42 that have been reported to exert multiple cellular functions in osteoclasts, the most prominent of which includes regulating the dynamic actin cytoskeleton rearrangements. In addition, natural products and their molecular frameworks have a long tradition as valuable starting points for medicinal chemistry and drug discovery. Although currently, there are reports about the natural product, which could play a therapeutic role in bone loss diseases (osteoporosis and osteolysis) through the regulation of Rac1/2 and Cdc42 during osteoclasts cytoskeletal structuring. There have been several excellent studies for exploring the therapeutic potentials of various natural products for their role in inhibiting cancer cells migration and function via regulating the Rac1/2 and Cdc42. Herein in this review, we try to focus on recent advancement studies for extensively understanding the role of Rho GTPases substrates Rac1, Rac2 and Cdc42 in osteoclastogenesis, as well as therapeutic potentials of natural medicinal products for their properties on the regulation of Rac1, and/or Rac2 and Cdc42, which is in order to inspire drug discovery in regulating osteoclastogenesis.

Vascular endothelial growth factors (VEGFs) consist of five molecules (VEGFA through D as well as placental growth factor) which are crucial for regulating key cellular and tissue functions. The role of VEGF and its intracellular signaling and downstream molecular pathways have been thoroughly studied. Activation of VEGF signal transduction can be initiated by the molecules’ binding to two classes of transmembrane receptors: (1) the VEGF tyrosine kinase receptors (VEGF receptors 1 through 3) and (2) the neuropilins (NRP1 and 2). The involvement of Rho GTPases in modulating VEGFA signaling in both cancer cells and endothelial cells has also been well established. Additionally, different isoforms of Rho GTPases, namely, RhoA, RhoC, and RhoG, have been shown to regulate VEGF expression as well as blood vessel formation. This review article will explore how Rho GTPases modulate VEGF signaling and the consequences of such interaction on cancer progression.

Memo1deletion in mice causes premature aging and an unbalanced metabolism partially resemblingFgf23andKlotholoss-of-function animals. We report a role for Memo’s redox function in renal FGF23-Klotho signaling using mice with postnatally induced Memo deficiency in the whole body (cKO). Memo cKO mice showed impaired FGF23-driven renal ERK phosphorylation and transcriptional responses. FGF23 actions involved activation of oxidation-sensitive protein phosphotyrosyl phosphatases (PTP) in the kidney. Redox proteomics revealed excessive thiols of Rho-GDP dissociation inhibitor 1 (Rho-GDI1) in Memo cKO, and we detected a functional interaction between Memo’s redox function and oxidation at Rho-GDI1 Cys79. In isolated cellular systems, Rho-GDI1 did not directly affect FGF23-driven cell signaling, but we detected disturbed Rho-GDI1 dependent small Rho-GTPase protein abundance and activity in the kidney of Memo cKO mice. Collectively, this study reveals previously unknown layers in the regulation of renal FGF23 signaling and connects Memo with the network of small Rho-GTPases.

Rho GTPases have many and diverse roles in cell physiology, and some family members are very well studied, including RhoA, Rac1 and Cdc42. But many are relatively neglected, and fundamental questions about their mechanisms and functions remain open.

Neuronal differentiation is a complex biological process accompanying cytoskeletal reorganization, including neurite outgrowth and growth cone formation. Therefore, neuronal differentiation is critically regulated by actin-related signaling proteins, such as small Rho GTPases, guanine nucleotide exchange factors (GEFs), and myosins. This study will demonstrate the change in activity of three small Rho GTPases, Rac, Cdc42, and Rho A, by treatment with blebbistatin (BBS), a specific inhibitor for myosin, during bFGF-induced neurite outgrowth in PC12 cells. Treatment with BBS induced morphological changes in growth cones and neurites during differentiation. A marked increase in protrusion and filopodia structures in growth cones, the shaft of neuritis, and cell membranes was observed in the cells treated with BBS. Activity of Rho GTPases showed the alterations in response to BBS. Activities of both Rac and Rho A were inhibited by BBS in a time-dependent manner. By contrast, Cdc42 activity was not changed by BBS. These results suggest that inactivation of myosin II by BBS induced morphological changes in neurites and growth cones and distinct regulation of three Rho GTPases during differentiation of PC12 cells.

PDZ domains represent one group of the major structural units that mediate protein interactions in intercellular contact, signal transduction and assembly of biological machineries. Tax-interacting protein (TIP)-1 protein is composed of a single PDZ domain that distinguishes TIP-1 from other PDZ domain proteins that more often contain multiple protein domains and function as scaffolds for protein complex assembly. However, the biological functions of TIP-1, especially in cell transformation and tumor progression, are still controversial as observed in a variety of cell types. In this study, we have identified ARHGEF7, a guanine nucleotide exchange factor for Rho GTPases, as one novel TIP-1-interacting protein in human glioblastoma cells. We found that the presence of TIP-1 protein is essential to the intracellular redistribution of ARHGEF7 and rhotekin, one Rho effector and the spatiotemporally coordinated activation of Rho GTPases (RhoA, Cdc42 and Rac1) in migrating glioblastoma cells. TIP-1 knockdown resulted in both aberrant localization of ARHGEF7 and rhotekin, as well as abnormal activation of Rho GTPases that was accompanied with impaired motility of glioblastoma cells. Furthermore, TIP-1 knockdown suppressed tumor cell dispersal in orthotopic glioblastoma murine models. We also observed high levels of TIP-1 expression in human glioblastoma specimens, and the elevated TIP-1 levels are associated with advanced staging and poor prognosis in glioma patients. Although more studies are needed to further dissect the mechanism(s) by which TIP-1 modulates the intracellular redistribution and activation of Rho GTPases, this study suggests that TIP-1 holds potential as both a prognostic biomarker and a therapeutic target of malignant gliomas.

© 2021 by the authors., Rho guanosine triphosphate hydrolases (GTPases) are molecular switches that cycle between an inactive guanosine diphosphate (GDP)-bound and an active guanosine triphosphate (GTP)-bound state during signal transduction. As such, they regulate a wide range of both cellular and physiological processes. In this review, we will summarize recent work on the role of Rho GTPase-regulated pathways in skeletal muscle development, regeneration, tissue mass homeostatic balance, and metabolism. In addition, we will present current evidence that links the dysregulation of these GTPases with diseases caused by skeletal muscle dysfunction. Overall, this information underscores the critical role of a number of members of the Rho GTPase subfamily in muscle development and the overall metabolic balance of mammalian species., The X.R.B.’s lab is funded by the Spanish Association against Cancer (GC16173472GARC), the Castilla-León autonomous government (CSI252P18, CSI145P20, CLC-2017-01), the Spanish Ministry of Science and Innovation (MSI) (RTI2018-096481-B-100), and “la Caixa” Banking Foundation (HR20-00164). X.R.B.’s institution is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Castilla-León autonomous government (CLC-2017-01). SR–F’s contract has been supported by the MISIU (BES–2013–063573) and a fellowship from the Fundación Ramón Areces (BEVP32P01S10090). The funding from both the Spanish and Castilla-León governments has been partially supported by the European Regional Development Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Tunneling nanotubes (TNTs) emerged as important specialized actin-rich membrane protrusions for cell-to-cell communication. These structures allow the intercellular exchange of material, such as ions, soluble proteins, receptors, vesicles and organelles, therefore exerting critical roles in normal cell function. Indeed, TNTs participate in a number of physiological processes, including embryogenesis, immune response, and osteoclastogenesis. TNTs have been also shown to contribute to the transmission of retroviruses (e.g., human immunodeficiency virus-1, HIV-1) and coronaviruses. As with other membrane protrusions, the involvement of Rho GTPases in the formation of these elongated structures is undisputable, although the mechanisms involved are not yet fully elucidated. The tight control of Rho GTPase function by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) strongly suggests that localized control of these Rho regulators may contribute to TNT assembly and disassembly. Deciphering the intricacies of the complex signaling mechanisms leading to actin reorganization and TNT development would reveal important information about their involvement in normal cellular physiology as well as unveil potential targets for disease management.

Astrocytomas are primary malignant brain tumors that originate from astrocytes. Grade IV astrocytoma or glioblastoma is a highly invasive tumor that occur within the brain parenchyma. The Rho family of small GTPases, which includes Rac1, Cdc42, and RhoA, is an important family whose members are key regulators of the invasion and migration of glioblastoma cells. In this review, we describe the role played by the Rho family of GTPases in the regulation of the invasion and migration of glioblastoma cells. Specifically, we focus on the role played by RhoA, Rac1, RhoG, and Cdc42 in cell migration through rearrangement of actin cytoskeleton, cell adhesion, and invasion. Finally, we highlight the importance of potentially targeting Rho GTPases in the treatment of glioblastoma.

The Rho GTPases regulate many cellular signaling cascades that modulate cell motility, migration, morphology and cell division. A large body of work has now delineated the biochemical cues and pathways, which stimulate the GTPases and their downstream effectors. However, cells also respond exquisitely to biophysical and mechanical cues such as stiffness and topography of the extracellular matrix that profoundly influence cell migration, proliferation and differentiation. As these cellular responses are mediated by the actin cytoskeleton, an involvement of Rho GTPases in the transduction of such cues is not unexpected. In this review, we discuss an emerging role of Rho GTPase proteins in the regulation of the responses elicited by biophysical and mechanical stimuli. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Published version

The histone demethylase KDM2B is an epigenetic factor with oncogenic properties that is regulated by the basic fibroblasts growth factor (FGF-2). It has recently been shown that KDM2B co-operates with Polycomb Group proteins to promote cell migration and angiogenesis in tumors. In the present study we addressed the role of KDM2B in regulating actin cytoskeleton signaling, cell-cell adhesion and migration of prostate tumor cells. We report here that KDM2B is functionally expressed in DU-145 prostate cancer cells, activated by FGF-2 and regulates EZH2. KDM2B knockdown induced potent up-regulation of gene transcription and protein expression of the epithelial markers E-cadherin and ZO-1, while KDM2B overexpression down-regulated the levels of both markers, suggesting control of cell adhesion by KDM2B. RhoA and RhoB protein expression and activity were diminished upon KDM2B-knockdown and upregulated in KDM2B-overexpressing cell clones. In accordance, actin reorganization with formation of stress fibers became evident in KDM2B-overexpressing cells and abolished in the presence of the Rho inhibitor C3 transferase. DU-145 cell migration was significantly enhanced in KDM2B overexpressing cells and abolished in C3-pretreated cells. Conversely, the retardation of cell migration observed in KDM2B knockdown cells was enhanced in C3-pretreated cells. These results establish a clear functional link between the epigenetic factor KDM2B and the regulation of cell adhesion and Rho-GTPases signaling that controls actin reorganization and cell migration.

Rho family GTPases belong to the Ras GTPase superfamily and transduce intracellular signals known to regulate a variety of cellular processes, including cell polarity, morphogenesis, migration, apoptosis, vesicle trafficking, viral transport and cellular transformation. The three best-characterized Rho family members are Cdc42, RhoA and Rac1. Cdc42 regulates endocytosis, the transport between the endoplasmic reticulum and Golgi apparatus, post-Golgi transport and exocytosis. Cdc42 influences trafficking through interaction with Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, leading to changes in actin dynamics. Rac1 mediates endocytic and exocytic vesicle trafficking by interaction with its effectors, PI3kinase, synaptojanin 2, IQGAP1 and phospholipase D1. RhoA participates in the regulation of endocytosis through controlling its downstream target, Rho kinase. Interestingly, these GTPases play important roles at different stages of viral protein and genome transport in infected host cells. Importantly, dysregulation of Cdc42, Rac1 and RhoA leads to numerous disorders, including malignant transformation. In some cases, hyperactivation of Rho GTPases is required for cellular transformation. In this article, we review a number of findings related to Rho GTPase function in intracellular transport and cellular transformation.

Myeloid cells form a first tine of defense against infections. They migrate from the circulation to the infected tissues by adhering to and subsequently crossing the vascular wall. This process requires precise control and proper regulation of these interactions with the environment is therefore crucial. Podosomes are the most prominent adhesion structures in myeloid cells. Podosomes control both the adhesive and migratory properties of myeloid cells and the regulation of podosomes is key to the proper functioning of these cells. Here we discuss the regulation of podosomes by Rho GTPases, well known regulators of adhesion and migration, focusing on myeloid cells. In addition, the regulation of podosomes by GTPase regulators such as GEFs and GAPs, as well as the effects of some Rho GTPase effector pathways, will be discussed. (C) 2010 Elsevier GmbH. All rights reserved

Pollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is enriched in the subapical plasma membrane of pollen tubes of Arabidopsis thaliana and tobacco (Nicotiana tabacum) and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P2 effects are specified is unclear. In tobacco pollen tubes, spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P2-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labeling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P2. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P2 in pollen tubes. Despite localizing to one membrane region, the PIP5Ks AtPIP5K2-EYFP and NtPIP5K6-EYFP display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2-EYFP and NtPIP5K6-EYFP variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, active PI4P 5-kinase variants stabilized actin when targeted to nanodomains, suggesting a role of nanodomain-associated PtdIns(4,5)P2 in actin regulation. This notion is further supported by interaction and proximity of nanodomain-associated AtPIP5K2 with the Rho-GTPase NtRac5, and by its functional interplay with elements of Rho of plants signaling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P2 functions to coordinate cytoskeletal dynamics and secretion.

Peroxisomes are essential for a number of cellular functions, including reactive oxygen species metabolism, fatty acid β‐oxidation and lipid synthesis. To ensure optimal functionality, peroxisomal size, shape and number must be dynamically maintained; however, many aspects of how this is regulated remain poorly characterised. Here, we show that the localisation of Miro1 and Miro2—outer mitochondrial membrane proteins essential for mitochondrial trafficking—to peroxisomes is not required for basal peroxisomal distribution and long‐range trafficking, but rather for the maintenance of peroxisomal size and morphology through peroxisomal fission. Mechanistically, this is achieved by Miro negatively regulating Drp1‐dependent fission, a function that is shared with the mitochondria. We further find that the peroxisomal localisation of Miro is regulated by its first GTPase domain and is mediated by an interaction through its transmembrane domain with the peroxisomal‐membrane protein chaperone, Pex19. Our work highlights a shared regulatory role of Miro in maintaining the morphology of both peroxisomes and mitochondria, supporting a crosstalk between peroxisomal and mitochondrial biology., Miro1 and Miro2 localise to peroxisomes and negatively regulate peroxisomal fission rather than long‐range transport and distribution.

Primary immunodeficiencies (PIDs) are associated with deleterious mutations of genes that encode proteins involved in actin cytoskeleton reorganisation. This deficiency affects haematopoietic cells. PID results in the defective function of immune cells, such as impaired chemokine-induced motility, receptor signalling, development and maturation. Some of the genes mutated in PIDs are related to small Ras homologous (Rho) guanosine triphosphatase (GTPase), one of the families of the Ras superfamily. Most of these genes act as molecular switches by cycling between active guanosine triphosphate-bound and inactive guanosine diphosphate-bound forms to control multiple cellular functions. They are best studied for their role in promoting cytoskeleton reorganisation, cell adhesion and motility. Currently, only three small Rho GTPases, namely, Rac2, Cdc42 and RhoH, have been identified in PIDs. However, several other Rho small G proteins might also contribute to the deregulation and phenotype observed in PIDs. Their contribution in PIDs may involve their main regulator, Rho guanine nucleotide exchange factors such as DOCK2 and DOCK8, wherein mutations may result in the impairment of small Rho GTPase activation. Thus, this review outlines the potential contribution of several small Rho GTPases to the promotion of PIDs.

International audience; Cells from the myeloid lineage, namely macrophages, dendritic cells and osteoclasts, develop podosomes instead of stress fibers and focal adhesions to adhere and migrate. Podosomes share many components with focal adhesions but differ in their molecular organization, with a dense core of polymerized actin surrounded by scaffolding proteins, kinases and integrins. Podosomes are found either isolated both in macrophages and dendritic cells or arranged into superstructures in osteoclasts. When osteoclasts resorb bone, they form an F-actin rich sealing zone, which is a dense array of connected podosomes that firmly anchors osteoclasts to bone. It delineates a compartment in which protons and proteases are secreted to dissolve and degrade the mineralized matrix. Since Rho GTPases have been shown to control F-actin stress fibers and focal adhesions in mesenchymal cells, the question of whether they could also control podosome formation and arrangement in cells from the myeloid lineage, and particularly in osteoclasts, rapidly emerged. This article considers recent advances made in our understanding of podosome arrangements in osteoclasts and how Rho GTPases may control it.

Epithelial tissues are defined by polarized epithelial cells that are integrated into tissues and exhibit barrier function in order to regulate what is allowed to pass between cells. Cell-cell junctions must be stable enough to promote barrier function and tissue integrity, yet plastic enough to remodel when necessary. This remarkable ability to dynamically sense and respond to changes in cell shape and tissue tension allows cell-cell junctions to remain functional during events that disrupt epithelial homeostasis including morphogenesis, wound healing, and cell division. In order to achieve this plasticity, both tight junctions and adherens junctions are coupled to the underlying actomyosin cytoskeleton. Here, we discuss the importance of the junctional linkage to actomyosin and how a localized zone of active RhoA along with other Rho GTPases work together to orchestrate junctional actomyosin dynamics. We focus on how scaffold proteins help coordinate Rho GTPases, their upstream regulators, and their downstream effectors for efficient, localized Rho GTPase signaling output. Additionally, we highlight important roles junctional actin-binding proteins play in addition to their traditional roles in organizing actin. Together, Rho GTPases, their regulators, and effectors form compartmentalized signaling modules that regulate actomyosin structure and contractility to achieve proper cell-cell adhesion and tissue barriers.

Rho GTPases family members influenced the filopodia, lamellipodia, stress fiber and adhesion plaque of melanoma cells through regulating cytoskeleton recombination. The role of Rho GTPases family in the migration and invasion of melanoma and its molecular mechanism were explored. The morphological difference between three types of melanoma cells (M14, A375 and MV3) and human melanocyte (MC) was observed by the Hoffman microscope. Cells were stained by phalloidin labeled by rhodamine. The differences between 4 types of cells in filopodia, lamellipodia, stress fiber and adhesion plaque (microfilament is the main constituent) were observed under the super-high resolution microscope. The migration ability of 4 types of cells was detected by Transwell migration assay. QPCR was used to detect the mRNA transcription level of Rho GTPases family. WB was adopted to detect the expression of RhoD and DIAPH2 proteins. There were significant differences in filopodia, lamellipodia, stress fiber and adhesion plaque between MC and 3 types of melanoma cells (M14, A375 and MV3). MC did not have stress fiber or adhesion plaque, while M14, A375 and MV3 had stress fiber and adhesion plaque. All 4 types of cells had thin and short filopodia. MV3 had fewer but thicker stress fibers than the latter two. Transwell migration test indicated the followings: M14 and A375 had a similar high migration rate; the migration rate of MV3 was slightly low; MC did not have the ability of transmembrane migration. QPCR results of Rho GTPases family in 4 types of cells showed the change corresponding to immunofluorescence. WB results showed that RhoD was barely expressed in M14, A375 or MV3. DIAPH2, the downstream effector molecule of RhoD, had the corresponding change. Rho GTPases influences the migration and invasion of melanoma cells through regulating filopodia, lamellipodia, stress fiber and adhesion plaque (microfilament is the main constituent).

Microtubule (MT) organization and dynamics downstream of external cues is crucial for maintaining cellular architecture and the generation of cell asymmetries. In interphase cells RhoA, Rac, and Cdc42, conspicuous members of the family of small Rho GTPases, have major roles in modulating MT stability, and hence polarized cell behaviors. However, MTs are not mere targets of Rho GTPases, but also serve as signaling platforms coupling MT dynamics to Rho GTPase activation in a variety of cellular conditions. In this article, we review some of the key studies describing the reciprocal relationship between small Rho-GTPases and MTs during migration and polarization. Fil: Wojnacki Fonseca, José Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Quassollo Infanzon, Gonzalo Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Marzolo, Maria Paz. Pontificia Universidad Católica de Chile; Chile Fil: Caceres, Alfredo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina

Cell migration and invasion play an important role in the development of cancer. Cell migration is associated with several specific actin filament-based structures, including lamellipodia, filopodia, invadopodia and blebs, and with cell-cell adhesion, cell-extracellular matrix adhesion. Migration occurs via different modes, human epithelial cancer cells mainly migrate collectively, while in vivo imaging studies in laboratory animals have found that most cells migrate as single cells. Rho GTPases play an important role in the process of cell migration, and several Rho GTPase-related signaling complexes are also involved. However, the exact mechanism by which these signaling complexes act remains unclear. This paper reviews how Rho GTPases and related signaling complexes interact with other proteins, how their expression is regulated, how tumor microenvironment-related factors play a role in invasion and metastasis, and the mechanism of these complex signaling networks in cell migration and invasion.

Neuronal migration is essential for the orchestration of brain development and involves several contiguous steps: interkinetic nuclear movement (INM), multipolar–bipolar transition, locomotion, and translocation. Growing evidence suggests that Rho GTPases, including RhoA, Rac, Cdc42, and the atypical Rnd members, play critical roles in neuronal migration by regulating both actin and microtubule cytoskeletal components. This review focuses on the spatiotemporal-specific regulation of Rho GTPases as well as their regulators and effectors in distinct steps during the neuronal migration process. Their roles in bridging extracellular signals and cytoskeletal dynamics to provide optimal structural support to the migrating neurons will also be discussed.

Rho guanosine triphosphatases (GTPases) are key regulators in a number of cellular functions, including actin cytoskeleton remodeling and vesicle traffic. Traditionally, Rho GTPases are studied because of their function in cell migration and cancer, while their roles in metabolism are less documented. However, emerging evidence implicates Rho GTPases as regulators of processes of crucial importance for maintaining metabolic homeostasis. Thus, the time is now ripe for reviewing Rho GTPases in the context of metabolic health. Rho GTPase-mediated key processes include the release of insulin from pancreatic β-cells, glucose uptake into skeletal muscle and adipose tissue, and muscle mass regulation. Through the current review, we cast light on the important role of Rho GTPases in skeletal muscle, adipose tissue, and the pancreas and mechanisms by which Rho GTPases act to regulate glucose metabolism in health and disease. We also describe challenges and goals for future research.

The posttranslational lipid modification of Rho GTPases is important for their proper subcellular localization and signal transduction. Rho GTPases terminate in a CaaX motif, in which the cysteine residue is modified with either a farnesyl or geranylgeranyl isoprenoid. RhoGDI renders Rho GTPases soluble by masking their lipid moieties. We recently identified that the brain-specific splice variant of Cdc42 (bCdc42) containing a noncanonical CCaX motif harbors a dual prenyl-palmitoyl modification that prevents its binding to RhoGDI. This chapter describes a method to analyze RhoGDI extraction of Rho GTPases containing different lipid modifications from membranes using a liposome reconstitution assay and click chemistry.

Rho GTPases, which control processes such as cell proliferation and cytoskeleton remodeling, are often hyper-expressed 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.

In the version of this article originally published, the values for time shown on the x axis of Figure 5c were incorrect. The error has been corrected in all versions of the paper.

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.

The following collection of reviews concerning Rho GTPases was elaborated in the perspective of a special anniversary. 30 years ago, Pascal Madaule, postdoc in Richard Axel's laboratory at Columbia...

Accumulating evidence suggests that Rho GTPases, together with scaffolding SHANK proteins, and associated signaling pathways play a role in the development of autism symptoms in various conditions. Research data have brought information on multiple intracellular signaling pathways, including Rho-associated protein kinases and serine/threonine-protein kinases involved in cytoskeleton rearranging. Alterations in downstream effectors of GTPase signaling pathways are associated with neurodevelopmental disorders. Bioinformatics and experimental data show that complex genetic and molecular defects (GTPases, actin-binding proteins, kinases, neuropeptides) can result in neuronal remodeling, leading to the functional connectivity deficits that manifest as the heterogeneous autism spectrum phenotype. Finally, the known hormone and neuropeptide oxytocin appears to be a factor for consideration in therapeutic intervention.

Neutrophils are short-lived, abundant peripheral blood leukocytes that provide a first line of defense against bacterial and fungal infections whilst also being a key part of the inflammatory response. Chemokines induce neutrophil recruitment to inflammatory sites, where neutrophils perform a number of diverse functions that are aimed at fighting infections. Neutrophil effector functions are tightly regulated processes that are governed by an array of intracellular signaling pathways and initiated by receptor-ligand binding events. Dysregulated, neutrophil activation can result in excessive inflammation and host damage, as is evident in a number of autoimmune diseases. Rho family small GTPases and agonist-activated phosphoinositide 3-kinases (PI3Ks) represent two classes of key regulators of the highly specialized neutrophil. Here we review cross-talk between these important signaling intermediates in the context of neutrophil functions. We include PI3K-dependent activation of Rho family small GTPases and of their guanine nucleotide exchange factors and GTPase activating proteins, as well as Rho GTPase-dependent regulation of PI3K.

Members of the Rho family of GTPases act as molecular switches that control a variety of signal transduction pathways. Although discovered in 1985, it was not until the early 1990s that cellular functions were assigned to Rho GTPases. The finding that Rho family proteins regulate actin dynamics revolutionized the field of cell biology, and opened the doors to studying Rho GTPases in a variety of cellular contexts. Over the years, new members of the Rho family of GTPases have been identified as well as their impact on new areas of investigation including cell migration, cancer, microbial infection, developmental biology, and neurobiology.

We recently described the atypical Rho GTPases Miro-1 and Miro-2. These proteins have tandem GTP-binding domains separated by a linker region with putative calcium-binding motives. In addition, the Miro GTPases have a C-terminal transmembrane domain, which confers targeting to the mitochondria. It was reported previously that a constitutively active mutant of Miro-1 induced a clustering of the mitochondria. This response can be separated into two distinct phenotypes: a formation of aggregated mitochondria and the appearance of thread-like mitochondria probably caused by defects in mitochondrial trafficking. The first GTPase domain is required for the clustering of the mitochondria, but the effect is not dependent on the EF-hands. Miro-2 only induces aggregation and not the formation of thread-like mitochondria. Moreover, we show that Miro interacts with the Kinesin-binding proteins, GRIF-1 and OIP106, suggesting that the Miro GTPases form a link between the mitochondria and the trafficking apparatus of the microtubules.

Certain bacterial toxins and type-III-translocated virulence factors have a peculiar property: they exert part of their actions by modulating Rho GTPases. These toxins target the actin cytoskeleton of host cells and reorganize it to their own advantage, either to facilitate macropinocytosis, which is required for invasive bacteria to enter cells, or to block pathogen sequestration by macrophages. In addition, by acting on Rho GTPases, bacteria may also interfere with the fate of host cells, favoring survival or death depending on their needs. Rho GTPases control the activation of NF-kappaB, which is involved in the expression of antiapoptotic proteins and mediates immunological responses as well. Here, we give a perspective on how NF-kappaB may participate in linking Rho-acting toxins and apoptosis.

Oncogenic protein kinase C epsilon (PKCe) promotes the formation of membrane ruffles and motility in non-small cell lung cancer (NSCLC) cells. We found that PKCe is down-regulated when NSCLC cells undergo epithelial-to-mesenchymal transition (EMT) in response to TGF-β, thus becoming dispensable for migration and invasion in the mesenchymal state. PKCe silencing or inhibition leads to stress fibre formation, suggesting that this kinase negatively regulates RhoA activity. Ruffle formation induced by PKCe activation in the epithelial state is dependent on PI3K, but does not involve the PI3K-dependent Rac-GEFs Ect2, Trio, Vav2 or Tiam1, suggesting alternative Rac-GEFs as mediators of this response. In the proposed model, PKCe acts as a rheostat for Rho GTPases that differs in the epithelial and mesenchymal states.

During development, growth cones are essential for axon pathfinding by sensing numerous guidance cues in their environment. Retinoic acid, the metabolite of vitamin A, is important for neurite outgrowth during vertebrate development, but may also play a role in axon guidance, though little is known of the cellular mechanisms involved. Our previous studies showed that retinoid-induced growth cone turning of invertebrate motorneurons requires local protein synthesis and calcium influx. However, the signalling pathways that link calcium influx to cytoskeletal dynamics involved in retinoid-mediated growth cone turning are not currently known. The Rho GTPases, Cdc42 and Rac, are known regulators of the growth cone cytoskeleton. Here, we demonstrated that inhibition of Cdc42 or Rac not only prevented growth cone turning toward retinoic acid but could also induce a switch in growth cone responsiveness to chemorepulsion or growth cone collapse. However, the effects of Cdc42 or Rac inhibition on growth cone responsiveness differed, depending on whether the turning was induced by the all-trans or 9-cis retinoid isomer. The effects also differed depending on whether the growth cones maintained communication with the cell body. These data strongly suggest that Cdc42 and Rac are downstream effectors of retinoic acid during growth cone guidance.

Malignant gliomas are one of the most aggressive and deadly forms of cancer and can affect any age group. In glioblastoma multiforme (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Patients with malignant gliomas respond poorly to the standard therapeutic regimen of radiotherapy and chemotherapy that follow tumor resection and have only a 16-month median survival. It is therefore imperative to identify new approaches to specifically attack GBM cell survival, proliferation and invasion. The cellular mechanisms driving GBM-mediated migration and invasion are not fully understood. RSK2 (p90 ribosomal S6 kinase 2) is a kinase that regulates proliferation and adhesion and can promote metastasis. We find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. We demonstrate that active RSK2 regulates GBM adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. We further show that the effects of RSK2 on GBM invasion are mediated in part through activation of Rho GTPases. Rho family of GTPases are key regulators of cell polarity, migration and invasion. Here, we identify Rho A, B and C as downstream effectors of (RSK2) in GBM cell migration and invasion. Here, we identified a unique signaling pathway in which RSK2 confers invasiveness through leukemia-associated RhoGEF (LARG)-dependent Rho GTPase activation. RSK2 directly interacts with LARG and nucleotide-bound Rho isoforms, but not Rac1 and Cdc42. Our data support a fundamental importance of residue of Thr577 to the invasive signaling of RSK2. The invasive signaling of RSK2 appears to depend on RhoA and B, but not RhoC. RSK2 activates RhoA through activation of LARG by phosphorylation at Ser1288. Intriguingly, LARG activity is only required for RSK2-T577E, but not RSK2-Y707A-mediated RhoA activation and cell invasion. This further supports a pivotal role of Thr577 in invasive RSK2 signaling. These results establish a unique RSK2-dependent LARG-Rho signaling cascade as an uncompensated factor key to GBM cell migration and invasion. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy. Citation Format: Geng-Xian Shi, Ling Jin, Michelle L. Matter, Santosh Kesari, Joe W. Ramos. RSK2 provokes invasive signaling in glioblastoma through LARG-dependent activation of Rho GTPases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1363. doi:10.1158/1538-7445.AM2017-1363

Dendritic morphology has an important influence on neuronal information processing. Multiple environmental cues, including neuronal activity, the neurotrophin family of growth factors, and extracellular guidance molecules have been shown to influence dendritic size, shape, and development. The Rho GTPases have emerged as key integrators of these environmental cues to regulate the underlying dendritic cytoskeleton.

Since their discovery in the late eighties, the role of Rho GTPases in the regulation of cell migration has been extensively studied and has mainly focused on the hallmark family members Rho, Rac, and Cdc42. Recent technological advances in cell biology, such as Rho-family GTPase activity biosensors, studies in 3D, and unbiased RNAi-based screens, have revealed an increasingly complex role for Rho GTPases during cell migration, with many inter-connected functions and a strong dependency on the physical and chemical properties of the surrounding environment. This review aims to give an overview of recent studies on the role of Rho-family GTPase members in the modulation of cell migration in different environments, and discuss future directions.

Alzheimer's disease (AD) is characterized by a wide loss of synapses and dendritic spines. Despite extensive efforts, the molecular mechanisms driving this detrimental alteration have not yet been determined. Among the factors potentially mediating this loss of neuronal connectivity, the contribution of Rho GTPases is of particular interest. This family of proteins is classically considered a key regulator of actin cytoskeleton remodeling and dendritic spine maintenance, but new insights into the complex dynamics of its regulation have recently determined how its signaling cascade is still largely unknown, both in physiological and pathological conditions. Here, we review the growing evidence supporting the potential involvement of Rho GTPases in spine loss, which is a unanimously recognized hallmark of early AD pathogenesis. We also discuss some new insights into Rho GTPase signaling framework that might explain several controversial results that have been published. The study of the connection between AD and Rho GTPases represents a quite unchartered avenue that holds therapeutic potential.