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Rho GTPases, master spatial regulators of a wide range of cellular processes, are orchestrated by complex formation with guanine nucleotide dissociation inhibitors (RhoGDIs). These have been thought to possess an unstructured N‐terminus that inhibits nucleotide exchange of their client upon binding/folding. Via NMR analyses, molecular dynamics simulations, and biochemical assays, we reveal instead pertinent structural properties transiently maintained both, in the presence and absence of the client, imposed onto the terminus context‐specifically by modulating interactions with the surface of the folded C‐terminal domain. These observations revise the long‐standing textbook picture of the GTPases' mechanism of membrane extraction. Rather than by a disorder‐to‐order transition upon binding of an inhibitory peptide, the intricate and highly selective extraction process of RhoGTPases is orchestrated via a dynamic ensemble bearing preformed transient structural properties, suitably modulated by the specific surrounding along the multi‐step process. [ABSTRACT FROM AUTHOR]

The mevalonate pathway plays an important role in breast cancer and other tumor types. However, many issues remain obscure as yet regarding its mechanism of regulation and action. In the present study, we report that the expression of mevalonate pathway enzymes is mediated by the RHO guanosine nucleotide exchange factors VAV2 and VAV3 in a RAC1‐ and sterol regulatory element‐binding factor (SREBF)‐dependent manner in breast cancer cells. Furthermore, in vivo tumorigenesis experiments indicated that the two most upstream steps of this metabolic pathway [3‐hydroxy‐3‐methylglutaryl‐coenzyme A synthase 1 (HMGCS1) and 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (HMGCR)] are important for primary tumorigenesis, angiogenesis, and cell survival in breast cancer cells. HMGCR, but not HMGCS1, is also important for the extravasation and subsequent fitness of breast cancer cells in the lung parenchyma. Genome‐wide expression analyses revealed that HMGCR influences the expression of gene signatures linked to proliferation, metabolism, and immune responses. The HMGCR‐regulated gene signature predicts long‐term tumor recurrence but not metastasis in cohorts of nonsegregated and chemotherapy‐resistant breast cancer patients. These results reveal a hitherto unknown, VAV‐catalysis‐dependent mechanism involved in the regulation of the mevalonate pathway in breast cancer cells. They also identify specific mevalonate‐pathway‐dependent processes that contribute to the malignant features of breast cancer cells. [ABSTRACT FROM AUTHOR]

Metastatic cutaneous melanoma is a fatal skin cancer. Resistance to targeted and immune therapies limits the benefits of current treatments. Identifying and adding anti‐resistance agents to current treatment protocols can potentially improve clinical responses. Myocardin‐related transcription factor (MRTF) is a transcriptional coactivator whose activity is indirectly regulated by actin and the Rho family of GTPases. We previously demonstrated that development of BRAF inhibitor (BRAFi) resistance frequently activates the Rho/MRTF pathway in human and mouse BRAFV600E melanomas. In clinical trials, pretreatment with BRAFi reduces the benefit of immune therapies. We aimed to test the efficacy of concurrent treatment with our MRTF pathway inhibitor CCG‐257081 and anti‐PD1 in vivo and to examine its effects on the melanoma immune microenvironment. Because MRTF pathway activation upregulates the expression of immune checkpoint inhibitor genes/proteins, we asked whether CCG‐257081 can improve the response to immune checkpoint blockade. CCG‐257081 reduced the expression of PDL1 in BRAFi‐resistant melanoma cells and decreased surface PDL1 levels on both BRAFi‐sensitive and ‐resistant melanoma cells. Using our recently described murine vemurafenib‐resistant melanoma model, we found that CCG‐257081, in combination with anti‐PD1 immune therapy, reduced tumor growth and increased survival. Moreover, anti‐PD1/CCG‐257081 co‐treatment increased infiltration of CD8+ T cells and B cells into the tumor microenvironment and reduced tumor‐associated macrophages. Here, we propose CCG‐257081 as an anti‐resistance and immune therapy‐enhancing anti‐melanoma agent. [ABSTRACT FROM AUTHOR]

The small Rho GTP‐binding proteins are important cell morphology, function, and apoptosis regulators. Unlike other Rho proteins, RhoB can be subjected to either geranylgeranylation (RhoB‐GG) or farnesylation (RhoB‐F), making that the only target of the farnesyltransferase inhibitor (FTI). Fluorescence resonance energy transfer experiments revealed that RhoB is activated by hyperosmolarity. By contrast, hyposmolarity did not affect RhoB activity. Interestingly, treatment with farnesyltransferase inhibitor‐277 (FTI‐277) decreased the cell size. To evaluate whether RhoB plays a role in volume reduction, renal collecting duct MCD4 cells and Human Kidney, HK‐2 were transiently transfected with RhoB‐wildtype‐Enhance Green Fluorescence Protein (RhoB‐wt‐EGFP) and RhoB‐CLLL‐EGFP which cannot undergo farnesylation. A calcein‐based fluorescent assay revealed that hyperosmolarity caused a significant reduction of cell volume in mock and RhoB‐wt‐EGFP‐expressing cells. By contrast, cells treated with FTI‐277 or expressing the RhoB‐CLLL‐EGFP mutant did not properly respond to hyperosmolarity with respect to mock and RhoB‐wt‐EGFP expressing cells. These findings were further confirmed by 3D‐LSCM showing that RhoB‐CLLL‐EGFP cells displayed a significant reduction in cell size compared to cells expressing RhoB‐wt‐EGFP. Moreover, flow cytometry analysis revealed that RhoB‐CLLL‐EGFP expressing cells as well as FTI‐277‐treated cells showed a significant increase in cell apoptosis. Together, these data suggested that: (i) RhoB is sensitive to hyperosmolarity and not to hyposmolarity; (ii) inhibition of RhoB farnesylation associates with an increase in cell apoptosis, likely suggesting that RhoB might be a paramount player controlling apoptosis by interfering with responses to cell volume change. [ABSTRACT FROM AUTHOR]

Renewable energy resources such as biomass are crucial for a sustainable global society. Trees are a major source of lignocellulosic biomass, which can vary in response to different environmental factors owing to epigenetic regulation, such as DNA C‐methylation. To investigate the effects of DNA methylation on plant development and wood formation, and its impacts on gene expression, with a focus on secondary cell wall (SCW)‐associated genes, Salix purpurea plantlets were cloned from buds derived from a single hybrid tree for both treatment and control conditions. For the treatment condition, buds were exposed to 50 μM zebularine in vitro and a combined strategy of whole‐genome bisulfite sequencing (WGBS) and RNA‐seq was employed to examine the methylome and transcriptome profiles of different tissues collected at various time points under both conditions. Transcriptomic and methylome data revealed that most of the promoter and gene body demethylation had no marked effects on the expression profiles of genes. Nevertheless, gene expression tended to decrease with the increased methylation levels of genes with highly methylated promoters. Results indicated that demethylation is less evident in centromeric regions and sex chromosomes. Promoters of secondary cell wall‐associated genes, such as 4‐coumarate‐CoA ligase‐like and Rac‐like GTP‐binding protein RHO, were differentially methylated in the secondary xylem samples collected from two‐month potted treated plants compared to control samples. Our results provide novel insights into DNA methylation and gene expression landscapes and a basis for investigating the epigenetic regulation of wood formation in S. purpurea as a model plant for bioenergy species. [ABSTRACT FROM AUTHOR]

Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN‐7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella‐containing vacuoles after long‐term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread. [ABSTRACT FROM AUTHOR]

Basal cell carcinoma (BCC) is the most common malignant tumour arising from the basal cells of the epidermis or follicular structures. The aetiology of BCC is a multifactorial combination of genotype, phenotype and environmental factors. The pathogenesis of BCC remains unclear, with diverse and complex signalling pathways involved. ARHGAP40 is a Rho GTPase‐activating protein (RhoGAP). Rho GTPases play a crucial role in the formation and progression of numerous cancers. The expression levels and roles of ARHGAP40 in BCC have not been explored. Here, ARHGAP40 expression was detected in a set of formalin‐fixed, paraffin‐embedded (FFPE) samples of basal cell carcinoma, paracancerous normal skin and benign skin lesions. The epigenetic mechanism that downregulates ARHGAP40 in basal cell carcinoma was investigated. We found that ARHGAP40 is expressed in normal basal cells and most benign skin lesions but lost in most basal cell carcinomas. We detected CpG island hypermethylation at the promoter‐associated region of ARHGAP40. Our data suggest that ARHGAP40 is downregulated in BCC due to hypermethylation. ARHGAP40 protein is a potential novel biomarker for distinguishing trichoblastoma from BCC. This report is preliminary, and extensive research into the role of ARHGAP40 in BCC carcinogenesis and its potential as a treatment target is required in the future. [ABSTRACT FROM AUTHOR]

The bacterial product CNF1, through its action on the Rho GTPases, is emerging as a modulator of crucial signalling pathways involved in selected neurological diseases characterized by mitochondrial dysfunctions. Mitochondrial impairment has been hypothesized to have a key role in paramount mechanisms underlying Rett syndrome (RTT), a severe neurologic rare disorder. CNF1 has been already reported to have beneficial effects in mouse models of RTT. Using human RTT fibroblasts from four patients carrying different mutations, as a reliable disease‐in‐a‐dish model, we explored the cellular and molecular mechanisms, which can underlie the CNF1‐induced amelioration of RTT deficits. We found that CNF1 treatment modulates the Rho GTPases activity of RTT fibroblasts and induces a considerable re‐organization of the actin cytoskeleton, mainly in stress fibres. Mitochondria of RTT fibroblasts show a hyperfused morphology and CNF1 decreases the mitochondrial mass leaving substantially unaltered the mitochondrial dynamic. From a functional perspective, CNF1 induces mitochondrial membrane potential depolarization and activation of AKT in RTT fibroblasts. Given that mitochondrial quality control is altered in RTT, our results are suggestive of a reactivation of the damaged mitochondria removal via mitophagy restoration. These effects can be at the basis of the beneficial effects of CNF1 in RTT. [ABSTRACT FROM AUTHOR]

The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase‐activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy‐based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP‐RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking. [ABSTRACT FROM AUTHOR]

Although forgetting was once regarded as a passive decline in memory and an occasional source of embarrassment, recent research suggests that it is an active biological process of removing outdated or irrelevant memories via activation of specific genes and signal transduction pathways. Rho family G proteins are known to have a role in synaptic plasticity mediated by the actin cytoskeleton. However, the current study reveals that another Rho guanosine triphosphate enzyme (GTPase), RAC‐2, facilitates the occurrence of forgetting in Caenorhabditis elegans independent of actin dynamics. Functioning downstream of RAC‐2 in the same signalling pathway, JNK‐1 and its phosphorylated protein are required to positively regulate forgetting. The pan‐neuronal rescue of RAC‐2 or JNK‐1, instead of AWC neuron‐specific expression, reverses the delayed forgetting caused by the rac‐2 mutation, which indicates that the involvement of RAC‐2/JNK‐1 in more than AWCs must be required. In summary, our work elucidates the action of the Rho GTPase RAC‐2 and downstream JNK‐1 as a potential novel pathway in forgetting in C. elegans. [ABSTRACT FROM AUTHOR]

Metastatic disease is the leading cause of death in children suffering from medulloblastoma and a major treatment challenge. The evidence of leptomeningeal dissemination defines the most aggressive tumours and is associated with increased mortality; thus, inhibition of migration as a factor involved in the process of metastatic disease is fundamental for the treatment and prevention of metastatic dissemination. Targeting the small Rho GTPases Rac1 has been shown to effectively impair medulloblastoma cell migration in vitro. Yet clinically applicable selective Rac1 inhibitors are still lacking. In view of the pertinent oncogenic role of the PI3K signalling cascade and tyrosine kinase‐mediated signalling pathways in medulloblastoma, we explored clinically available targeted therapeutics to this effect. Here, we show that Rac1 is expressed in both the cytoplasm and nucleus in the medulloblastoma cell lines Daoy and MEB‐Med‐8A representative of two high risk medulloblastoma entities. We demonstrate that activated Rac1 is subject to substantial downmodulation following administration of the clinically available inhibitor of the PI3K pathway Pictilisib (GDC‐0941) and the multityrosine kinase inhibitors Pazopanib and Sorafenib. The application of those drugs was associated with reduced mobility of the medulloblastoma cells and alterations of the actin skeleton. Of note, PI3K inhibition reveals the strongest anti‐migratory effect in Daoy cells. Thus, our in vitro observations provide new insights into different strategies of blocking Rac1 and inhibiting migration in medulloblastoma employing clinically available agents paving the way for confirmatory studies in in vivo models. [ABSTRACT FROM AUTHOR]

Rho GTPases and Hippo kinases are key regulators of cardiomyoblast differentiation. However, how these signaling axes are coordinated spatiotemporally remains unclear. Here, the central and multifaceted roles of the BCH domain containing protein, BNIP‐2, in orchestrating the expression of two key cardiac genes (cardiac troponin T [cTnT] and cardiac myosin light chain [Myl2]) in H9c2 and human embryonic stem cell‐derived cardiomyocytes are delineated. This study shows that BNIP‐2 mRNA and protein expression increase with the onset of cTnT and Myl2 and promote the alignment of H9c2 cardiomyocytes. Mechanistically, BNIP‐2 is required for the inactivation of YAP through YAP phosphorylation and its cytosolic retention. Turbo‐ID proximity labeling corroborated by super‐resolution analyses and biochemical pulldown data reveals a scaffolding role of BNIP‐2 for LATS1 to phosphorylate and inactivate YAP in a process that requires BNIP‐2 activation of cellular contractility. The findings identify BNIP‐2 as a pivotal signaling scaffold that spatiotemporally integrates RhoA/Myosin II and LATS1/YAP mechanotransduction signaling to drive cardiomyoblast differentiation, by switching the genetic programming from YAP‐dependent growth to YAP‐silenced differentiation. These findings offer insights into the importance of scaffolding proteins in bridging the gap between mechanical and biochemical signals in cell growth and differentiation and the prospects in translational applications. [ABSTRACT FROM AUTHOR]

The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor axon and a skeletal muscle fibre that allows muscle contraction and the coordinated movement in many species. A main hallmark of the mature NMJ is the assembly of nicotinic acetylcholine receptor (nAChR) aggregates in the muscle postsynaptic domain, that distributes in perfect apposition to presynaptic motor terminals. To assemble its unique functional architecture, initial embryonic NMJs undergo an early postnatal maturation process characterised by the transformation of homogenous nAChR‐containing plaques to elaborate and branched pretzel‐like structures. In spite of a detailed morphological characterisation, the molecular mechanisms controlling the intracellular scaffolding that organises a postsynaptic domain at the mature NMJ have not been fully elucidated. In this review, we integrate evidence of key processes and molecules that have shed light on our current understanding of the NMJ maturation process. On the one hand, we consider in vitro studies revealing the potential role of podosome‐like structures to define discrete low nAChR‐containing regions to consolidate a plaque‐to‐pretzel transition at the NMJ. On the other hand, we focus on in vitro and in vivo evidence demonstrating that members of the Ras homologous (Rho) protein family of small GTPases (small Rho GTPases) play indispensable roles on NMJ maturation by regulating the stability of nAChR aggregates. We combine this evidence to propose that small Rho GTPases are key players in the assembly of podosome‐like structures that drive the postsynaptic maturation of vertebrate NMJs. [ABSTRACT FROM AUTHOR]

Osteoclasts are the key target cells for cadmium (Cd)‐induced bone metabolism diseases, while Rho GTPases play an important role in osteoclast differentiation and bone resorption. To identify new therapeutic targets of Cd‐induced bone diseases; we evaluated signal transduction through Rho GTPases during osteoclast differentiation under the influence of Cd. In osteoclastic precursor cells, 10 nM Cd induced pseudopodia stretching, promoted cell migration, upregulated the levels of Cdc42, and RhoQ mRNAs and downstream Rho‐associated coiled‐coil kinase 1 (ROCK1) and ROCK2 proteins, and downregulated the actin‐related protein 2/3 (ARP2/3) levels. Cd at 2 and 5 μM shortened the pseudopodia, inhibited cell migration, and decreased ROCK1, ROCK2, and ARP2/3 protein levels; Cd at 5 μM also reduced the mRNA expression levels of Rac1, Rac2, and RhoU mRNAs and decreased the level of phosphorylated (p)‐cofilin. In osteoclasts, 10 nM Cd induced the formation of sealing zones, slightly upregulated Cdc42 mRNA levels and ROCK2 and ARP2/3 protein levels and significantly reduced p‐cofilin levels. Cd at 2 μM and 5 μM Cd blocked the fusion of precursor cells; and 5 μM Cd downregulated the expression levels of RhoB, Rac1, Rac3, and RhoU mRNAs, and ROCK1, p‐cofilin and ARP2/3 protein levels, significantly. In vivo, Cd (at 5 or 25 mg/L) increased the levels of key proteins RhoA, Rac1/2/3, Cdc42, and RhoU and their mRNAs in bone marrow cells. In summary, the results suggested that Cd affected the differentiation process of osteoclast and altered the expression of several Rho GTPases, which might be crucial targets of Cd during the differentiation of osteoclasts. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica causes amoebiasis which is a major health concern in developing countries. E. histolytica pathogenicity has been implicated to a large repertoire of small GTPases which switch between the inactive GDP bound state and the active GTP bound state with the help of guanine nucleotide exchange factors (GEFs) and GTPase activating protein (GAPs). Rho family of small GTPases are well known to modulate the actin cytoskeletal dynamics which plays a major role in E. histolytica pathogenicity. Here, we report an atypical amoebic RhoGEF, and its preferred substrate EhRho6, which, upon overexpression abrogated the pathogenic behavior of the amoeba such as adhesion to host cell, monolayer destruction, erythrophagocytosis, and formation of actin dots. A causative immunoblot analysis revealed actin degradation in the EhRho6 overexpressing trophozoites that could be inhibited by blocking the amoebic proteasomal pathway. A careful analysis of the results from a previously published transcriptomics study, in conjunction with our observations, led to the identification of a clade of Rho GTPases in this pathogenic amoeba which we hypothesize to have implications during the amoebic encystation. [ABSTRACT FROM AUTHOR]

Cellular invasion by Trypanosoma cruzi metacyclic trypomastigotes (MTs) or tissue culture trypomastigotes (TCTs) is a complex process involving host–parasite cellular and molecular interactions. Particularly, the involvement of host cell actin cytoskeleton during trypomastigote invasion is poorly investigated, and still, the results are controversial. In the present work, we compare side by side both trypomastigote forms and employ state‐of‐the‐art live‐cell imaging showing for the first time the dynamic mobilization of host cell actin cytoskeleton to MT and TCT invasion sites. Moreover, cytochalasin D, latrunculin B, and jasplakinolide‐pretreated cells inhibited MT and TCT invasion. Furthermore, our results demonstrated that TCT invasion decreased in RhoA, Rac1, and Cdc‐42 GTPase‐depleted cells, whereas MT invasion decreased only in Cdc42‐and RhoA‐depleted cells. Interestingly, depletion of the three studied GTPases induced a scattered lysosomal distribution throughout the cytosol. These observations indicate that GTPase depletion is sufficient to impair parasite invasion despite the importance of lysosome spread in trypomastigote invasion. Together, our results demonstrate that the host cell actin cytoskeleton plays a direct role during TCT and MT invasion. [ABSTRACT FROM AUTHOR]

For the discovery of novel chemical matter generally endowed with bioactivity, strategies may be particularly efficient that combine previous insight about biological relevance, e.g. natural product (NP) structure, with methods that enable efficient coverage of chemical space, such as fragment‐based design. We describe the de novo combination of different 5‐membered NP‐derived N‐heteroatom fragments to structurally unprecedented "pseudo‐natural products" in an efficient complexity‐generating and enantioselective one‐pot synthesis sequence. The pseudo‐NPs inherit characteristic elements of NP structure but occupy areas of chemical space not covered by NP‐derived chemotypes, and may have novel biological targets. Investigation of the pseudo‐NPs in unbiased phenotypic assays and target identification led to the discovery of the first small‐molecule ligand of the RHO GDP‐dissociation inhibitor 1 (RHOGDI1), termed Rhonin. Rhonin inhibits the binding of the RHOGDI1 chaperone to GDP‐bound RHO GTPases and alters the subcellular localization of RHO GTPases. [ABSTRACT FROM AUTHOR]

Rho GTPases are critically important and are centrally positioned regulators of the actomyosin cytoskeleton. By influencing the organization and architecture of the cytoskeleton, Rho proteins play prominent roles in many cellular processes including adhesion, migration, intra‐cellular transportation, and proliferation. The most important method of Rho GTPase regulation is via the GTPase cycle; however, post‐translational modifications (PTMs) also play critical roles in Rho protein regulation. Relative to other PTMs such as lipidation or phosphorylation that have been extensively characterized, protein oxidation is a regulatory PTM that has been poorly studied. Protein oxidation primarily occurs from the reaction of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), with amino acid side chain thiols on cysteine (Cys) and methionine (Met) residues. The versatile redox modifications of cysteine residues exemplify their integral role in cell signalling processes. Here we review prominent members of the Rho GTPase family and discuss how lipidation, phosphorylation, and oxidation on conserved cysteine residues affects their regulation and function. [ABSTRACT FROM AUTHOR]

Plant Rho small GTPases (Rop/Rac) are versatile molecular switches regulating many plant developmental processes. Particularly, their important functions in regulating pollen development have been demonstrated in Arabidopsis. A group of conserved Rop/Rac activators RopGEFs were recently reported to regulate rice (Oryza sativa) pollen tube germination, indicating that rice and Arabidopsis may have a conserved Rop/Rac mediated signaling pathway in regulating pollen tube growth. However, the Rop/Rac activated by the rice pollen specific RopGEFs remains to be identified. Here we demonstrated a Rop/Rac gene, OsRacB, co‐expressed with the mature pollen expressed OsRopGEF2/3/6/8. The knockout mutants were normal in anther and pollen development but defective in the pollen grain germination, suggesting a specific and non‐redundant role of OsRacB in the mature pollen. We further demonstrated that OsRacB is directly activated by the pollen specific expressing OsRopGEFs in vitro. Together with the previous study, we establish a RopGEF‐Rop/Rac regulon which plays essential roles in rice pollen grain germination. Our data encourage further identification of the upstream and downstream players of RopGEF‐Rop/Rac signaling in pollen germination and have agricultural implications for breeding robust seed yielding cultivars. [ABSTRACT FROM AUTHOR]

TcdB is a potent cytotoxin produced by pathogenic Clostridioides difficile that inhibits Rho GTPases by mono-glucosylation. TcdB enters cells via receptor-mediated endocytosis. The pathogenic glucosyltransferase domain (GTD) egresses endosomes by pH-mediated conformational changes, and is subsequently released in an autoproteolytic manner. We here investigated the uptake, localization and degradation of TcdB. TcdB colocalized with lysosomal marker protein LAMP1, verifying the endosomallysosomal route of the toxin. In pulse assays endocytosed TcdB declined to a limit of detection within 2 hr, whereas the released GTD accumulated for up to 8 hr. We observed that autoproteolytic deficient TcdB NXN C698S was degraded significantly faster than wildtype TcdB, suggesting interference of TcdB with lysosomal degradation process. In fact, TcdB reduced lysosomal degradation of endosome cargo as tested with DQ-Green BSA. Lysosomal dysfunction was accompanied by perinuclear accumulation of LAMP1 and a weaker detection in immunoblots. Galectin-8 or galectin-3 was not recruited to lysosomes speaking against lysosome membrane damage. Changes in the autophagosomal marker LC3B suggested additional indirect effect of lysosomal dysfunction on the autophagic flux. In contrast to necrotic signaling induced in by TcdB, lysosomal dysfunction was not abolished by calcium channel blocker nifedipin, indicating separate cytopathogenic effects induced by TcdB during endo-lysosomal trafficking. [ABSTRACT FROM AUTHOR]

Cell polarity is achieved by regulators such as small G proteins, exocyst members and phosphoinositides, with the latter playing a key role when bound to the exocyst proteins Sec3p and Exo70p, and Rho GTPases. This ensures asymmetric growth via the routing of proteins and lipids to the cell surface using actin cables. Previously, using a yeast mutant for a lysophosphatidylinositol acyl transferase encoded by the PSI1 gene, we demonstrated the role of stearic acid in the acyl chain of phosphoinositides in cytoskeletal organization and secretion. Here, we use a genetic approach to characterize the effect on late steps of the secretory pathway. The constitutive overexpression of PSI1 in mutants affecting kinases involved in the phosphoinositide pathway demonstrated the role of molecular species containing stearic acid in bypassing a lack of phosphatidylinositol‐4‐phosphate (PI(4)P) at the plasma membrane, which is essential for the function of the Cdc42p module. Decreasing the levels of stearic acid‐containing phosphoinositides modifies the environment of the actors involved in the control of late steps in the secretory pathway. This leads to decreased interactions between Exo70p and Sec3p, with Cdc42p, Rho1p and Rho3p, because of disruption of the GTP/GDP ratio of at least Rho1p and Rho3p GTPases, thereby preventing activation of the exocyst. [ABSTRACT FROM AUTHOR]

Background: The breakpoint cluster region (BCR) is a protein that originally forms a fusion protein with c‐Abl tyrosine kinase and induces leukemia. Researchers have shown that BCR is enriched in the central nervous system and may contribute to neurological disorders. We aimed to investigate the physiological function of BCR in neural development in the gastrointestinal (GI) tract and brain. Methods: Whole‐exome sequencing was used to screen for mutations in the BCR. Bcr knockout mice (Bcr−/−, ΔExon 2–22) were generated using the CRISPR/Cas9 system. Transit of carmine red dye and glass bead expulsion assays were used to record total and proximal GI transit and distal colonic transit. Key Results: In an infant with pediatric intestinal pseudo‐obstruction, we found a heterozygous de novo mutation (NM_004327.3:c.3072+1G>A) in BCR. Bcr deficiency mice (Bcr−/−) exhibited growth retardation and impaired gastrointestinal motility. Bcr−/− mice had a prolonged average total GI transit time with increased distal colonic transit and proximal GI transit in isolation. Morphology analysis indicated that Bcr−/− mice had a less number of neurons in the submucosal plexus and myenteric plexus. Bcr−/− mice exhibited apparent structural defects in the brain, particularly in the cortex. Additionally, Bcr− depletion in the mouse cortex altered the expression of Ras homologous (Rho) family small GTPases. Conclusions and Inferences: BCR mutations are associated with intestinal obstruction in children. Loss of Bcr can cause intestinal dysmotility and brain developmental defects may via regulation of Rho GTPases. [ABSTRACT FROM AUTHOR]

Dendritic spines are small, actin‐rich protrusions that act as the receiving sites of most excitatory inputs in the central nervous system. The remodeling of the synapse architecture is mediated by actin cytoskeleton dynamics, a process precisely regulated by the small Rho GTPase family. Wnt ligands exert their presynaptic and postsynaptic effects during formation and consolidation of the synaptic structure. Specifically, Wnt5a has been identified as an indispensable synaptogenic factor for the regulation and organization of the postsynaptic side; however, the molecular mechanisms through which Wnt5a induces morphological changes resulting from actin cytoskeleton dynamics within dendritic spines remain unclear. In this work, we employ primary rat hippocampal cultures and HT22 murine hippocampal neuronal cell models, molecular and pharmacological tools, and fluorescence microscopy (laser confocal and epifluorescence) to define the Wnt5a‐induced molecular signaling involved in postsynaptic remodeling mediated via the regulation of the small Rho GTPase family. We report that Wnt5a differentially regulates the phosphorylation of Cofilin in neurons through both Ras‐related C3 botulinum toxin substrate 1 and cell division cycle 42 depending on the subcellular compartment and the extracellular calcium levels. Additionally, we demonstrate that Wnt5a increases the density of dendritic spines and promotes their maturation via Ras‐related C3 botulinum toxin substrate 1. Accordingly, we find that Wnt5a requires the combined activation of small Rho GTPases to increase the levels of filamentous actin, thus promoting the stability of actin filaments. Altogether, these results provide evidence for a new mechanism by which Wnt5a may target actin dynamics, thereby regulating the subsequent morphological changes in dendritic spine architecture. [ABSTRACT FROM AUTHOR]

Intracellular signaling pathways that promote axon regeneration are closely linked to the mechanism of neurite outgrowth. TC10, a signaling molecule that acts on neurite outgrowth through membrane transport, is a member of the Rho family G proteins. Axon injury increases the TC10 levels in motor neurons, suggesting that TC10 may be involved in axon regeneration. In this study, we tried to understand the roles of TC10 in the nervous system using TC10 knock‐out mice. In cultured hippocampal neurons, TC10 ablation significantly reduced axon elongation without affecting ordinary polarization. We determined a role of TC10 in microtubule stabilization at the growth cone neck; therefore, we assume that TC10 limits axon retraction and promotes in vitro axon outgrowth. In addition, there were no notable differences in the size and structure of brains during prenatal and postnatal development between wild‐type and TC10 knock‐out mice. In motor neurons, axon regeneration after injury was strongly suppressed in mice lacking TC10 (both in conventional and injured nerve specific deletion). In retinal ganglion cells, TC10 ablation suppressed the axon regeneration stimulated by intraocular inflammation and cAMP after optic nerve crush. These results show that TC10 plays an important role in axon regeneration in both the peripheral and central nervous systems, and the role of TC10 in peripheral axon regeneration is neuron‐intrinsic. [ABSTRACT FROM AUTHOR]

Cytotoxic necrotizing factors (CNFs) are bacterial single‐chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three‐dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full‐length Yersinia pseudotuberculosis CNFY. CNFY consists of five domains (D1–D5), and by integrating structural and functional data, we demonstrate that D1–3 act as export and translocation module for the catalytic unit (D4–5) and for a fused β‐lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP‐ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4–5 fragment. This liberates D5 from a semi‐blocked conformation in full‐length CNFY, leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad‐specificity protein delivery tool. SYNOPSIS: Single‐chain bacterial cytotoxic necrotizing factor (CNF) toxins constitutively activate small Rho GTPases in host cells, leading to actin rearrangements and multinucleation. Here, crystal structures and accompanying cell biological experiments using CNFY from Yersinia pseudotuberculosis reveal the molecular basis of CNFY secretion, host cell binding, and translocation into the host cell cytosol. The CNFY structure reveals five domains, D1–5, with distinct functions in cell entry and RhoA activation.Domains D1–3 constitute a minimal translocation unit that can be employed as broad‐specificity protein delivery tool.A structure of the enzymatically‐active fragment D4–5 reveals structural rearrangements that accompany its release into the host cell cytosol.D4 constitutes a DUF4765 domain and displays structural similarity to ADP‐ribosyl transferases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Antigen recognition by B‐cell antigen receptors (BCRs) activates distinct intracellular signaling pathways that control the differentiation fate of activated B lymphocytes. BCR‐proximal signaling enzymes comprise protein tyrosine kinases, phosphatases, and plasma membrane lipid‐modifying enzymes, whose function is furthermore coordinated by catalytically inert adaptor proteins. Here, we show that an additional class of enzymatic activity provided by guanine‐nucleotide exchange factors (GEFs) of the Vav family controls BCR‐proximal Ca2+ mobilization, cytoskeletal actin reorganization, and activation of the PI3 kinase/Akt pathway. Whereas Vav1 and Vav3 supported all of those signaling processes to different extents in a human B‐cell model system, Vav2 facilitated Actin remodeling, and activation of Akt but did not promote Ca2+ signaling. On BCR activation, Vav1 was directly recruited to the phosphorylated BCR and to the central adaptor protein SLP65 via its Src homology 2 domain. Pharmacological inhibition or genetic inactivation of the substrates of Vav GEFs, small G proteins of the Rho/Rac family, impaired BCR‐induced Ca2+ mobilization, probably because phospholipase Cγ2 requires activated Rac proteins for optimal activity. Our findings show that Vav family members are key relays of the BCR signalosome that differentially control distinct signaling pathways both in a catalysis‐dependent and ‐independent manner. [ABSTRACT FROM AUTHOR]

To undergo mitosis successfully, most animal cells need to acquire a round shape to provide space for the mitotic spindle. This mitotic rounding relies on mechanical deformation of surrounding tissue and is driven by forces emanating from actomyosin contractility. Cancer cells are able to maintain successful mitosis in mechanically challenging environments such as the increasingly crowded environment of a growing tumor, thus, suggesting an enhanced ability of mitotic rounding in cancer. Here, it is shown that the epithelial–mesenchymal transition (EMT), a hallmark of cancer progression and metastasis, gives rise to cell‐mechanical changes in breast epithelial cells. These changes are opposite in interphase and mitosis and correspond to an enhanced mitotic rounding strength. Furthermore, it is shown that cell‐mechanical changes correlate with a strong EMT‐induced change in the activity of Rho GTPases RhoA and Rac1. Accordingly, it is found that Rac1 inhibition rescues the EMT‐induced cortex‐mechanical phenotype. The findings hint at a new role of EMT in successful mitotic rounding and division in mechanically confined environments such as a growing tumor. [ABSTRACT FROM AUTHOR]

Most eukaryotic cells are polarized. Common toolbox regulating cell polarization includes Rho guanosine triphosphatases (GTPases), in which spatiotemporal activation is regulated by a plethora of regulators. Rho of plants (ROPs) are the only Rho GTPases in plants. Although vesicular trafficking was hinted in the regulation of ROPs, it was unclear where vesicle‐carried ROP starts, whether it is dynamically regulated, and which components participate in vesicle‐mediated ROP targeting. In addition, although vesicle trafficking and guanine nucleotide inhibitor (GDI) pathways in Rho signaling have been extensively studied in yeast, it is unknown whether the two pathways interplay. Unclear are also cellular and developmental consequences of their interaction in multicellular organisms. Here, we show that the dynamic targeting of ROP through vesicles requires coat protein complex II and ADP‐ribosylation factor 1‐mediated post‐Golgi trafficking. Trafficking of vesicle‐carried ROPs between the plasma membrane and the trans‐Golgi network is mediated through adaptor protein 1 and sterol‐mediated endocytosis. Finally, we show that GDI and vesicle trafficking synergistically regulate cell polarization and ROP targeting, suggesting that the establishment and maintenance of cell polarity is regulated by an evolutionarily conserved mechanism. [ABSTRACT FROM AUTHOR]

The activity of Rho‐specific guanine nucleotide dissociation inhibitor α (RhoGDIα) is regulated by its own phosphorylation at different amino acid sites. These phosphorylation sites may have a crucial role in local Rho GTPases activation during cell migration. This paper is designed to explore the influence of phosphorylation on shear stress‐induced spatial RhoGDIα activation. Based on the fluorescence resonance energy transfer biosensor sl‐RhoGDIα, which was constructed to test the RhoGDIα activity in living cells, new RhoGDIα phosphomimetic mutation (sl‐S101E/S174E, sl‐Y156E, sl‐S101E, sl‐S174E) and phosphorylation‐deficient mutation (sl‐S101A/S174A, sl‐Y156A, sl‐S101A, sl‐S174A) biosensors were designed to test their effects on RhoGDIα activation upon shear stress application in human umbilical vein endothelial cells (HUVECs). The results showed lower RhoGDIα activity at the downstream of HUVECs (the region from the edge of the nucleus to the edge of the cell along with the flow). The overall decrease in RhoGDIα activity was inhibited by Y156A‐mutant, whereas the polarized RhoGDIα and Rac1 activity were blocked by S101A/S174A mutant. It is concluded that the Tyr156 phosphorylation mainly mediates shear stress‐induced overall RhoGDIα activity, while Ser101/Ser174 phosphorylation mediates its polarization. This study demonstrates that differential phosphorylation of RhoGDIα regulates shear stress‐induced spatial RhoGDIα activation, which could be a potential target to control cell migration. [ABSTRACT FROM AUTHOR]

The Rho family GTPases are crucial drivers of tumor growth and metastasis. However, it is difficult to develop GTPases inhibitors due to a lack of well‐characterized binding pockets for compounds. Here, through molecular dynamics simulation of the RhoA protein, a groove around cysteine 107 (Cys107) that is relatively well‐conserved within the Rho family is discovered. Using a combined strategy, the novel inhibitor DC‐Rhoin is discovered, which disrupts interaction of Rho proteins with guanine nucleotide exchange factors (GEFs) and guanine nucleotide dissociation inhibitors (GDIs). Crystallographic studies reveal that the covalent binding of DC‐Rhoin to the Cys107 residue stabilizes and captures a novel allosteric pocket. Moreover, the derivative compound DC‐Rhoin04 inhibits the migration and invasion of cancer cells, through targeting this allosteric pocket of RhoA. The study reveals a novel allosteric regulatory site within the Rho family, which can be exploited for anti‐metastasis drug development, and also provides a novel strategy for inhibitor discovery toward "undruggable" protein targets. [ABSTRACT FROM AUTHOR]

During angiogenesis, VEGF acts as an attractive cue for endothelial cells (ECs), while Sema3E mediates repulsive cues. Here, we show that the small GTPase RhoJ integrates these opposing signals in directional EC migration. In the GTP‐bound state, RhoJ interacts with the cytoplasmic domain of PlexinD1. Upon Sema3E stimulation, RhoJ released from PlexinD1 induces cell contraction. PlexinD1‐bound RhoJ further facilitates Sema3E‐induced PlexinD1‐VEGFR2 association, VEGFR2 transphosphorylation at Y1214, and p38 MAPK activation, leading to reverse EC migration. Upon VEGF stimulation, RhoJ is required for the formation of the holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin‐1, thereby preventing degradation of internalized VEGFR2, prolonging downstream signal transductions via PLCγ, Erk, and Akt, and promoting forward EC migration. After conversion to the GDP‐bound state, RhoJ shifts from PlexinD1 to VEGFR2, which then terminates the VEGFR2 signals. RhoJ deficiency in ECs efficiently suppressed aberrant angiogenesis in ischemic retina. These findings suggest that distinct Rho GTPases may act as context‐dependent integrators of chemotactic cues in directional cell migration and may serve as candidate therapeutic targets to manipulate cell motility in disease or tissue regeneration. Synopsis: Semaphorin 3E (Sema3E)/PlexinD1 and VEGF/VEGFR2 signalling pathways regulate endothelial cell migration in an opposite fashion. In this study, GTPase RhoJ is shown to integrate attractive and repulsive chemotactic cues by mediating interaction between VEGFR2 and PlexinD1. Sema3E releases GTP‐RhoJ from PlexinD1 to promote cell contractility.RhoJ facilitates Sema3E‐induced VEGFR2‐PlexinD1 association and p38 MAPK activation.GTP‐RhoJ sustains VEGF‐induced signalling by mediating VEFR2 interaction with PlexinD1/Neuropilin 1 complex and preventing VEGFR2 degradation.GDP‐RhoJ promotes VEGFR2 degradation, thus terminating signalling via internalized VEGFR2.RhoJ deficiency suppresses aberrant angiogenesis in ischemic retina. [ABSTRACT FROM AUTHOR]

Asthma is a chronic and heterogeneous disease characterised by airway inflammation and intermittent airway narrowing. The key obstacle in the prevention and treatment of asthma has been our incomplete understanding of its aetiology and biological mechanisms. The ras homolog family member A (RhoA) of the Rho family GTPases has been considered to be one of the most promising and novel therapeutic targets for asthma. It is well known that RhoA/Rho‐kinases play an important role in the pathophysiology of asthma, including airway smooth muscle contraction, airway hyper‐responsiveness, β‐adrenergic desensitisation and airway remodelling. However, recent advances have suggested novel roles for RhoA in regulating allergic airway inflammation. Specifically, RhoA has been shown to regulate allergic airway inflammation through controlling Th2 or Th17 cell differentiation and to regulate airway remodelling through regulating mesenchymal stem cell (MSC) differentiation. In this review, we evaluate the literature regarding the recent advances in the activation of RhoA/Rho‐kinase, cytokine and epigenetic regulation of RhoA/Rho‐kinase, and the role of RhoA/Rho‐kinase in regulating major features of asthma, such as airway hyper‐responsiveness, remodelling and inflammation. We also discuss the importance of the newly identified role of RhoA/Rho‐kinase signalling in MSC differentiation and bronchial epithelial barrier dysfunction. These findings indicate the functional significance of the RhoA/Rho‐kinase pathway in the pathophysiology of asthma and suggest that RhoA/Rho‐kinase signalling may be a promising therapeutic target for the treatment of asthma. [ABSTRACT FROM AUTHOR]

Emerging epidemiological and preclinical studies have focused on statins and mevalonate pathway to identify potential therapeutic target and clarify the underlying mechanism of the anti‐neoplastic effects. Reductions of mevalonate or isoprenoids, caused by statins, would further decrease the isoprenylation of Rho GTPases which is the crucial step for Rho GTPases to anchor on inner cellular membrane. Following anchoring, activated Rho GTPases can mediate a series of cellular activities such as cytoskeleton reprogramming, front–rear polarity, and cell‐ECM adhesion. These changes not only facilitate tumor cell detachment and migration but also bring great mechanical changes to directly activate YAP, the major nuclear mechanotransducer, to translocate into nucleus. Recently, statins have been identified as potent inhibitors of YAP. Once entering nucleus, YAP would combine TEADs to promote the transcription of about 100 genes, which are involved in cell proliferation, cell cycle regulation, stemness, invasion, and metastasis. Besides, statins are able to promote the degradation of misfolded mutant p53 (mutp53), which is an oncogene in a variety of human malignancies. Reduction in mevalonate‐5‐phosphate (MVP), also induced by statins, would impair the stability of DNAJA1‐mutp53 complex; then, elevated C terminus of Hsc70‐interacting protein (CHIP) mediates the nuclear export and degradation of misfolded mutp53 through ubiquitin–proteasome pathway. It is worth noted that YAP, mutp53, and mevalonate pathway form two positive feedback loops. It is reasonable to believe that Rho GTPases, YAP, and mutp53 are determinants for statins as anti‐cancer agents: tumor cells harboring mutp53 and nuclear‐located YAP would be more sensitive to statins. [ABSTRACT FROM AUTHOR]

The p21‐activated kinase (PAK) family of proteins function as key effectors of RHO family GTPases in mammalian cells to regulate many pathways including Ras/Raf/MEK/ERK and Wnt/β‐catenin, amongst others. Here we report an individual with a novel autosomal dominant disorder characterized by severe regressive autism, intellectual disability, and epilepsy. Exome sequencing of the proband and her parents revealed a de novo variant in the PAK1 gene ([NM_001128620] c.362C>T/p.Pro121Leu). Studies in patient cells showed a clear effect on PAK1 protein function, including altered phosphorylation of targets (JNK and ERK), decreased abundance of β‐catenin, and concomitant altered expression downstream of these key regulators. Our findings add PAK1 to the list of PAK proteins and kinases which when mutated cause rare genetic diseases. [ABSTRACT FROM AUTHOR]

The molecular signals that regulate mitotic spindle orientation to determine the proper division axis play a critical role in the development and maintenance of tissue homeostasis. However, deregulation of signaling events can result in spindle misorientation, which in turn can trigger developmental defects and cancer progression. Little is known about the cellular signaling pathway involved in the misorientation of proliferating cells that evade apoptosis after DNA damage. In this study, we found that perturbations to spindle orientation were induced in ultraviolet C (UVC)‐irradiated surviving cells. N‐terminal truncated Rho GDP‐dissociation inhibitor β (RhoGDIβ), which is produced by UVC irradiation, distorted the spindle orientation of HeLa cells cultured on Matrigel. The short hairpin RNA‐mediated knockdown of RhoGDIβ significantly attenuated UVC‐induced misorientation. Subsequent expression of wild‐type RhoGDIβ, but not a noncleavable mutant, RhoGDIβ (D19A), again led to a relative increase in spindle misorientation in response to UVC. Our findings revealed that RhoGDIβ impacts spindle orientation in response to DNA damage. [ABSTRACT FROM AUTHOR]

The hippocampus is generally considered as a brain center for learning and memory. We have recently established an electroporation‐mediated gene transfer method to investigate the development of neonatal dentate granule cells in vivo. Using this new technique, we introduced knockdown vectors against Rac1 small GTPase into precursors for dentate granule cells at postnatal day 0. After 21 days, Rac1‐deficient cells were frequently mispositioned between the granule cell layer (GCL) and hilus. About 60% of these mislocalized cells expressed a dentate granule cell marker, Prox1. Both the dendritic spine density and the ratio of mature spine were reduced when Rac1 was silenced. Notably, the deficient cells have immature thin processes during migrating in the early neonatal period. Knockdown of another Rac isoform, Rac3, also resulted in mislocalization of neonatally born dentate granule cells. In addition, knockdown of Cdc42, another Rho family protein, also caused mislocalization of the cell, although the effects were moderate compared to Rac1 and 3. Despite the ectopic localization, Rac3‐ or Cdc42‐disrupted mispositioned cells expressed Prox1. These results indicate that Rho signaling pathways differentially regulate the proper localization and differentiation of dentate granule cells. [ABSTRACT FROM AUTHOR]

We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE‐214 by macropinocytosis. In this study, we have extended our investigation into SHK‐1 cells, a macrophage‐like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK‐1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK‐1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK‐1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis‐mediated entry of IPNV into its target cells. [ABSTRACT FROM AUTHOR]

Background: The mammalian gut epithelium displays among the highest rates of self‐renewal, with a turnover time of less than 5 days. Renewal involves concerted proliferation at the bottom of the crypt, migration and differentiation along the crypt‐villus axis and anoïkis/shedding in the luminal epithelium. Renewal is controlled by interplay between signalling pathways, among which canonical and non‐canonical Wnt signals play prominent roles. Overall 92% of colon tumours show increased canonical Wnt signalling resulting from mutations, established as major driver steps towards carcinogenesis. Results: Here, we examined the physiological role of RhoU/Wrch1 in gut homeostasis. RhoU is an atypical Rho GTPase related to Cdc42/Rac1 and identified as a transcriptional target of non‐canonical Wnt signalling. We found that RHOU expression is reduced in human colorectal tumour samples. We show that RhoU is mainly expressed in the differentiated compartment of the gut epithelium. Rhou specific invalidation in the mouse gut elicits cell hyperplasia and is associated in the colon with a highly disorganized luminal epithelium. Hyperplasia affects all cell types in the small intestine and colon and has a higher impact on goblet cells. Hyperplasia is associated with a reduction of apoptosis and an increased proliferation. RhoU knockdown in human DLD‐1 colon cancer cells also elicits a higher growth index and reduces cell apoptosis. Last, loss of RhoU function in the mouse gut epithelium or in DLD‐1 cells increases RhoA activity and the level of phosphorylated Myosin Light Chain‐2, which may functionally link RhoU activity to apoptosis. Conclusion: RhoU is mostly expressed in the differentiated compartment of the gut. It plays a role in homeostasis as its specific invalidation elicits hyperplasia of all cell types. This mainly results from a reduction of apoptosis, through actomyosin‐dependent mechanisms. Significance: RhoU negatively controls cell growth in the intestinal epithelium. Since its expression is sensitive to non‐canonical Wnt signals and is reduced in colorectal tumours, downregulating RhoU may thus have an instrumental role in tumour progression. [ABSTRACT FROM AUTHOR]

RhoA GTPase plays a variety of functions in regulation of cytoskeletal proteins, cellular morphology, and migration along with various proliferation and transcriptional activity in cells. RhoA activity is regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and the guanine nucleotide dissociation factor (GDI). The RhoA‐RhoGDI complex exists in the cytosol and the active GTP‐bound form of RhoA is located to the membrane. GDI displacement factors (GDFs) including IκB kinase γ (IKKγ) dissociate the RhoA‐GDI complex, allowing activation of RhoA through GEFs. In addition, modifications of Tyr42 phosphorylation and Cys16/20 oxidation in RhoA and Tyr156 phosphorylation and oxidation of RhoGDI promote the dissociation of the RhoA‐RhoGDI complex. The expression of RhoA is regulated through transcriptional factors such as c‐Myc, HIF‐1α/2α, Stat 6, and NF‐κB along with several reported microRNAs. As the role of RhoA in regulating actin‐filament formation and myosin‐actin interaction has been well described, in this review we focus on the transcriptional activity of RhoA and also the regulation of RhoA message itself. Of interest, in the cytosol, activated RhoA induces transcriptional changes through filamentous actin (F‐actin)‐dependent (“actin switch”) or—independent means. RhoA regulates the activity of several transcription regulators such as serum response factor (SRF)/MAL, AP‐1, NF‐κB, YAP/TAZ, β‐catenin, and hypoxia inducible factor (HIF)‐1α. Interestingly, RhoA also itself is localized to the nucleus by an as‐yet‐undiscovered mechanism. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: Rho GTPase‐activating protein 42 was identified as an inhibitor of RhoA to maintain normal blood pressure homeostasis. However, the effect of ARHGAP42 in promoting cell malignancy in nasopharyngeal carcinoma is demonstrated in this study. Microarray and real‐time quantitative PCR were used for a mRNA profiling of ARHGAP42 in nasopharyngeal primary and metastatic carcinoma tissues. Western blot and immunohistochemical staining were used for detecting the expression of ARHGAP42 protein in nasopharyngeal carcinoma tissues and cell lines. The overexpression and silence experiments of ARHGAP42 were performed in NPC cell lines using siRNA and expressive plasmid for evaluating cancer cell migration and invasion in vitro. Real‐time quantitative PCR, western blot, and transwell test were employed for with the function of ARHGAP42 and its antisense lncRNA uc010rul. We confirmed the elevated expression of ARHGAP42 in metastatic NPC tissues of mRNA and protein for the first time. Immunohistochemical analysis indicated that NPC patients with highly ARHGAP42 expression were significantly associated with shorter metastasis‐free survival. Knockdown of ARHGAP42 resulted in significant inhibition of nasopharyngeal cancer cell migration and invasion in vitro, and the overexpression of ARHGAP42 showed the opposite effects. In addition, the silence of uc010rul resulted in ARHGAP42 expression decrease and significant inhibition of nasopharyngeal cancer cell migration and invasion. High expression of ARHGAP42 is associated with poor metastasis‐free survival of nasopharyngeal carcinoma patients. ARHGAP42 promotes migration and invasion of nasopharyngeal carcinoma cells in vitro; the antisense lncRNA may be involved in this effect. [ABSTRACT FROM AUTHOR]

Abstract: The Rho GTPase Rac1 is a multifunctional protein involved in distinct pathways ranging from development to pathology. The aim of the present study was to unravel the contribution of neuronal Rac1 in regulating the response to brain injury induced by permanent focal cerebral ischemia (pMCAO). Our results show that pMCAO significantly increased total Rac1 levels in wild type mice, mainly through rising nuclear Rac1, while a reduction in Rac1 activation was observed. Such changes preceded cell death induced by excitotoxic stress. Pharmacological inhibition of Rac1 in primary neuronal cortical cells prevented the increase in oxidative stress induced after overactivation of glutamate receptors. However, this was not sufficient to prevent the associated neuronal cell death. In contrast, RNAi‐mediated knock down of Rac1 in primary cortical neurons prevented cell death elicited by glutamate excitotoxicity and decreased the activity of NADPH oxidase. To test whether in vivo down regulation of neuronal Rac1 was neuroprotective after pMCAO, we used tamoxifen‐inducible neuron‐specific conditional Rac1‐knockout mice. We observed a significant 50% decrease in brain infarct volume of knockout mice and a concomitant increase in HIF‐1α expression compared to littermate control mice, demonstrating that ablation of Rac1 in neurons is neuroprotective. Transmission electron microscopy performed in the ischemic brain showed that lysosomes in the infarct of Rac1‐ knockout mice were preserved at similar levels to those of non‐infarcted tissue, while littermate mice displayed a decrease in the number of lysosomes, further corroborating the notion that Rac1 ablation in neurons is neuroprotective. Our results demonstrate that Rac1 plays important roles in the ischemic pathological cascade and that modulation of its levels is of therapeutic interest. [ABSTRACT FROM AUTHOR]

Peroxisomes are dynamic organelles which fulfil essential roles in lipid and ROS metabolism. Peroxisome movement and positioning allows interaction with other organelles and is crucial for their cellular function. In mammalian cells, such movement is microtubule‐dependent and mediated by kinesin and dynein motors. The mechanisms of motor recruitment to peroxisomes are largely unknown, as well as the role this plays in peroxisome membrane dynamics and proliferation. Here, using a combination of microscopy, live‐cell imaging analysis and mathematical modelling, we identify a role for Mitochondrial Rho GTPase 1 (MIRO1) as an adaptor for microtubule‐dependent peroxisome motility in mammalian cells. We show that MIRO1 is targeted to peroxisomes and alters their distribution and motility. Using a peroxisome‐targeted MIRO1 fusion protein, we demonstrate that MIRO1‐mediated pulling forces contribute to peroxisome membrane elongation and proliferation in cellular models of peroxisome disease. Our findings reveal a molecular mechanism for establishing peroxisome‐motor protein associations in mammalian cells and provide new insights into peroxisome membrane dynamics in health and disease. [ABSTRACT FROM AUTHOR]

This article focuses on the role of Rho family GTPases, particularly Rac1 and Rac1b in TGF‐β‐induced epithelial‐mesenchymal transition (EMT) and EMT‐associated responses such as cell migration, invasion, and metastasis in cancer. EMT is considered a prerequisite for cells to adopt a motile and invasive phenotype and eventually become metastatic. A major regulator of EMT and metastasis in cancer is TGF‐β, and its specific functions on tumor cells are mediated beside Smad proteins and mitogen‐activated protein kinases (MAPKs) by small GTPases of the Rho/Rac1 family. Available data point to extensive signaling crosstalk between TGF‐β and various Rho GTPases, and in particular a synergistic role of Rho and Rac1 during EMT and cell motility in normal and neoplastic epithelial cells. In contrast, the Rac1‐related isoform, Rac1b, emerges as an endogenous inhibitor of Rac1 in TGF‐β signaling, at least in pancreatic carcinoma cells. Given the tumor‐promoting role of TGF‐β in late‐stage carcinomas and the intimate crosstalk of Rho/Rac1/Rac1b and TGF‐β signaling in various tumor cell responses, targeting specific Rho GTPases may allow for selective interference with prooncogenic TGF‐β responses to aid in anticancer treatments. Developmental Dynamics 247:451–461, 2018. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho‐GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double‐knockout mouse embryos and single‐ and double‐knockout embryonic fibroblasts, we demonstrate the essential and non‐redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double‐knockout cells. In contrast, we show that TRAK2‐mediated retrograde mitochondrial transport is Miro1‐dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin‐dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double‐knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine‐tune actin‐ and tubulin‐dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation. [ABSTRACT FROM AUTHOR]

Abstract: The endothelial barrier consists of intercellular contacts localized in the cleft between endothelial cells, which is covered by the glycocalyx in a sievelike manner. Both types of barrier‐forming junctions, i.e. the adherens junction (AJ) serving mechanical anchorage and mechanotransduction and the tight junction (TJ) sealing the intercellular space to limit paracellular permeability, are tethered to the actin cytoskeleton. Under resting conditions, the endothelium thereby builds a selective layer controlling the exchange of fluid and solutes with the surrounding tissue. However, in the situation of an inflammatory response such as in anaphylaxis or sepsis intercellular contacts disintegrate in post‐capillary venules leading to intercellular gap formation. The resulting oedema can cause shock and multi‐organ failure. Therefore, maintenance as well as coordinated opening and closure of interendothelial junctions is tightly regulated. The two principle underlying mechanisms comprise spatiotemporal activity control of the small GTPases Rac1 and RhoA and the balance of the phosphorylation state of AJ proteins. In the resting state, junctional Rac1 and RhoA activity is enhanced by junctional components, actin‐binding proteins, cAMP signalling and extracellular cues such as sphingosine‐1‐phosphate (S1P) and angiopoietin‐1 (Ang‐1). In addition, phosphorylation of AJ components is prevented by junction‐associated phosphatases including vascular endothelial protein tyrosine phosphatase (VE‐PTP). In contrast, inflammatory mediators inhibiting cAMP/Rac1 signalling cause strong activation of RhoA and induce AJ phosphorylation finally leading to endocytosis and cleavage of VE‐cadherin. This results in dissolution of TJs the outcome of which is endothelial barrier breakdown. [ABSTRACT FROM AUTHOR]

Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood-brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin-mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin-mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells. [ABSTRACT FROM AUTHOR]

Acute subcellular protein targeting is a powerful tool to study biological networks. However, signaling at the plasma membrane is highly dynamic, making it difficult to study in space and time. In particular, sustained local control of molecular function is challenging owing to the lateral diffusion of plasma membrane targeted molecules. Herein we present 'molecular activity painting' (MAP), a novel technology which combines photoactivatable chemically induced dimerization (pCID) with immobilized artificial receptors. The immobilization of artificial receptors by surface-immobilized antibodies blocks lateral diffusion, enabling rapid and stable 'painting' of signaling molecules and their activity at the plasma membrane with micrometer precision. Using this method, we show that painting of the RhoA-myosin activator GEF-H1 induces patterned acto-myosin contraction inside living cells. [ABSTRACT FROM AUTHOR]