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Much of our current knowledge of Rho GTPase networks and the regulation by Rho guanine exchange factors (Rho GEFs) and Rho GTPase activating proteins (Rho GAPs) is based on population-based techniques. Over the last decades, technologies that enable single cell analysis with high spatial and temporal resolution have revealed that Rho GTPase activity in cells is regulated on second timescales and at submicrometer length scales. Therefore, perturbation methods with matching spatial and temporal resolution are crucial to further our understanding of Rho GTPase signaling. Here, we give a brief overview of the components of Rho GTPase signaling networks and review a range of existing perturbation strategies that target a specific component of the Rho GTPase signaling module. The advantages and limitations of each perturbation method are discussed. Several recommendations are formulated to guide future studies aimed at addressing spatiotemporal aspects of Rho GEF and Rho GTPase signaling.

Oscillations occur in a wide variety of essential cellular processes, such as cell cycle progression, circadian clocks and calcium signaling in response to stimuli. It remains unclear how intrinsic stochasticity can influence these oscillatory systems. Here, we focus on oscillations of Cdc42 GTPase in fission yeast. We extend our previous deterministic model by Xu and Jilkine to construct a stochastic model, focusing on the fast diffusion case. We use SSA (Gillespie's algorithm) to numerically explore the low copy number regime in this model, and use analytical techniques to study the long-time behavior of the stochastic model and compare it to the equilibria of its deterministic counterpart. Numerical solutions suggest noisy limit cycles exist in the parameter regime in which the deterministic system converges to a stable limit cycle, and quasi-cycles exist in the parameter regime where the deterministic model has a damped oscillation. Near an infinite period bifurcation point, the deterministic model has a sustained oscillation, while stochastic trajectories start with an oscillatory mode and tend to approach deterministic steady states. In the low copy number regime, metastable transitions from oscillatory to steady behavior occur in the stochastic model. Our work contributes to the understanding of how stochastic chemical kinetics can affect a finite-dimensional dynamical system, and destabilize a deterministic steady state leading to oscillations.

Small GTPases play crucial roles in the maintenance of a homeostatic environment and appropriate movements of the cell. In these processes, the direct or indirect interaction between distinct small GTPases could be required for regulating mutual signaling pathways. In our recent study, ARHGEF10, known as a guanine nucleotide exchange factor (GEF) for RhoA, was indicated to interact with Rab6A and Rab8A, which are known to function in the exocytotic pathway, and colocalized with these Rabs at exocytotic vesicles. Moreover, it was suggested that ARHGEF10 is involved in the regulation of Rab6A and Rab8A localization and invasion of breast carcinoma cells, in which Rab8 also acts via regulation of membrane trafficking. These results may reveal the existence of a novel small GTPase cascade which connects the signaling of these Rabs with RhoA during membrane trafficking. In this mini-review, we consider the possible functions of ARHGEF10 and RhoA in the Rab6- and Rab8-mediated membrane trafficking pathway.

Colorectal cancer (CRC) is the third most common malignancy and a leading cause of cancer-related death worldwide. GEF-H1 is considered a RhoA-specific guanine nucleotide exchange factor. GEF-H1 upregulation may contribute to cancer cell migration and invasion and tumor progression. However, the expression and role of GEF-H1 in CRC have not yet been elucidated. This study attempted to elucidate how GEF-H1 drives tumor formation, motility, invasion and metastasis in colon cancer (CC). The expression of GEF-H1 in CC tissue microarrays (TMAs) was analyzed by immunohistochemistry (IHC). GEF-H1 was upregulated in CC tissues compared with adjacent non-tumoral tissues. In addition, we found that high GEF-H1 expression correlated with shorter overall survival and distant metastasis. Migration and invasion assays showed that GEF-H1 upregulation increased CC cell motility, invasion and metastasis. In contrast, functional knockdown of GEF-H1 by RNAi rescued the effects caused by GEF-H1 overexpression in CC cells. Overexpression of GEF-H1 re-organized the actin cytoskeleton, with increased punctate paxillin staining and F-actin stress fibers. Furthermore, western blotting showed that RhoA activation triggered by GEF-H1 overexpression caused phosphorylation of its downstream target, MLC2, in CC cells. In summary, the present study revealed that GEF-H1 is upregulated in CC tissues and plays a key role in CC metastasis through the GEF-H1-RhoA-MLC2 signaling pathway., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Proper function of GABAergic synapses depends upon the postsynaptic compartment anchoring of neurotransmitter receptors to the membrane by gephyrin and collybistin (Cb). In humans, Cb is encoded by ARHGEF9 on Xq11.1. ARHGEF9 alterations, some inherited from unaffected mothers, have been reported in males with autism, seizures and severe neurodevelopmental abnormalities. In females, a spectrum of mild to moderate phenotype has been detected. We report two unrelated females with autism and mild intellectual disability. High resolution X-chromosome microarray analysis revealed de novo intragenic deletions in ARHGEF9 of 24 kb and 56 kb involving exons 5-8 and exons 3-8 and leading to truncated forms of collybistin. Peripheral blood samples revealed random X-chromosome inactivation in both patients. To explain phenotypic variability in female patients, we propose a model for disruption of collybistin and various irregular interactions in post-synaptic neurons based on X inactivation patterns. Our findings highlight the importance of ARHGEF9 integrity and suggest further research on its correlation with autism and neurobehavioral problems., (Copyright © 2018. Published by Elsevier Masson SAS.)

During infection, transcription factor interferon regulatory factor 5 (IRF5) is essential for the control of host defense. Here we show that the microtubule-associated guanine nucleotide exchange factor (GEF)-H1, is required for the phosphorylation of IRF5 by microbial muramyl-dipeptides (MDP), the minimal structural motif of peptidoglycan of both Gram-positive and Gram-negative bacteria. Specifically, GEF-H1 functions in a microtubule based recognition system for microbial peptidoglycans that mediates the activation of IKKε which we identify as a new upstream IKKα/β and IRF5 kinase. The deletion of GEF-H1 or dominant-negative variants of GEF-H1 prevent activation of IKKε and phosphorylation of IRF5. The GEF-H1-IKKε-IRF5 signaling axis functions independent of NOD-like receptors and is critically required for the recognition of intracellular peptidoglycans and host defenses against Listeria monocytogenes.

Staphylococcus aureus is a major etiological agent of sepsis and induces endothelial cell (EC) barrier dysfunction and inflammation, two major hallmarks of acute lung injury. However, the molecular mechanisms of bacterial pathogen-induced EC barrier disruption are incompletely understood. Here, we investigated the role of microtubules (MT) in the mechanisms of EC barrier compromise caused by heat-killed S. aureus (HKSA). Using a customized monolayer permeability assay in human pulmonary EC and MT fractionation, we observed that HKSA-induced barrier disruption is accompanied by MT destabilization and increased histone deacetylase-6 (HDAC6) activity resulting from elevated reactive oxygen species (ROS) production. Molecular or pharmacological HDAC6 inhibition rescued barrier function in HKSA-challenged vascular endothelium. The HKSA-induced EC permeability was associated with impaired MT-mediated delivery of cytoplasmic linker-associated protein 2 (CLASP2) to the cell periphery, limiting its interaction with adherens junction proteins. HKSA-induced EC barrier dysfunction was also associated with increased Rho GTPase activity via activation of MT-bound Rho-specific guanine nucleotide exchange factor-H1 (GEF-H1) and was abolished by HDAC6 down-regulation. HKSA activated the NF-κB proinflammatory pathway and increased the expression of intercellular and vascular cell adhesion molecules in EC, an effect that was also HDAC6-dependent and mediated, at least in part, by a GEF-H1/Rho-dependent mechanism. Of note, HDAC6 knockout mice or HDAC6 inhibitor-treated WT mice were partially protected from vascular leakage and inflammation caused by both HKSA or methicillin-resistant S. aureus (MRSA). Our results indicate that S. aureus -induced, ROS-dependent up-regulation of HDAC6 activity destabilizes MT and thereby activates the GEF-H1/Rho pathway, increasing both EC permeability and inflammation., (© 2019 Karki et al.)

Chronic lymphocytic leukemia cells (CLL) migrate between the blood and lymphoid tissues in response to chemokines. Such migration requires structured cytoskeletal-actin polymerization, which may involve the protein cortactin. We discovered that treatment of CLL cells with Wnt5a causes Receptor tyosin kinase-like orphan receptor 1 (ROR1) to bind cortactin, which undergoes tyrosine phosphorylation at Y421, recruits ARHGEF1, and activates RhoA, thereby enhancing leukemia-cell migration; such effects could be inhibited by cirmtuzumab, a humanized mAb specific for ROR1. We transfected the CLL-cell-line MEC1 with either full-length ROR1 or various mutant forms of ROR1 to examine the structural features required for binding cortactin. We found that the proline-rich domain (PRD) was necessary for ROR1 to recruit cortactin. We generated MEC1 cells that each expressed a mutant form of ROR1 with a single amino-acid substitution of alanine (A) for proline (P) in potential SH3-binding sites in the ROR1-PRD at positions 784, 808, 826, 841, or 850. In contrast to wild-type ROR1, or other ROR1 P=>A mutants, ROR1 P(841)A failed to complex with cortactin or ARHGEF1 in response to Wnt5a. Moreover, Wnt5a could not induce MEC1-ROR1 P(841)A to phosphorylate cortactin or enhance CLL-cell F-actin polymerization. Taken together, these studies show that cortactin plays an important role in ROR1-dependent Wnt5a-enhanced CLL-cell migration.

During cytokinesis, fission yeast coordinates actomyosin ring constriction with septum ingression, resulting in concentric furrow formation by a poorly defined mechanism. We report that Schizosaccharomyces pombe cells lacking the Cdc42 activator Gef1, combined with an activated allele of the formin, Cdc12, display non-concentric furrowing. Non-concentrically furrowing cells display uneven distribution of the scaffold Cdc15 along the ring. This suggests that, after ring assembly, uniform Cdc15 distribution along the ring enables proper furrow formation. We find that, after assembly, Cdc15 is recruited to the ring in an Arp2/3 complex-dependent manner and is decreased in the activated cdc12 mutant. Cdc15 at cortical endocytic patches shows increased levels and extended lifetimes in gef1 and activated cdc12 mutants. We hypothesize endocytosis helps recruit Cdc15 to assembled rings; uneven Cdc15 distribution at the ring occurs when endocytic patches contain increased Cdc15 levels and the patch-association rate is slow. Based on this, we developed a mathematical model that captures experimentally observed Cdc15 distributions along the ring. We propose that, at the ring, Gef1 and endocytic events promote uniform Cdc15 organization to enable proper septum ingression and concentric furrow formation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

PAK-interacting guanine nucleotide exchange factor (βPix; also known as Arhgef7) has been implicated in many actin-based cellular processes, including spine morphogenesis in neurons. However, the molecular mechanisms by which βPix controls spine morphology remain elusive. Previously, we have reported the expression of several alternative spliced βPix isoforms in the brain. Here, we report a novel finding that the b isoform of βPix (βPix-b) mediates the regulation of spine and synapse formation. We found that βPix-b, which is mainly expressed in neurons, enhances spine and synapse formation through preferential localization at spines. In neurons, glutamate treatment efficiently stimulates Rac1 GEF activity of βPix-b. The glutamate stimulation also promotes Src-mediated phosphorylation of βPix-b in both an AMPA receptor- and NMDA receptor-dependent manner. Tyrosine 598 (Y598) of βPix-b is identified as the major Src-mediated phosphorylation site. Finally, Y598 phosphorylation of βPix-b enhances its Rac1 GEF activity that is critical for spine and synapse formation. In conclusion, we provide a novel mechanism by which βPix-b regulates activity-dependent spinogenesis and synaptogenesis via Src-mediated phosphorylation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The C-terminal guanine nucleotide exchange factor (GEF) module of Trio (TrioC) transfers signals from the Gα q/11 subfamily of heterotrimeric G proteins to the small guanosine triphosphatase (GTPase) RhoA, enabling Gα q/11 -coupled G protein-coupled receptors (GPCRs) to control downstream events, such as cell motility and gene transcription. This conserved signal transduction axis is crucial for tumor growth in uveal melanoma. Previous studies indicate that the GEF activity of the TrioC module is autoinhibited, with release of autoinhibition upon Gα q/11 binding. Here, we determined the crystal structure of TrioC in its basal state and found that the pleckstrin homology (PH) domain interacts with the Dbl homology (DH) domain in a manner that occludes the Rho GTPase binding site, thereby suggesting the molecular basis of TrioC autoinhibition. Biochemical and biophysical assays revealed that disruption of the autoinhibited conformation destabilized and activated the TrioC module in vitro. Last, mutations in the DH-PH interface found in patients with cancer activated TrioC and, in the context of full-length Trio, led to increased abundance of guanosine triphosphate-bound RhoA (RhoA·GTP) in human cells. These mutations increase mitogenic signaling through the RhoA axis and, therefore, may represent cancer drivers operating in a Gα q/11 -independent manner., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Motility and phagocytosis are key processes that are involved in invasive amoebiasis disease caused by intestinal parasite Entamoeba histolytica. Previous studies have reported unconventional myosins to play significant role in membrane based motility as well as endocytic processes. EhMyosin IB is the only unconventional myosin present in E. histolytica, is thought to be involved in both of these processes. Here, we report an interaction between the SH3 domain of EhMyosin IB and c-terminal domain of EhFP10, a Rho guanine nucleotide exchange factor. EhFP10 was found to be confined to Entamoeba species only, and to contain a c-terminal domain that binds and bundles actin filaments. EhFP10 was observed to localize in the membrane ruffles, phagocytic and macropinocytic cups of E. histolytica trophozoites. It was also found in early pinosomes but not early phagosomes. A crystal structure of the c-terminal SH3 domain of EhMyosin IB (EhMySH3) in complex with an EhFP10 peptide and co-localization studies established the interaction of EhMySH3 with EhFP10. This interaction was shown to lead to inhibition of actin bundling activity and to thereby regulate actin dynamics during endocytosis. We hypothesize that unique domain architecture of EhFP10 might be compensating the absence of Wasp and related proteins in Entamoeba, which are known partners of myosin SH3 domains in other eukaryotes. Our findings also highlights the role of actin bundling during endocytosis., Competing Interests: The authors have declared that no competing interests exist.

NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for exocyst localization to cell tips and for exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to exocyst function in concert with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Aberrant activity of Rho small G-proteins and their regulators plays an important role in tumorigenesis. Rho guanine nucleotide exchange factor 10-Like (ARHGEF10L) is a member of the RhoGEF family that promotes the active GTP-bound state of Rho GTPases. This study used the Illumina GoldenGate microassay, Sequenom MassARRAY and TaqMan to analyze possible correlations between tag single nucleotide polymorphisms (tag SNPs) in the ARHGEF10L locus and various tumor risks. The genotyping analyses demonstrated a strong association of rs2244444 and rs12732894 with liver cancer. Western blotting and immunohistochemistry also revealed increased expression of ARHGEF10L in hepatocellular carcinoma tissues. Furthermore, increased cell proliferation, cell migration and RhoA activity; increased expression of Rho-associated coiled-coil kinase-1 (ROCK1), phospho- Ezrin/Radixin/Moesin (ERM), vimentin, N-cadherin and Slug, and decreased E-cadherin expression were detected in hepatocellular carcinoma cell Bel-7402 and HepG2 cells with transfection of ARHGEF10L-expressing plasmids. Opposite results were obtained in the two cell lines with transfection of anti-ARHGEF10L siRNA. Tumor-bearing mice were generated with Bel-7402 cells transfected with lentivirus vectors packaging short hairpin ARHGEF10L RNA. The xenograft tumors with the inhibited ARHGEF10L expression showed decreased tumor growth and expression of vimentin, N-cadherin and Slug. Additionally, decreased phospho-ERM expression was detected in Bel-7402 and HepG2 cells with transfection of anti-ROCK1 siRNA and increased expression of ROCK1 was detected in hepatocellular carcinoma tissues. E-cadherin, vimentin, N-cadherin and Slug are markers of the epithelial-to-mesenchymal transition (EMT). ROCK1, phospho-ERM and EMT have been reported to promote tumor cell proliferation, metastasis and angiogenesis. Our study suggests that increased expression of ARHGEF10L stimulates hepatocellular tumorigenesis by activating the RhoA-ROCK1- phospho ERM pathway and EMT., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Loss-of-function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early-onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule-associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3-labelled plus-end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAP1S dissociation from microtubules. Additionally, anterograde cargo trafficking is compromised in CDKL5 knockout mouse dendrites. Finally, EB2 phosphorylation is reduced in patient-derived human neurons. Our results reveal a novel activity-dependent molecular pathway in dendritic microtubule regulation and suggest a pathological mechanism which may contribute to CDKL5 deficiency disorder., (© 2018 The Francis Crick Institute. Published under the terms of the CC BY 4.0 license.)

Apical constriction regulates epithelial morphogenesis during embryonic development, but how this process is controlled is not understood completely. Here, we identify a Rho guanine nucleotide exchange factor (GEF) gene plekhg5 as an essential regulator of apical constriction of bottle cells during Xenopus gastrulation. plekhg5 is expressed in the blastopore lip and its expression is sufficient to induce ectopic bottle cells in epithelia of different germ layers in a Rho-dependent manner. This activity is not shared by arhgef3 , which encodes another organizer-specific RhoGEF. Plekhg5 protein is localized in the apical cell cortex via its pleckstrin homology domain, and the GEF activity enhances its apical recruitment. Plekhg5 induces apical actomyosin accumulation and cell elongation. Knockdown of plekhg5 inhibits activin-induced bottle cell formation and endogenous blastopore lip formation in gastrulating frog embryos. Apical accumulation of actomyosin, apical constriction and bottle cell formation fail to occur in these embryos. Taken together, our data indicate that transcriptional regulation of plekhg5 expression at the blastopore lip determines bottle cell morphology via local polarized activation of Rho by Plekhg5, which stimulates apical actomyosin activity to induce apical constriction., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Background: Colorectal cancer (CRC) is one of the most common digestive malignant tumors, and DMTN is a transcriptionally differentially expressed gene that was identified using CRC mRNA sequencing data from The Cancer Genome Atlas (TCGA). Our preliminary work suggested that the expression of DMTN was downregulated in CRC, and the Rac1 signaling pathway was significantly enriched in CRC tissues with low DMTN expression. However, the specific functions and underlying molecular mechanisms of DMTN in the progression of CRC and the upstream factors regulating the downregulation of the gene remain unclear., Methods: DMTN expression was analyzed in CRC tissues, and the relationship between DMTN expression and the clinicopathological parameters was analyzed. In vitro and in vivo experimental models were used to detect the effects of DMTN dysregulation on invasion and metastasis of CRC cells. GSEA assay was performed to explore the mechanism of DMTN in invasion and metastasis of CRC. Westernblot, Co-IP and GST-Pull-Down assay were used to detect the interaction between DMTN and ARHGEF2, as well as the activation of the RAC1 signaling. Bisulfite genomic sequence (BSP) assay was used to test the degree of methylation of DMTN gene promoter in CRC tissues., Results: We found that the expression of DMTN was significantly decreased in CRC tissues, and the downregulation of DMTN was associated with advanced progression and poor survival and was regarded as an independent predictive factor of CRC patient prognosis. The overexpression of DMTN inhibited, while the knockdown of DMTN promoted, invasion and metastasis in CRC cells. Moreover, hypermethylation and the deletion of DMTN relieved binding to the ARHGEF2 protein, activated the Rac1 signaling pathway, regulated actin cytoskeletal rearrangements, and promoted the invasion and metastasis of CRC cells., Conclusion: Our study demonstrated that the downregulation of DMTN promoted the metastasis of colorectal cancer cells by regulating the actin cytoskeleton through RAC1 signaling activation, potentially providing a new therapeutic target to enable cancer precision medicine for CRC patients.

The epithelial-specific splicing regulators Esrp1 and Esrp2 are required for mammalian development, including establishment of epidermal barrier functions. However, the mechanisms by which Esrp ablation causes defects in epithelial barriers remain undefined. We determined that the ablation of Esrp1 and Esrp2 impairs epithelial tight junction (TJ) integrity through loss of the epithelial isoform of Rho GTP exchange factor Arhgef11. Arhgef11 is required for the maintenance of TJs via RhoA activation and myosin light chain (MLC) phosphorylation. Ablation or depletion of Esrp1/2 or Arhgef11 inhibits MLC phosphorylation and only the epithelial Arhgef11 isoform rescues MLC phosphorylation in Arhgef11 KO epithelial cells. Mesenchymal Arhgef11 transcripts contain a C-terminal exon that binds to PAK4 and inhibits RhoA activation byArhgef11. Deletion of the mesenchymal-specific Arhgef11 exon in Esrp1/2 KO epithelial cells using CRISPR/Cas9 restored TJ function, illustrating how splicing alterations can be mechanistically linked to disease phenotypes that result from impaired functions of splicing regulators., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Mechanical and metabolic cues independently contribute to the regulation of cell and tissue homeostasis. However, how they cross-regulate each other during this process remains largely unknown. Here, we show that cellular metabolism can regulate integrin rigidity-sensing via the sphingolipid metabolic pathway controlled by the amino acid transporter and integrin coreceptor CD98hc (SLC3A2). Genetic invalidation of CD98hc in dermal cells and tissue impairs rigidity sensing and mechanical signaling downstream of integrins, including RhoA activation, resulting in aberrant tissue mechanical homeostasis. Unexpectedly, we found that this regulation does not occur directly through regulation of integrins by CD98hc but indirectly, via the regulation of sphingolipid synthesis and the delta-4-desaturase DES2. Loss of CD98hc decreases sphingolipid availability preventing proper membrane recruitment, shuttling and activation of upstream regulators of RhoA including Src kinases and GEF-H1. Altogether, our results unravel a novel cross-talk regulation between integrin mechanosensing and cellular metabolism which may constitute an important new regulatory framework contributing to mechanical homeostasis.

Patients with severe, treatment-refractory asthma are at risk for death from acute exacerbations. The cytokine IL17A has been associated with airway inflammation in severe asthma, and novel therapeutic targets within this pathway are urgently needed. We recently showed that IL17A increases airway contractility by activating the procontractile GTPase RhoA. Here, we explore the therapeutic potential of targeting the RhoA pathway activated by IL17A by inhibiting RhoA guanine nucleotide exchange factors (RhoGEFs), intracellular activators of RhoA. We first used a ribosomal pulldown approach to profile mouse airway smooth muscle by qPCR and identified Arhgef12 as highly expressed among a panel of RhoGEFs. ARHGEF12 was also the most highly expressed RhoGEF in patients with asthma, as found by RNA sequencing. Tracheal rings from Arhgef12-KO mice and WT rings treated with a RhoGEF inhibitor had evidence of decreased contractility and RhoA activation in response to IL17A treatment. In a house dust mite model of allergic sensitization, Arhgef12-KO mice had decreased airway hyperresponsiveness without effects on airway inflammation. Taken together, our results show that Arhgef12 is necessary for IL17A-induced airway contractility and identify a therapeutic target for severe asthma.

The sodium iodide symporter (NIS) is a classical iodide pump typically localized within the cell plasma membrane in thyroid cells, where NIS expression is believed to ensure success of mainstay radioiodide therapy in thyroid cancers. Although radioiodide uptake is generally reduced in thyroid cancer tissue, intracellular nonmembranous NIS has been reported to increase, suggesting that NIS serves a pump-independent function. Thyroid cancer is one of the major component cancers of Cowden syndrome, a subset of which is caused by germline mutations in PTEN In this study, we explored the noncanonical tumorigenic role of NIS in thyroid cancer cells in relation to PTEN signaling. PTEN knockdown in thyroid cancer cell lines stabilized intracellular NIS protein by promoting an interaction with NIS-LARG (leukemia-associated RhoA guanine exchange factor). Increased protein levels of cytoplasmic NIS enhanced RhoA activation and resulted in a promigration tumorigenic phenotype. Inhibition of NIS glycosylation through activation of the PI3K/AKT/mTOR signaling pathway contributed to mislocalization of NIS in the cytoplasm, facilitating its nonpump tumorigenic function through an interaction with LARG, which predominantly localized in the cytoplasm. Moreover, PTEN or PI3K/AKT/mTOR signaling could affect DPAGT1, a glycosylating enzyme involved in the initial step of N-linked glycosylation, to inhibit glycosylation of NIS. In summary, our results elucidate a pump-independent, protumorigenic role for NIS in thyroid cancer via its cross-talk with PTEN signaling. Significance: A novel pump-independent protumorigenic role of nonmembranous NIS challenges the presumption that radioiodine treatment of thyroid cancer is ineffective when transmembrane NIS is not expressed. Cancer Res; 78(21); 6121-33. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Rho guanine nucleotide exchange factor 4 (ARHGEF4) is a guanine nucleotide exchange factor that is specific for Rac1 and Cdc42. The aim of the present study was to investigate the role of ARHGEF4 in the motility and invasiveness of pancreatic cancer cells. Evaluation of an immunohistochemical staining of 102 resected pancreatic cancer samples demonstrated that high ARHGEF4 expression was correlated with an independent predictor of worse overall survival in univariate and multivariate analyses. Immunofluorescence analyses and Matrigel invasion assays demonstrated that suppression of ARHGEF4 inhibited the formation of membrane protrusions, and in turn inhibited cell motility and invasion. A phosphoprotein array analysis demonstrated that knockdown of ARHGEF4 decreased phosphorylated extracellular signal-regulated kinase (ERK)1/2 and glycogen synthase kinase-3 (GSK-3)α/β in pancreatic cancer cells, and ERK1/2 and GSK-3α/β were associated with ARHGEF4-related motility and invasiveness through an increase in cell protrusions. These results suggested that ARHGEF4 stimulates ERK1/2 and GSK-3α/β, and provided evidence that ARHGEF4 promotes cell motility and invasiveness. Inhibition of ARHGEF4 may be a novel approach to a targeted molecular therapy, as any such therapy would limit the motility and invasiveness of pancreatic cancer cells.

Deep layer III pyramidal cells in the dorsolateral prefrontal cortex (DLPFC) from subjects with schizophrenia and bipolar disorder previously were shown to exhibit dendritic arbor pathology. This study sought to determine whether MARCKS, its regulatory protein dysbindin-1, and two proteins, identified using microarray data, CDC42BPA and ARHGEF6, were associated with dendritic arbor pathology in the DLPFC from schizophrenia and bipolar disorder subjects. Using western blotting, relative protein expression was assessed in the DLPFC (BA 46) grey matter from subjects with schizophrenia (n = 19), bipolar disorder (n = 17) and unaffected control subjects (n = 19). Protein expression data were then correlated with dendritic parameter data obtained previously. MARCKS and dysbindin-1a expression levels did not differ among the three groups. Dysbindin-1b expression was 26% higher in schizophrenia subjects (p = 0.01) and correlated inversely with basilar dendrite length (r = -0.31, p = 0.048) and the number of spines per basilar dendrite (r = -0.31, p = 0.048), but not with dendritic spine density (r = -0.16, p = 0.32). The protein expression of CDC42BPA was 33% higher in schizophrenia subjects (p = 0.03) but, did not correlate with any dendritic parameter (p > 0.05). ARHGEF6 87 kDa isoform expression did not differ among the groups. CDC42BPA expression was not altered in frontal cortex from rats chronically administered haloperidol or clozapine. Dysbindin-1b appears to play a role in dendritic arbor pathology observed previously in the DLPFC in schizophrenia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Previous studies have shown that Rho guanine nucleotide exchange factor 7 (ARHGEF7) is implicated in cytoskeleton remodelling, which is important for cell motility and invasiveness, and exhibits frequent high-level genetic amplification in metastatic lesions of colorectal adenocarcinoma. Therefore, it was hypothesized that ARHGEF7 may be involved in the metastasis of colorectal adenocarcinoma. In the present study, it was demonstrated that the expression level of ARHGEF7 was significantly upregulated in colorectal adenocarcinoma tumor tissues compared with matched nontumorous tissues, and its expression level correlated with colorectal adenocarcinoma metastasis. In vitro assays showed that the overexpression of ARHGEF7 in CRC cells significantly enhanced cell migration and invasion, whereas the knockdown of ARHGEF7 in colorectal adenocarcinoma cells significantly decreased cell migration and invasion. In vivo assays showed that the overexpression of ARHGEF7 in CRC cells facilitated tumor metastasis, whereas the knockdown of ARHGEF7 in CRC cells significantly inhibited tumor metastasis. Furthermore, it was demonstrated that ARHGEF7 promoted cell motility by regulating the actin cytoskeleton. Finally, according to ReMARK guidelines for reporting prognostic biomarkers in cancer, it was found that a high expression of ARHGEF7 was significantly correlated with lymph node, mesenteric and distant metastasis. Patients with colorectal adenocarcinoma with a high expression of ARHGEF7 had shorter disease-free survival (DFS) and shorter overall survival (OS) rates, compared with those with a low expression of ARHGEF7, as determined by the Kaplan-Meier method with a log-rank test. Cox regression analysis showed that a high expression of ARHGEF7 was an independent risk factor for DFS and OS rates in colorectal adenocarcinoma.

miR-30d has been shown to play pivotal roles in cancer development, and has the potential to act as a diagnostic biomarker and therapeutic target in breast cancer. However, the specific function and molecular mechanism of miR-30d in breast cancer cell growth and metastasis is still unknown. The present study seeks to shed light on the potential contribution of the MiR-30d-KLF-11-STAT3 pathway in breast cancer. The results revealed that miR-30d levels were markedly increased in the breast cancer cell lines BT474, MDA-MB-231, HCC197, and MDA-MB-468 compared with the non-tumor mammary gland MCF10A cell line. Furthermore, the miR-30d mimic increased BT474 and MDA-MB-231 breast cancer cell survival, inhibited apoptosis and increased Bcl-2 expression, whilst inhibited Bax protein levels. miR-30d mimics promote BT474 and MDA-MB-231 cell migration, invasion, and mediate the EMT phenotype. However, miR-30d inhibitors reverse all of the effects of miR-30d mimics on breast cancer cell biology. Also, we observed that KLF-11 is a direct target of miR-30d and KLF-11 and pSTAT3 expression are determined by miR-30d. Finally, the results suggest that miR-30d plays essential roles in breast cancer cells in a manner that is dependent on the levels of KLF-1 and pSTAT3. In summary, miR-30d appears to be a novel diagnostic biomarker and treatment target in breast cancer., (© 2018 Wiley Periodicals, Inc.)

Background: By allowing intercellular communication between cells, tunneling nanotubes (TNTs) could play critical role in cancer progression. If TNT formation is known to require cytoskeleton remodeling, key mechanism controlling their formation remains poorly understood., Methods: The cells of human bronchial (HBEC-3, A549) or mesothelial (H2452, H28) lines are transfected with different siRNAs (inactive, anti-RASSF1A, anti-GEFH1 and / or anti-Rab11). At 48 h post-transfection, i) the number and length of the nanotubes per cell are quantified, ii) the organelles, previously labeled with specific tracers, exchanged via these structures are monitored in real time between cells cultured in 2D or 3D and in normoxia, hypoxia or in serum deprivation condition., Results: We report that RASSF1A, a key-regulator of cytoskeleton encoded by a tumor-suppressor gene on 3p chromosome, is involved in TNTs formation in bronchial and pleural cells since controlling proper activity of RhoB guanine nucleotide exchange factor, GEF-H1. Indeed, the GEF-H1 inactivation induced by RASSF1A silencing, leads to Rab11 accumulation and subsequent exosome releasing, which in turn contribute to TNTs formation. Finally, we provide evidence involving TNT formation in bronchial carcinogenesis, by reporting that hypoxia or nutriment privation, two almost universal conditions in human cancers, fail to prevent TNTs induced by the oncogenic RASSF1A loss of expression., Conclusions: This finding suggests for the first time that loss of RASSF1A expression could be a potential biomarker for TNTs formation, such TNTs facilitating intercellular communication favoring multistep progression of bronchial epithelial cells toward overt malignancy.

Glioblastoma multiforme (GBM) is the most common type of malignant brain tumors in adults and has a dismal prognosis. The highly aggressive invasion of malignant cells into the normal brain parenchyma renders complete surgical resection of GBM tumors impossible, increases resistance to therapeutic treatment, and leads to near-universal tumor recurrence. We have previously demonstrated that TROY (TNFRSF19) plays an important role in glioblastoma cell invasion and therapeutic resistance. However, the potential downstream effectors of TROY signaling have not been fully characterized. Here, we identified PDZ-RhoGEF as a binding partner for TROY that potentiated TROY-induced nuclear factor kappa B activation which is necessary for both cell invasion and survival. In addition, PDZ-RhoGEF also interacts with Pyk2, indicating that PDZ-RhoGEF is a component of a signalsome that includes TROY and Pyk2. PDZ-RhoGEF is overexpressed in glioblastoma tumors and stimulates glioma cell invasion via Rho activation. Increased PDZ-RhoGEF expression enhanced TROY-induced glioma cell migration. Conversely, silencing PDZ-RhoGEF expression inhibited TROY-induced glioma cell migration, increased sensitivity to temozolomide treatment, and extended survival of orthotopic xenograft mice. Furthermore, depletion of RhoC or RhoA inhibited TROY- and PDZ-RhoGEF-induced cell migration. Mechanistically, increased TROY expression stimulated Rho activation, and depletion of PDZ-RhoGEF expression reduced this activation. Taken together, these data suggest that PDZ-RhoGEF plays an important role in TROY signaling and provides insights into a potential node of vulnerability to limit GBM cell invasion and decrease therapeutic resistance., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

We attempted to investigate the mechanism and susceptibility genes for diabetes in Han and Kazak ethnic individuals.The abdominal omental adipose tissues were obtained from diabetic cases and healthy controls. The gene expression and methylation data were produced for Kazak and Han individuals, respectively, and analyzed by bioinformatics analysis.We obtained 921 differentially expressed genes (DEGs) in Han group and 1772 in Kazak group. DEGs in Han group were significantly related with type 2 diabetes mellitus, and biosynthesis of amino acids, while the DEGs specific to Kazak patients were significantly enriched in metabolism-related pathways such as carbon metabolism, propanoate metabolism, and 2-oxocarboxylic acid metabolism. Major facilitator superfamily domain containing 1 (MFSD1) was found to be a methylation associated gene at hypermethylation site of cg16289538 in Han group. Rho guanine nucleotide exchange factor 1 (ARHGEF1) was the susceptible gene corresponding to the methylation sites of cg18800192 and cg00759295 in Kazak group. ARHGEF1 was also a node in protein-protein interaction network and significantly enriched in hsa04270: vascular smooth muscle contraction pathways.The molecular mechanism of diabetes may be different in Han and Kazak patients. MFSD1 and ARHGEF1 may be the diabetes susceptible genes.

Adenomatous polyposis coli (APC) is a key molecule to maintain cellular homeostasis in colonic epithelium by regulating cell-cell adhesion, cell polarity, and cell migration through activating the APC-stimulated guanine nucleotide-exchange factor (Asef). The APC-activated Asef stimulates the small GTPase, which leads to decreased cell-cell adherence and cell polarity, and enhanced cell migration. In colorectal cancers, while truncated APC constitutively activates Asef and promotes cancer initiation and progression, regulation of Asef by full-length APC is still unclear. Here, we report the autoinhibition mechanism of full-length APC. We found that the armadillo repeats in full-length APC interact with the APC residues 1362 to 1540 (APC-2,3 repeats), and this interaction competes off and inhibits Asef. Deletion of APC-2,3 repeats permits Asef interactions leading to downstream signaling events, including the induction of Golgi fragmentation through the activation of the Asef-ROCK-MLC2. Truncated APC also disrupts protein trafficking and cholesterol homeostasis by inhibition of SREBP2 activity in a Golgi fragmentation-dependent manner. Our study thus uncovers the autoinhibition mechanism of full-length APC and a novel gain of function of truncated APC in regulating Golgi structure, as well as cholesterol homeostasis, which provides a potential target for pharmaceutical intervention against colon cancers., (Copyright © 2018 American Society for Microbiology.)

Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABA A Rs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABA A Rs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2-1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.

Protein kinase D (PKD) is a family of serine/threonine kinases that is required for the structural integrity and function of the Golgi complex. Despite its importance in the regulation of Golgi function, the molecular mechanisms regulating PKD activity are still incompletely understood. Using the genetically encoded PKD activity reporter G-PKDrep we now uncover a Rho signaling network comprising GEF-H1, the RhoGAP DLC3, and the Rho effector PLCε that regulate the activation of PKD at trans-Golgi membranes. We further show that this molecular network coordinates the formation of TGN-derived Rab6-positive transport carriers delivering cargo for localized exocytosis at focal adhesions., Competing Interests: SE, FC, GL, SS, MN, MS, MO, AH No competing interests declared, (© 2018, Eisler et al.)

Our previous work has established that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na + channel (ENaC) by promoting its binding to neural precursor cell-expressed, developmentally down-regulated 4-2, E3 ubiquitin protein ligase (Nedd4-2). Here, using MS analysis and in vitro phosphorylation, we show that AMPK phosphorylates Nedd4-2 at the Ser-444 ( Xenopus Nedd4-2) site critical for Nedd4-2 stability. We further demonstrate that the Pak-interacting exchange factor β 1 Pix is required for AMPK-mediated inhibition of ENaC-dependent currents in both CHO and murine kidney cortical collecting duct (CCD) cells. Short hairpin RNA-mediated knockdown of β 1 Pix expression in CCD cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β 1 Pix dimerization-deficient mutant unable to bind 14-3-3 proteins (Δ602-611) increased ENaC currents in CCD cells, whereas overexpression of WT β 1 Pix had the opposite effect. Using additional immunoblotting and co-immunoprecipitation experiments, we found that treatment with AMPK activators promoted the binding of β 1 Pix to 14-3-3 proteins in CCD cells. However, the association between Nedd4-2 and 14-3-3 proteins was not consistently affected by AMPK activation, β 1 Pix knockdown, or overexpression of WT β 1 Pix or the β 1 Pix-Δ602-611 mutant. Moreover, we found that β 1 Pix is important for phosphorylation of the aforementioned Nedd4-2 site critical for its stability. Overall, these findings elucidate novel molecular mechanisms by which AMPK regulates ENaC. Specifically, they indicate that AMPK promotes the assembly of β 1 Pix, 14-3-3 proteins, and Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and its degradation., (© 2018 Ho et al.)

The conserved Rho-family GTPase Cdc42 plays a central role in eukaryotic cell polarity. The rod-shaped fission yeast Schizosaccharomyces pombe has two Cdc42 guanine nucleotide exchange factors (GEFs), Scd1 and Gef1, but little is known about how they are coordinated in polarized growth. Although the microtubule cytoskeleton is normally not required for polarity maintenance in fission yeast, we show here that when scd1 function is compromised, disruption of microtubules or the polarity landmark proteins Tea1, Tea4 or Pom1 leads to disruption of polarized growth. Instead, cells adopt an isotropic-like pattern of growth, which we term PORTLI growth. Surprisingly, PORTLI growth is caused by spatially inappropriate activity of Gef1. Although most Cdc42 GEFs are membrane associated, we find that Gef1 is a broadly distributed cytosolic protein rather than a membrane-associated protein at cell tips like Scd1. Microtubules and the Tea1-Tea4-Pom1 axis counteract inappropriate Gef1 activity by regulating the localization of the Cdc42 GTPase-activating protein Rga4. Our results suggest a new model of fission yeast cell polarity regulation, involving coordination of 'local' (Scd1) and 'global' (Gef1) Cdc42 GEFs via microtubules and microtubule-dependent polarity landmarks., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The characteristic six layers of the mammalian neocortex develop sequentially as neurons are generated by neural progenitors and subsequently migrate past older neurons to their final position in the cortical plate. One of the earliest steps of neuronal differentiation is the formation of an axon. Small GTPases play essential roles during this process by regulating cytoskeletal dynamics and intracellular trafficking. While the function of GTPases has been studied extensively in cultured neurons and in vivo much less is known about their upstream regulators. Here we show that Arhgef7 (also called βPix or Cool1) is essential for axon formation during cortical development. The loss of Arhgef7 results in an extensive loss of axons in cultured neurons and in the developing cortex. Arhgef7 is a guanine-nucleotide exchange factor (GEF) for Cdc42, a GTPase that has a central role in directing the formation of axons during brain development. However, active Cdc42 was not able to rescue the knockdown of Arhgef7. We show that Arhgef7 interacts with the GTPase TC10 that is closely related to Cdc42. Expression of active TC10 can restore the ability to extend axons in Arhgef7-deficient neurons. Our results identify an essential role of Arhgef7 during neuronal development that promotes axon formation upstream of TC10.

Human huntingtin (Htt) contains 3144 amino acids and has an expanded polyglutamine region near the NH 2 -terminus in patients with Huntington's disease. While numerous binding partners have been identified to NH 2 -terminal Htt, fewer proteins are known to interact with C-terminal domains of Htt. Here we report that kalirin, a Rac1 activator, is a binding partner to C-terminal Htt. Kalirin and Htt co-precipitated from mouse brain endosomes and co-localized at puncta in NRK and immortalized striatal cells and primary cortical neurons. We mapped the interaction domains to kalirin674-1272 and Htt2568-3144 and determined that the interaction between kalirin and Htt was independent of HAP1, a known interactor for Htt and kalirin. Kalirin precipitated with mutant Htt was more abundant than with wild-type Htt and had a reduced capacity to activate Rac1 when mutant Htt was present. Expression of Htt2568-3144 caused cytotoxicity, partially rescued by co-expressing kalirin674-1272 but not other regions of kalirin. Our study suggests that the interaction of kalirin with the C-terminal region of Htt influences the function of kalirin and modulates the cytotoxicity induced by C-terminal Htt.

The active form of the small GTPase RhoA is necessary and sufficient for formation of a cytokinetic furrow in animal cells. Despite the conceptual simplicity of the process, the molecular mechanisms that control it are intricate and involve redundancy at multiple levels. Here, we discuss our current knowledge of the mechanisms underlying spatiotemporal regulation of RhoA during cytokinesis by upstream activators. The direct upstream activator, the RhoGEF Ect2, requires activation due to autoinhibition. Ect2 is primarily activated by the centralspindlin complex, which contains numerous domains that regulate its subcellular localization, oligomeric state, and Ect2 activation. We review the functions of these domains and how centralspindlin is regulated to ensure correctly timed, equatorial RhoA activation. Highlighting recent evidence, we propose that although centralspindlin does not always prominently accumulate on the plasma membrane, it is the site where it promotes RhoA activation during cytokinesis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In order to metastasize, tumor cells need to migrate and invade the surrounding tissues. It is important to identify compound(s) capable of disrupting the metastasis of invasive cancer cells, especially for hindering invadopodia formation, so as to provide anti-metastasis targeted therapy. Invadopodia are thought to be specialized actin-rich protrusions formed by highly invasive cancer cells to degrade the extracellular matrix (ECM). A curcuminoid analogue known as 2,6-bis-(4-hydroxy-3-methoxybenzylidine)cyclohexanone or BHMC has shown good potential in inhibiting inflammation and hyperalgesia. It also possesses an anti-tumor effects on 4T1 murine breast cancer cells in vivo. However, there is still a lack of empirical evidence on how BHMC works in preventing human breast cancer invasion. In this study, we investigated the effect of BHMC on MDA-MB-231 breast cancer cells and its underlying mechanism of action to prevent breast cancer invasion, especially during the formation of invadopodia. All MDA-MB-231 cells, which were exposed to the non-cytotoxic concentrations of BHMC, expressed the proliferating cell nuclear antigen (PCNA), which indicate that the anti-proliferative effects of BHMC did not interfere in the subsequent experiments. By using a scratch migration assay, transwell migration and invasion assays, we determined that BHMC reduces the percentage of migration and invasion of MDA-MB-231 cells. The gelatin degradation assay showed that BHMC reduced the number of cells with invadopodia. Analysis of the proteins involved in the invasion showed that there is a significant reduction in the expressions of Rho guanine nucleotide exchange factor 7 (β-PIX), matrix metalloproteinase-9 (MMP-9), and membrane type 1 matrix metalloproteinase (MT1-MMP) in the presence of BHMC treatment at 12.5 µM. Therefore, it can be postulated that BHMC at 12.5 µM is the optimal concentration for preventing breast cancer invasion., Competing Interests: The authors declare no conflict of interest.

Antiestrogen resistance in estrogen receptor positive (ER + ) breast cancer is associated with increased expression and activity of insulin-like growth factor 1 receptor (IGF1R). Here, a kinome siRNA screen has identified 10 regulators of IGF1R-mediated antiestrogen with clinical significance. These include the tamoxifen resistance suppressors BMPR1B, CDK10, CDK5, EIF2AK1, and MAP2K5, and the tamoxifen resistance inducers CHEK1, PAK2, RPS6KC1, TTK, and TXK. The p21-activated kinase 2, PAK2, is the strongest resistance inducer. Silencing of the tamoxifen resistance inducing genes, particularly PAK2, attenuates IGF1R-mediated resistance to tamoxifen and fulvestrant. High expression of PAK2 in ER + metastatic breast cancer patients is correlated with unfavorable outcome after first-line tamoxifen monotherapy. Phospho-proteomics has defined PAK2 and the PAK-interacting exchange factors PIXα/β as downstream targets of IGF1R signaling, which are independent from PI3K/ATK and MAPK/ERK pathways. PAK2 and PIXα/β modulate IGF1R signaling-driven cell scattering. Targeting PIXα/β entirely mimics the effect of PAK2 silencing on antiestrogen re-sensitization. These data indicate PAK2/PIX as an effector pathway in IGF1R-mediated antiestrogen resistance.

Purpose: Gitelman's syndrome (GS) presents normo-hypotension and absence of cardiovascular-renal remodeling despite high angiotensin II (Ang II), activation of renin-angiotensin-aldosterone system and is a human model of endogenous antagonism of Ang II signaling, opposite to hypertension. GS's clinical presentation leads to questions regarding what features might be responsible. One area of investigation involves Ang II signaling. In hypertensive patients, RhoA/Rho kinase (RhoA/ROCK) pathway activation by Ang II is involved in hypertension development/maintenance and induction of long-term consequences (cardiovascular-renal remodeling), while GS has reduced p63RhoGEF gene and protein levels and ROCK activity. Ang II signaling is mediated by Gαq, which interacts with p63RhoGEF via the α6-αN linker connecting p63RhoGEF's DH and PH domains acting as a conformational switch to activate RhoA/ROCK signaling., Methods: We have investigated in GS patients, the presence of mutations in either p63RhoGEF's α6-αN linker domain and in Gαq's Ala253, Trp263, and Tyr356 residues, crucial for p63RhoGEF-Gαq interplay., Results: No mutations have been found in specific aminoacids of p63RhoGEF α6-αN linker and Gαq, key for p63RhoGEF/Gαq interplay., Conclusions: Gitelman's syndrome normo/hypotension and lack of cardiovascular-renal remodeling are not due to mutations of p63RhoGEF α6-αN linker and Gαq interactions. This opens the way for investigations on different coding and no-coding regions (p63RhoGEF and Gαq promoters) and on altered transcriptional/post-transcriptional regulation. Clarification of how these biochemical/molecular mechanisms work/interact would provide insights into mechanisms involved in the GS's Ang II signaling fine tuning, in human physiology/pathophysiology in general and could also identify significant targets for intervention in the treatments of hypertension.

Early gut mucosal restitution is a process by which intestinal epithelial cells (IECs) migrate over the wounded area, and its defective regulation occurs commonly in various critical pathological conditions. This rapid reepithelialization is mediated by different activating small GTP-binding proteins, but the exact mechanism underlying this process remains largely unknown. Recently, it has been reported that interaction between p21-activated kinase-interacting exchange factor (β-PIX) and G protein-coupled receptor kinase-interacting protein 1 (GIT1) activates small GTPases and plays an important role in the regulation of cell motility. Here, we show that induced association of β-PIX with GIT1 is essential for the stimulation of IEC migration after wounding by activating Rac1. Levels of β-PIX and GIT1 proteins and their association in differentiated IECs (line of IEC-Cdx2L1) were much higher than those observed in undifferentiated IECs (line of IEC-6), which was associated with an increase in IEC migration after wounding. Decreased levels of endogenous β-PIX by its gene-silencing destabilized β-PIX/GIT1 complexes, repressed Rac1 activity and inhibited cell migration over the wounded area. In contrast, ectopic overexpression of β-PIX increased the levels of β-PIX/GIT1 complexes, stimulated Rac1 activity, and enhanced intestinal epithelial restitution. Increased levels of cellular polyamines also stimulated β-PIX/GIT1 association, increased Rac1 activity, and promoted the epithelial restitution. Moreover, polyamine depletion decreased cellular abundances of β-PIX/GIT1 complex and repressed IEC migration after wounding, which was rescued by ectopic overexpression of β-PIX or GIT1. These results indicate that β-PIX/GIT1/Rac1 association is necessary for stimulation of IEC migration after wounding and that this signaling pathway is tightly regulated by cellular polyamines. NEW & NOTEWORTHY Our current study demonstrates that induced association of β-PIX with GIT1 is essential for the stimulation of intestinal epithelial restitution by activating Rac1, and this signaling pathway is tightly regulated by cellular polyamines.

Introduction: Fetal macrosomia has confirmed be related to multiple labor complications and metabolism syndromes later in life. However, the mechanism of fetal macrosomia in normal glucose tolerance (NGT) and gestational diabetes mellitus (GDM) pregnant women is still obscure. This study aimed to investigate the mechanism of Rho guanine nucleotide exchange factor 11 (ARHGEF11) and the insulin signaling pathway in placenta affecting fetal overgrowth in NGT and GDM pregnant women., Methods: Eighty-nine pregnant women with paired antepartum BMI were recruited and divided into four groups: NGT with normal birth weight (NGT-N, n = 30) or macrosomia (NGT-M, n = 22) and GDM with normal birth weight (GDM-N, n = 22) or macrosomia (GDM-M, n = 15). Placenta tissue was collected to examine the expression of ARHGEF11, ROCK1, the phosphorylation of Tyr612 IRS-1 (p-Y612), Ser307 IRS-1 (p-S307), PI3K, AKT, pAKT, GLUT4 and S6K., Results: Gene expression of ARHGEF11, ROCK1, p-S307 and S6K in placenta was significantly increased in the NGT-M group (p < .05). The protein density of ARHGEF11 and ROCK1 was positively correlated with birth weight (p < .001) in the NGT groups. p-Y612, PI3K, pAKT and GLUT4 were significantly decreased in NGT-M, GDM-N and GDM-M group (p < .05). But there was no statistical difference between the two GDM groups., Discussion: ARHGRF11 inhibited the insulin signaling pathway in placenta may participated in fetal macrosomia in NGT pregnant women. The insulin signaling pathway was suppressed in GDM placenta, but was not closely related to fetal macrosomia, indicated different mechanism in fetal overgrowth in NGT and GDM pregnant women., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Epithelia are layers of polarised cells tightly bound to each other by adhesive contacts. Epithelia act as barriers between an organism and its external environment. Understanding how epithelia maintain their essential integrity while remaining sufficiently plastic to allow events such as cytokinesis to take place is a key biological problem. In vertebrates, the remodelling and reinforcement of adherens junctions maintains epithelial integrity during cytokinesis. The involvement of tight junctions in cell division, however, has remained unexplored. Here, we examine the role of tight junctions during cytokinesis in the epithelium of the Xenopus laevis embryo. Depletion of the tight junction-associated proteins ZO-1 and GEF-H1 leads to altered cytokinesis duration and contractile ring geometry. Using a tension biosensor, we show that cytokinesis defects originate from misregulation of tensile forces applied to adherens junctions. Our results reveal that tight junctions regulate mechanical tension applied to adherens junctions, which in turn impacts cytokinesis.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The quantity and quality of bone depends on osteoblastic differentiation of mesenchymal stem cells (MSCs), where adipogenic commitment depletes the available pool for osteogenesis. Cell architecture influences lineage decisions, where interfering with cytoskeletal structure promotes adipogenesis. Mechanical strain suppresses MSC adipogenesis partially through RhoA driven enhancement of cytoskeletal structure. To understand the basis of force-driven RhoA activation, we considered critical GEFs (activators) and GAPs (inactivators) on bone marrow MSC lineage fate. Knockdown of LARG accelerated adipogenesis and repressed basal RhoA activity. Importantly, mechanical activation of RhoA was almost entirely inhibited following LARG depletion, and the ability of strain to inhibit adipogenesis was impaired. Knockdown of ARHGAP18 increased basal RhoA activity and actin stress fiber formation, but did not enhance mechanical strain activation of RhoA. ARHGAP18 null MSCs exhibited suppressed adipogenesis assessed by Oil-Red-O staining and Western blot of adipogenic markers. Furthermore, ARHGAP18 knockdown enhanced osteogenic commitment, confirmed by alkaline phosphatase staining and qPCR of Sp7, Alpl, and Bglap genes. This suggests that ARHGAP18 conveys tonic inhibition of MSC cytoskeletal assembly, returning RhoA to an "off state" and affecting cell lineage in the static state. In contrast, LARG is recruited during dynamic mechanical strain, and is necessary for mechanical suppression of adipogenesis. In summary, mechanical activation of RhoA in mesenchymal progenitors is dependent on LARG, while ARHGAP18 limits RhoA delineated cytoskeletal structure in static cultures. Thus, on and off GTP exchangers work through RhoA to influence MSC fate and responses to static and dynamic physical factors in the microenvironment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

βPix activates Nox1, an O 2 - -generating NADPH oxidase, through Rac activation. In this study, we found that S525E mutation of βPix eliminated its Nox1-activating ability in transfected Caco-2 cells. Unexpectedly, affinity for Rac was not diminished but rather enhanced by S525E mutation, and guanine nucleotide exchange factor (GEF) activity was not altered. The N-terminal fragment (amino acids 1-400) showed similar Rac-binding and GEF activity to wild-type βPix. In contrast, the C-terminal fragment (amino acids 408-646) had higher Rac-binding activity, particularly for Rac-GTP, than wild-type βPix, and showed no GEF activity. These data suggest that a second Rac-binding site within the C-terminal region is opened by phosphorylation of Ser-525. The site may bind not only Rac-GDP but also Rac-GTP released from the N-terminal catalytic region, which interrupts Rac-GTP translocation to the membrane where Nox1 resides. If one considers that S340E mutation enhances Nox1 activation (Kaito et al., 2014), the present study suggests that βPix can also play an inhibitory role in O 2 - production, depending on the sites of phosphorylation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Directed migration is essential for cell motility in many processes, including development and cancer cell invasion. RSKs (p90 ribosomal S6 kinases) have emerged as central regulators of cell migration; however, the mechanisms mediating RSK-dependent motility remain incompletely understood. We have identified a unique signaling mechanism by which RSK2 promotes cell motility through leukemia-associated RhoGEF (LARG)-dependent Rho GTPase activation. RSK2 directly interacts with LARG and nucleotide-bound Rho isoforms, but not Rac1 or Cdc42. We further show that epidermal growth factor or FBS stimulation induces association of endogenous RSK2 with LARG and LARG with RhoA. In response to these stimuli, RSK2 phosphorylates LARG at Ser1288 and thereby activates RhoA. Phosphorylation of RSK2 at threonine 577 is essential for activation of LARG-RhoA. Moreover, RSK2-mediated motility signaling depends on RhoA and -B, but not RhoC. These results establish a unique RSK2-dependent LARG-RhoA signaling module as a central organizer of directed cell migration and invasion., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Rho GTPases, a family of molecular switches, are essential for the assembly and rearrangement of the cellular actin network. Actin remodeling is a central component of many important biological phenomena including chemotaxis, immunological synapse formation, and phagocytosis. Proper execution of these processes requires careful modulation of Rho GTPase activity in space and time. This is accomplished by delicate coordination of Rho GTPase activation and inactivation by Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GTPase-activating proteins (RhoGAPs), respectively. Elucidating the function of these Rho GTPase modulators is complicated by their diversity, varied expression across different tissues, and multiplicity of substrates. To overcome some of these hurdles, we describe here a systematic and unbiased screening approach consisting of three sequential steps: (1) monitoring the subcellular localization of a library of Rho GTPase modulators; (2) assessing endogenous levels of expression of the suitably localized candidates in the cell type of interest; and (3) validating the functional relevance of the identified candidates by siRNA, followed by determining the effects of gene silencing on Rho GTPase activity and actin polymerization. To this end, we describe the expression and visualization of fluorescent Rho GTPase modulators, and the use of genetically encoded biosensors for active Rac/Cdc42 and of fluorescent probes of polymerized actin. Phagocytosis by macrophages is used in this chapter as an experimental paradigm, but the methods described herein can be easily extended to other cells and actin-dependent processes.

MYO18A is a divergent member of the myosin family characterized by the presence of an amino-terminal PDZ domain. MYO18A has been found in a few different complexes involved in intracellular transport processes. MYO18A is found in a complex with LURAP1 and MRCK that functions in retrograde treadmilling of actin. It also has been found in a complex with PAK2, βPIX, and GIT1, functioning to transport that protein complex from focal adhesions to the leading edge. Finally, a high proportion of MYO18A is found in complex with GOLPH3 at the trans Golgi, where it functions to promote vesicle budding for Golgi-to-plasma membrane trafficking. Interestingly, MYO18A has been implicated as a cancer driver, as have other components of the GOLPH3 pathway. It remains uncertain as to whether or not MYO18A has intrinsic motor activity. While many questions remain, MYO18A is a fascinatingly unique myosin that is essential in higher organisms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Key Points: Transforming growth-factor-β (TGF-β) and RhoA/Rho-kinase are independently implicated in the airway hyper-responsiveness associated with asthma, but how these proteins interact is not fully understood. We examined the effects of pre-treatment with TGF-β on expression and activity of RhoA, Rho-kinase and ARHGEF1, an activator of RhoA, as well as on bradykinin-induced contraction, in airway smooth muscle. TGF-β enhanced bradykinin-induced RhoA translocation, Rho-kinase-dependent phosphorylation and contraction, but partially suppressed bradykinin-induced RhoA activity (RhoA-GTP content). TGF-β enhanced the expression of ARHGEF1, while a small interfering RNA against ARHGEF1 and a RhoGEF inhibitor prevented the effects of TGF-β on RhoA and Rho-kinase activity and contraction, respectively. ARHGEF1 expression was also enhanced in airway smooth muscle from asthmatic patients and ovalbumin-sensitized mice. ARHGEF1 is a key TGF-β target gene, an important regulator of Rho-kinase activity and therefore a potential therapeutic target for the treatment of asthmatic airway hyper-responsiveness., Abstract: Transforming growth factor-β (TGF-β), RhoA/Rho-kinase and Src-family kinases (SrcFK) have independently been implicated in airway hyper-responsiveness, but how they interact to regulate airway smooth muscle contractility is not fully understood. We found that TGF-β pre-treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho-kinase (Y27632), but not the SrcFK inhibitor PP2, prevented this enhancement. In cultured human airway smooth muscle cells (hASMCs), TGF-β pre-treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC 20 , MYPT-1 and the actin-severing protein cofilin, but not of RhoA, ROCK2 or c-Src. In hASMCs, acute treatment with BK triggered subcellular translocation of ARHGEF1 and RhoA and enhanced auto-phosphorylation of SrcFK and phosphorylation of MYPT1 and MLC 20 , but induced de-phosphorylation of cofilin. TGF-β pre-treatment amplified the effects of BK on RhoA translocation and MYPT1/MLC 20 phosphorylation, but suppressed the effects of BK on RhoA-GTP content, SrcFK auto-phosphorylation and cofilin de-phosphorylation. In hASMCs, an ARHGEF1 small interfering RNA suppressed the effects of BK and TGF-β on RhoA-GTP content, RhoA translocation and MYPT1 and MLC 20 phosphorylation, but minimally influenced the effects of TGF-β on cofilin expression and phosphorylation. ARHGEF1 expression was also enhanced in ASMCs of asthmatic patients and in lungs of ovalbumin-sensitized mice. Our data indicate that TGF-β enhances BK-induced contraction, RhoA translocation and Rho-kinase activity in airway smooth muscle largely via ARHGEF1, but independently of SrcFK and total RhoA-GTP content. A role for smooth muscle ARHGEF1 in asthmatic airway hyper-responsiveness is worthy of further investigation., (© 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

We describe a high-throughput screening (HTS)-compatible method for detecting GTPase exchange factor (GEF) activity based on stimulation of GDP formation by Rho GTPases. The method is based on the fact that GDP dissociation is the rate-limiting step in the Rho GTPase catalytic cycle, so by accelerating its release a GEF causes an increase in the steady-state rate of GDP formation. The Transcreener ® GDP GTPase Assay, a fluorescence polarization immunoassay (FPIA), is used to detect GDP formation in a homogeneous format.

p63, more specifically its ΔNp63α isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type-specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to maintain the undifferentiated status is associated with defects in ΔNp63α nuclear transport. We further find that differentiation-inducible Rho kinase reduces the interaction between NUP62 and ΔNp63α by phosphorylation of phenylalanine-glycine regions of NUP62, attenuating ΔNp63α nuclear import. Our results characterize NUP62 as a gatekeeper for ΔNp63α and uncover its role in the control of cell fate through regulation of ΔNp63α nuclear transport in SCC., (© 2017 The Authors.)