Rho Guanine Nucleotide Exchange Factor Research Articles

Dexamethasone induces trabecular meshwork cell myofibroblast transdifferentiation through ARHGEF26.

Glucocorticoid use may cause elevated intraocular pressure, leading to the development of glucocorticoid-induced glaucoma (GIG). However, the mechanism of GIG development remains incompletely understood. In this study, we subjected primary human trabecular meshwork cells (TMCs) and mice to dexamethasone treatment to mimic glucocorticoid exposure. The myofibroblast transdifferentiation of TMCs was observed in cellular and mouse models, as well as in human trabecular mesh specimens. This was demonstrated by the cytoskeletal reorganization, alterations in cell morphology, heightened transdifferentiation markers, increased extracellular matrix deposition, and cellular dysfunction. Knockdown of Rho guanine nucleotide exchange factor 26 (ARHGEF26) expression ameliorated dexamethasone-induced changes in cell morphology and upregulation of myofibroblast markers, reversed dysfunction and extracellular matrix deposition in TMCs, and prevented the development of dexamethasone-induced intraocular hypertension. And, this process may be related to the TGF-β pathway. In conclusion, glucocorticoids induced the myofibroblast transdifferentiation in TMCs, which played a crucial role in the pathogenesis of GIG. Inhibition of ARHGEF26 expression protected TMCs by reversing myofibroblast transdifferentiation. This study demonstrated the potential of reversing the myofibroblast transdifferentiation of TMCs as a new target for treating GIG.

Molecular mechanism of bitter taste receptor agonist-mediated relaxation of airway smooth muscle.

G-protein-coupled receptors (GPCRs) belonging to the type 2 taste receptors (TAS2Rs) family are predominantly present in taste cells to allow the perception of bitter-tasting compounds. TAS2Rs have also been shown to be expressed in human airway smooth muscle (ASM), and TAS2R agonists relax ASM cells and bronchodilate airways despite elevating intracellular calcium. This calcium "paradox" (calcium mediates contraction by pro-contractile Gq-coupled GPCRs) and the mechanisms by which TAS2R agonists relax ASM remain poorly understood. To gain insight into pro-relaxant mechanisms effected by TAS2Rs, we employed an unbiased phosphoproteomic approach involving dual-mass spectrometry to determine differences in the phosphorylation of contractile-related proteins in ASM following the stimulation of cells with TAS2R agonists, histamine (an agonist of the Gq-coupled H1 histamine receptor) or isoproterenol (an agonist of the Gs-coupled β2-adrenoceptor) alone or in combination. Our study identified differential phosphorylation of proteins regulating contraction, including A-kinase anchoring protein (AKAP)2, AKAP12, and RhoA guanine nucleotide exchange factor (ARHGEF)12. Subsequent signaling analyses revealed RhoA and the T853 residue on myosin light chain phosphatase (MYPT)1 as points of mechanistic divergence between TAS2R and Gs-coupled GPCR pathways. Unlike Gs-coupled receptor signaling, which inhibits histamine-induced myosin light chain (MLC)20 phosphorylation via protein kinase A (PKA)-dependent inhibition of intracellular calcium mobilization, HSP20 and ERK1/2 activity, TAS2Rs are shown to inhibit histamine-induced pMLC20 via inhibition of RhoA activity and MYPT1 phosphorylation at the T853 residue. These findings provide insight into the TAS2R signaling in ASM by defining a distinct signaling mechanism modulating inhibition of pMLC20 to relax contracted ASM.

RhoGDI1 regulates cell-cell junctions in polarized epithelial cells.

Cell-cell contact formation of polarized epithelial cells is a multi-step process that involves the co-ordinated activities of Rho family small GTPases. Consistent with the central role of Rho GTPases, a number of Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase-activating proteins (GAPs) have been identified at cell-cell junctions at various stages of junction maturation. As opposed to RhoGEFs and RhoGAPs, the role of Rho GDP dissociation inhibitors (GDIs) during cell-cell contact formation is poorly understood. Here, we have analyzed the role of RhoGDI1/ARHGDIA, a member of the RhoGDI family, during cell-cell contact formation of polarized epithelial cells. Depletion of RhoGDI1 delays the development of linear cell-cell junctions and the formation of barrier-forming tight junctions. In addition, RhoGDI1 depletion impairs the ability of cells to stop migration in response to cell collision and increases the migration velocity of collectively migrating cells. We also find that the cell adhesion receptor JAM-A promotes the recruitment of RhoGDI1 to cell-cell contacts. Our findings implicate RhoGDI1 in various processes involving the dynamic reorganization of cell-cell junctions.

RhoGEF Tiam2 Regulates Glutamatergic Synaptic Transmission in Hippocampal CA1 Pyramidal Neurons.

Glutamatergic synapses exhibit significant molecular diversity, but circuit-specific mechanisms that underlie synaptic regulation are not well characterized. Prior reports show that Rho-guanine nucleotide exchange factor (RhoGEF) Tiam1 regulates perforant path→dentate gyrus granule neuron synapses. In the present study, we report Tiam1's homolog Tiam2 is implicated in glutamatergic neurotransmission in CA1 pyramidal neurons. We find that Tiam2 regulates evoked excitatory glutamatergic currents via a postsynaptic mechanism mediated by the catalytic Dbl-homology domain. Overall, we present evidence for RhoGEF Tiam2's role in glutamatergic synapse function at Schaffer collateral→CA1 pyramidal neuron synapses.

New mechanisms underlying oncogenesis in Dbl family Rho guanine nucleotide exchange factors.

Transmembrane signaling is a critical process by which changes in the extracellular environment are relayed to intracellular systems that induce changes in homeostasis. One common intracellular system involves guanine nucleotide exchange factors (GEFs), which catalyzes the exchange of GTP for GDP bound to inactive guanine nucleotide binding proteins (G proteins). The resulting active G proteins then interact with downstream targets that control cell proliferation, growth, shape, migration, adhesion, and transcription. Dysregulation of any of these processes is a hallmark of cancer. The Dbl family of GEFs activate Rho family G proteins, which in turn alter the actin cytoskeleton and promote gene transcription. Although they have a common catalytic mechanism exercised by their conserved Dbl homology (DH) domains, Dbl GEFs are regulated in very diverse ways. Often, this regulation involves the release of autoinhibition imposed by accessory domains. Amongst these domains, the pleckstrin homology (PH) domain is the most conserved and is almost always found immediately C-terminal to the DH domain. The domain been associated with both positive and negative regulation. Recently, some atomic structures of Dbl GEFs have been determined which reemphasize the complex and central role that the PH domain can play in orchestrating regulation of the DH domain. Here we discuss these newer structures, put them into context by cataloging the various ways that PH domains are known to contribute to signaling across the Dbl family, and discuss how the PH might be exploited to achieve selective inhibition of this protein family by small molecule therapeutics. Significance Statement Dysregulation via overexpression or mutation of Dbl family RhoGEFs contributes disease. Targeting the Dbl homology (DH) catalytic domain by small molecule therapeutics has been challenging due to its high conservation and the lack of a discrete binding pocket. By evaluating some new autoinhibitory mechanisms in the Dbl family, we demonstrate the great diversity of roles played by the regulatory domains, in particular the PH domain, and how this holds tremendous potential for development of selective therapeutics that modulate GEF activity.

ARHGEF5 binds Drebrin and affects α-tubulin acetylation to direct neuronal morphogenesis and migration during mouse brain development.

Rho guanine nucleotide exchange factors (Rho GEFs) activate Rho GTPases, which act as molecular switches regulating various essential cellular functions. This study investigated the role of ARHGEF5, a Rho GEF known for its involvement in cell migration and invasion processes, in the context of brain development. We found that ARHGEF5 is essential for dendrite development during the early stages of neuronal growth. We also discovered that ARHGEF5 binds to Drebrin E, which is vital for coordinating actin and microtubule dynamics, and facilitates the interaction between Drebrin E and Cyclin-dependent kinase 5, which phosphorylates Drebrin E. Notably, ARHGEF5 deficiency resulted in a decrease in acetylated α-tubulin levels, and the expression of an α-tubulin acetylation mimetic mutant (K40Q) rescued the defects in dendrite development and neuronal migration, suggesting ARHGEF5's role in modulating microtubule stability. Additionally, ARHGEF5 was shown to influence Golgi positioning in the leading processes of migrating cortical neurons during brain development. Our study suggests that ARHGEF5 plays a crucial role in integrating cytoskeletal dynamics with neuronal morphogenesis and migration processes during brain development.

Decreased Tiam1-mediated Rac1 activation is responsible for impaired directional persistence of chondrocyte migration in microtia.

The human auricle has a complex structure, and microtia is a congenital malformation characterized by decreased size and loss of elaborate structure in the affected ear with a high incidence. Our previous studies suggest that inadequate cell migration is the primary cytological basis for the pathogenesis of microtia, however, the underlying mechanism is unclear. Here, we further demonstrate that microtia chondrocytes show a decreased directional persistence during cell migration. Directional persistence can define a leading edge associated with oriented movement, and any mistakes would affect cell function and tissue morphology. By the screening of motility-related genes and subsequent confirmations, active Rac1 (Rac1-GTP) is identified to be critical for the impaired directional persistence of microtia chondrocytes migration. Moreover, Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase-activating proteins (GAPs) are detected, and overexpression of Tiam1 significantly upregulates the level of Rac1-GTP and improves directional migration in microtia chondrocytes. Consistently, decreased expression patterns of Tiam1 and active Rac1 are found in microtia mouse models, Bmp5se/J and Prkralear-3J/GrsrJ. Collectively, our results provide new insights into microtia development and therapeutic strategies of tissue engineering for microtia patients.

Mitigation of TDP-43 toxic phenotype by an RGNEF fragment in amyotrophic lateral sclerosis models.

Aggregation of the RNA-binding protein TAR DNA binding protein (TDP-43) is a hallmark of TDP-proteinopathies including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As TDP-43 aggregation and dysregulation are causative of neuronal death, there is a special interest in targeting this protein as a therapeutic approach. Previously, we found that TDP-43 extensively co-aggregated with the dual function protein GEF (guanine exchange factor) and RNA-binding protein rho guanine nucleotide exchange factor (RGNEF) in ALS patients. Here, we show that an N-terminal fragment of RGNEF (NF242) interacts directly with the RNA recognition motifs of TDP-43 competing with RNA and that the IPT/TIG domain of NF242 is essential for this interaction. Genetic expression of NF242 in a fruit fly ALS model overexpressing TDP-43 suppressed the neuropathological phenotype increasing lifespan, abolishing motor defects and preventing neurodegeneration. Intracerebroventricular injections of AAV9/NF242 in a severe TDP-43 murine model (rNLS8) improved lifespan and motor phenotype, and decreased neuroinflammation markers. Our results demonstrate an innovative way to target TDP-43 proteinopathies using a protein fragment with a strong affinity for TDP-43 aggregates and a mechanism that includes competition with RNA sequestration, suggesting a promising therapeutic strategy for TDP-43 proteinopathies such as ALS and FTD.

Change in RhoGAP and RhoGEF availability drives transitions in cortical patterning and excitability in Drosophila

Actin cortex patterning and dynamics are critical for cell shape changes. These dynamics undergo transitions during development, often accompanying changes in collective cell behavior. Although mechanisms have been established for individual cells' dynamic behaviors, the mechanisms and specific molecules that result in developmental transitions invivo are still poorly understood. Here, we took advantage of two developmental systems in Drosophila melanogaster to identify conditions that altered cortical patterning and dynamics. We identified a Rho guanine nucleotide exchange factor (RhoGEF) and Rho GTPase activating protein (RhoGAP) pair required for actomyosin waves in egg chambers. Specifically, depletion of the RhoGEF, Ect2, or the RhoGAP, RhoGAP15B, disrupted actomyosin wave induction, and both proteins relocalized from the nucleus to the cortex preceding wave formation. Furthermore, we found that overexpression of a different RhoGEF and RhoGAP pair, RhoGEF2 and Cumberland GAP (C-GAP), resulted in actomyosin waves in the early embryo, during which RhoA activation precedes actomyosin assembly by ∼4 s. We found that C-GAP was recruited to actomyosin waves, and disrupting F-actin polymerization altered the spatial organization of both RhoA signaling and the cytoskeleton in waves. In addition, disrupting F-actin dynamics increased wave period and width, consistent with a possible role for F-actin in promoting delayed negative feedback. Overall, we showed a mechanism involved in inducing actomyosin waves that is essential for oocyte development and is general to other cell types, such as epithelial and syncytial cells.

Presenilin2 D439A Mutation Induces Dysfunction of Mitochondrial Fusion/Fission Dynamics and Abnormal Regulation of GTPase Activity

Alzheimer’s disease (AD) is an age-related progressive neurodegenerative disease, and approximately 10% of AD cases are early-onset familial AD (EOFAD), which is mainly linked to point mutations in genes encoding presenilins (PS1 and PS2). Mutations in PS2 are extremely rare and have not received enough attention. Recently, studies have found that Rho GTPase activity is closely related to the pathogenesis of AD. In this study, we used transcriptome sequencing in PS2 siRNA-transfected SH-SY5Y cells and found a group of differentially expressed genes (DEGs) related to the regulation of GTPase activity. Among those DEGs, the most significantly downregulated was Rho guanine nucleotide exchange factor 5 (ARHGEF5). GTPase activity in PS2 siRNA-transfected cells was significantly decreased. Then, we found that the expression of ARHGEF5 and the GTPase activity of Mitochondrial Rho GTPase 2 (Miro2) in PS2 D439A mutant SH-SY5Y cells were significantly decreased. We found for the first time that PS2 can bind to Miro2, and the PS2 D439A mutation reduced the binding between PS2 and Miro2, reduced the expression of Miro2, and resulted in an imbalance in mitochondrial fusion/fission dynamics. In conclusion, PS2 gene knockdown may participate in the pathogenesis of AD through the regulation of GTPase activity. The imbalance in mitochondrial dynamics mediated by the PS2 D439A mutation through regulation of the expression and GTPase activity of Miro2 may be a potential pathogenic mechanism of AD.

The RhoGEF Trio is transported by microtubules and affects microtubule stability in migrating neural crest cells

Directed cell migration requires a local fine-tuning of Rho GTPase activity to control protrusion formation, cell-cell contraction, and turnover of cellular adhesions. The Rho guanine nucleotide exchange factor (GEF) TRIO is ideally suited to control RhoGTPase activity because it combines two distinct catalytic domains to control Rac1 and RhoA activity in one molecule. However, at the cellular level, this molecular feature also requires a tight spatiotemporal control of TRIO activity. Here, we analyze the dynamic localization of Trio in Xenopus cranial neural crest (NC) cells, where we have recently shown that Trio is required for protrusion formation and migration. Using live cell imaging, we find that the GEF2 domain, but not the GEF1 domain of Trio, dynamically colocalizes with EB3 at microtubule plus-ends. Microtubule-mediated transport of Trio appears to be relevant for its function in NC migration, as a mutant GEF2 construct lacking the SxIP motif responsible for microtubule plus-end localization was significantly impaired in its ability to rescue the Trio loss-of-function phenotype compared to wild-type GEF2. Furthermore, by analyzing microtubule dynamics in migrating NC cells, we observed that loss of Trio function stabilized microtubules at cell-cell contact sites compared to controls, whereas they were destabilized at the leading edge of NC cells. Our data suggest that Trio is transported by microtubules to distinct subcellular locations where it has different functions in controlling microtubule stability, cell morphology, and cell-cell interaction during directed NC migration.

TRIOBP modulates β-catenin signaling by regulation of miR-29b in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal and devastating lung disease of unknown etiology, described as the result of multiple cycles of epithelial cell injury and fibroblast activation. Despite this impressive increase in understanding, a therapy that reverses this form of fibrosis remains elusive. In our previous study, we found that miR-29b has a therapeutic effect on pulmonary fibrosis. However, its anti-fibrotic mechanism is not yet clear. Recently, our study identified that F-Actin Binding Protein (TRIOBP) is one of the target genes of miR-29b and found that deficiency of TRIOBP increases resistance to lung fibrosis in vivo. TRIOBP knockdown inhibited the proliferation of epithelial cells and attenuated the activation of fibroblasts. In addition, deficiency of Trio Rho Guanine Nucleotide Exchange Factor (TRIO) in epithelial cells and fibroblasts decreases susceptibility to lung fibrosis. TRIOBP interacting with TRIO promoted abnormal epithelial–mesenchymal crosstalk and modulated the nucleocytoplasmic translocation of β-catenin. We concluded that the miR-29b‒TRIOBP–TRIO–β-catenin axis might be a key anti-fibrotic axis in IPF to regulate lung regeneration and fibrosis, which may provide a promising treatment strategy for lung fibrosis.Graphical

Role and mechanism of EphB3 in epileptic seizures and epileptogenesis through Kalirin

BackgroundThe EphB receptor tyrosine kinase family participates in intricate signaling pathways that orchestrate neural networks, guide neuronal axon development, and modulate synaptic plasticity through interactions with surface-bound ephrinB ligands. Additionally, Kalirin, a Rho guanine nucleotide exchange factor, is notably expressed in the postsynaptic membrane of excitatory neurons and plays a role in synaptic morphogenesis. This study postulates that Kalirin may act as a downstream effector of EphB3 in epilepsy. This investigation focuses on understanding the link between EphB3 and epilepsy. Materials and methodsChronic seizure models using LiCl-pilocarpine (LiCl/Pilo) and pentylenetetrazol were developed in rats. Neuronal excitability was gauged through whole-cell patch clamp recordings on rat hippocampal slices. Real-time PCR determined Kalirin's mRNA expression, and Western blotting was employed to quantify EphB3 and Kalirin protein levels. Moreover, dendritic spine density in epileptic rats was evaluated using Golgi staining. ResultsModulation of EphB3 functionality influenced acute seizure severity, latency duration, and frequency of spontaneous recurrent seizures. Golgi staining disclosed an EphB3-driven alteration in dendritic spine density within the hippocampus of epileptic rats, underscoring its pivotal role in the reconfiguration of hippocampal neural circuits. Furthermore, our data propose Kalirin as a prospective downstream mediator of the EphB3 receptor. ConclusionsOur findings elucidate that EphB3 impacts the action potential dynamics in isolated rat hippocampal slices and alters dendritic spine density in the inner molecular layer of epileptic rat hippocampi, likely through Kalirin-mediated pathways. This hints at EphB3's significant role in shaping excitatory circuit loops and recurrent seizure activity via Kalirin.

A chromosome-level genome of electric catfish (Malapterurus electricus) provided new insights into order Siluriformes evolution.

The online version contains supplementary material available at 10.1007/s42995-023-00197-8.

Spatiotemporal Optical Control of Gαq-PLCβ Interactions.

Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with broad physiological and pathological significance. Compared with other Gα members, GαqGTP activates many crucial effectors, including PLCβ (Phospholipase Cβ) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCβ regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal-debilitating diseases, including cancer, epilepsy, Huntington's Disease, and Alzheimer's Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCβ signaling controls cellular physiology has been difficult. Since activated PLCβ induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCβ interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCβ signaling control, our data indicate that the molecular competition between RhoGEFs and PLCβ for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

ARHGEF16 expression correlates with proliferation, migration and invasion of colon cancer cells.

Rho guanine nucleotide exchange factor 16 (ARHGEF16) is a newly discovered Rho-family guanine nucleotide exchange factor (GEF) involved in the activation of Rho-family GTPases. However, its roles in colon cancer cell proliferation, migration, and invasion remain unknown. This study analyzed the expression of ARHGEF16 in colon cancer and explored its biological effects on colon cancer cells, so as to find new therapeutic targets for the treatment of colon cancer. The expression of ARHGEF16 in colon cancer tissues and cells was detected by bioinformatics analysis, western blot, and quantitative real time polymerase chain reaction (qRT-PCR) assays. The effects of overexpression and silencing of ARHGEF16 on the biological behavior of colon cancer cells were examined by Cell Counting Kit-8 (CCK-8), cell scratching and Transwell assays. The database showed that ARHGEF16 was highly expressed in colon cancer tissues. Validation with clinical fresh tissue specimens and colon cancer cell lines revealed that ARHGEF16 was highly expressed in both. The proliferation, growth, migration, and invasion ability of colon cancer cell lines increased significantly with the overexpression of ARHGEF16, while silencing ARHGEF16 showed the opposite effect. The expression of ARHGEF16 is closely related to the migration and invasive ability of colon cancer cells, and overexpression of ARHGEF16 promotes the migration and invasion of colon cancer cells and correlates with the metastatic potential of colon cancer.

ARHGEF18 can promote BVDV NS5B activation of the host NF-κB signaling pathway by combining with the NS5B-palm domain

Rho guanine nucleotide exchange factor 18 (ARHGEF18) is a member of the Rho guanine nucleotide exchange factor (RhoGEF) family. RhoGEF plays an important role in the occurrence of tumors and neurological diseases; however, its involvement in host cell resistance against pathogenic microorganisms is mostly unknown. Herein, we report that bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) can activate the nuclear factor kappa B (NF-κB) signaling pathway to induce an immune response. To clarify the functional domains of NS5B that activate NF-κB signaling, the six structural domains of NS5B were expressed separately: NS5B-core, NS5B-finger, NS5B-palm, NS5B-thumb, NS5B-N and NS5B-c domain. We preliminarily determined that the functional domains of NS5B that activate NF-κB signaling are the finger and palm domains. We used a bovine kidney cell cDNA library and yeast two-hybrid technology to identify that the host protein ARHGEF18 interacts with NS5B. Co-immunoprecipitation assays showed that ARHGEF18 interacts strongly with NS5B-palm. Interestingly ARHGEF18 could promote NF-κB signaling activation by BVDV NS5B. In addition silencing ARHGEF18 significantly inhibited NS5B-palm activation of NF-κB signaling. We concluded that ARHGEF18 can bind to BVDV NS5B through the palm domain to activate the NF-κB pathway. These findings provide direct evidence that BVDV NS5B induces immune responses by activating NF-κB signaling.

Traditional halal meat production without stunning versus commercial slaughter with electrical stunning of slow-growing broiler chicken: impact on meat quality and proteome changes

Impact of traditional halal meat production without stunning (NST) and commercial slaughter with electrical stunning (ST) of 100 slow-growing broiler chicken on blood plasma and different biochemical, enzymatic, hormonal, meat quality, and proteomic changes was evaluated. The results revealed lower (P < 0.05) postmortem pH values and higher redness (a*) scores for ST samples relative to NST group. Myofibrillar fragmentation index and bleeding efficiency (%) were lower (P < 0.05) in ST compared to NST samples. The ST group had higher (P < 0.05) creatinine, total protein, alanine aminotransferase (ALT), and triiodothyronine (T3) than NST group, however, no difference (P > 0.05) in blood glucose, lactate dehydrogenase (LDH), creatine kinase (CK), thyroxine (T4), cortisol, and aspartate aminotransferase (AST) was observed relative to NST samples. The 2-dimensional gel electrophoresis (2-DE) coupled to MALDI-TOF MS of meat samples has identified 14 differentially abundant proteins between 2 groups. Proteins demonstrating positive correlation with stress namely adenylate kinase isoenzyme-1, Rho guanine nucleotide exchange factor (NST), and apolipoprotein A-I (ST) were overabundant. From the current study, it is concluded that electrical stunning of broilers prior to slaughter or traditional halal slaughter without stunning does not adversely affect the meat quality.

Centralspindlin-mediated transport of RhoGEF positions the cleavage plane for cytokinesis.

During cytokinesis, the cell membrane furrows inward along a cleavage plane. The positioning of the cleavage plane is critical to faithful cell division and is determined by the Rho guanine nucleotide exchange factor (RhoGEF)-mediated activation of the small guanosine triphosphatase RhoA and the conserved motor protein complex centralspindlin. Here, we explored whether and how centralspindlin mediates the positioning of RhoGEF. In dividing neuroblasts from Drosophila melanogaster, we observed that immediately before cleavage, first centralspindlin and then RhoGEF localized to the sites where cleavage subsequently initiated. Using in vitro assays with purified Drosophila proteins and stabilized microtubules, we found that centralspindlin directly transported RhoGEF as cargo along single microtubules and sequestered it at microtubule plus-ends for prolonged periods of time. In addition, the binding of RhoGEF to centralspindlin appeared to stimulate centralspindlin motor activity. Thus, the motor activity and microtubule association of centralspindlin can translocate RhoGEF to areas where microtubule plus-ends are abundant, such as at overlapping astral microtubules, to locally activate RhoA and accurately position the cleavage plane during cell division.

RhoA/ROCK signalling activated by ARHGEF3 promotes muscle weakness via autophagy in dystrophic mdx mice.

Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, leads to progressive and fatal muscle weakness through yet-to-be-fully deciphered molecular perturbations. Emerging evidence implicates RhoA/Rho-associated protein kinase (ROCK) signalling in DMD pathology, yet its direct role in DMD muscle function, and related mechanisms, are unknown. Three-dimensionally engineered dystrophin-deficient mdx skeletal muscles and mdx mice were used to test the role of ROCK in DMD muscle function in vitro and in situ, respectively. The role of ARHGEF3, one of the RhoA guanine nucleotide exchange factors (GEFs), in RhoA/ROCK signalling and DMD pathology was examined by generating Arhgef3 knockout mdx mice. The role of RhoA/ROCK signalling in mediating the function of ARHGEF3 was determined by evaluating the effects of wild-type or GEF-inactive ARHGEF3 overexpression with ROCK inhibitor treatment. To gain more mechanistic insights, autophagy flux and the role of autophagy were assessed in various conditions with chloroquine. Inhibition of ROCK with Y-27632 improved muscle force production in 3D-engineered mdx muscles (+25% from three independent experiments, P<0.05) and in mice (+25%, P<0.001). Unlike suggested by previous studies, this improvement was independent of muscle differentiation or quantity and instead related to increased muscle quality. We found that ARHGEF3 was elevated and responsible for RhoA/ROCK activation in mdx muscles, and that depleting ARHGEF3 in mdx mice restored muscle quality (up to +36%, P<0.01) and morphology without affecting regeneration. Conversely, overexpressing ARHGEF3 further compromised mdx muscle quality (-13% vs. empty vector control, P<0.01) in GEF activity- and ROCK-dependent manner. Notably, ARHGEF3/ROCK inhibition exerted the effects by rescuing autophagy which is commonly impaired in dystrophic muscles. Our findings uncover a new pathological mechanism of muscle weakness in DMD involving the ARHGEF3-ROCK-autophagy pathway and the therapeutic potential of targeting ARHGEF3 in DMD.