RhoA Activation Research Articles

The Rho1 GTPase controls anillo-septin assembly to facilitate contractile ring closure during cytokinesis

Animal cell cytokinesis requires activation of the GTPase RhoA (Rho1 in Drosophila), which assembles an F-actin- and myosin II-dependent contractile ring (CR) at the equatorial plasma membrane. CR closure is poorly understood, but involves the multidomain scaffold protein, Anillin. Anillin binds many CR components including F-actin and myosin II (collectively actomyosin), RhoA and the septins. Anillin recruits septins to the CR but the mechanism is unclear. Live imaging of Drosophila S2 cells and HeLa cells revealed that the Anillin N-terminus, which scaffolds actomyosin, cannot recruit septins to the CR. Rather, septin recruitment required the ability of the Anillin C-terminus to bind Rho1-GTP and the presence of the Anillin PH domain, in a sequential mechanism occurring at the plasma membrane, independently of F-actin. Anillin mutations that blocked septin recruitment, but not actomyosin scaffolding, slowed CR closure and disrupted cytokinesis. Thus, CR closure requires coordination of two Rho1-dependent networks: actomyosin and anillo-septin.

Nuclear softening mediated by Sun2 suppression delays mechanical stress-induced cellular senescence

Nuclear decoupling and softening are the main cellular mechanisms to resist mechanical stress-induced nuclear/DNA damage, however, its molecular mechanisms remain much unknown. Our recent study of Hutchinson-Gilford progeria syndrome (HGPS) disease revealed the role of nuclear membrane protein Sun2 in mediating nuclear damages and cellular senescence in progeria cells. However, the potential role of Sun2 in mechanical stress-induced nuclear damage and its correlation with nuclear decoupling and softening is still not clear. By applying cyclic mechanical stretch to mesenchymal stromal cells (MSCs) of WT and Zmpset24−/− mice (Z24−/−, a model for HGPS), we observed much increased nuclear damage in Z24−/− MSCs, which also featured elevated Sun2 expression, RhoA activation, F-actin polymerization and nuclear stiffness, indicating the compromised nuclear decoupling capacity. Suppression of Sun2 with siRNA effectively reduced nuclear/DNA damages caused by mechanical stretch, which was mediated by increased nuclear decoupling and softening, and consequently improved nuclear deformability. Our results reveal that Sun2 is greatly involved in mediating mechanical stress-induced nuclear damage by regulating nuclear mechanical properties, and Sun2 suppression can be a novel therapeutic target for treating progeria aging or aging-related diseases.

Cell-based optimization and characterization of genetically encoded location-based biosensors for Cdc42 or Rac activity.

Rac (herein referring to the Rac family) and Cdc42 are Rho GTPases that regulate the formation of lamellipoda and filopodia, and are therefore crucial in processes such as cell migration. Relocation-based biosensors for Rac and Cdc42 have not been characterized well in terms of their specificity or affinity. In this study, we identify relocation sensor candidates for both Rac and Cdc42. We compared their (1) ability to bind the constitutively active Rho GTPases, (2) specificity for Rac and Cdc42, and (3) relocation efficiency in cell-based assays. Subsequently, the relocation efficiency was improved by a multi-domain approach. For Rac1, we found a sensor candidate with low relocation efficiency. For Cdc42, we found several sensors with sufficient relocation efficiency and specificity. These optimized sensors enable the wider application of Rho GTPase relocation sensors, which was showcased by the detection of local endogenous Cdc42 activity at assembling invadopodia. Moreover, we tested several fluorescent proteins and HaloTag for their influence on the recruitment efficiency of the Rho location sensor, to find optimal conditions for a multiplexing experiment. This characterization and optimization of relocation sensors will broaden their application and acceptance.

The RhoA-ROCK1/ROCK2 Pathway Exacerbates Inflammatory Signaling in Immortalized and Primary Microglia.

Neuroinflammation is a unifying factor among all acute central nervous system (CNS) injuries and chronic neurodegenerative disorders. Here, we used immortalized microglial (IMG) cells and primary microglia (PMg) to understand the roles of the GTPase Ras homolog gene family member A (RhoA) and its downstream targets Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) in neuroinflammation. We used a pan-kinase inhibitor (Y27632) and a ROCK1- and ROCK2-specific inhibitor (RKI1447) to mitigate a lipopolysaccharide (LPS) challenge. In both the IMG cells and PMg, each drug significantly inhibited pro-inflammatory protein production detected in media (TNF-α, IL-6, KC/GRO, and IL-12p70). In the IMG cells, this resulted from the inhibition of NF-κB nuclear translocation and the blocking of neuroinflammatory gene transcription (iNOS, TNF-α, and IL-6). Additionally, we demonstrated the ability of both compounds to block the dephosphorylation and activation of cofilin. In the IMG cells, RhoA activation with Nogo-P4 or narciclasine (Narc) exacerbated the inflammatory response to the LPS challenge. We utilized a siRNA approach to differentiate ROCK1 and ROCK2 activity during the LPS challenges and showed that the blockade of both proteins may mediate the anti-inflammatory effects of Y27632 and RKI1447. Using previously published data, we show that genes in the RhoA/ROCK signaling cascade are highly upregulated in the neurodegenerative microglia (MGnD) from APP/PS-1 transgenic Alzheimer's disease (AD) mice. In addition to illuminating the specific roles of RhoA/ROCK signaling in neuroinflammation, we demonstrate the utility of using IMG cells as a model for primary microglia in cellular studies.

Vangl-dependent Wnt/planar cell polarity signaling mediates collective breast carcinoma motility and distant metastasis

BackgroundIn light of the growing appreciation for the role of collective cell motility in metastasis, a deeper understanding of the underlying signaling pathways will be critical to translating these observations to the treatment of advanced cancers. Here, we examine the contribution of Wnt/planar cell polarity (Wnt/PCP), one of the non-canonical Wnt signaling pathways and defined by the involvement of the tetraspanin-like proteins Vangl1 and Vangl2, to breast tumor cell motility, collective cell invasiveness and mammary tumor metastasis.MethodsVangl1 and Vangl2 knockdown and overexpression and Wnt5a stimulation were employed to manipulate Wnt/PCP signaling in a battery of breast cancer cell lines representing all breast cancer subtypes, and in tumor organoids from MMTV-PyMT mice. Cell migration was assessed by scratch and organoid invasion assays, Vangl protein subcellular localization was assessed by confocal fluorescence microscopy, and RhoA activation was assessed in real time by fluorescence imaging with an advanced FRET biosensor. The impact of Wnt/PCP suppression on mammary tumor growth and metastasis was assessed by determining the effect of conditional Vangl2 knockout on the MMTV-NDL mouse mammary tumor model.ResultsWe observed that Vangl2 knockdown suppresses the motility of all breast cancer cell lines examined, and overexpression drives the invasiveness of collectively migrating MMTV-PyMT organoids. Vangl2-dependent RhoA activity is localized in real time to a subpopulation of motile leader cells displaying a hyper-protrusive leading edge, Vangl protein is localized to leader cell protrusions within leader cells, and actin cytoskeletal regulator RhoA is preferentially activated in the leader cells of a migrating collective. Mammary gland-specific knockout of Vangl2 results in a striking decrease in lung metastases in MMTV-NDL mice, but does not impact primary tumor growth characteristics.ConclusionsWe conclude that Vangl-dependent Wnt/PCP signaling promotes breast cancer collective cell migration independent of breast tumor subtype and facilitates distant metastasis in a genetically engineered mouse model of breast cancer. Our observations are consistent with a model whereby Vangl proteins localized at the leading edge of leader cells in a migrating collective act through RhoA to mediate the cytoskeletal rearrangements required for pro-migratory protrusion formation.

Signal transduction pathways of muscarinic receptors in the murine urinary bladder smooth muscle

Overactive bladder (OAB) is a clinical condition characterized by symptoms of frequency, urgency with/without incontinence. Despite of the fact that OAB affects millions of patients’ quality of life significantly, its etiology is still poorly understood. Currently, antimuscarinics are the first-line medical therapy for the management of OAB. However, their application is limited due to the decrease of patients’ compliance because of several adverse effects (e.g. dry mouth, obstipation).Our aim was to further analyze the signaling pathways of muscarinic receptors in detrusor muscle contraction with the goal of better understanding the intracellular regulation of micturition and identifying potential novel therapeutic targets for OAB.Experiments were performed on adult, male, wild-type (WT), M 2 , M 3 , M 2 /M 3 , Gα q/11 knockout (KO) and pertussis toxin (PTX)-treated mice. Contraction force and RhoA activity were measured in the urinary bladder smooth muscle (UBSM).Contractile responses induced by the muscarinic receptor agonist carbamoylcholine (CCh) in UBSM associated with increased activity of RhoA and were reduced in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632. The CCh-induced contractions were markedly reduced in detrusor strips lacking either M 2 or M 3 receptors and were abolished in UBSM from M 2 /M 3 KO mice. The RhoA activation was decreased in both M 2 KO and M 3 KO bladders and it was completely reduced in M 2 /M 3 KO UBSM. Inhibition of Gα i -coupled signaling by PTX-treatment shifted the concentration-response curve of CCh to the right and diminished RhoA activation. CCh-induced contractile responses were markedly decreased in Gα q/11 KO mice, however, RhoA activation was unaffected.In conclusion, cholinergic detrusor contraction and RhoA activation are mediated by both M 2 and M 3 receptors. Furthermore, CCh-induced contractions involve simultaneously the classical Gα q/11 - and the Gα i -coupled signaling pathways, but the RhoA activation appears to be mediated exclusively by the Gα i proteins. These findings may aid the identification of more specific therapeutic targets for OAB. Hungarian NRDIO K-125174, K-135683, K-139230 and PD-132851, Ministry for Innovation and Technology from the Hungarian NRDI fund (2020-1.1.6-JÖVÖ-2021-00010, TKP2021-EGA-25), STIA-KFI-2021, EFOP-3.6.3-VEKOP-16-2017-00009 grants and Gedeon Richter Plc Talent Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lfc subcellular localization and activity is controlled by αv-class integrin.

Fibronectin (FN)-binding integrins control a variety of cellular responses through Rho GTPases. The FN-binding integrins, αvβ3 and α5β1, are known to induce different effects on cell morphology and motility. Here, we report that FN-bound αvβ3 integrin, but not FN-bound α5β1 integrin, triggers the dissociation of the RhoA GEF Lfc (also known as GEF-H1 and ARHGEF2 in humans) from microtubules (MTs), leading to the activation of RhoA, formation of stress fibres and maturation of focal adhesions (FAs). Conversely, loss of Lfc expression decreases RhoA activity, stress fibre formation and FA size, suggesting that Lfc is the major GEF downstream of FN-bound αvβ3 that controls RhoA activity. Mechanistically, FN-engaged αvβ3 integrin activates a kinase cascade involving MARK2 and MARK3, which in turn leads to phosphorylation of several phospho-sites on Lfc. In particular, S151 was identified as the main site involved in the regulation of Lfc localization and activity. Our findings indicate that activation of Lfc and RhoA is orchestrated in FN-adherent cells in an integrin-specific manner.

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking.

LRRC8A Anion Channel Associates with Myosin Phosphatase Rho Interacting Protein (MPRIP) to Regulate Vascular Function

Volume-regulated anion channels (VRACs) are activated by tumor necrosis factor alpha (TNFα) and angiotensin II. VRACs are composed of Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E) and LRRC8A is required for all VRACs. LRRC8A associates with NADPH oxidase 1 (Nox1) and modulates production of extracellular superoxide in vascular smooth muscle cells (VSMCs). We investigated vascular reactivity in mesenteric arteries from wild type (WT) and smooth muscle-specific LRRC8A knockout (KO) mice. KO vessels displayed enhanced responsiveness to multiple vasodilator agents; acetylcholine, sodium nitroprusside, PKA (forskolin) or PKG (BAY60-2770) activators and a Rho kinase inhibitor (Y-27632), compared to WT. This was associated with decreased RhoA activity and reduced ROCK-dependent MYPT1 phosphorylation at T853. To explore the mechanism of enhanced relaxation we immunoprecipitated LRRC8A-associated proteins from WT and KO VSMCs and analyzed these by mass spectroscopy. Myosin Phosphatase Rho-Interacting protein (MPRIP) was identified only in WT lysates. MPRIP is a scaffolding protein that binds to RhoA, MYPT1, and actin and regulates activity of the myosin light chain phosphatase complex. Association of LRRC8A and MPRIP was confirmed using IP/western blot, proximity ligation assay (PLA) and immunohistochemistry. LRRC8A binds to the second plextrin homology domain of MPRIP. Knockdown of MPRIP (siRNA) increased protein expression of LRRC8A in cultured VSMCs. To explore the relationship between Nox1/LRRC8A, superoxide and intracellular redox signaling events sulfenylation of MPRIP cysteine residues (-SOH) was quantified using DCP-Bio1 (a biotin-labeled dimedone derivative) pulldown with streptavidin beads and western blotting. Sulfenylation was significantly increased 3 min following TNFα stimulation (2.3 fold, n = 3, p < 0.05). It remains to be determined how this impacts association of MPRIP with its four binding partners. Disruption of interaction between Nox1/LRRC8A and MPRIP/RhoA/MYPT1/actin may underly enhanced vasodilation in VSMC-specific LRRC8A null blood vessels. This multi-protein complex provides a potential mechanism by which oxidants produced by Nox1 impact vasomotor function, linking inflammation to increased vascular reactivity and hypertension. NIGMS (5R35GM138191-02), NIDDK (1R01DK132948-01), NHLBI (1R01HL160975-01A1) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

SVEP1 influences monocyte to macrophage differentiation via integrin α4β1/α9β1 and Rho/Rac signalling

BackgroundThe large extracellular matrix protein SVEP1 mediates cell adhesion via integrin α9β1. Recent studies have identified an association between a missense variant in SVEP1 and increased risk of coronary artery disease (CAD) in humans and in mice Svep1 deficiency alters the development of atherosclerotic plaques. However how SVEP1 functionally contributes to CAD pathogenesis is not fully understood. Monocyte recruitment and differentiation to macrophages is a key step in the development of atherosclerosis. Here, we investigated the requirement for SVEP1 in this process. MethodsSVEP1 expression was measured during monocyte–macrophage differentiation in primary monocytes and THP-1 human monocytic cells. SVEP1 knockout THP-1 cell lines and the dual integrin α4β1/α9β1 inhibitor, BOP, were utilised to investigate the effect of these proteins in THP-1 cell adhesion, migration and cell spreading assays. Subsequent activation of downstream integrin signalling intermediaries was quantified by western blotting. ResultsSVEP1 gene expression increases in monocyte to macrophage differentiation in human primary monocytes and THP-1 cells. Using two SVEP1 knockout THP-1 cells we observed reduction in monocyte adhesion, migration, and cell spreading compared to control cells. Similar results were found with integrin α4β1/α9β1 inhibition. We demonstrate reduced activity of Rho and Rac1 in SVEP1 knockout THP-1 cells. ConclusionsSVEP1 regulates monocyte recruitment and differentiation phenotypes through an integrin α4β1/α9β1 dependent mechanism. General significanceThese results describe a novel role for SVEP1 in monocyte behaviour relevant to CAD pathophysiology.

Septin11 promotes hepatocellular carcinoma cell motility by activating RhoA to regulate cytoskeleton and cell adhesion

Septins as GTPases in the cytoskeleton, are linked to a broad spectrum of cellular functions, including cell migration and the progression of hepatocellular carcinoma (HCC). However, roles of SEPT11, the new member of septin, have been hardly understood in HCC. In the study, the clinical significance and biological function of SEPT11 in HCC was explored. SEPT11 was screened out by combining ATAC-seq with mRNA-seq. Role of SEPT11 in HCC was further investigated by using overexpression, shRNA and CRISPR/Cas9-mediated SEPT11-knockout cells or in vivo models. We found RNA-seq and ATAC-seq highlights LncRNA AY927503 (AY) induced SEPT11 transcription, resulting in Rho GTPase activation and cytoskeleton actin aggregation. The GTP-binding protein SEPT11 is thus considered, as a downstream factor of AY, highly expressed in various tumors, including HCC, and associated with poor prognosis of the patients. In vitro, SEPT11 overexpression promotes the migration and invasion of HCC cells, while SEPT11-knockout inhibits migration and invasion. In vivo, SEPT11-overexpressed HCC cells show high metastasis incidents but don’t significantly affect proliferation. Meanwhile, we found SEPT11 targets RhoA, thereby regulating cytoskeleton rearrangement and abnormal cell adhesion through ROCK1/cofilin and FAK/paxillin signaling pathways, promoting invasion and migration of HCC. Further, we found SEPT11 facilitates the binding of GEF-H1 to RhoA, which enhances the activity of RhoA. Overall, our study confirmed function of SEPT11 in promoting metastasis in HCC, and preliminarily explored its related molecular mechanism. SEPT11 acts as an oncogene in HCC, also draws further interest regarding its clinical application as a potential therapeutic target.

Cdc42 prevents precocious Rho1 activation during cytokinesis in a Pak1-dependent manner.

During cytokinesis, a series of coordinated events partition a dividing cell. Accurate regulation of cytokinesis is essential for proliferation and genome integrity. In fission yeast, these coordinated events ensure that the actomyosin ring and septum start ingressing only after chromosome segregation. How cytokinetic events are coordinated remains unclear. The GTPase Cdc42 promotes recruitment of certain cell wall-building enzymes whereas the GTPase Rho1 activates these enzymes. We show that Cdc42 prevents early Rho1 activation during fission yeast cytokinesis. Using an active Rho probe, we find that although the Rho1 activators Rgf1 and Rgf3 localize to the division site in early anaphase, Rho1 is not activated until late anaphase, just before the onset of ring constriction. We find that loss of Cdc42 activation enables precocious Rho1 activation in early anaphase. Furthermore, we provide functional and genetic evidence that Cdc42-dependent Rho1 inhibition is mediated by the Cdc42 target Pak1 kinase. Our work proposes a mechanism of Rho1 regulation by active Cdc42 to coordinate timely septum formation and cytokinesis fidelity.

Targeting the RhoA-GEF-H1 pathway of mast cells attenuates experimental airway allergy

Mast cells are the major effector cells in allergic diseases. RhoA and its downstream pathway is associated with the pathogenesis of airway allergy. The objective of this study is to test a hypothesis that modulating the RhoA-GEF-H1 axis in mast cells can attenuate airway allergy. An airway allergic disorder (AAD) mouse model was employed. Mast cells were isolated from AAD mouse airway tissues to be analyzed by RNA sequencing. We observed that mast cells isolated from the respiratory tract of AAD mice were resistant to apoptosis. Mast cell mediator levels in nasal lavage fluid were correlated with apoptosis resistance in AAD mice. Activation of RhoA in AAD mast cells was related to resistance to apoptosis. Mast cells isolated from the airway tissues in AAD mouse exhibited strong RhoA-GEF-H1 expression. The RhoA-GEF-H1 axis was associated with the lower FasL expression in AAD mast cells. Activation of the RhoA-GEF-H1 axis promoted the production of mediators in mast cells. Inhibition of GEF-H1 facilitated the SIT-induced mast cell apoptosis and enhanced the therapeutic efficacy of AAD. In conclusion, RhoA-GEF-H1 activities are associated with resistance to apoptosis in mast cells isolated from sites of allergic lesions. The state of apoptosis resistance in mast cells is associated with the state of AAD disease. Inhibition of GEF-H1 restores the sensitivity of mast cells to apoptosis inducers, and alleviates experimental AAD in mice.

RhoA/ROCK inhibition attenuates endothelin-1–induced glomerulopathy in the rats

Endothelin-1 (ET-1) contributes to the development of kidney diseases. However, the underlying molecular mechanism is largely undefined. Here we sought to investigate the potential role of ET-1 receptors, ETA and ETB in the regulation of increased glomerular permeability and underlying signaling pathways post-ET-1 infusion. Male Sprague-Dawley rats were infused with ET-1 (2 pmol/kg per minute, i.v.) for four weeks, and the effect on glomerular permeability to albumin (Palb) and albuminuria was measured. The selective ROCK-1/2 inhibitor, Y-27632, was administered to a separate group of rats to determine its effect on ET-1-induced Palb and albuminuria. The role of ETA and ETB receptors in regulating RhoA/ROCK activity was determined by incubating isolated glomeruli from normal rats with ET-1 and with selective ETA and ETB receptor antagonists. ET-1 infusion for four weeks significantly elevated Palb and albuminuria. Y-27632 significantly reduced the elevation of Palb and albuminuria. The activities of both RhoA and ROCK-1/2 were increased by ET-1 infusion. Selective ETB receptor antagonism had no effect on the elevated activity of both RhoA and ROCK-1/2 enzymes. Selective ETA receptor and combined ETA/ETB receptors blockade restored the activity of RhoA and ROCK-1/2 to normal levels. In addition, chronic ET-1 infusion increased the levels of glomerular inflammatory and fibrotic markers. These effects were all attenuated in rats following ROCK-1/2 inhibition. These observations suggest that ET-1 contributes to increased albuminuria, inflammation, and fibrosis by modulating the activity of the ETA-RhoA/ROCK-1/2 pathway. Selective ETA receptor blockade may represent a potential therapeutic strategy to limit glomerular injury and albuminuria in kidney disease.

RhoA promotes osteoclastogenesis and regulates bone remodeling through mTOR-NFATc1 signaling

BackgroundThe cytoskeletal architecture of osteoclasts (OCs) and bone resorption activity must be appropriately controlled for proper bone remodeling, which is associated with osteoporosis. The RhoA protein of GTPase plays a regulatory role in cytoskeletal components and contributes to osteoclast adhesion, podosome positioning, and differentiation. Although osteoclast investigations have traditionally been performed by in vitro analysis, however, the results have been inconsistent, and the significance of RhoA in bone physiology and pathology is still unknown.MethodsWe generated RhoA knockout mice by specifically deleting RhoA in the osteoclast lineage to understand more about RhoA’s involvement in bone remodeling. The function of RhoA in osteoclast differentiation and bone resorption and the mechanisms were assessed using bone marrow macrophages (BMMs) in vitro. The ovariectomized (OVX) mouse model was adopted to examine the pathological effect of RhoA in bone loss.ResultsConditional deletion of RhoA in the osteoclast lineage causes a severe osteopetrosis phenotype, which is attributable to a bone resorption suppression. Further mechanistic studies suggest that RhoA deficiency suppresses Akt-mTOR-NFATc1 signaling during osteoclast differentiation. Additionally, RhoA activation is consistently related to the significant enhancement the osteoclast activity, which culminates in the development of an osteoporotic bone phenotype. Furthermore, in mice, the absence of RhoA in osteoclast precursors prevented occurring OVX-induced bone loss.ConclusionRhoA promoted osteoclast development via the Akt-mTOR-NFATc1 signaling pathway, resulting a osteoporosis phenotype, and that manipulating RhoA activity might be a therapeutic strategy for osteoporotic bone loss.

Abstract 2547: YAP1 associates with the EPHA3 receptor tyrosine kinase to promote prostate cancer progression

Abstract Metastatic castration-resistant prostate cancer is lethal, and the mechanisms contributing to this deadly disease are largely unknown. The disparity in our knowledge poses a significant barrier to developing effective therapeutics against advanced prostate cancer in humans. Evidence suggests that the ephrin family of receptor tyrosine kinase signaling may play a critical role in the pathobiology of prostate cancer progression. However, the mechanism is elusive. Our initial study showed that YAP1, in concert with the TEAD1 transcription factor, regulated EPHA3 expression. However, the significance of EPHA3 signaling in prostate cancer remains elusive. Here we investigated the role of EPHA3 signaling in prostate cancer cells. We showed that the expression of EPHA3 correlated with YAP1 in prostate cancer clinical samples. We also showed that EPHA3 knockout reduced cell growth in 2D and 3D cultures. In addition, EPHA3 loss sensitized castration-resistant prostate cancer cells to enzalutamide. Moreover, EPHA3 knockout reduced the GTPase activity of RHOA compared to cells with EPHA3-WT, which coincided with reduced phospho-ERK1/2 levels. Consistently, EPHA3 knockout reduces phosphor-ERK1/2, similar to the inhibition of phospho-ERK1/2 by an inhibitor of ROCK1 (Rho-associated coiled-coil-containing protein kinase 1). Besides, EPHA3 knockout reduced the expression of EMT markers ZEB1/2 and the cancer stem cell marker CD44 compared to the wild type. Furthermore, the levels of EPAH3 transcripts were higher in high-grade PC of AA patients compared to other races. Finally, AA and Caucasian Americans (CA) PC cell lines differentially express the member of ephrin-A and ephrin-B receptors. These observations suggest that the YAP1-EPHA3 signaling is a viable therapeutic target for patients with advanced prostate cancer. Citation Format: Marwah M. Al-Mathkour, Abdulrahman M. Dwead, Kezhan Khazaw, Bekir Cinar. YAP1 associates with the EPHA3 receptor tyrosine kinase to promote prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2547.

Abstract 2461: The cell migration inhibitor dihydromotuporamine C regulates actin-myosin contractility and actin polymerization

Abstract The motuporamines are a promising class of anti-metastatic compounds. Dihydromotuporamine C (Motu33) has been shown to activate the small GTPase RhoA, however, little is known about subsequent downstream events leading to cell migration inhibition. In the present study, we investigated the mechanism of action of Motu33 and a synthetic derivative, Motu-(CH2)-33, in Drosophila by manipulating the gene dose of positive and negative regulators of actin dynamics. Consistent with previous findings, reduced gene dose of Rho1 (the Drosophila RhoA ortholog) attenuates motuporamine activity, confirming that RhoA/Rho1 is targeted by these compounds. Actin-myosin contraction is controlled by the RhoA-ROCK-myosin regulatory light chain (MRLC) pathway. Reduced gene dose of the myosin binding subunit of myosin phosphatase, a negative regulator of the RhoA-ROCK-MRLC pathway, enhances motuporamine activity indicating that the motuporamines stimulate actin-myosin contraction through activation of the myosin regulatory light chain. RhoA also activates diaphanous (dia) to control actin polymerization. Surprisingly, reduced gene dose of dia enhances motuporamine activity, suggesting that the motuporamines act on dia in a RhoA-independent manner. Reduction in gene dose of the Drosophila Rac orthologs also enhances motuporamine activity. In contrast, motuporamine activity is unaltered by reduced gene dose of slingshot (ssh) which acts to trigger actin severing and depolymerization. Since ssh is directly regulated by Rac, the enhanced activity of motuporamines observed when Rac gene dose is reduced may reflect an indirect mode of action on the Rac GTPases leading to increased RhoA activity. In summary, these findings demonstrate that motuporamines act through RhoA and diaphanous to regulate actin-myosin contractility and actin polymerization. Citation Format: Laurence von Kalm, Corey Seavey. The cell migration inhibitor dihydromotuporamine C regulates actin-myosin contractility and actin polymerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2461.

Nuclear SUN1 stabilizes endothelial cell junctions via microtubules to regulate blood vessel formation.

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here, we show that the nuclear-localized linker of the nucleoskeleton and cytoskeleton (LINC) complex protein SUN1 regulates vascular sprouting and endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted junction function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts, and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and junction integrity.

Rho GTPase regulatory proteins contribute to podocyte morphology and function

Podocytes are a critical cellular component of the glomerular filtration barrier, whose strict permselectivity prohibits the passage of large proteins and charged species into the urine. Phenotypic variability or injury of these highly specialized cells can lead to proteinuria and has been linked with altered activity of Rho GTPases, which are strongly associated with the actin cytoskeleton. Notable regulators of these intracellular molecular switches are called guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). In this study, the roles of several GEFs in podocyte morphology and activity were investigated, including ECT2, ARHGEF2, ARHGEF26, and ARHGEF40. Results from RhoA and Rac1 G-LISA Activation Assays indicated that the absence of ARHGEF40 impairs epidermal growth factor (EGF)-stimulated RhoA and Rac1 activation, whereas knockout of ARHGEF2 and ARHGEF26 may selectively diminish RhoA activation. Furthermore, filopodia formation was hindered for the ARHGEF40 knockout. There are a number of additional investigations underway to understand Rho GTPase regulatory proteins, including the elimination of new sets of GEFs and GAPs in vivo. It is hopeful that these studies can provide insights into potential novel therapeutic strategies for proteinuria.

Actin-regulated Siglec-1 nanoclustering influences HIV-1 capture and virus-containing compartment formation in dendritic cells.

The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DCs) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single-particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment.