Increased Actin Polymerization Research Articles

The G Protein-Coupled Receptor GPR56 Is an Inhibitory Checkpoint for NK Cell Migration.

G protein-coupled receptors (GPCRs) represent the largest family of surface receptors and are responsible for key physiological functions, including cell growth, neurotransmission, hormone release, and cell migration. The GPCR 56 (GPR56), encoded by ADGRG1, is an adhesion GPCR found on diverse cell types, including neural progenitor cells, melanoma cells, and lymphocytes, such as effector memory T cells, γδ T cells, and NK cells. Using RNA-sequencing and high-resolution flow cytometry, we found that GPR56 mRNA and protein expression increased with NK cell differentiation, reaching its peak in adaptive NK cells. Small interfering RNA silencing of GPR56 led to increased spontaneous and chemokine-induced migration, suggesting that GPR56 functions as an upstream checkpoint for migration of highly differentiated NK cells. Increased NK cell migration could also be induced by agonistic stimulation of GPR56 leading to rapid internalization and deactivation of the receptor. Mechanistically, GPR56 ligation and downregulation were associated with transcriptional coactivator with PDZ-binding motif translocation to the nucleus and increased actin polymerization. Together, these data provide insights into the role of GPR56 in the migratory behavior of human NK cell subsets and may open possibilities to improve NK cell infiltration into cancer tissues by releasing a migratory checkpoint.

PIP5K-Ras bistability initiates plasma membrane symmetry breaking to regulate cell polarity and migration.

Symmetry breaking, polarity establishment, and spontaneous cell protrusion formation are fundamental but poorly explained cell behaviors. Here, we demonstrate that a biochemical network, where the mutually inhibitory localization of PIP5K and Ras activities plays a central role, governs these processes. First, in resting cells devoid of cytoskeletal activity, PIP5K is uniformly elevated on the plasma membrane, while Ras activity remains minimal. Symmetry is broken by spontaneous local displacements of PIP5K, coupled with simultaneous activations of Ras and downstream signaling events, including PI3K activation. Second, knockout of PIP5K dramatically increases both the incidence and size of Ras-PI3K activation patches, accompanied by branched F-actin assembly. This leads to enhanced cortical wave formation, increased protrusive activity, and a shift in migration mode. Third, high inducible overexpression of PIP5K virtually eliminates Ras-PI3K signaling, cytoskeletal activity, and cell migration, while acute recruitment of cytosolic PIP5K to the membrane induces contraction and blebs in cancer cells. These arrested phenotypes are reversed by reducing myosin II activity, indicating myosin's involvement in the PIP5K-Ras-centered regulatory network. Remarkably, low inducible overexpression of PIP5K unexpectedly facilitates polarity establishment, highlighting PIP5K as a highly sensitive master regulator of these processes. Simulations of a computational model combining an excitable system, cytoskeletal loops, and dynamic partitioning of PIP5K recreates the experimental observations. Taken together, our results reveal that a bistable, mutually exclusive localization of PIP5K and active Ras on the plasma membrane triggers the initial symmetry breaking. Coupled actomyosin reduction and increased actin polymerization lead to intermittently extended protrusions and, with feedback from the cytoskeleton, self-organizing, complementary gradients of PIP5K versus Ras steepen, raising the threshold of the networks at the rear and lowering it at the front to generate polarity for cell migration.

Diazoxon exposure increases susceptibility to infection by Salmonella Typhimurium

ABSTRACT Various exogenous factors, such as microbiological and chemical contamination condition food security. Salmonella Typhimurium (S. Typhimurium) is the cause of salmonellosis. This bacterium utilizes phagocytosis to create bacterial reservoirs. On the other hand, exposure to chemical contaminants, such as pesticides, increases susceptibility to numerous infections. Therefore, this research aims to evaluate the effect of co-exposure to diazoxon and S. Typhimurium on the in vitro infection dynamics. For this purpose, human mononuclear cells were pre-exposed in vitro to diazoxon and then challenged with S. Typhimurium at 1, 8, and 24 h. Bacterial internalization, actin polymerization, and reactive oxygen species (ROS) were analyzed. Obtained data show that mononuclear cells previously exposed to diazoxon exhibit greater internalization of S. Typhimurium. Likewise, greater ROS production and an increase in actin polymerization were observed. Therefore, in the proposed scenario, obtained data suggest that co-exposure to diazoxon and S. Typhimurium increases susceptibility to acquiring an illness.

Does actin polymerization contribute to closure of the avian ductus arteriosus (Gallus gallus)?

The ductus arteriosus (DA) is a cardiovascular embryonic shunt present in all developing land vertebrates. This oxygen-sensitive blood vessel plays the important role of connecting the pulmonary artery to the aorta during development and directs blood flow toward the fetal respiratory organ (placenta in mammals or chorioallantois in birds and reptiles). Permanent vessel closure upon birth or hatch is necessary to establish proper circulation in the neonate. Increased arterial PO2 at birth or hatch stimulates DA closure via smooth muscle contraction. Smooth muscle contraction, such as in the DA, is governed by myosin light chain kinase (MLCK) stimulating increased cross-bridge cycling, and through increases in actin polymerization. The Ras homologous protein family (Rho) of GTPases are involved in calcium sensitization and contraction of vascular smooth muscle but have mostly been studied for their role in cell migration and signaling. Rho GTPases activate the Rho activated kinase (ROCK) pathway, which works in conjunction with MLCK to generate smooth muscle contraction as well as activating LIM kinase which inhibits the actin depolymerizer cofilin. Rac and Cdc42 GTPase pathways increase contractile strength in smooth muscle cells by activating the nucleator Arp2/3, leading to increased actin polymerization. The role of these GTPase pathways during closure of the DA is unknown. We examined the role of the Rho, Rac, and Cdc42 GTPase pathways leading to actin polymerization for their role in the closure of the avian DA in late-term chicken ( Gallus gallus) embryos. We hypothesized that inhibiting the pathways that lead to actin polymerization will inhibit tension development in the DA. Using a Danish Myo Technology wire myograph, DA physiology was examined when exposed to 25% oxygen followed by activators and inhibitors of the Rho, Rac, and Cdc42 GTPase pathways. Activation of Rho A pathways with CN01 (0.2 units/ml) produced contraction of the DA. Blocking ROCK pathways with Y-27632 (10 mM) removed the O2-sensitivity of the vessel, while the vessel was still able to respond to phenylephrine (100 mM). This suggests the Rho pathway is involved in maintaining baseline tension and contractile response to O2. Exposure to inhibitors of Rac (10 uM EHT 1864) and Cdc42 (via ML141) GTPases blocked the ability of vessels to contract in response to 25% oxygen, even after 3 hours of constant O2. Similarly, inhibition of the Arp2/3 complex (100 uM CK666) reduced the ability of the vessel to respond to oxygen. These findings suggest that Rho A, Rac, and Cdc42 pathways play a role in active tension of the chicken DA during development by potentially increasing actin polymerization. This research was funded by Grant R15HL14887 from NIH-NHLBI. This is the full abstract presented at the American Physiology Summit 2024 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.

Focal adhesion kinase (FAK) inhibition induces aquaporin 2 (AQP2) plasma membrane accumulation in renal epithelial cells by remodeling the actin cytoskeleton

The actin cytoskeleton is involved in the cellular traffcking of the AQP2 water channel in kidney collecting duct (CD) principal cells (PC): this plays an important role in water homeostasis. Focal adhesion kinase (FAK) is known to modulate the actin cytoskeleton through small GTPase-mediated pathways and we, therefore, investigated the potential role of FAK in AQP2 traffcking. We and others have previously shown that AQP2 constitutively traffcs and recycles independently of vasopressin (VP) and that this involves actin depolymerization/polymerization; therefore, FAK could regulate AQP2 traffcking through actin remodeling, bypassing the VP-V2R (cAMP/PKA) signaling pathway. To examine this, we used small molecule FAK inhibitors, VS-4718, GSK2256098 (GSK), or Defactinib-Hydrochloride (DH). We used immunofluorescence staining, a transferrin endocytosis assay ± cold block treatment, a fluorometric exocytosis assay using cells expressing secreted yellow fluorescent protein (ssYFP), a RhoA activity assay, and western blotting. Inhibiting FAK with 1mM VS-4718 for 30 min increased AQP2 membrane accumulation in LLCPK1 and IMCD kidney epithelial cells expressing AQP2. VS-4718 resulted in reduced clathrin-mediated endocytosis, as well as increased vesicle exocytosis, both mechanisms contributing to increased AQP2 membrane accumulation. This effect of FAK inhibition was independent of vasopressin/cAMP signaling and did not require phosphorylation of the Serine 256 residue of AQP2, since AQP2 membrane accumulation also occurred in cells expressing the constitutive dephosphorylation AQP2 S256A mutant. The FAK inhibitor VS-4718 promoted actin depolymerization (phalloidin staining assay) which would facilitate AQP2 vesicle exocytosis into the plasma membrane. We then examined the activity of the small GTPase, RhoA, whose activity increases actin polymerization and vesicle traffcking in the exocytic and endocytic pathways. We found that FAK inhibition by VS-4718 downregulated RhoA activity using GST-RBD, a substrate that binds to active RhoA, as seen by western blotting using phospho-specific antibodies. Similar results were obtained with GSK and DH. In summary, our study revealed that the interaction between FAK and cytoskeletal architecture plays a crucial role in regulating AQP2 recycling and its subsequent entry into the endocytic pathway. Thus, targeting FAK could be a potential therapeutic strategy for water balance disorders involving defective AQP2 reabsorption, such as nephrogenic diabetes insipidus. This work was supported by NIDDK R01DK096015 (JHL) and DK096586 (DB). This is the full abstract presented at the American Physiology Summit 2024 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.

Oligomeric Amyloid-β and Tau Alter Cell Adhesion Properties and Induce Inflammatory Responses in Cerebral Endothelial Cells Through the RhoA/ROCK Pathway.

Dysfunction of cerebral endothelial cells (CECs) has been implicated in the pathology of Alzheimer's disease (AD). Despite evidence showing cytotoxic effects of oligomeric amyloid-β (oAβ) and Tau (oTau) in the central nervous system, their direct effects on CECs have not been fully investigated. In this study, we examined the direct effects of oAβ, oTau, and their combination on cell adhesion properties and inflammatory responses in CECs. We found that both oAβ and oTau increased cell stiffness, as well as the p-selectin/Sialyl-LewisX (sLeX) bonding-mediated membrane tether force and probability of adhesion in CECs. Consistent with these biomechanical alterations, treatments with oAβ or oTau also increased actin polymerization and the expression of p-selectin at the cell surface. These toxic oligomeric peptides also triggered inflammatory responses, including upregulations of p-NF-kB p65, IL-1β, and TNF-α. In addition, they rapidly activated the RhoA/ROCK pathway. These biochemical and biomechanical changes were further enhanced by the treatment with the combination of oAβ and oTau, which were significantly suppressed by Fasudil, a specific inhibitor for the RhoA/ROCK pathway. In conclusion, our data suggest that oAβ, oTau, and their combination triggered subcellular mechanical alterations and inflammatory responses in CECs through the RhoA/ROCK pathway.

SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, SCV2), which has resulted in higher morbidity and mortality rate than other respiratory viral infections, such as Influenza A virus (IAV) infection. Investigating the molecular mechanisms of SCV2-host infection vs IAV is vital in exploring antiviral drug targets against SCV2. We assessed differential gene expression in human nasal cells upon SCV2 or IAV infection using RNA sequencing. Compared to IAV, we observed alterations in both metabolic and cytoskeletal pathways suggestive of epithelial remodeling in the SCV2-infected cells, reminiscent of pathways activated as a response to chronic injury. We found that spike protein interaction with the epithelium was sufficient to instigate these epithelial responses using a SCV2 spike pseudovirus. Specifically, we found downregulation of the mitochondrial markers SIRT3 and TOMM22. Moreover, SCV2 spike infection increased extracellular acidification and decreased oxygen consumption rate in the epithelium. In addition, we observed cytoskeletal rearrangements with a reduction in the actin-severing protein cofilin-1 and an increase in polymerized actin, indicating epithelial cytoskeletal rearrangements. This study revealed distinct epithelial responses to SCV2 infection, with early mitochondrial dysfunction in the host cells and evidence of cytoskeletal remodeling that could contribute to the worsened outcome in COVID-19 patients compared to IAV patients. These changes in cell structure and energetics could contribute to cellular resilience early during infection, allowing for prolonged cell survival and potentially paving the way for more chronic symptoms. IMPORTANCE COVID-19 has caused a global pandemic affecting millions of people worldwide, resulting in a higher mortality rate and concerns of more persistent symptoms compared to influenza A. To study this, we compare lung epithelial responses to both viruses. Interestingly, we found that in response to SARS-CoV-2 infection, the cellular energetics changed and there were cell structural rearrangements. These changes in cell structure could lead to prolonged epithelial cell survival, even in the face of not working well, potentially contributing to the development of chronic symptoms. In summary, these findings represent strategies utilized by the cell to survive the infection but result in a fundamental shift in the epithelial phenotype, with potential long-term consequences, which could set the stage for the development of chronic lung disease or long COVID-19.

Actin Polymerization Increases in The Ductus Arteriosus During The Developmental Transition to Lung Ventilation

The Ductus Arteriosus (DA) is an embryonic blood vessel that connects the descending aorta and the pulmonary artery during fetal development. It functions as a right-to-left shunt diverting blood away from the nonventilated lungs and to the descending aorta. The DA serves a comparable physiological role in all terrestrial vertebrates during their embryonic development. Following birth or hatching, the DA constricts in response to increase in arterial oxygen tension brought on by initiation of lung ventilation, and it closes to stop the embryonic shunt. We examined the extent of actin polymerization in the DA during the transition to lung ventilation at hatching in the chicken. We isolated and fixed DA from developing chickens on embryonic days 15 and 19 (ED15 and ED19), during internal pipping (IP) and external pipping (EP), and in hatchlings. Vessel sections were fluorescently stained with Alexa Fluor 488 (DNase 1) for G-actin, Alexa Fluor 568 (Phalloidin) for F-actin, and TOPRO-3 for nuclei staining. Fluorescence microscopy was used to image the sections and quantify F-actin to G-actin levels within the DA for each age. From the images, F-actin to G-actin ratios were determined. Embryonic stages exhibited strong G-actin staining with minimal F-actin staining. During hatching, F-actin staining became more pronounced, exhibiting clearer fiber patterns in the tunica media. F-actin to G-actin ratios increased significantly in the DA from ED15 through hatching, suggesting actin polymerization in the DA increased as the chickens progressed from day 19 to external pipping (EP). These results suggest that increases in actin polymerization play a role in ductus arteriosus constriction at hatching in chickens. This research is funded by grant R15HL14887 from the NIH-NHLBI. 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.

Visfatin-Induced Smooth Muscle Cell Stiffness Syndrome during Lysosomal Acid Ceramidase Deficiency

Recent studies have reported that intrinsic stiffness of smooth muscle cells (SMCs) plays an important role in contributing to arterial wall stiffness, which led to the concept of smooth muscle cell stiffness syndrome (SMCSS). However, the molecular mechanisms regulating the intrinsic stiffness of SMCs are still poorly understood. The present study tested whether lysosomal acid ceramidase (AC) determines intrinsic stiffness of mouse aortic SMCs via regulation of actin polymerization and integrin recycling. Atom force microscopy was used to study mechanical properties of SMCs. As an injurious adipokine, visfatin was found to reduce SMC height, but elevate cortical stiffness and focal adhesion in SMCs, indicating the augmentation of intrinsic stiffness. Moreover, visfatin-induced elevation of cortical stiffness was associated with increased actin polymerization in peripheral areas of SMCs. These pathological changes were much more remarkable in SMCs from smooth muscle-specific Asah1 gene (gene code of AC) knockout (Asah1fl/fl/SMcre) mice compared to SMCs from WT/WT mice. Super-resolution microscopy showed that multivesicular bodies (MVBs) as integrin degrading organelles were much less interacted or fused with lysosomes in SMCs from Asah1fl/fl/SMcre mice than WT/WT mice upon visfatin stimulation. In contrast, MVB-α5 integrin encounters increased more in SMCs from Asah1fl/fl/SMcre mice compared to SMCs from control littermates, suggesting that AC deficiency increases α5 integrin accumulation in MVB, which may enhance recycling endosome moving to cell membrane. Given the vital role of lysosomal transient receptor potential mucolipin 1 (TRPML1) channel in the regulation of lysosome trafficking and its interaction with MVB, we tested whether this lysosomal channel is disturbed by visfatin. By GCaMP3 Ca2+ imaging and whole-lysosome patch clamp recording, we found that ML-SA5 (TRPML1 channel agonist) induced remarkable lysosomal Ca2+ release in WT/WT SMCs, which was attenuated by visfatin. In SMCs lacking Asah1 gene, however, ML-SA5 induced much less Ca2+ release from lysosomes, which was almost abolished by visfatin. Lysosome trafficking analysis showed that ML-SA5 activation of lysosomal TRPML1 channels markedly increased lysosome movement in SMCs, which was significantly inhibited by visfatin and Asah1 gene deletion. Based on these results, we concluded that lysosomal AC deficiency leads to dysregulation of actin polymerization and integrin recycling in SMCs, which may be a novel molecular mechanism of SMCSS and ultimate arterial wall stiffening during obesity. This study was supported by NIH grants HL057244 and HL075316. 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.

Abstract 2966: Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells

Abstract Objectives of the Study: Bispecific antibodies (BsAbs) are a class of immunotherapy drugs that facilitate targeted killing of cancer cells. Here, we report therapeutic potential of a novel Siglec-6/CD3 targeted bsAb against chronic lymphocytic leukemia (CLL) cells. We show here for the first time the functional and therapeutic relevance of Siglec-6 in CLL cell adhesion and migration. Mechanistically, we have shown Siglec-6 mediated cell adhesion and migration through Siglec-6 ligand (sialyl Tn) mediated DOCK8 dependent activation of Cdc42 associated with actin polymerization. To evaluate the therapeutic potential of Siglec-6, we generated a human (hu) Siglec-6 transgenic mouse model and crossed it with the TCL1 CLL mouse model to obtain Siglec-6 x TCL1 mice which develop Siglec-6+ leukemia. A pilot in-vivo study with anti-Siglec-6/CD3 targeted bsAb showed a survival benefit in humanized CD3 (huCD3) mice engrafted with Siglec-6+ leukemic cells. Methods used: Flow cytometry was used to analyze cell surface expression of Siglec-6 and sialyl Tn (sTn). Mass spectrometry analysis was performed to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. For in-vivo BsAb treatment experiments, huCD3 mice were engrafted with 5 million Siglec-6+ leukemic cells isolated from Siglec-6 x TCL1 mouse splenocytes. Weekly treatment (i.v) was started after mice display 5% circulating leukemia. Results and Conclusion: Compared to Siglec-6+ CLL cells, Siglec-6- CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) CLL-BMSCs. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation and WASP protein recruitment, followed by increased actin polymerization, that were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Siglec-6+ leukemia engrafted huCD3 mice treated with anti-Siglec-6/CD3 BsAb has so far demonstrated a survival benefit with median survival of 18 weeks versus a median survival of 8 weeks in the control group that received a non-targeting BsAb (n=4/group). Significance: We have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. An anti-Siglec-6/CD3 BsAb provides a survival benefit against Siglec-6+ leukemia, paving the way for development of Siglec-6 targeted therapeutics to treat CLL. Citation Format: Jessica Nunes, Charlene Mao, Matthew Purcell, Elizabeth Perry, Liwen Zhang, Xiaokui Mo, Matthew Cyr, Meixiao Long, John Byrd, Christoph Rader, Natarajan Muthusamy. Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells [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 2966.

PI4P-Containing Vesicles from Golgi Contribute to Mitochondrial Division by Coordinating with Polymerized Actin.

Golgi-derived PI4P-containing vesicles play important roles in mitochondrial division, which is essential for maintaining cellular homeostasis. However, the mechanism of the PI4P-containing vesicle effect on mitochondrial division is unclear. Here, we found that actin appeared to polymerize at the contact site between PI4P-containing vesicles and mitochondria, causing mitochondrial division. Increasing the content of PI4P derived from the Golgi apparatus increased actin polymerization and reduced the length of the mitochondria, suggesting that actin polymerization through PI4P-containing vesicles is involved in PI4P vesicle-related mitochondrial division. Collectively, our results support a model in which PI4P-containing vesicles derived from the Golgi apparatus cooperate with actin filaments to participate in mitochondrial division by contributing to actin polymerization, which regulates mitochondrial dynamics. This study enriches the understanding of the pathways that regulate mitochondrial division and provides new insight into mitochondrial dynamics.

Negatively curved cellular membranes promote BAIAP2 signaling hub assembly.

Plasma membrane deformations are associated with curvature-dependent protein enrichment that contributes to a wide array of cellular functions. While the spatio-temporal protein dynamics at membrane indentations is well characterized, relatively little is known about protein kinetics at outwardly deforming membrane sites. This is in part due to the lack of high throughput approaches to systematically probe the curvature-dependence of protein-membrane interactions. Here, we developed a nanopatterned array for multiplexed analysis of protein dynamics at negatively curved cellular membranes. Taking advantage of this robust and versatile platform, we explored how membrane shape influences the prototypic negative curvature sensing protein BAIAP2 and its effector proteins. We find assembly of multi-protein signaling hubs and increased actin polymerization at outwardly deformed membrane sections, indicative of curvature-dependent BAIAP2 activation. Collectively, this study presents technical and conceptual advancements towards a quantitative understanding of spatio-temporal protein dynamics at negatively curved membranes.

The Soluble Fms-like Tyrosine Kinase-1 Contributes to Structural and Functional Changes in Endothelial Cells in Chronic Kidney Disease.

Endothelial cells are a critical target of the soluble Fms-like tyrosine kinase-1 (sFlt-1), a soluble factor increased in different diseases with varying degrees of renal impairment and endothelial dysfunction, including chronic kidney disease (CKD). Although the mechanisms underlying endothelial dysfunction are multifactorial and complex, herein, we investigated the damaging effects of sFlt-1 on structural and functional changes in endothelial cells. Our results evidenced that sera from patients with CKD stiffen the endothelial cell cortex in vitro, an effect correlated with sFlt-1 levels and prevented by sFlt-1 neutralization. Besides, we could show that recombinant sFlt-1 leads to endothelial stiffening in vitro and in vivo. This was accompanied by cytoskeleton reorganization and changes in the endothelial barrier function, as observed by increased actin polymerization and endothelial cell permeability, respectively. These results depended on the activation of the p38 MAPK and were blocked by the specific inhibitor SB203580. However, sFlt-1 only minimally affected the expression of stiffness-sensitive genes. These findings bring new insight into the mechanism of action of sFlt-1 and its biological effects that cannot be exclusively ascribed to the regulation of angiogenesis.

Comparative Analysis of Small-Molecule LIMK1/2 Inhibitors: Chemical Synthesis, Biochemistry, and Cellular Activity.

LIM domain kinases 1 and 2 (LIMK1 and LIMK2) regulate actin dynamics and subsequently key cellular functions such as proliferation and migration. LIMK1 and LIMK2 phosphorylate and inactivate cofilin leading to increased actin polymerization. As a result, LIMK inhibitors are emerging as a promising treatment strategy for certain cancers and neurological disorders. High-quality chemical probes are required if the role of these kinases in health and disease is to be understood. To that end, we report the results of a comparative assessment of 17 reported LIMK1/2 inhibitors in a variety of in vitro enzymatic and cellular assays. Our evaluation has identified three compounds (TH-257, LIJTF500025, and LIMKi3) as potent and selective inhibitors suitable for use as in vitro and in vivo pharmacological tools for the study of LIMK function in cell biology.

Statin-induced increase in actin polymerization modulates GPCR dynamics and compartmentalization

The function of the actin cytoskeleton in cellular motility and trafficking has been widely studied. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol and its consequences on membrane dynamics are addressed only rarely. In a recent work, we reported that chronic cholesterol depletion using statins leads to significant polymerization of the actin cytoskeleton. In this work, we explore the effect of reorganization of the actin cytoskeleton on membrane dynamics under cholesterol-depleted condition. Specifically, we explore the role of actin cytoskeleton in regulating the dynamics of the serotonin1A receptor, a crucial neurotransmitter G protein-coupled receptor (GPCR) that plays a major role in the generation and modulation of cognitive and behavioral functions. For this, we analyzed the lateral dynamics of the serotonin1A receptor in cholesterol-depleted cells (using statins) by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) measurements. Our results indicate that lateral diffusion parameters of serotonin1A receptors in normal cells are consistent with models describing the diffusion of molecules in a homogeneous membrane. Interestingly, these parameters are altered in cholesterol-depleted cells and the receptor exhibits dynamic confinement. Notably, our results show that statin-induced dynamic confinement could be reversed by destabilization of the actin cytoskeleton. On a broader perspective, these results assume significance in understanding the modulatory role of the membrane environment on the organization and dynamics of GPCRs in diseases caused by altered cholesterol biosynthesis.

RhoBTB1 reverses established arterial stiffness in angiotensin II-induced hypertension by promoting actin depolymerization.

Arterial stiffness predicts cardiovascular disease and all-cause mortality, but its treatment remains challenging. Mice treated with angiotensin II (Ang II) develop hypertension, arterial stiffness, vascular dysfunction, and a downregulation of Rho-related BTB domain–containing protein 1 (RhoBTB1) in the vasculature. RhoBTB1 is associated with blood pressure regulation, but its function is poorly understood. We tested the hypothesis that restoring RhoBTB1 can attenuate arterial stiffness, hypertension, and vascular dysfunction in Ang II–treated mice. Genetic complementation of RhoBTB1 in the vasculature was achieved using mice expressing a tamoxifen-inducible, smooth muscle–specific RhoBTB1 transgene. RhoBTB1 restoration efficiently and rapidly alleviated arterial stiffness but not hypertension or vascular dysfunction. Mechanistic studies revealed that RhoBTB1 had no substantial effect on several classical arterial stiffness contributors, such as collagen deposition, elastin content, and vascular smooth muscle remodeling. Instead, Ang II increased actin polymerization in the aorta, which was reversed by RhoBTB1. Changes in the levels of 2 regulators of actin polymerization, cofilin and vasodilator-stimulated phosphoprotein, in response to RhoBTB1 were consistent with an actin depolymerization mechanism. Our study reveals an important function of RhoBTB1, demonstrates its vital role in antagonizing established arterial stiffness, and further supports a functional and mechanistic separation among hypertension, vascular dysfunction, and arterial stiffness.

First person – Baishakhi Ghosh and Kristine Nishida

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Baishakhi Ghosh and Kristine Nishida are co-first authors on ‘ Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin’, published in JCS. Baishakhi is a post-doctoral fellow in the lab of Venkataramana K. Sidhaye at Johns Hopkins Bloomberg School of Public Health, Maryland, USA, evaluating the mechanisms by which inhaled pollutants can cause chronic injury to model chronic lung diseases. Kristine is a Research Associate in the FastForward Facility at Maryland, where she is interested in increasing the accessibility of cell and gene therapies to patients.

Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin.

ABSTRACTThe airway epithelium is subjected to insults such as cigarette smoke (CS), a primary cause of chronic obstructive pulmonary disease (COPD) and serves as an excellent model to study cell plasticity. Here, we show that both CS-exposed and COPD-patient derived epithelia (CHBE) display quantitative evidence of cellular plasticity, with loss of specialized apical features and a transcriptional profile suggestive of partial epithelial-to-mesenchymal transition (pEMT), albeit with distinct cell motion indicative of cellular unjamming. These injured/diseased cells have an increased fraction of polymerized actin, due to loss of the actin-severing protein cofilin-1. We observed that decreasing polymerized actin restores the jammed state in both CHBE and CS-exposed epithelia, indicating that the fraction of polymerized actin is critical in unjamming the epithelia. Our kinetic energy spectral analysis suggests that loss of cofilin-1 results in unjamming, similar to that seen with both CS exposure and in CHBE cells. The findings suggest that in response to chronic injury, although epithelial cells display evidence of pEMT, their movement is more consistent with cellular unjamming. Inhibitors of actin polymerization rectify the unjamming features of the monolayer. This article has an associated First Person interview with the first author of the paper.

The missing 'link'? β-Catenin's role in skeletal muscle glucose uptake during exercise and contraction.

The missing 'link'? β-Catenin's role in skeletal muscle glucose uptake during exercise and contraction.

G protein coupled estrogen receptor attenuates mechanical stress-mediated apoptosis of chondrocyte in osteoarthritis via suppression of Piezo1

BackgroundApoptosis of chondrocyte is involved in osteoarthritis (OA) pathogenesis, and mechanical stress plays a key role in this process by activation of Piezo1. However, the negative regulation of signal conduction mediated by mechanical stress is still unclear. Here, we elucidate that the critical role of G protein coupled estrogen receptor (GPER) in the regulation of mechanical stress-mediated signal transduction and chondrocyte apoptosis.MethodsThe gene expression profile was detected by gene chip upon silencing Piezo1. The expression of GPER in cartilage tissue taken from the clinical patients was detected by RT-PCR and Western blot as well as immunohistochemistry, and the correlation between GPER expression and OA was also investigated. The chondrocytes exposed to mechanical stress were treated with estrogen, G-1, G15, GPER-siRNA and YAP (Yes-associated protein)-siRNA. The cell viability of chondrocytes was measured. The expression of polymerized actin and Piezo1 as well as the subcellular localization of YAP was observed under laser confocal microscope. Western blot confirmed the changes of YAP/ Rho GTPase activating protein 29 (ARHGAP29) /RhoA/LIMK /Cofilin pathway. The knee specimens of osteoarthritis model were stained with safranin and green. OARSI score was used to evaluate the joint lesions. The expressions of GPER and YAP were detected by immunochemistry.ResultsExpression profiles of Piezo1- silenced chondrocytes showed that GPER expression was significantly upregulated. Moreover, GPER was negatively correlated with cartilage degeneration during OA pathogenesis. In addition, we uncovered that GPER directly targeted YAP and broadly restrained mechanical stress-triggered actin polymerization. Mechanism studies revealed that GPER inhibited mechanical stress-mediated RhoA/LIMK/cofilin pathway, as well as the actin polymerization, by promoting expression of YAP and ARHGAP29, and the YAP nuclear localization, eventually causing the inhibition of Piezo1. YAP was obviously decreased in degenerated cartilage. Silencing YAP caused significantly increased actin polymerization and activation of Piezo1, and an increase of chondrocyte apoptosis. In addition, intra-articular injection of G-1 to OA rat effectively attenuated cartilage degeneration.ConclusionWe propose a novel regulatory mechanism underlying mechanical stress-mediated apoptosis of chondrocyte and elucidate the potential application value of GPER as therapy targets for OA.