Lamellipodia Formation Research Articles

Baicalein disturbs the morphological plasticity and motility of breast adenocarcinoma cells depending on the tumor microenvironment.

During tumor invasion, cancer cells change their morphology and mode of migration based on communication with the surrounding environment. Numerous studies have indicated that paracrine interactions from non-neoplastic cells impact the migratory and invasive properties of cancer cells. Thus, these interactions are potential targets for anticancer therapies. In this study, we showed that the flavones member baicalein suppresses the motility of breast cancer cells that is promoted by paracrine interactions. First, we identified laminin-332 (LN-332) as a principle paracrine factor in conditioned medium from mammary epithelium-derived MCF10A cells that regulates the morphology and motility of breast adenocarcinoma MDA-MB-231 cells. Then, we carried out a morphology-based screen for small compounds, which showed that baicalein suppressed the morphological changes and migratory activity of MDA-MB-231 cells that were induced by conditioned medium from MCF10A cells and LN-332. We also found that baicalein caused narrower and incomplete lamellipodia formation in conditioned medium-treated MDA-MB-231 cells, although actin dynamics downstream of Rho family small GTPases were unaffected. These results suggest the importance of mammary epithelial cells in the cancer microenvironment promoting the migratory activity of breast adenocarcinoma cells and show a novel mechanism through which baicalein inhibits cancer cell motility.

Transcription Factor SOX5 Promotes the Migration and Invasion of Fibroblast-Like Synoviocytes in Part by Regulating MMP-9 Expression in Collagen-Induced Arthritis.

Fibroblast-like synoviocytes (FLS) exhibit a unique aggressive phenotype in rheumatoid arthritis (RA). Increased FLS migration and subsequent invasion of the extracellular matrix are essential to joint destruction in RA. Our previous research reported that transcription factor SOX5 was highly expressed in RA-FLS. Here, the effects of SOX5 in RA-FLS migration and invasion will be investigated. The migration and invasion of RA-FLS were evaluated using a transwell chamber assay. The expression of several potential SOX5-targeted genes, including matrix metalloproteinases (MMP-1, 2, 3 and 9), chemokines (CCL4, CCL2, CCR5 and CCR2), and pro-inflammatory cytokines (TNF-α and IL-6), were examined in RA-FLS using SOX5 gain- and loss-of-function study. The molecular mechanisms of SOX5-mediated MMP-9 expressions were assayed by luciferase reporter gene and chromatin immunoprecipitation (ChIP) studies. The in vivo effect of SOX5 on FLS migration and invasion was examined using collagen-induced arthritis (CIA) in DBA/1J mice. Knockdown SOX5 decreased lamellipodium formation, migration, and invasion of RA-FLS. The expression of MMP-9 was the only gene tested to be concomitantly affected by silencing or overexpressing SOX5. ChIP assay revealed that SOX5 was bound to the MMP-9 promoter in RA-FLS. The overexpression of SOX5 markedly enhanced the MMP-9 promoter activity, and specific deletion of a putative SOX5-binding site in MMP-9 promoter diminished this promoter-driven transcription in FLS. Locally knocked down SOX5 inhibited MMP-9 expression in the joint tissue and reduced pannus migration and invasion into the cartilage in CIA mice. SOX5 plays a novel role in mediating migration and invasion of FLS in part by regulating MMP-9 expression in RA.

Effect of Ginger Powder Extracts on Actin Assembly In Vitro

ObjectiveThe components of ginger root (Zingiber officinale Roscoe) have been widely used for various medicinal purposes in Asia. Several bioactive compounds have been identified in ginger, including 6‐ and 8‐gingerols and 6‐shogaol, which are agonists of the capsaicin receptor TRPV1[1, 2]. Recently, we demonstrated that ginger powder might affect human metabolism in vivo[3]. However, the effects of the bioactive compounds of ginger on live cells have not been completely elucidated. In this study, we investigated whether ginger powder extracts (GPE) induced signal transduction, especially actin assembly, in vitro.MethodThe active components of GPE were characterized by high‐performance liquid chromatography. NIH3T3 mouse fibroblast cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. These cells were treated with GPE (10 ng/mL) for 10 min, followed by analysis of the activation of mitogen‐activated protein kinases (MAPKs) ERK and p38 MAPK, and actin assembly.ResultsThe levels of phosphorylated ERK and p38 MAPK increased significantly after a 10‐ min stimulation with GPE. In addition, lamellipodia formation, which is known to facilitate cell migration[4], occurred at cell edges after GPE stimulation.ConclusionsGPE activated MAPKs and increased lamellipodia formation in mouse fibroblasts. These results suggest that GPE modifies signal transduction, including actin assembly, in vitro.Support or Funding InformationKAKENHI, JapanThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NAA80 is actin’s N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility

Actin, one of the most abundant proteins in nature, participates in countless cellular functions ranging from organelle trafficking and pathogen motility to cell migration and regulation of gene transcription. Actin's cellular activities depend on the dynamic transition between its monomeric and filamentous forms, a process exquisitely regulated in cells by a large number of actin-binding and signaling proteins. Additionally, several posttranslational modifications control the cellular functions of actin, including most notably N-terminal (Nt)-acetylation, a prevalent modification throughout the animal kingdom. However, the biological role and mechanism of actin Nt-acetylation are poorly understood, and the identity of actin's N-terminal acetyltransferase (NAT) has remained a mystery. Here, we reveal that NAA80, a suggested NAT enzyme whose substrate specificity had not been characterized, is Nt-acetylating actin. We further show that actin Nt-acetylation plays crucial roles in cytoskeletal assembly in vitro and in cells. The absence of Nt-acetylation leads to significant differences in the rates of actin filament depolymerization and elongation, including elongation driven by formins, whereas filament nucleation by the Arp2/3 complex is mostly unaffected. NAA80-knockout cells display severely altered cytoskeletal organization, including an increase in the ratio of filamentous to globular actin, increased filopodia and lamellipodia formation, and accelerated cell motility. Together, the results demonstrate NAA80's role as actin's NAT and reveal a crucial role for actin Nt-acetylation in the control of cytoskeleton structure and dynamics.

Transforming growth factor-β modulates pancreatic cancer associated fibroblasts cell shape, stiffness and invasion

BackgroundTumor microenvironment consists of the extracellular matrix (ECM), stromal cells, such as fibroblasts (FBs) and cancer associated fibroblasts (CAFs), and a myriad of soluble factors. In many tumor types, including pancreatic tumors, the interplay between stromal cells and the other tumor microenvironment components leads to desmoplasia, a cancer-specific type of fibrosis that hinders treatment. Transforming growth factor beta (TGF-β) and CAFs are thought to play a crucial role in this tumor desmoplastic reaction, although the involved mechanisms are unknown. MethodsOptical/fluorescence microscopy, atomic force microscopy, image processing techniques, invasion assay in 3D collagen I gels and real-time PCR were employed to investigate the effect of TGF-β on normal pancreatic FBs and CAFs with regard to crucial cellular morphodynamic characteristics and relevant gene expression involved in tumor progression and metastasis. ResultsCAFs present specific myofibroblast-like characteristics, such as α-smooth muscle actin expression and cell elongation, they also form more lamellipodia and are softer than FBs. TGF-β treatment increases cell stiffness (Young's modulus) of both FBs and CAFs and increases CAF's (but not FB's) elongation, cell spreading, lamellipodia formation and spheroid invasion. Gene expression analysis shows that these morphodynamic characteristics are mediated by Rac, RhoA and ROCK expression in CAFs treated with TGF-β. ConclusionsTGF-β modulates CAFs', but not FBs', cell shape, stiffness and invasion. General SignificanceOur findings elucidate on the effects of TGF-β on CAFs' behavior and stiffness providing new insights into the mechanisms involved.

P66Shc regulates migration of castration-resistant prostate cancer cells

Metastatic castration-resistant (CR) prostate cancer (PCa) is a lethal disease for which no effective treatment is currently available. p66Shc is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species (ROS) and is elevated in clinical PCa and multiple CR PCa cell lines. We hypothesize p66Shc also increases the migratory activity of PCa cells through ROS and investigate the associated mechanism. Using the transwell assay, our study reveals that the level of p66Shc protein correlates with cell migratory ability across several PCa cell lines. Furthermore, we show hydrogen peroxide treatment induces migration of PCa cells that express low levels of p66Shc in a dose-dependent manner, while antioxidants inhibit migration. Conversely, PCa cells that express high levels of endogenous p66Shc or by cDNA transfection possess increased cell migration which is mitigated upon p66Shc shRNA transfection or expression of oxidase-deficient dominant-negative p66Shc W134F mutant. Protein microarray and immunoblot analyses reveal multiple proteins, including ErbB-2, AKT, mTOR, ERK, FOXM1, PYK2 and Rac1, are activated in p66Shc-elevated cells. Their involvement in PCa migration was examined using respective small-molecule inhibitors. The role of Rac1 was further validated using cDNA transfection and, significantly, p66Shc is found to promote lamellipodia formation through Rac1 activation. In summary, the results of our current studies clearly indicate p66Shc also regulates PCa cell migration through ROS-mediated activation of migration-associated proteins, notably Rac1.

Yap regulates gastric cancer survival and migration via SIRT1/Mfn2/mitophagy.

Gastric cancer is the fifth most common cancer worldwide and Hippo-Yap is the novel signaling pathway which plays an important role in gastric cancer tumor development and progression. However, little insight is available to date regarding the specific role of Yes-associated protein (Yap) in gastric cancer. In the present study, we identified the mechanism through which Yap sustains gastric cancer viability and migration. Yap was greatly upregulated in gastric cancer cells and its expression promoted cellular migration and survival. Functional studies found that knockdown of Yap reduced the mitophagy activity, which subsequently caused mitochondrial apoptosis and cellular oxidative stress. The latter impaired adhesive protein expression, alleviated F-actin expression, blunted lamellipodium formation, leading to inhibition of cancer cell motility. Mechanistically, Yap preserved Sirtuin 1 (SIRT1) activity which manipulated mitofusin 2 (Mfn2) expression and subsequent mitophagy. Loss of Yap reduced SIRT1 expression and inhibited Mfn2-mediated mitophagy. Collectively, our results identified Hippo-Yap as a tumor promoter in gastric cancer that was mediated via activation of the SIRT1/Mfn2/mitophagy axis, with potential applications to gastric cancer therapy involving cancer survival and migration.

Decreased TGFBR3/betaglycan expression enhances the metastatic abilities of renal cell carcinoma cells through TGF-\u03b2-dependent and -independent mechanisms

TGF-β regulates both the tumor-forming and migratory abilities of various types of cancer cells. However, it is unclear how the loss of TGF-β signaling components affects these abilities in clear-cell renal cell carcinoma (ccRCC). In this study, we investigated the role of TGFBR3 (TGF-β type III receptor, also known as betaglycan) in ccRCC. Database analysis revealed decreased expression of TGFBR3 in ccRCC tissues, which correlated with poor prognosis in patients. Orthotopic inoculation experiments using immunocompromised mice indicated that low TGFBR3 expression in ccRCC cells enhanced primary tumor formation and lung metastasis. In the presence of TGFBR3, TGF-β2 decreased the aldehyde dehydrogenase (ALDH)-positive ccRCC cell population, in which renal cancer-initiating cells are enriched. Loss of TGFBR3 also enhanced cell migration in cell culture and induced expression of several mesenchymal markers in a TGF-β-independent manner. Increased lamellipodium formation by FAK-PI3K signaling was observed with TGFBR3 downregulation, and this contributed to TGF-β-independent cell migration in ccRCC cells. Taken together, our findings reveal that loss of TGFBR3 endows ccRCC cells with multiple metastatic abilities through TGF-β-dependent and independent pathways.

The SH3 Domain of Unconventional Myosin IB from E. histolytica Interacts With a GEF (EhFP10) and Regulates Phagocytosis by Affecting Actin Bundling

Unconventional myosins form the largest class of myosin Superfamily. Unlike conventional two-headed myosins, they are involved in organelle movement, vesicle trafficking, phagocytosis, pinocytosis, membrane ruffling and the formation of lamellipodia. Several isoforms of class I myosins differing in structure and function have been reported in lower as well as higher eukaryotes. E.histolytica has only two myosins. The sole unconventional EhMyosin IB is a long-tailed myosin I with a c-terminal SH3. In the present work, we have determined the crystal structure of the Sh3 domain of EhMyosin IB, in native and PEG-bound form. Structural studies of PEG-bound EhMySh3 domain revealed characteristics of its probable ligand. We have identified a GTPase exchange factor, EhFP10 with APC basic domain to be one of the binding partner of EhMySH3 and validated it through SPR studies and co-crystallization of the target peptide with the EhMySH3 domain. EhMyosin IB co-localizes with EhFP10 during pinocytosis and phagocytosis. Interaction with EhMySh3 was observed to inhibit the actin bundling activity by APC domain at high EhMySH3: APC ratios while as the concentration of APC increases, it was able to overcome the inhibition. Our findings reveal a new mode of regulation of endocytic processes in E. histolytica, a highly motile gastric pathogen.

Mitochondrial fission promotes cell migration by Ca2+ /CaMKII/ERK/FAK pathway in hepatocellular carcinoma.

Mitochondrial dynamics of fission and fusion plays critical roles in a diverse range of important cellular functions, and its deregulation has been increasingly implicated in human diseases. Previous studies have shown that increased mitochondrial fission significantly promoted the proliferation of hepatocellular carcinoma (HCC) cells. However, how they influence the migration of tumour cells remained largely unknown. In the present study, we further investigated the effect of mitochondrial fission on the migration and metastasis of hepatocellular carcinoma cells. Moreover, the underlying molecular mechanisms and therapeutic application were explored. Our data showed that dynamin-1-like protein expression was strongly increased in distant metastasis of hepatocellular carcinoma when compared to primary hepatocellular carcinoma. In contrast, the mitochondrial fusion protein mitofusin 1 showed an opposite trend. Moreover, the expression of dynamin-1-like protein and mitofusin 1 was significantly associated with the disease-free survival of hepatocellular carcinoma patients. In addition, our data further showed that mitochondrial fission significantly promoted the reprogramming of focal-adhesion dynamics and lamellipodia formation in hepatocellular carcinoma cells mainly by activating typical Ca2+ /CaMKII/ERK/FAK pathway. Importantly, treatment with mitochondrial division inhibitor-1 significantly decreased calcium signalling in hepatocellular carcinoma cells and had a potential treatment effect for hepatocellular carcinoma metastasis in vivo. Taken together, our findings demonstrate that mitochondrial fission plays a critical role in the regulation of hepatocellular carcinoma cell migration, which provides strong evidence for this process as a drug target in hepatocellular carcinoma metastasis treatment.

Glycolysis, but not Mitochondria, responsible for intracellular ATP distribution in cortical area of podocytes

Differentiated podocytes, a type of renal glomerular cells, require substantial levels of energy to maintain glomerular physiology. Mitochondria and glycolysis are two major producers of ATP, but the precise roles of each in podocytes remain unknown. This study evaluated the roles of mitochondria and glycolysis in differentiated and differentiating podocytes. Mitochondria in differentiated podocytes are located in the central part of cell body while blocking mitochondria had minor effects on cell shape and migratory ability. In contrast, blocking glycolysis significantly reduced the formation of lamellipodia, a cortical area of these cells, decreased the cell migratory ability and induced the apoptosis. Consistently, the local ATP production in lamellipodia was predominantly regulated by glycolysis. In turn, synaptopodin expression was ameliorated by blocking either mitochondrial respiration or glycolysis. Similar to differentiated podocytes, the differentiating podocytes utilized the glycolysis for regulating apoptosis and lamellipodia formation while synaptopodin expression was likely involved in both mitochondrial OXPHOS and glycolysis. Finally, adult mouse podocytes have most of mitochondria predominantly in the center of the cytosol whereas phosphofructokinase, a rate limiting enzyme for glycolysis, was expressed in foot processes. These data suggest that mitochondria and glycolysis play parallel but distinct roles in differentiated and differentiating podocytes.

Sphingolipids Signaling in Lamellipodia Formation and Enhancement of Endothelial Barrier Function.

Sphingolipids, first described in the brain in 1884, are important structural components of biological membranes of all eukaryotic cells. In recent years, several lines of evidence support the critical role of sphingolipids such as sphingosine, sphingosine-1-phosphate (S1P), and ceramide as anti- or pro-inflammatory bioactive lipid mediators in a variety of human pathologies including pulmonary and vascular disorders. Among the sphingolipids, S1P is a naturally occurring agonist that exhibits potent barrier enhancing property in the endothelium by signaling via G protein-coupled S1P1 receptor. S1P, S1P analogs, and other barrier enhancing agents such as HGF, oxidized phospholipids, and statins also utilize the S1P/S1P1 signaling pathway to generate membrane protrusions or lamellipodia, which have been implicated in resealing of endothelial gaps and maintenance of barrier integrity. A better understanding of sphingolipids mediated regulation of lamellipodia formation and barrier enhancement of the endothelium will be critical for the development of sphingolipid-based therapies to alleviate pulmonary disorders such as sepsis-, radiation-, and mechanical ventilation-induced acute lung injury.

Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Nonnredundant Functions in Vivo

Cell migration often involves the formation of sheet‐like lamellipodia, generated by actin filament branching through Arp2/3 complex. In these structures, the latter is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family. Recent structural studies defined two independent Rac1 binding sites on WRC within the Sra‐1/PIR121 subunit of the pentameric WRC, but the functions of these sites in vivo have remained unknown. Here we used CRISPR/Cas9‐mediated gene disruption of Sra‐1 and its paralogue PIR121 in murine B16‐F1 cells combined with Sra‐1 mutant rescue to dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra‐1. We found that the A site, positioned adjacent to the binding region of WAVE‐WCA mediating actin‐ and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site towards the C‐terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium amoebas. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants , we conclude these residues are important for Rac/D site interaction. Finally, lamellipodia formation was observable even after disruption of both Rac interaction sites in constitutively active WRC, showing Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but not mandatory for WRC accumulation in the lamellipodium.

Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling

Background/Aims: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. Methods: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (V_mem) and swelling-dependent chloride (Cl^-) currents (ICl_swell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. Results: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl^- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl^- current under isotonic conditions resembling features of the swelling-activated Cl^- current (ICl_swell). The cell membrane potential (V_mem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na⁺-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. Conclusion: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.

Identification of ARL4C as a Peritoneal Dissemination-Associated Gene and Its Clinical Significance in Gastric Cancer.

In gastric cancer (GC), peritoneal dissemination (PD) occurs frequently and is incurable. In this study, we aimed to identify PD-associated genes in GC. We identified a PD-associated gene using three GC datasets: highly disseminated peritoneal GC cell lines, the Singapore dataset and The Cancer Genome Atlas (TCGA) dataset. We assessed the clinicopathological significance of the gene expression using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and performed immunohistochemical analysis for the gene in our patient cohort. We also performed survival analyses of the gene in our patient cohort, the Singapore dataset and the GSE62254 datasets. Moreover, gene set enrichment analysis (GSEA) was performed using the Singapore and TCGA datasets. Finally, in vitro experiments such as invasion/migration assays, immunofluorescence staining of actin filaments, epidermal growth factor (EGF) treatment analysis, and gene expression analysis were conducted using three gene-knockdown GC cell lines (AGS, 58As9, MKN45). ADP-ribosylation factor-like 4c (ARL4C) was identified as a PD-associated gene, and immunohistochemical analysis showed that ARL4C was overexpressed in GC cells. High ARL4C expression was associated with the depth of invasion (p<0.01) and PD (p<0.05) and was a poor prognostic factor (p<0.05) in our patient cohort, the Singapore dataset and the GSE62254 dataset. ARL4C expression positively correlated with the epithelial-mesenchymal transition (EMT) gene set in GSEA. Moreover, ARL4C knockdown reduced invasion/migration capacity, SLUG expression, and the formation of lamellipodia or filopodia in AGS and 58As9 cells. Finally, EGF treatment increased ARL4C expression in MKN45 cells. ARL4C was associated with PD and was a poor prognostic factor in GC, possibly through promoting invasive capacity by activation of both EMT and motility.

Apolipoprotein A-1 mimetic peptide 4F promotes endothelial repairing and compromises reendothelialization impaired by oxidized HDL through SR-B1

Disruption of endothelial monolayer integrity is the primary instigating factor for many cardiovascular diseases. High density lipoprotein (HDL) oxidized by heme enzyme myeloperoxidase (MPO) is dysfunctional in promoting endothelial repair. Apolipoprotein A-1 mimetic 4F with its pleiotropic benefits has been proven effective in many in vivo models. In this study we investigated whether 4F promotes endothelial repair and restores the impaired function of oxidized HDL (Cl/NO2-HDL) in promoting re-endothelialization. We demonstrate that 4F and Cl/NO2-HDL act on scavenger receptor type I (SR-B1) using human aorta endothelial cells (HAEC) and SR-B1 (-/-) mouse aortic endothelial cells. Wound healing, transwell migration, lamellipodia formation and single cell migration assay experiments show that 4F treatment is associated with a recovery of endothelial cell migration and associated with significantly increased endothelial nitric oxide synthase (eNOS) activity, Akt phosphorylation and SR-B1 expression. 4F increases NO generation and diminishes oxidative stress. In vivo, 4F can stimulate cell proliferation and re-endothelialization in the carotid artery after treatment with Cl/NO2-HDL in a carotid artery electric injury model but fails to do so in SR-B1(-/-) mice. These findings demonstrate that 4F promotes endothelial cell migration and has a potential therapeutic benefit against early endothelial injury in cardiovascular diseases.

Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.

In vivo imaging of epithelial wound healing in the cnidarian Clytia hemisphaerica demonstrates early evolution of purse string and cell crawling closure mechanisms

BackgroundAll animals have mechanisms for healing damage to the epithelial sheets that cover the body and line internal cavities. Epithelial wounds heal either by cells crawling over the wound gap, by contraction of a super-cellular actin cable (“purse string”) that surrounds the wound, or some combination of the two mechanisms. Both cell crawling and purse string closure of epithelial wounds are widely observed across vertebrates and invertebrates, suggesting early evolution of these mechanisms. Cnidarians evolved ~600 million years ago and are considered a sister group to the Bilateria. They have been much studied for their tremendous regenerative potential, but epithelial wound healing has not been characterized in detail. Conserved elements of wound healing in bilaterians and cnidarians would suggest an evolutionary origin in a common ancestor. Here we test this idea by characterizing epithelial wound healing in live medusae of Clytia hemisphaerica.ResultsWe identified cell crawling and purse string-mediated mechanisms of healing in Clytia epithelium that appear highly analogous of those seen in higher animals, suggesting that these mechanisms may have emerged in a common ancestor. Interestingly, we found that epithelial wound healing in Clytia is 75 to >600 times faster than in cultured cells or embryos of other animals previously studied, suggesting that Clytia may provide valuable clues about optimized healing efficiency. Finally, in Clytia, we show that damage to the basement membrane in a wound gap causes a rapid shift between the cell crawling and purse string mechanisms for wound closure. This is consistent with work in other systems showing that cells marginal to a wound choose between a super-cellular actin cable or lamellipodia formation to close wounds, and suggests a mechanism underlying this decision.Conclusions1. Cell crawling and purse string mechanisms of epithelial wound healing likely evolved before the divergence of Cnidaria from the bilaterian lineage ~ 600mya 2. In Clytia, the choice between cell crawling and purse string mechanisms of wound healing depends on interactions between the epithelial cells and the basement membrane.

MeHg affects the activation of FAK, Src, Rac1 and Cdc42, critical proteins for cell movement in PDGF-stimulated SH-SY5Y neuroblastoma cells

Methylmercury (MeHg) is an environmental neurotoxicant that inhibits neuronal migration. This process requires several cyclic steps involving the formation of membrane protrusions (lamellipodia and filopodia) and focal adhesion turnover. FAK and Src are critical proteins that regulate both processes. The FAK-Src complex promotes the activation of Rac1 and Cdc42, two GTPases involved in the remodeling of the actin cytoskeletal network. Here, we studied the effect of MeHg (1, 10, 100, 500 and 1000nM) on cell migration, the formation of cell protrusions, focal adhesion location and the activation of FAK, Src, Rac1 and Cdc42 using the SH-SY5Y neuroblastoma cell line stimulated with PDGF-BB (PDGF). The data show that MeHg (1–500nM) inhibited PDGF-stimulated cell migration. In PDGF-stimulated cells, MeHg (100–1000nM) decreased protrusions and increased the size of the p-FAKY397 clusters. MeHg also inhibited PDGF-induced FAK and Src activation and, at 100nM, MeHg inhibited the activation of Rac1 and Cdc42. Altogether, the findings show that low concentrations of MeHg inhibit SH-SY5Y cell migration by disrupting the activation and disassembly of FAK. This negatively affects the activation of Src, Rac1 and Cdc42, all of which are critical proteins for the regulation of cell movement. These effects could be related to the MeHg-mediated inhibition of PDGF-induced formation of lamellipodia and filopodia, focal adhesion disassembly and PDGF-induced movement.

Advillin acts upstream of phospholipase C ϵ1 in steroid-resistant nephrotic syndrome.

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of chronic kidney disease. Here, we identified recessive mutations in the gene encoding the actin-binding protein advillin (AVIL) in 3 unrelated families with SRNS. While all AVIL mutations resulted in a marked loss of its actin-bundling ability, truncation of AVIL also disrupted colocalization with F-actin, thereby leading to impaired actin binding and severing. Additionally, AVIL colocalized and interacted with the phospholipase enzyme PLCE1 and with the ARP2/3 actin-modulating complex. Knockdown of AVIL in human podocytes reduced actin stress fibers at the cell periphery, prevented recruitment of PLCE1 to the ARP3-rich lamellipodia, blocked EGF-induced generation of diacylglycerol (DAG) by PLCE1, and attenuated the podocyte migration rate (PMR). These effects were reversed by overexpression of WT AVIL but not by overexpression of any of the 3 patient-derived AVIL mutants. The PMR was increased by overexpression of WT Avil or PLCE1, or by EGF stimulation; however, this increased PMR was ameliorated by inhibition of the ARP2/3 complex, indicating that ARP-dependent lamellipodia formation occurs downstream of AVIL and PLCE1 function. Together, these results delineate a comprehensive pathogenic axis of SRNS that integrates loss of AVIL function with alterations in the action of PLCE1, an established SRNS protein.