Pioneer statoacoustic neurons guide neuroblast behaviour during otic ganglion assembly.

Cranial ganglia are aggregates of sensory neurons that mediate distinct types of sensation. The statoacoustic ganglion (SAG) develops into several lobes that are spatially arranged to connect appropriately with hair cells of the inner ear. To investigate the cellular behaviours involved in the 3D organization of the SAG, we use high-resolution confocal imaging of single-cell, labelled zebrafish neuroblasts (NBs), photoconversion, photoablation, and genetic perturbations. We show that otic NBs delaminate out of the otic epithelium in an epithelial-mesenchymal transition-like manner, rearranging apical polarity and primary cilia proteins. We also show that, once delaminated, NBs require RhoGTPases in order to perform active migration. Furthermore, tracking of recently delaminated NBs revealed their directed migration and coalescence around a small population of pioneer SAG neurons. These pioneer SAG neurons, not from otic placode origin, populate the coalescence region before otic neurogenesis begins and their ablation disrupts delaminated NB migratory pathways, consequentially affecting SAG shape. Altogether, this work shows for the first time the role of pioneer SAG neurons in orchestrating SAG development.

CD93 maintains endothelial barrier function by limiting the phosphorylation and turnover of VE-cadherin.

Regulation of vascular permeability to plasma is essential for tissue and organ homeostasis and is mediated by endothelial cell-to-cell junctions that tightly regulate the trafficking of molecules between blood and tissue. The single-pass transmembrane glycoprotein CD93 is upregulated in endothelial cells during angiogenesis and controls cytoskeletal dynamics. However, its role in maintaining homeostasis by regulating endothelial barrier function has not been elucidated yet. Here, we demonstrate that CD93 interacts with vascular endothelial (VE)-cadherin and limits its phosphorylation and turnover. CD93 deficiency in vitro and in vivo induces phosphorylation of VE-cadherin under basal conditions, displacing it from endothelial cell-cell contacts. Consistent with this, endothelial junctions are defective in CD93-/- mice, and the blood-brain barrier permeability is enhanced. Mechanistically, CD93 regulates VE-cadherin phosphorylation and turnover at endothelial junctions through the Rho/Rho kinase-dependent pathway. In conclusion, our results identify CD93 as a key regulator of VE-cadherin stability at endothelial junctions, opening up possibilities for therapeutic strategies directed to control vascular permeability.

Structural basis for selective AMPylation of Rac-subfamily GTPases by Bartonella effector protein 1 (Bep1).

Small GTPases of the Ras-homology (Rho) family are conserved molecular switches that control fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, the molecular mechanism of target selectivity has remained largely elusive. Here we report a bacterial effector protein that selectively targets members of the Rac subfamily in the Rho family of small GTPases but none in the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding G4 motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 enable modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural understanding of target selectivity toward Rac-subfamily GTPases and provides a highly selective tool for their functional analysis.

Transient Structural Properties of the Rho GDP-Dissociation Inhibitor.

Rho GTPases, master spatial regulators of a wide range of cellular processes, are orchestrated by complex formation with guanine nucleotide dissociation inhibitors (RhoGDIs). These have been thought to possess an unstructured N-terminus that inhibits nucleotide exchange of their client upon binding/folding. Via NMR analyses, molecular dynamics simulations, and biochemical assays, we reveal instead pertinent structural properties transiently maintained both, in the presence and absence of the client, imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain. These observations revise the long-standing textbook picture of the GTPases' mechanism of membrane extraction. Rather than by a disorder-to-order transition upon binding of an inhibitory peptide, the intricate and highly selective extraction process of RhoGTPases is orchestrated via a dynamic ensemble bearing preformed transient structural properties, suitably modulated by the specific surrounding along the multi-step process.

Pleckstrin homology and RhoGEF domain containing G4 (PLEKHG4) leads to the activation of RhoGTPases promoting the malignant phenotypes of thyroid cancer.

Thyroid cancer (TC) is one of the most common endocrine system cancers, and its incidence is elevating. There is an urgent need to develop a deeper understanding of TC pathogenesis and explore new therapeutic target for its treatment. This study aimed to investigate the effects of pleckstrin homology and RhoGEF domain containing G4 (PLEKHG4) on the progression of TC. Herein, 29 pairs of TC and adjacent tissues were used to assess the expression of PLEKHG4. A xenograft model of mouse was established by subcutaneously injected with TC cells. Lung metastasis model was established through left ventricular injection. The results revealed that PLEKHG4 was up-regulated in human TC tissues. PLEKHG4 level was correlated with clinicopathological parameters of TC patients. In vitro assays revealed that PLEKHG4 promoted TC cell proliferation, migration, invasion, and epithelial-mesenchymal transformation. Knockdown of PLEKHG4 led to the opposite effects, and the loss of PLEKHG4 enhanced the apoptosis ability and inhibited the stemness properties of TC cells. These findings were further confirmed by the in vivo growth and lung metastasis of TC tumor. Mechanistically, PLEKHG4 promoted the activation of RhoGTPases RhoA, Cdc42, and Rac1. The inhibitors of these RhoGTPases reversed the PLEKHG4-induced malignant phenotypes. Additionally, ubiquitin-conjugating enzyme E2O (UBE2O), a large E2 ubiquitin-conjugating enzyme acted as an ubiquitin enzyme of PLEKHG4, facilitated its ubiquitination and degradation. In conclusion, PLEKHG4, regulated by UBE2O, promoted the thyroid cancer progression via activating the RhoGTPases pathway. UBE2O/PLEKHG4/RhoGTPases axis is expected to be a novel a therapeutic target for TC treatment.

Rho-GTPase dependent leukocyte interaction generates pro-inflammatory thymic Tregs and causes arthritis.

Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+GLCs). Spleen and joint draining lymph nodes (dLN) CD4+GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4+GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXP3+ regulatory T cells (Tregs) of thymic origin. Activation of the ß-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype.

The emerging roles of srGAPs in cancer.

GTPase activating proteins (GAPs) were initially considered as the inhibitors of cell signaling pathways because of their nature to activate the intrinsic GTPase activity of the RhoGTPases. But recent studies of dysregulated GAPs in many cancers such as glioblastoma, colorectal cancer, breast cancer, and renal cancer have elucidated the important roles of GAPs in carcinogenesis and GAPs have been shown to perform multiple nonconventional functions in different contexts. We have discussed the recent developments in the roles played by different types of srGAPs (SLIT-ROBO Rho GTPase-activating proteins) in cancer.

A MZB Cell Activation Profile Present in the Lacrimal Glands of Sjögren's Syndrome-Susceptible C57BL/6.NOD-Aec1Aec2 Mice Defined by Global RNA Transcriptomic Analyses.

The C57BL/6.NOD-Aec1Aec2 mouse has been extensively studied to define the underlying cellular and molecular basis for the onset and development of Sjögren's syndrome (SS), a human systemic autoimmune disease characterized clinically as the loss of normal lacrimal and salivary gland functions leading respectively to dry eye and dry mouth pathologies. While an overwhelming majority of SS studies in both humans and rodent models have long focused primarily on pathophysiological events and the potential role of T lymphocytes in these events, recent studies in our murine models have indicated that marginal zone B (MZB) lymphocytes are critical for both development and onset of SS disease. Although migration and function of MZB cells are difficult to study in vivo and in vitro, we have carried out ex vivo investigations that use temporal global RNA transcriptomic analyses to track early cellular and molecular events in these exocrine glands of C57BL/6.NOD-Aec1Aec2 mice. In the present report, genome-wide transcriptome analyses of lacrimal glands indicate that genes and gene-sets temporally upregulated during early onset of disease define the Notch2/NF-kß14 and Type1 interferon signal transduction pathways, as well as identify chemokines, especially Cxcl13, and Rho-GTPases, including DOCK molecules, in the cellular migration of immune cells to the lacrimal glands. We discuss how the current results compare with our recently published salivary gland data obtained from similar studies carried out in our C57BL/6.NOD-Aec1Aec2 mice, pointing out both similarities and differences in the etiopathogeneses underlying the autoimmune response within the two glands. Overall, this study uses the power of transcriptomic analyses to identify temporal molecular bioprocesses activated during the preclinical covert pathogenic stage(s) of SS disease and how these findings may impact future intervention therapies as the disease within the two exocrine glands may not be identical.

Microtubules at focal adhesions - a double-edged sword.

Cell adhesion to the extracellular matrix is essential for cellular processes, such as migration and invasion. In response to cues from the microenvironment, integrin-mediated adhesions alter cellular behaviour through cytoskeletal rearrangements. The tight association of the actin cytoskeleton with adhesive structures has been extensively studied, whereas the microtubule network in this context has gathered far less attention. In recent years, however, microtubules have emerged as key regulators of cell adhesion and migration through their participation in adhesion turnover and cellular signalling. In this Review, we focus on the interactions between microtubules and integrin-mediated adhesions, in particular, focal adhesions and podosomes. Starting with the association of microtubules with these adhesive structures, we describe the classical role of microtubules in vesicular trafficking, which is involved in the turnover of cell adhesions, before discussing how microtubules can also influence the actin-focal adhesion interplay through RhoGTPase signalling, thereby orchestrating a very crucial crosstalk between the cytoskeletal networks and adhesions.

Upregulated Chemokine and Rho-GTPase Genes Define Immune Cell Emigration into Salivary Glands of Sjögren's Syndrome-Susceptible C57BL/6.NOD-Aec1Aec2 Mice.

The C57BL/6.NOD-Aec1Aec2 mouse is considered a highly appropriate model of Sjögren's Syndrome (SS), a human systemic autoimmune disease characterized primarily as the loss of lacrimal and salivary gland functions. This mouse model, as well as other mouse models of SS, have shown that B lymphocytes are essential for the development and onset of observed clinical manifestations. More recently, studies carried out in the C57BL/6.IL14α transgenic mouse have indicated that the marginal zone B (MZB) cell population is responsible for development of SS disease, reflecting recent observations that MZB cells are present in the salivary glands of SS patients and most likely initiate the subsequent loss of exocrine functions. Although MZB cells are difficult to study in vivo and in vitro, we have carried out an ex vivo investigation that uses temporal global RNA transcriptomic analyses to profile differentially expressed genes known to be associated with cell migration. Results indicate a temporal upregulation of specific chemokine, chemokine receptor, and Rho-GTPase genes in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice that correlate with the early appearance of periductal lymphocyte infiltrations. Using the power of transcriptomic analyses to better define the genetic profile of lymphocytic emigration into the salivary glands of SS mice, new insights into the underlying mechanisms of SS disease development and onset begin to come into focus, thereby establishing a foundation for further in-depth and novel investigations of the covert and early overt phases of SS disease at the cellular level.

Arginine deprivation: a potential therapeutic for cancer cell metastasis? A review.

Arginine is a semi essential amino acid that is used in protein biosynthesis. It can be obtained from daily food intake or synthesized in the body through the urea cycle using l-citrulline as a substrate. Arginine has a versatile role in the body because it helps in cell division, wound healing, ammonia disposal, immune system, and hormone biosynthesis. It is noteworthy that l-arginine is the precursor for the biosynthesis of nitric oxide (NO) and polyamines. In the case of cancer cells, arginine de novo synthesis is not enough to compensate for their high nutritional needs, forcing them to rely on extracellular supply of arginine. In this review, we will go through the importance of arginine deprivation as a novel targeting therapy by discussing the different arginine deprivation agents and their mechanism of action. We will also focus on the factors that affect cell migration and on the influence of arginine on metastases through polyamine and NO.

Three-dimensional biomimetic vascular model reveals a RhoA, Rac1, and N-cadherin balance in mural cell-endothelial cell-regulated barrier function.

The integrity of the endothelial barrier between circulating blood and tissue is important for blood vessel function and, ultimately, for organ homeostasis. Here, we developed a vessel-on-a-chip with perfused endothelialized channels lined with human bone marrow stromal cells, which adopt a mural cell-like phenotype that recapitulates barrier function of the vasculature. In this model, barrier function is compromised upon exposure to inflammatory factors such as LPS, thrombin, and TNFα, as has been observed in vivo. Interestingly, we observed a rapid physical withdrawal of mural cells from the endothelium that was accompanied by an inhibition of endogenous Rac1 activity and increase in RhoA activity in the mural cells themselves upon inflammation. Using a system to chemically induce activity in exogenously expressed Rac1 or RhoA within minutes of stimulation, we demonstrated RhoA activation induced loss of mural cell coverage on the endothelium and reduced endothelial barrier function, and this effect was abrogated when Rac1 was simultaneously activated. We further showed that N-cadherin expression in mural cells plays a key role in barrier function, as CRISPR-mediated knockout of N-cadherin in the mural cells led to loss of barrier function, and overexpression of N-cadherin in CHO cells promoted barrier function. In summary, this bicellular model demonstrates the continuous and rapid modulation of adhesive interactions between endothelial and mural cells and its impact on vascular barrier function and highlights an in vitro platform to study the biology of perivascular-endothelial interactions.

From miRNA Target Gene Network to miRNA Function: miR-375 Might Regulate Apoptosis and Actin Dynamics in the Heart Muscle via Rho-GTPases-Dependent Pathways.

MicroRNAs (miRNAs) are short, single-stranded, non-coding ribonucleic acid (RNA) molecules, which are involved in the regulation of main biological processes, such as apoptosis or cell proliferation and differentiation, through sequence-specific interaction with target mRNAs. In this study, we propose a workflow for predicting miRNAs function by analyzing the structure of the network of their target genes. This workflow was applied to study the functional role of miR-375 in the heart muscle (myocardium), since this miRNA was previously shown to be associated with heart diseases, and data on its function in the myocardium are mostly unclear. We identified PIK3CA, RHOA, MAPK3, PAFAH1B1, CTNNB1, MYC, PRKCA, ERBB2, and CDC42 as key genes in the miR-375 regulated network and predicted the possible function of miR-375 in the heart muscle, consisting mainly in the regulation of the Rho-GTPases-dependent signaling pathways. We implemented our algorithm for miRNA function prediction into a Python module, which is available at GitHub.

Mechanotransduction and Stiffness-Sensing: Mechanisms and Opportunities to Control Multiple Molecular Aspects of Cell Phenotype as a Design Cornerstone of Cell-Instructive Biomaterials for Articular Cartilage Repair.

Since material stiffness controls many cell functions, we reviewed the currently available knowledge on stiffness sensing and elucidated what is known in the context of clinical and experimental articular cartilage (AC) repair. Remarkably, no stiffness information on the various biomaterials for clinical AC repair was accessible. Using mRNA expression profiles and morphology as surrogate markers of stiffness-related effects, we deduced that the various clinically available biomaterials control chondrocyte (CH) phenotype well, but not to equal extents, and only in non-degenerative settings. Ample evidence demonstrates that multiple molecular aspects of CH and mesenchymal stromal cell (MSC) phenotype are susceptible to material stiffness, because proliferation, migration, lineage determination, shape, cytoskeletal properties, expression profiles, cell surface receptor composition, integrin subunit expression, and nuclear shape and composition of CHs and/or MSCs are stiffness-regulated. Moreover, material stiffness modulates MSC immuno-modulatory and angiogenic properties, transforming growth factor beta 1 (TGF-ß1)-induced lineage determination, and CH re-differentiation/de-differentiation, collagen type II fragment production, and TGF-ß1- and interleukin 1 beta (IL-1ß)-induced changes in cell stiffness and traction force. We then integrated the available molecular signaling data into a stiffness-regulated CH phenotype model. Overall, we recommend using material stiffness for controlling cell phenotype, as this would be a promising design cornerstone for novel future-oriented, cell-instructive biomaterials for clinical high-quality AC repair tissue.

The epigenetic factor KDM2B regulates cell adhesion, small rho GTPases, actin cytoskeleton and migration in prostate cancer cells.

The histone demethylase KDM2B is an epigenetic factor with oncogenic properties that is regulated by the basic fibroblasts growth factor (FGF-2). It has recently been shown that KDM2B co-operates with Polycomb Group proteins to promote cell migration and angiogenesis in tumors. In the present study we addressed the role of KDM2B in regulating actin cytoskeleton signaling, cell-cell adhesion and migration of prostate tumor cells. We report here that KDM2B is functionally expressed in DU-145 prostate cancer cells, activated by FGF-2 and regulates EZH2. KDM2B knockdown induced potent up-regulation of gene transcription and protein expression of the epithelial markers E-cadherin and ZO-1, while KDM2B overexpression down-regulated the levels of both markers, suggesting control of cell adhesion by KDM2B. RhoA and RhoB protein expression and activity were diminished upon KDM2B-knockdown and upregulated in KDM2B-overexpressing cell clones. In accordance, actin reorganization with formation of stress fibers became evident in KDM2B-overexpressing cells and abolished in the presence of the Rho inhibitor C3 transferase. DU-145 cell migration was significantly enhanced in KDM2B overexpressing cells and abolished in C3-pretreated cells. Conversely, the retardation of cell migration observed in KDM2B knockdown cells was enhanced in C3-pretreated cells. These results establish a clear functional link between the epigenetic factor KDM2B and the regulation of cell adhesion and Rho-GTPases signaling that controls actin reorganization and cell migration.

Mechanical strain induces a pro-fibrotic phenotype in human mitral valvular interstitial cells through RhoC/ROCK/MRTF-A and Erk1/2 signaling pathways.

The mitral valve is a complex multilayered structure populated by fibroblast-like cells, valvular interstitial cells (VIC) which are embedded in an extracellular matrix (ECM) scaffold and are submitted to the mechanical deformations affecting valve at each heartbeat, for an average of 40 million times per year. Myxomatous mitral valve (MMV) is the most frequent heart valve disease characterized by disruption of several valvular structures due to alterations of their ECM preventing the complete closure of the valve resulting in symptoms of prolapse and regurgitation. VIC and their ECM exhibit reciprocal dynamic processes between the mechanical signals issued from the ECM and the modulation of VIC phenotype responsible for ECM homeostasis of the valve. Abnormal perception and responsiveness of VIC to mechanical stress may induce an inappropriate adaptative remodeling of the valve progressively leading to MMV. To investigate the response of human VIC to mechanical strain and identify the molecular mechanisms of mechano-transduction in these cells, a cyclic equibiaxial elongation of 14% at the cardiac frequency of 1.16 Hz was applied to VIC by using a Flexercell-4000 T™ apparatus for increasing time (from 1 h to 8 h). We showed that cyclic stretch induces an early (1 h) and transient over-expression of TGFß2 and αSMA. CTGF, a profibrotic growth factor promoting the synthesis of ECM components, was strongly induced after 1 and 2 h of stretching and still upregulated at 8 h. The mechanical stress-induced CTGF up-regulation was dependent on RhoC, but not RhoA, as demonstrated by siRNA-mediated silencing approaches, and further supported by evidencing RhoC activation upon cell stretching and suppression of cell response by pharmacological inhibition of the effector ROCK1/2. It was also dependent on the MEK/Erk1/2 pathway which was activated by mechanical stress independently of RhoC and ROCK. Finally, mechanical stretching induced the nuclear translocation of myocardin related transcription factor-A (MRTF-A) which forms a transcriptional complex with SRF to promote the expression of target genes, notably CTGF. Treatment of stretched cultures with inhibitors of the identified pathways (ROCK1/2, MEK/Erk1/2, MRTF-A translocation) blocked CTGF overexpression and abrogated the increased MRTF-A nuclear translocation. CTGF is up-regulated in many pathological processes involving mechanically challenged organs, promotes ECM accumulation and is considered as a hallmark of fibrotic diseases. Pharmacological targeting of MRTF-A by newly developed inhibitors may represent a relevant therapy for MMV.

N-myc downstream-regulated gene 2 controls astrocyte morphology via Rho-GTPase signaling.

Astrocyte undergoes morphology changes that are closely associated with the signaling communications at synapses. N-myc downstream-regulated gene 2 (NDRG2) is specifically expressed in astrocytes and is associated with several important astrocyte functions, but its potential role(s) relating to astrocyte morphological changes remain unknown. Here, primary astrocytes were prepared from neonatal Ndrg2+/+ and Ndrg2-/- pups, and the drug Y27632 was used to induce stellation. We then used a variety of methods to measure the levels of NDRG2, α-Actinin4, and glial fibrillary acidic protein (GFAP), and the activity of RhoA, Rac1, and Cdc42 in Y27632-treated astrocytes as well as in Ndrg2+/+ , Ndrg2-/- , or Ndrg2-/- + lentivirus (restore NDRG2 expression) astrocytes. We also conducted live-imaging and proteomics studies of the cultured astrocytes. We found that induction of astrocytes stellation (characterized by cytoplasmic retraction and process outgrowth) resulted in increased NDRG2 protein expression and Rac1 activity and in reduced α-Actinin4 protein expression and RhoA activity. Ndrg2 deletion induced astrocyte flattening, whereas the restoration of NDRG2 expression induced stellation. Ndrg2 deletion also significantly increased α-Actinin4 protein expression and RhoA activity yet reduced GFAP protein expression and Rac1 activity, and these trends were reversed by restoration of NDRG2 expression. Collectively, our results showed that Ndrg2 deletion promoted cell proliferation, interrupted stellation capability, and extensively altered the protein expression profiles of proteins that function in Rho-GTPase signaling. These findings suggest that NDRG2 functions to regulate astrocytes morphology via altering the accumulation of the Rho-GTPase signaling pathway components, thereby supporting that NDRG2 should be understood as a regulator of synaptic plasticity and thus neuronal communications.

Analysis of Functional Domains in Rho5, the Yeast Homolog of Human Rac1 GTPase, in Oxidative Stress Response.

The small GTPase Rho5 of Saccharomyces cerevisiae is required for proper regulation of different signaling pathways, which includes the response to cell wall, osmotic, nutrient, and oxidative stress. We here show that proper in vivo function and intracellular distribution of Rho5 depends on its hypervariable region at the carboxyterminal end, which includes the CAAX box for lipid modification, a preceding polybasic region (PBR) carrying a serine residue, and a 98 amino acid-specific insertion only present in Rho5 of S. cerevisiae but not in its human homolog Rac1. Results from trapping GFP-Rho5 variants to the mitochondrial surface suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation to mitochondria in order to fulfil its role in oxidative stress response. These findings are supported by heterologous expression of a codon-optimized human RAC1 gene, which can only complement a yeast rho5 deletion in a chimeric fusion with RHO5 sequences that restore the correct spatiotemporal distribution of the encoded protein.

Inhibition of Breast Cancer Cell Invasion by Ras Suppressor-1 (RSU-1) Silencing Is Reversed by Growth Differentiation Factor-15 (GDF-15).

Extracellular matrix (ECM)-related adhesion proteins are important in metastasis. Ras suppressor-1 (RSU-1), a suppressor of Ras-transformation, is localized to cell⁻ECM adhesions where it interacts with the Particularly Interesting New Cysteine-Histidine rich protein (PINCH-1), being connected to Integrin Linked Kinase (ILK) and alpha-parvin (PARVA), a direct actin-binding protein. RSU-1 was also found upregulated in metastatic breast cancer (BC) samples and was recently demonstrated to have metastasis-promoting properties. In the present study, we transiently silenced RSU-1 in BC cells, MCF-7 and MDA-MB-231. We found that RSU-1 silencing leads to downregulation of Growth Differentiation Factor-15 (GDF-15), which has been associated with both actin cytoskeleton reorganization and metastasis. RSU-1 silencing also reduced the mRNA expression of PINCH-1 and cell division control protein-42 (Cdc42), while increasing that of ILK and Rac regardless of the presence of GDF-15. However, the downregulation of actin-modulating genes PARVA, RhoA, Rho associated kinase-1 (ROCK-1), and Fascin-1 following RSU-1 depletion was completely reversed by GDF-15 treatment in both cell lines. Moreover, complete rescue of the inhibitory effect of RSU-1 silencing on cell invasion was achieved by GDF-15 treatment, which also correlated with matrix metalloproteinase-2 expression. Finally, using a graph clustering approach, we corroborated our findings. This is the first study providing evidence of a functional association between RSU-1 and GDF-15 with regard to cancer cell invasion.

Secretome of Cultured Human Endothelial Cells in Simulated Microgravity.

The secretome of human umbilical vein endothelial cells (HUVEC) cultured under static conditions and in modeled microgravity for 24 h was studied by chromatography-mass spectrometry. In the secretome of cells exposed to microgravity, we identified a group of microtubule proteins including many structural elements of microtubules and regulatory proteins interacting with Rho-GTPases. Hence, reorganization of actin cytoskeleton and microtubules induced by microgravity is under complex regulation mediated by Rho proteins.