Rearrangement Of Actin Cytoskeleton Research Articles

A Dual Role for Melatonin in Medaka Ovulation: Ensuring Prostaglandin Synthesis and Actin Cytoskeleton Rearrangement in Follicular Cells.

Understanding the direct effects of melatonin on vertebrate ovulation remains a challenge. The present study provides the first characterization of the role of melatonin in ovulation using the teleost medaka. The melatonin receptor antagonist luzindole inhibited in vitro follicle ovulation. In the preovulatory follicles, arylalkylamine N-acetyltransferase 1a and hydroxyindole-O-methyltransferase 2, the enzymes responsible for melatonin synthesis, were expressed in the granulosa cells throughout the 24 h spawning cycle. The granulosa cells of the follicle also expressed the melatonin receptor 1a-a. An in vitro characterization study using medaka OLHNI-2 cells revealed that melatonin and luzindole act as an agonist and an antagonist, respectively, of the melatonin receptor. The intracellular cAMP levels in these cells were reduced after melatonin treatment. The expression of cytosolic phospholipase A2 group 4a (Pla2g4a), the enzyme producing arachidonic acid (cyclooxygenase-2 substrate), was inhibited in the granulosa cells in luzindole-treated follicles. Follicular prostaglandin E2 levels and in vitro follicle ovulation were suppressed in follicles isolated at 12 h prior to ovulation and incubated with the Pla2g4a inhibitor AACOCF3. The G-actin:F-actin ratios in follicular cells increased with approaching ovulation, but this increase was suppressed after luzindole treatment. The phosphorylation of moesin, an ezrin-radixin-moesin protein, was inhibited in the follicular cells in luzindole-treated follicles. These results indicate a dual role for melatonin in medaka ovulation: melatonin ensures prostaglandin E2 synthesis throughout the spawning cycle and induces actin cytoskeleton rearrangement in the follicular cells at ovulation.

Phosphatase 2A Inhibition Affects Endoplasmic Reticulum and Mitochondria Homeostasis Via Cytoskeletal Alterations in Brain Endothelial Cells.

The loss of endothelial cells (ECs) homeostasis is a trigger for cerebrovascular dysfunction that is a common event in several neurodegenerative disorders such as Alzheimer's disease (AD). The present work addressed the role of phosphatase 2A (PP2A) in cytoskeleton rearrangement, endoplasmic reticulum (ER) homeostasis, ER-mitochondria communication and mitochondrial dynamics in brain ECs. For this purpose, rat brain endothelial (RBE4) cells were exposed to okadaic acid, a well-known inhibitor of PP2A activity. An increase in the levels of tau phosphorylated on Ser396 and Thr181 residues was observed upon PP2A inhibition, concomitantly with the rearrangement of microtubules and actin cytoskeleton. Under these conditions, an increase in the levels of ER stress markers, namely GRP78, XBP1, p-eIF2αSer51, and ERO1α, was observed. Moreover, PP2A inhibition upregulated the Sigma-1 receptor, an ER chaperone located at the ER-mitochondria interface, and enhanced inter-organelle Ca2+ transfer, culminating in mitochondrial Ca2+ overload and activation of mitochondria-dependent apoptosis. The inhibition of PP2A activity also promoted an alteration of the structural and spatial mitochondria network due to upregulation of mitochondrial fission (Drp1 and Fis1) and fusion (Mfn1, Mfn2 and OPA1) proteins, suggesting detrimental changes in mitochondrial dynamics. In accordance with our in vitro observations, brain vessels from 3xTg-AD mice showed a significant decrease in PP2A protein levels accompanied by an increase in tau phosphorylated on Ser396 and GRP78 protein levels. Collectively, these results suggest that the loss of cerebrovascular homeostasis that occurs in AD might be a downstream event of the compromised activity and/or expression of PP2A, which is observed in the brain of individuals affected with this devastating neurodegenerative disorder.

A novel high-content analysis tool reveals Rab8-driven cytoskeletal reorganization through Rho GTPases, calpain and MT1-MMP.

Rab8 is a small Ras-related GTPase that regulates polarized membrane transport to the plasma membrane. Here, we developed a high-content analysis (HCA) tool to dissect Rab8-mediated actin and focal adhesion reorganization that revealed that Rab8 activation significantly induced Rac1 and Tiam1 to mediate cortical actin polymerization and RhoA-dependent stress fibre disassembly. Rab8 activation increased Rac1 activity, whereas its depletion activated RhoA, which led to reorganization of the actin cytoskeleton. Rab8 was also associated with focal adhesions, promoting their disassembly in a microtubule-dependent manner. This Rab8 effect involved calpain, MT1-MMP (also known as MMP14) and Rho GTPases. Moreover, we demonstrate the role of Rab8 in the cell migration process. Indeed, Rab8 is required for EGF-induced cell polarization and chemotaxis, as well as for the directional persistency of intrinsic cell motility. These data reveal that Rab8 drives cell motility by mechanisms both dependent and independent of Rho GTPases, thereby regulating the establishment of cell polarity, turnover of focal adhesions and actin cytoskeleton rearrangements, thus determining the directionality of cell migration.

GPCRs and actin-cytoskeleton dynamics.

A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others. This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study.

Retinoids and glucocorticoids have opposite effects on actin cytoskeleton rearrangement in hippocampal HT22 cells

A chronic excess of glucocorticoids elicits deleterious effects in the hippocampus. Conversely, retinoic acid plays a major role in aging brain plasticity. As synaptic plasticity depends on mechanisms related to cell morphology, we investigated the involvement of retinoic acid and glucocorticoids in the remodelling of the HT22 neurons actin cytoskeleton.Cells exhibited a significantly more elongated shape with retinoic acid and a rounder shape with dexamethasone; retinoic acid reversed the effects of dexamethasone.Actin expression and abundance were unchanged by retinoic acid or dexamethasone but F-actin organization was dramatically modified. Indeed, retinoic acid and dexamethasone increased (70±7% and 176±5%) cortical actin while retinoic acid suppressed the effect of dexamethasone (90±6%). Retinoic acid decreased (−22±9%) and dexamethasone increased (134±16%) actin stress fibres. Retinoic acid also suppressed the effect of dexamethasone (−21±7%).Spectrin is a key protein in the actin network remodelling. Its abundance was decreased by retinoic acid and increased by dexamethasone (−21±11% and 52±10%). However, retinoic acid did not modify the effect of dexamethasone (48±7%). Calpain activity on spectrin was increased by retinoic acid and decreased by dexamethasone (26±14% and −57±5%); retinoic acid mildly but significantly modified the effect of dexamethasone (−44±7%). The calpain inhibitor calpeptin suppressed the effects of retinoic acid and dexamethasone on cell shape and actin stress fibres remodelling but did not modify the effects on cortical actin.Retinoic acid and dexamethasone have a dramatic but mainly opposite effect on actin cytoskeleton remodelling. These effects originate, at least partly, from calpain activity.

Regulation of Macropinocytosis by Diacylglycerol Kinase ζ.

Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis.

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK.

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that process.

Neurofibromin - protein structure and cellular functions in the context of neurofibromatosis type I pathogenesis

Neurofibromatosis type I (NF1) is multisystemic disease characterized by pigmentary skin changes, increased susceptibility to tumor formation, neurological deficits and skeletal defects. The disease is a monogenic, autosomal dominant disorder, caused by the presence of mutations in the NF1 gene encoding neurofibromin - a multifunctional regulatory protein. The basic function of neurofibromin protein is modulation of the RAS protein activity necessary for regulation of cell proliferation and differentiation by the RAS/MAPK and RAS/PI3K/AKT signal transduction pathways. In addition, neurofibromin is a regulator of adenylate cyclase activity and therefore may interfere with signaling by the cAMP/protein kinase A pathway that regulates cell cycle progression or learning and memory formation processes. Neurofibromin also interacts with many other proteins that are engaged in intracellular transport (tubulin, kinesin), actin cytoskeleton rearrangements (LIMK2, Rho and Rac) or morphogenesis of neural cells (syndecans, CRMP proteins). The activity of neurofibromin is strictly regulated by the expression of different NF1 mRNA isoforms depending on tissue type or period in organism development, the protein localization, posttranslational modifications (phosphorylation, ubiquitination) or interactions with other proteins (e.g. 14-3-3). The fact that neurofibromin is engaged in many cellular processes has significant consequences when the proper protein functioning is impaired due to decreased protein level or activity. It affects the normal cell function and results in disturbances of organism development that lead to the occurrence of clinical signs specific for NF1. In the article, the basic neurofibromin functions are presented in the context of the molecular pathogenesis of NF1.

Leukocyte adhesion and polarization: Role of glycosylphosphatidylinositol-anchored proteins.

ABSTRACTLeukocyte traffic out of the blood stream is crucial for an adequate immune response. Leukocyte extravasation is critically dependent on the binding of leukocyte integrins to their endothelial counterreceptors. This interaction enables the firm adhesion of leukocytes to the luminal side of the vascular wall and allows for leukocyte polarization, crawling and diapedesis. Leukocyte adhesion, polarization and migration requires the orchestrated regulation of integrin adhesion/de-adhesion dynamics and actin cytoskeleton rearrangements. Adhesion strength depends on conformational changes of integrin molecules (affinity) as well as the number of integrin molecules engaged at adhesion sites (valency). These two processes can be independently regulated and several molecules modulate either one or both processes. Cholesterol-rich membrane domains (lipid rafts) participate in integrin regulation and play an important role in leukocyte adhesion, polarization and motility. In particular, lipid raft-resident glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs) have been reported to regulate leukocyte adhesion, polarization and motility in both integrin-dependent and independent manners. Here, we present our recent discovery concerning the novel role of the GPI-AP prion protein (PrP) in the regulation of β1 integrin-mediated monocyte adhesion, migration and shape polarization in the context of existing literature on GPI-AP-dependent regulation of integrins.

Pertussis Toxin Exploits Specific Host Cell Signaling Pathways for Promoting Invasion and Translocation of Escherichia coli K1 RS218 in Human Brain-derived Microvascular Endothelial Cells

Pertussis toxin (PTx), an AB5 toxin and major virulence factor of the whooping cough-causing pathogen Bordetella pertussis, has been shown to affect the blood-brain barrier. Dysfunction of the blood-brain barrier may facilitate penetration of bacterial pathogens into the brain, such as Escherichia coli K1 (RS218). In this study, we investigated the influence of PTx on blood-brain barrier permissiveness to E. coli infection using human brain-derived endothelial HBMEC and TY10 cells as in vitro models. Our results indicate that PTx acts at several key points of host cell intracellular signaling pathways, which are also affected by E. coli K1 RS218 infection. Application of PTx increased the expression of the pathogen binding receptor gp96. Further, we found an activation of STAT3 and of the small GTPase Rac1, which have been described as being essential for bacterial invasion involving host cell actin cytoskeleton rearrangements at the bacterial entry site. In addition, we showed that PTx induces a remarkable relocation of VE-cadherin and β-catenin from intercellular junctions. The observed changes in host cell signaling molecules were accompanied by differences in intracellular calcium levels, which might act as a second messenger system for PTx. In summary, PTx not only facilitates invasion of E. coli K1 RS218 by activating essential signaling cascades; it also affects intercellular barriers to increase paracellular translocation.

Contractile actin cables induced by Bacillus anthracis lethal toxin depend on the histone acetylation machinery.

It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier.

The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity.

Multifactorial mechanisms comprising countless cellular factors and virus-encoded transactivators regulate the transcription of HIV-1 (HIV). Since poly(ADP-ribose) polymerase 1 (PARP-1) regulates numerous genes through its interaction with various transcription factors, inhibition of PARP-1 has surfaced recently as a powerful anti-inflammatory tool. We suggest a novel tactic to diminish HIV replication via PARP-1 inhibition in an in vitro model system, exploiting human primary monocyte-derived macrophages (MDM). PARP-1 inhibition was capable to lessen HIV replication in MDM by 60–80% after 7 days infection. Tat, tumor necrosis factor α (TNFα), and phorbol 12-myristate 13-acetate (PMA) are known triggers of the Long Terminal Repeat (LTR), which can switch virus replication. Tat overexpression in MDM transfected with an LTR reporter plasmid resulted in a 4.2-fold increase in LTR activation; PARP inhibition caused 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85–95%). PARP inhibition in MDM exhibited 90% diminution in NFκB activity (known to mediate TNFα- and PMA-induced HIV LTR activation). Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These discoveries suggest that inactivation of PARP suppresses HIV replication in MDM by via attenuation of LTR activation, NFκB suppression and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide an effective approach to management of HIV infection.

Identification of immediate early response protein 2 as a regulator of angiogenesis through the modulation of endothelial cell motility and adhesion.

Human immediate early response2 (IER2) has been characterized as a putative nuclear protein that functions as a transcription factor or transcriptional co‑activator in the regulation of cellular responses, and may be involved in the regulation of tumor progression and metastasis. Data from our previous gene expression profile of the human microvascular endothelial cells during capillary morphogenesis showed a significant alteration of IER2 expression, suggesting that IER2 may participate in the regulation of the endothelial cell morphogenesis and angiogenesis. The aim of the present study was to investigate the role of IER2 in cell motility, cell‑matrix adhesion and invitro capillary‑like structures formation of the human umbilical vein endothelium cells (HUVECs). IER2 was constitutively expressed in HUVECs, and lentiviral‑mediated depletion of IER2 significantly reduced the cell motility, cell‑matrix adhesion and capillary‑like structures formation of HUVECs. Results also showed that depletion and overexpression of IER2 altered the actin cytoskeleton rearrangement in HUVECs. Furthermore, results from western blot analysis showed that the activity of the focal adhesion kinase (FAK) can be regulated by IER2. These results indicated that IER2 regulates endothelial cell motility, adhesion on collagen typeI matrix and the capillary tube formation, as the result of the regulation of the actin cytoskeleton rearrangement presumably via a FAK‑dependent mechanism.

Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: Exploring a new role of haptoglobin in the tumoral microenvironment

Haptoglobin (Hp) is an acute-phase protein that is produced by the liver to capture the iron that is present in the blood circulation, thus avoiding its accumulation in the blood. Moreover, Hp has been detected in a wide variety of tissues, in which it performs various functions. In addition, this protein is considered a potential biomarker in many diseases, such as cancer, including ovarian carcinoma; however, its participation in the cancerous processes has not yet been determined. The objective of this work was to demonstrate the expression of Hp and its receptor CCR2 in the ovarian cancer cells and its possible involvement in the process of cell migration through changes in the rearrangement of the actin cytoskeleton using western blot and wound-healing assays and confirming by confocal microscopy. Ovarian cancer cells express both Hp and its receptor CCR2 but only after exposure to ascitic fluid, inducing moderated cell migration. However, when the cells are exposed to exogenous Hp, the expression of CCR2 is induced together with drastic changes in the actin cytoskeleton rearrangement. At the same time, Hp induced cell migration in a much more efficient manner than did ascitic fluid. These effects were blocked when the CCR2 synthetic antagonist RS102895 was used to pretreat the cells. These results suggest that Hp-induced changes in the cell morphology, actin cytoskeleton structure, and migration ability of tumor cells, is possibly “preparing” these cells for the potential induction of the metastatic phenotype.

Group B streptococcal infection and activation of human astrocytes.

Background Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of life-threatening meningitis in human newborns in industrialized countries. Meningitis results from neonatal infection that occurs when GBS leaves the bloodstream (bacteremia), crosses the blood-brain barrier (BBB), and enters the central nervous system (CNS), where the bacteria contact the meninges. Although GBS is known to invade the BBB, subsequent interaction with astrocytes that physically associate with brain endothelium has not been well studied.Methodology/Principal FindingsWe hypothesize that human astrocytes play a unique role in GBS infection and contribute to the development of meningitis. To address this, we used a well- characterized human fetal astrocyte cell line, SVG-A, and examined GBS infection in vitro. We observed that all GBS strains of representative clinically dominant serotypes (Ia, Ib, III, and V) were able to adhere to and invade astrocytes. Cellular invasion was dependent on host actin cytoskeleton rearrangements, and was specific to GBS as Streptococcus gordonii failed to enter astrocytes. Analysis of isogenic mutant GBS strains deficient in various cell surface organelles showed that anchored LTA, serine-rich repeat protein (Srr1) and fibronectin binding (SfbA) proteins all contribute to host cell internalization. Wild-type GBS also displayed an ability to persist and survive within an intracellular compartment for at least 12 h following invasion. Moreover, GBS infection resulted in increased astrocyte transcription of interleukin (IL)-1β, IL-6 and VEGF.Conclusions/SignificanceThis study has further characterized the interaction of GBS with human astrocytes, and has identified the importance of specific virulence factors in these interactions. Understanding the role of astrocytes during GBS infection will provide important information regarding BBB disruption and the development of neonatal meningitis.

Actin-cytoskeleton polymerization differentially controls the stability of Ski and SnoN co-repressors in normal but not in transformed hepatocytes

BackgroundSki and SnoN proteins function as transcriptional co-repressors in the TGF-β pathway. They regulate cell proliferation and differentiation, and their aberrant expression results in altered TGF-β signalling, malignant transformation, and alterations in cell proliferation. MethodsWe carried out a comparative characterization of the endogenous Ski and SnoN protein regulation by TGF-β, cell adhesion disruption and actin-cytoskeleton rearrangements between normal and transformed hepatocytes; we also analyzed Ski and SnoN protein stability, subcellular localization, and how their protein levels impact the TGF-β/Smad-driven gene transcription. ResultsSki and SnoN protein levels are lower in normal hepatocytes than in hepatoma cells. They exhibit a very short half-life and a nuclear/cytoplasmic distribution in normal hepatocytes opposed to a high stability and restricted nuclear localization in hepatoma cells. Interestingly, while normal cells exhibit a transient TGF-β-induced gene expression, the hepatoma cells are characterized by a strong and sustained TGF-β-induced gene expression. A novel finding is that Ski and SnoN stability is differentially regulated by cell adhesion and cytoskeleton rearrangements in the normal hepatocytes. The inhibition of protein turnover down-regulated both Ski and SnoN co-repressors impacting the kinetic of expression of TGF-β-target genes. ConclusionNormal regulatory mechanisms controlling Ski and SnoN stability, subcellular localization and expression are altered in hepatocarcinoma cells. General significanceThis work provides evidence that Ski and SnoN protein regulation is far more complex in normal than in transformed cells, since many of the normal regulatory mechanisms are lost in transformed cells.

ARP2/3-Mediated Actin Nucleation Associated With Symbiosome Membrane Is Essential for the Development of Symbiosomes in Infected Cells of Medicago truncatula Root Nodules.

The nitrogen-fixing rhizobia in the symbiotic infected cells of root nodules are kept in membrane compartments derived from the host cell plasma membrane, forming what are known as symbiosomes. These are maintained as individual units, with mature symbiosomes having a specific radial position in the host cell cytoplasm. The mechanisms that adapt the host cell architecture to accommodate intracellular bacteria are not clear. The intracellular organization of any cell depends heavily on the actin cytoskeleton. Dynamic rearrangement of the actin cytoskeleton is crucial for cytoplasm organization and intracellular trafficking of vesicles and organelles. A key component of the actin cytoskeleton rearrangement is the ARP2/3 complex, which nucleates new actin filaments and forms branched actin networks. To clarify the role of the ARP2/3 complex in the development of infected cells and symbiosomes, we analyzed the pattern of actin microfilaments and the functional role of ARP3 in Medicago truncatula root nodules. In infected cells, ARP3 protein and actin were spatially associated with maturing symbiosomes. Partial ARP3 silencing causes defects in symbiosome development; in particular, ARP3 silencing disrupts the final differentiation steps in functional maturation into nitrogen-fixing units.

Silencing of MICAL-L2 suppresses malignancy of ovarian cancer by inducing mesenchymal–epithelial transition

Ovarian cancer remains the disease with the highest associated mortality rate of gynecologic malignancy due to cancer metastasis. Rearrangement of actin cytoskeleton by cytoskeleton protein plays a critical role in tumor cell metastasis. MICAL-L2, a member of MICAL family, can interact with actin-binding proteins, regulate actin cross-linking and coordinate the assembly of adherens junctions and tight junctions. However, the roles of MICAL-L2 in tumors and diseases have not been explored. In this study, we found that MICAL-L2 protein is significantly up-regulated in ovarian cancer tissues along with FIGO stage and associated with histologic subgroups of ovarian cancer. Silencing of MICAL-L2 suppressed ovarian cancer cell proliferation, migration and invasion ability. Moreover, silencing of MICAL-L2 prevented nuclear translocation of β-catenin, inhibited canonical wnt/β-catenin signaling and induced the mesenchymal–epithelial transition (MET). Taken together, our data indicated that MICAL-L2 may be an important regulator of epithelial–mesenchymal transition (EMT) in ovarian cancer cells and a new therapeutic target for interventions against ovarian cancer invasion and metastasis.

α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation

Malformed α-Synuclein (α-syn) aggregates in neurons are released into the extracellular space, activating microglia to induce chronic neuroinflammation that further enhances neuronal damage in α-synucleinopathies, such as Parkinson's disease. The mechanisms by which α-syn aggregates activate and recruit microglia remain unclear, however. Here we show that α-syn aggregates act as chemoattractants to direct microglia toward damaged neurons. In addition, we describe a mechanism underlying this directional migration of microglia. Specifically, chemotaxis occurs when α-syn binds to integrin CD11b, leading to H2O2 production by NADPH oxidase. H2O2 directly attracts microglia via a process in which extracellularly generated H2O2 diffuses into the cytoplasm and tyrosine protein kinase Lyn, phosphorylates the F-actin-associated protein cortactin after sensing changes in the microglial intracellular concentration of H2O2. Finally, phosphorylated cortactin mediates actin cytoskeleton rearrangement and facilitates directional cell migration. These findings have significant implications, given that α-syn-mediated microglial migration reaches beyond Parkinson's disease.

Schip1 is a novel podocyte foot process protein that mediates actin cytoskeleton rearrangements and forms a complex with Nherf2 and ezrin.

BackgroundPodocyte foot process effacement accompanied by actin cytoskeleton rearrangements is a cardinal feature of many progressive human proteinuric diseases.ResultsBy microarray profiling of mouse glomerulus, SCHIP1 emerged as one of the most highly enriched transcripts. We detected Schip1 protein in the kidney glomerulus, specifically in podocytes foot processes. Functionally, Schip1 inactivation in zebrafish by morpholino knock-down results in foot process disorganization and podocyte loss leading to proteinuria. In cultured podocytes Schip1 localizes to cortical actin-rich regions of lamellipodia, where it forms a complex with Nherf2 and ezrin, proteins known to participate in actin remodeling stimulated by PDGFβ signaling. Mechanistically, overexpression of Schip1 in vitro causes accumulation of cortical F-actin with dissolution of transversal stress fibers and promotes cell migration in response to PDGF-BB stimulation. Upon actin disassembly by latrunculin A treatment, Schip1 remains associated with the residual F-actin-containing structures, suggesting a functional connection with actin cytoskeleton possibly via its interaction partners. A similar assay with cytochalasin D points to stabilization of cortical actin cytoskeleton in Schip1 overexpressing cells by attenuation of actin depolymerisation.ConclusionsSchip1 is a novel glomerular protein predominantly expressed in podocytes, necessary for the zebrafish pronephros development and function. Schip1 associates with the cortical actin cytoskeleton network and modulates its dynamics in response to PDGF signaling via interaction with the Nherf2/ezrin complex. Its implication in proteinuric diseases remains to be further investigated.