Cortical Actin Reorganization Research Articles

Formin-like 1β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

Formin-like 1β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes.

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

Green silver nanoparticles: Prospective nanotools against neurodegenerative cell line model.

Neurodegenerative diseases represent an increasingly burdensome challenge of the past decade, primarily driven by the global aging of the population. Ongoing efforts focus on implementing diverse strategies to mitigate the adverse effects of neurodegeneration, with the goal of decelerating the pathology progression. Notably, in recent years, it has emerged that the use of nanoparticles (NPs), particularly those obtained through green chemical processes, could constitute a promising therapeutic approach. Green NPs, exclusively sourced from phytochemicals, are deemed safer compared to NPs synthetized through conventional chemical route. In this study, the effects of green chemistry-derived silver NPs (AgNPs) were assessed in neuroblastoma cells, SHSY-5Y, which are considered a pivotal model for investigating neurodegenerative diseases. Specifically, we used two different concentrations (0.5 and 1 µM) of AgNPs and two time points (24 and 48 h) to evaluate the impact on neuroblastoma cells by observing viability reduction and intracellular calcium production, especially using 1 µM at 48 h. Furthermore, investigation using atomic force microscopy (AFM) unveiled an alteration in Young's modulus due to the reorganization of cortical actin following exposure to green AgNPs. This evidence was further corroborated by confocal microscopy acquisitions as well as coherency and density analyses on actin fibers. Our in vitro findings suggest the potential efficacy of green AgNPs against neurodegeneration; therefore, further in vivo studies are imperative to optimize possible therapeutic protocols.

Dynamic multimerization of Dab2–Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s−1) of the Dab2 MIR–myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 μM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and “stop-and-go” motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR–myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor–cargo association in enabling cargo transportation without disrupting cytoskeletal organization.

Bulk Actin Dynamics Drive Phase Segregation in Zebrafish Oocytes

Segregation of maternal determinants within the oocyte constitutes the first step in embryo patterning. In zebrafish oocytes, extensive ooplasmic streaming leads to the segregation of ooplasm from yolk granules along the animal-vegetal axis of the oocyte. Here, we show that this process does not rely on cortical actin reorganization, as previously thought, but instead on a cell-cycle-dependent bulk actin polymerization wave traveling from the animal to the vegetal pole of the oocyte. This wave functions in segregation by both pulling ooplasm animally and pushing yolk granules vegetally. Using biophysical experimentation and theory, we show that ooplasm pulling is mediated by bulk actin network flows exerting friction forces on the ooplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. Our study defines a novel role of cell-cycle-controlled bulk actin polymerization waves in oocyte polarization via ooplasmic segregation.

Abstract 31: Targeted dissociation of PF1 from SIN3A chromatin regulator complex inhibits tumor growth and metastasis in triple-negative breast cancer

Abstract Triple negative breast cancer (TNBC) is characterized by an aggressive, poorly differentiated phenotype associated with early recurrence. Chemotherapy is the only treatment option due to the lack of an effective molecular target. Targeting reversible epigenetic alterations in TNBC to reprogram the TNBC phenotype may increase chemosensitivity and identify effective targeted therapies. The SIN3 chromatin modifier complex is a multidomain scaffold protein which plays a key role in multiple cellular functions including epigenetic gene expression regulation, making SIN3 a potential therapeutic target. The PAH-2 domain of SIN3 binds with different affinities to a group of proteins that contain variations of an amino acid sequence known as the SIN3 interaction domain (SID). We previously reported the effects of blocking PAH-2 interaction with MAD-1, a SID sequence containing protein by using SID peptides and small molecule inhibitors (SMI) as decoys. We now report the effects of blocking the PAH-2 binding of PF1 and TGIF1, two other Sin3 interacting proteins with different amino acid sequence and PAH-2 binding affinities. Treatment with MAD-1 SID peptide and SMI revert the epithelial to mesenchymal transition (EMT) process, inhibit cancer stem cells expansion, cell invasion, tumor growth and metastasis development in human and mouse models of TNBC. TGIF1 interaction with SIN3 is associated with the invasive phenotype and EMT regulation by modulating wnt signaling and inhibiting beta catenin nuclear localization which is reverted by TGIF1 knockdown. PF1, which binds MRG15 and KDM5A/B, an epigenetic modifier, is involved in the regulation of cancer stem cell compartment expansion, DNA damage control, senescence, apoptosis and the expression of genes involved in metastatic progression. Interestingly, decrease in H3K4me3 is found in stem cell and EMT genes following 72 hour SID peptide treatment. PF1 dissociation from Sin3 by transfecting MDA-MB-231 and 4T1 cells with a PF1-SID expressing vector grown in 3D morphogenesis cultures display the formation of small organoids in the MDA-MB-231PF1-SID and tubular morphogenesis in the 4T1PF1-SID cells. There is increased activated caspase-3 and γH2AX, reduced Ki67, tenascin-C and marked cortical actin reorganization. This is accompanied by small organized colonies with loss of invasive and proliferative phenotype. In vivo, 4T1PF1-SID barely form tumors and have a 95% decrease in lung metastasis. These results show that decoys can be designed to block specific proteins that bind to the PAH-2 domain resulting in different outcomes which contribute to a common effect on inhibition of tumor progression and metastasis dissemination. Therefore, this strategy opens a potential therapeutic alternative for TNBC patients for whom there are no other therapeutic options besides chemotherapy. Citation Format: Rama Kadamb, Nidhi Bansal, Boris A Leibovitch, Yeon-Jin Kwon, Ming-Ming Zhou, Eduardo Farias, Samuel Waxman. Targeted dissociation of PF1 from SIN3A chromatin regulator complex inhibits tumor growth and metastasis in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 31.

Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction.

Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1 of lymphocytes was important for inducing the release of sequestered HIV-1 from epithelial cells and facilitating cell-to-cell spread of virus from epithelial cells to lymphocytes. This mechanism may serve as a pathway of HIV-1 mucosal transmission.

Bleb formation is induced by alkaline but not acidic pH in estrogen receptor silenced breast cancer cells.

De novo and acquired resistance to endocrine-based therapies in breast cancer occurs in parallel with epithelial to mesenchymal transition (EMT), which is associated with enhanced proliferative and metastatic potential, and poor clinical outcome. We have established several endocrine insensitive breast cancer lines by shRNA-induced depletion of estrogen receptor (ER) by transfection of MCF7 cells. All of these exhibit EMT. We have previously reported that brief exposure of specifically ER- breast cancer cells, to extracellular alkaline pH, results in cell rounding and segregation, and leads to enhanced invasive potential. In this study we describe more detailed morphological changes and compare these with cell exposure to acidic pH. Morphological changes and localization of various molecules critical for cell adhesion and motility, associated with pH effects, were assessed by live cell microscopy, electron microscopy, and immunofluorescence. Exposure of either ER- or ER+ breast cancer cells to extracellular acidic pH did not induce significant changes in morphological appearance. Conversely, brief exposure of specifically ER silenced cells, to alkaline pH, resulted in cell contractolation and formation of bleb-like actin-rich structures which were evenly distributed on the outer membrane. Integrin α2, FAK, and JAM-1 were found in the cytoplasm streaming into the newly formed blebs. These blebs appear to be related to cell polarity and movement. Pre-treatment with cytochalasin-D or inhibitors of Rho or MLCK prevented both contractolation and bleb formation. Our data suggest that the effect of pH on the microenvironment of endocrine resistant breast cancer cells needs to be more extensively investigated. Alkaline, rather than acidic pH, appears to induce dramatic morphological changes, and enhances their invasive capabilities, through re-organization of cortical actin.

Tropomodulin3 as the link between insulin-activated AKT2 and cortical actin remodeling in preparation of GLUT4 exocytosis.

It is well established that insulin-induced remodeling of actin filaments into a cortical mesh is required for insulin-stimulated GLUT4 exocytosis. Akt2 and its downstream effectors play a pivotal role in mediating the translocation and membrane fusion of GLUT4-storage vesicle (GSV). However, the direct downstream effector underlying the event of cortical actin reorganization has not been elucidated. In a recent study in Nature Communications, (1) Lim etal identify Tropomodulin3 (Tmod3) as a downstream target of the Akt2 kinase and describe the role of this pointed-end actin-capping protein in regulating insulin-dependent exocytosis of GSVs in adipocytes through the remodeling of the cortical actin network. Phosphorylation of Tmod3 by Akt2 on Ser71 modulates insulin-induced actin remodeling, a key step for GSV fusion with the plasma membrane (PM). Furthermore, the authors establish Tm5NM1 (Tpm3.1 in new nomenclature) (2) as the cognate tropomyosin partner of Tmod3, and an essential role of Tmod3-Tm5NM1 interaction for GSV exocytosis and glucose uptake. This study elucidates a novel effector of Akt2 that provides a direct mechanistic link between Akt2 signaling and actin reorganization essential for vesicle fusion, and suggests that a subset of actin filaments with specific molecular compositions may be dedicated for the process of vesicle fusion.

Rac1-Mediated Effects of HMG-CoA Reductase Inhibitors (Statins) in Cardiovascular Disease

HMG-CoA reductase inhibitors (statins) lower serum cholesterol concentrations and are beneficial in the primary and secondary prevention of coronary heart disease. The positive clinical effects have only partially been reproduced with other lipid-lowering interventions suggesting potential statin effects in addition to cholesterol lowering. In experimental models, direct beneficial cardiovascular effects that are mediated by the inhibition of isoprenoids have been documented, which serve as lipid attachments for intracellular signaling molecules such as small Rho guanosine triphosphate-binding proteins, whose membrane localization and function are dependent on isoprenylation. Rac1 GTPase is an established master regulator of cell motility through the cortical actin reorganization and of reactive oxygen species generation through the regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Observations in cells, animals, and humans have implicated the activation of Rac1 GTPase as a key component of cardiovascular pathologies, including the endothelial dysfunction, cardiac hypertrophy and fibrosis, atrial fibrillation, stroke, hypertension, and chronic kidney disease. However, the underlying signal transduction remains incompletely understood. Based on the recent advance made in Rac1 research in the cardiovascular system by using mouse models with transgenic overexpression of activated Rac1 or conditional knockout, as well as Rac1-specific small molecule inhibitor NSC 23766, the improved understanding of the Rac1-mediated effects statins may help to identify novel therapeutic targets and strategies.

Novel aspects of the roles of Rac1 GTPase in the cardiovascular system

Rac1 GTPase is an established master regulator of cell motility through cortical actin re-organization and of reactive oxygen species generation through regulation of NADPH oxidase activity. Numerous molecular and cellular studies have implicated Rac1 in various cardiovascular pathologies: vascular smooth muscle proliferation, cardiomyocyte hypertrophy, and endothelial cell shape change. The physiological relevance of these in vitro findings, however, is just beginning to be reassessed with the newly developed, conditional mouse mutagenesis technology. Conditional gene targeting has also revealed unexpected, cell type-specific roles of Rac1. The aim of this review is to summarize the recent advance made in Rac1 research in the cardiovascular system, with special focus on its novel roles in the regulation of endothelial function, angiogenesis, and endothelium-mediated neuroprotection.

Dual FGF-2 and Intergrin α5β1 Signaling Mediate GRAF-Induced RhoA Inactivation in a Model of Breast Cancer Dormancy

Interactions with the bone marrow stroma regulate dormancy and survival of breast cancer micrometastases. In an in vitro model of dormancy in the bone marrow, we previously demonstrated that estrogen-dependent breast cancer cells are partially re-differentiated by FGF-2, re-express integrin α5β1 lost with malignant transformation and acquire an activated PI3K/Akt pathway. Ligation of integrin α5β1 by fibronectin and activation of the PI3K pathway both contribute to survival of these dormant cells. Here, we investigated mechanisms responsible for the dormant phenotype. Experiments demonstrate that integrin α5β1 controls de novo cytoskeletal rearrangements, cell spreading, focal adhesion kinase rearrangement to the cell perimeter and recruitment of a RhoA GAP known as GRAF. This results in the inactivation of RhoA, an effect which is necessary for the stabilization of cortical actin. Experiments also demonstrate that activation of the PI3K pathway by FGF-2 is independent of integrin α5β1 and is also required for cortical actin reorganization, GRAF membrane relocalization and RhoA inactivation. These data suggest that GRAF-mediated RhoA inactivation and consequent phenotypic changes of dormancy depend on dual signaling by FGF-2-initiated PI3K activation and through ligation of integrin α5β1 by fibronectin.

CDK5-dependent Phosphorylation of the Rho Family GTPase TC10α Regulates Insulin-stimulated GLUT4 Translocation

Insulin stimulation results in the activation of cyclin-dependent kinase-5 (CDK5) in lipid raft domains via a Fyn-dependent phosphorylation on tyrosine residue 15. In turn, activated CDK5 phosphorylates the Rho family GTP-binding protein TC10alpha on threonine 197 that is sensitive to the CDK5 inhibitor olomoucine and blocked by small interfering RNA-mediated knockdown of CDK5. The phosphorylation deficient mutant T197A-TC10alpha was not phosphorylated and excluded from the lipid raft domain, whereas the phosphorylation mimetic mutant (T197D-TC10alpha) was lipid raft localized. Insulin resulted in the GTP loading of T197D-TC10alpha but not T197A-TC10alpha and in parallel, T197D-TC10alpha but not T197A-TC10alpha depolymerized cortical actin and inhibited insulin-stimulated GLUT4 translocation. These data demonstrate that CDK5-dependent phosphorylation maintains TC10alpha in lipid raft compartments thereby disrupting cortical actin, whereas subsequent dephosphorylation of TC10alpha through inactivation of CDK5 allows for the re-assembly of F-actin. Because cortical actin reorganization is required for insulin-stimulated GLUT4 translocation, these data are consistent with a CDK5-dependent TC10alpha cycling between lipid raft and non-lipid raft compartments.

Moesin stiffens up mitosis

Microtubule spindle dynamics and reorganization of cortical actin drive cell division in animals. Two reports suggest that the ERM (ezrin, radixin and moesin) protein moesin can coordinate these two processes (DOI 10.1016/j.cub.2007.12.051; DOI 10.1083/jcb.200709161). Moesin, the only known ERM protein in Drosophila melanogaster, crosslinks actin filaments to the plasma membrane and was shown previously to be activated by phosphorylation. Baum and colleagues noticed that phosphorylated moesin accumulated at the cortex of mitotic Drosophila cells in culture, whereas Payre and colleagues observed that moesin-mutant flies exhibited defects in cytokinesis. Both observations suggested a potential role for moesin in regulating cortical actin dynamics during cell division. Both groups identified Slik as the kinase responsible for moesin activation during mitosis and showed that inactivation of moesin, using RNAi or by silencing Slik, caused mitotic defects. Inactivation of moesin prevented dividing cells from retracting their margins and rounding up, and live analysis revealed that the cortex of moesin-inactivated cells was extremely unstable throughout mitosis. Surprisingly, the mitotic spindles in these cells had variable morphological defects, such as centrosome misplacement and an asymmetrical organization. Erratic changes in spindle positioning accompanied these defects, indicating that moesin also keeps the spindle in place. Using atomic force microscopy, Baum and colleagues showed that moesin-depleted cells did not undergo the increase in cortical stiffness usually seen in mitosis, and that expression of phosphomimetic moesin in interphase cells was sufficient to induce cortical stiffening and cell rounding. They also demonstrated that incubating moesindepleted cells with concavalin A, which increases cortical stiffness, rescued the defects in spindle morphogenesis and in the cortex during metaphase. Nevertheless, increasing overall cortical stiffness towards the end of mitosis prevented cell elongation, suggesting that moesin activity needs to be regulated precisely in time and space for completion of mitosis. Both groups showed that the dynamic distribution of F-actin and the non-muscle myosin II motor was impaired when moesin was depleted. Payre and colleagues found that inhibition of RhoA-mediated actin contractility suppressed erratic spindle movements, suggesting that the role of moesin during mitosis, in part, involves RhoA activity. In addition, Baum and colleagues observed that expression of phosphomimetic moesin in cells depleted of myosin II light chain (a known target of RhoA) could induce cortical tension, indicating a role for moesin in cortical stiffening that is independent of myosin II. Understanding how moesin and myosin II coordinate their efforts to regulate cortical tension, as well as how this, in turn, regulates normal mitotic spindle dynamics will be the challenge of future studies.

Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells

Cell division requires cell shape changes involving the localized reorganization of cortical actin, which must be tightly linked with chromosome segregation operated by the mitotic spindle. How this multistep process is coordinated remains poorly understood. In this study, we show that the actin/membrane linker moesin, the single ERM (ezrin, radixin, and moesin) protein in Drosophila melanogaster, is required to maintain cortical stability during mitosis. Mitosis onset is characterized by a burst of moesin activation mediated by a Slik kinase–dependent phosphorylation. Activated moesin homogenously localizes at the cortex in prometaphase and is progressively restricted at the equator in later stages. Lack of moesin or inhibition of its activation destabilized the cortex throughout mitosis, resulting in severe cortical deformations and abnormal distribution of actomyosin regulators. Inhibiting moesin activation also impaired microtubule organization and precluded stable positioning of the mitotic spindle. We propose that the spatiotemporal control of moesin activation at the mitotic cortex provides localized cues to coordinate cortical contractility and microtubule interactions during cell division.

Possible role of Rho/Rhotekin signaling in mammalian septin organization

There is growing evidence for crosstalk between septin filaments and actin cytoskeleton which is regulated by Rho family of GTPases. Here we show that active Rho disrupts septin filament structures in rat embryonic fibroblast REF52 cells. Among Rho effector molecules tested, Rhotekin induced morphological changes of septin filaments similar to those by activated Rho. The center region of Rhotekin was sufficient for the septin reorganization in the cells, and likely to interact indirectly with the C-terminal half of a septin Sept9b, where a GTPase domain is located. Rhotekin and Sept9b are colocalized mainly in perinuclear regions in serum-starved REF52 cells. Upon stimulation with lysophosphatidic acid, they translocated to actin stress fibers in 10 min and then redistributed again to cytoplasm after 90 min treatment. In neuroblastoma Neuro2a cells, Rhotekin and Sept9b were enriched in the tip of neurites, a location where cortical actin reorganization is induced upon stimulation with lysophosphatidic acid. Taken together, we propose that Rhotekin is a novel regulator organizing mammalian septin structures and provide a new link between the septin and Rho-signaling.

The Small G-protein Arf6GTP Recruits the AP-2 Adaptor Complex to Membranes

The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.

Allicin inhibits SDF-1alpha-induced T cell interactions with fibronectin and endothelial cells by down-regulating cytoskeleton rearrangement, Pyk-2 phosphorylation and VLA-4 expression.

Allicin, a major ingredient of fresh garlic extract that is produced during the crushing of garlic cloves, exerts various beneficial biological effects, including a broad spectrum of antimicrobial activity, antihyperlipidaemic and antihypertensive effects. However, how allicin affects the immune system is less well known, and its effect on human T cells has never been studied. Here, we examined the in-vitro effects of allicin on the functioning of T cells related to their entry to inflamed extravascular sites. We found that allicin (20-100 microm) inhibits the SDF-1alpha (CXCL12)-induced T cell migration through fibronectin (FN), and that this inhibition is mediated by the down-regulation of (i) the reorganization of cortical actin and the subsequent T cell polarization, and (ii) T cell adhesion to FN. Moreover, allicin also inhibited T cell adhesion to endothelial cells and transendothelial migration. The mechanisms underlying these inhibitory effects of allicin are associated with its ability to down-regulate the phosphorylation of Pyk2, an intracellular member of the focal adhesion kinases, and to reduce the expression of the VCAM-1- and FN-specific alpha4beta1-integrin (VLA-4). The ability of allicin to down-regulate these chemokine-induced and VLA-4-mediated T cell functions explains its beneficial biological effects in processes where T cells play an important role and suggests that allicin may be used therapeutically with chronic inflammatory diseases.

Calpain facilitates GLUT4 vesicle translocation during insulin-stimulated glucose uptake in adipocytes.

Calpains are a family of non-lysosomal cysteine proteases. Recent studies have identified a member of the calpain family of proteases, calpain 10, as a putative diabetes-susceptibility gene that may be involved in the development of type 2 diabetes. Inhibition of calpain activity has been shown to reduce insulin-stimulated glucose uptake in isolated rat-muscle strips and adipocytes. In this report, we examine the mechanism by which calpain affects insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Inhibition of calpain activity resulted in approx. a 60% decrease in insulin-stimulated glucose uptake. Furthermore, inhibition of calpain activity prevented the translocation of insulin-responsive glucose transporter 4 (GLUT4) vesicles to the plasma membrane, as demonstrated by fluorescent microscopy of whole cells and isolated plasma membranes; it did not, however, alter the total GLUT4 protein content. While inhibition of calpain did not affect the insulin-mediated proximal steps of the phosphoinositide 3-kinase pathway, it did prevent the insulin-stimulated cortical actin reorganization required for GLUT4 translocation. Specific inhibition of calpain 10 by antisense expression reduced insulin-stimulated GLUT4 translocation and actin reorganization. Based on these findings, we propose a role for calpain in the actin reorganization required for insulin-stimulated GLUT4 translocation to the plasma membrane in 3T3-L1 adipocytes. These studies identify calpain as a novel factor involved in GLUT4 vesicle trafficking and suggest a link between calpain activity and the development of type 2 diabetes.

Differential localization and function of ADP-ribosylation factor-6 in anergic human T cells: a potential marker for their identification.

Anergy is a state of immunologic tolerance in which T cells are viable but incapable of responding to antigenic stimulation. Recent data indicate that anergic cells have a distinct gene expression program that determines their unique function. In this study we show that anergic human T cells selectively express the small GTPase ADP-ribosylation factor-6 (ARF6), which is involved in membrane traffic and regulation of the cortical actin cytoskeleton. ARF6 was expressed in the GTP-bound form that localizes at the plasma membrane, resulting in a distinct morphologic appearance of anergic cells. Forced expression of ARF6-GTP in Jurkat T cells prevented TCR-mediated reorganization of cortical actin, extracellular signal-regulated kinase1/2 activation, and IL-2 transcription. Forced expression of ARF6-GTP in primary human T cells inhibited extracellular signal-regulated kinase1/2 activation and proliferative responses. Importantly, T cells with the distribution pattern of ARF6-GTP were detected in peripheral blood, suggesting that anergic T cells may constitutively exist in vivo.