Roles of p-ERM and Rho-ROCK signaling in lymphocyte polarity and uropod formation

Front-rear asymmetry in motile cells is crucial for efficient directional movement. The uropod in migrating lymphocytes is a posterior protrusion in which several proteins, including CD44 and ezrin/radixin/moesin (ERM), are concentrated. In EL4.G8 T-lymphoma cells, Thr567 phosphorylation in the COOH-terminal domain of ezrin regulates the selective localization of ezrin in the uropod. Overexpression of the phosphorylation-mimetic T567D ezrin enhances uropod size and cell migration. T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton-dependent manner. Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor Y-27632 disrupts the uropod but not the polar cap, indicating that Rho-ROCK signaling is required for posterior protrusion but not for ERM phosphorylation. Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation. Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells. Thus, phosphorylated ERM has a potential function in establishing plasma membrane "posteriority" in the induction of the uropod in T lymphocytes.     

Membrane lipid organization is critical for human neutrophil polarization

In response to chemoattractants neutrophils extend an actin-rich pseudopod, which imparts morphological polarity and is required for migration. Even when stimulated by an isotropic bath of chemoattractant, neutrophils exhibit persistent polarization and continued lamellipod formation at the front, suggesting that the cells establish an internal polarity. In this report, we show that perturbing lipid organization by depleting plasma membrane cholesterol levels reversibly inhibits cell polarization and migration. Among other receptor-mediated responses, beta(2) integrin up-regulation was unaffected, and initial calcium mobilization was only partially reduced by cholesterol depletion, indicating that this treatment did not abrogate initial receptor-mediated signal transduction. Interestingly, cholesterol depletion did not prevent initial activation of the GTPase Rac or an initial burst of actin polymerization, but rather it inhibited prolonged activation of Rac and sustained actin polymerization. Collectively, these findings support a model in which the plasma membrane is organized into domains that aid in amplifying the chemoattractant gradient and maintaining cell polarization.     

Dual Roles for RHOA/RHO-Kinase In the Regulated Trafficking of a Voltage-sensitive Potassium Channel

Kv1.2 is a member of the Shaker family of voltage-sensitive potassium channels and contributes to regulation of membrane excitability. The electrophysiological activity of Kv1.2 undergoes tyrosine kinase-dependent suppression in a process involving RhoA. We report that RhoA elicits suppression of Kv1.2 ionic current by modulating channel endocytosis. This occurs through two distinct pathways, one clathrin-dependent and the other cholesterol-dependent. Activation of Rho kinase (ROCK) via the lysophosphatidic acid (LPA) receptor elicits clathrin-dependent Kv1.2 endocytosis and consequent attenuation of its ionic current. LPA-induced channel endocytosis is blocked by the ROCK inhibitor Y27632 or by clathrin RNA interference. In contrast, steady-state endocytosis of Kv1.2 in unstimulated cells is cholesterol dependent. Inhibition of basal ROCK signaling with Y27632 increased surface Kv1.2, an effect that persists in the presence of clathrin small interfering RNA and that is not additive to the increase in surface channel levels elicited by the cholesterol sequestering drug filipin. Temperature block experiments show that ROCK affects cholesterol-dependent trafficking by modulating the recycling of endocytosed channel back to the plasma membrane. Both receptor-stimulated and steady-state Kv1.2 trafficking modulated by RhoA/ROCK required the activation of dynamin as well as the ROCK effector Lim-kinase, indicating a key role for actin remodeling in RhoA-dependent Kv1.2 regulation.     

Filamin-A Regulates Neutrophil Uropod Retraction through RhoA during Chemotaxis

Filamin-A (FLNa) has been shown to be a key cross-linker of actin filaments in the leading edge of a motile melanoma cell line, however its role in neutrophils undergoing chemotaxis is unknown. Using a murine transgenic model in which FLNa is selectively deleted in granulocytes, we report that, while neutrophils lacking FLNa show normal polarization and pseudopod extension, they exhibit obvious defects in uropod retraction. This uropod retraction defect was found to be a direct result of reduced FLNa mediated activation of the small GTPase RhoA and myosin mediated actin contraction in the FLNa null cells. This results in a neutrophil recruitment defect in FLNa null mice. The compensatory increase in FLNb levels that was observed in the FLNa null neutrophils may be sufficient to compensate for the lack of FLNa at the leading edge allowing for normal polarization, however this compensation is unable to regulate RhoA activated tail retraction at the rear of the cell.     

Microtubules Regulate Migratory Polarity through Rho/ROCK Signaling in T Cells

Background

Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known.

Methodology/Principal Findings

Here we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs.

Conclusions/Significance

Our results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning.     

Cdc42 and RhoA are differentially regulated during arachidonate-mediated HeLa cell adhesion

Cell adhesion to extracellular matrix requires stimulation of an eicosanoid signaling pathway through the metabolism of arachidonate by 5-lipoxygenase to leukotrienes and cyclooxygenase-1/2 to prostaglandins, as well as activation of the small GTPase signaling pathway involving Cdc42 and Rho. These signaling pathways direct remodeling of the actin cytoskeleton during the adhesion process, specifically the polymerization of actin during cell spreading and the bundling of actin filaments when cells migrate. However, few studies linking these signaling pathways have been described in the literature. We have previously shown that HeLa cell adhesion to collagen requires oxidation of arachidonic acid (AA) by lipoxygenase for actin polymerization and cell spreading, and cyclooxygenase for bundling actin filaments during cell migration. We demonstrate that small GTPase activity is required for HeLa cell spreading upon gelatin, and that Cdc42 is activated while Rho is downregulated during the spreading process. Using constitutively active and dominant negative expression studies, we show that Cdc42 is required for HeLa cell spreading and migration, while activated RhoA is antagonistic towards spreading. Constitutively active RhoA promotes cell migration and increases the degree of actin bundling in HeLa cells. Further, we demonstrate that activation of either the AA oxidation pathway or the small GTPase pathway cannot rescue inhibition of spreading when the alternate pathway is blocked. Our results suggest (1) both the eicosanoid signaling pathway and small GTPase activation are required during HeLa cell adhesion, and (2) these signaling pathways converge to properly direct remodeling of the actin cytoskeleton during HeLa cell spreading and migration upon collagen.     

Asymmetric localization of calpain 2 during neutrophil chemotaxis

Chemoattractants induce neutrophil polarization through localized polymerization of F-actin at the leading edge. The suppression of rear and lateral protrusions is required for efficient chemotaxis and involves the temporal and spatial segregation of signaling molecules. We have previously shown that the intracellular calcium-dependent protease calpain is required for cell migration and is involved in regulating neutrophil chemotaxis. Here, we show that primary neutrophils and neutrophil-like HL-60 cells express both calpain 1 and calpain 2 and that chemoattractants induce the asymmetric recruitment of calpain 2, but not calpain 1, to the leading edge of polarized neutrophils and differentiated HL-60 cells. Using time-lapse microscopy, we show that enrichment of calpain 2 at the leading edge occurs during early pseudopod formation and that its localization is sensitive to changes in the chemotactic gradient. We demonstrate that calpain 2 is recruited to lipid rafts and that cholesterol depletion perturbs calpain 2 localization, suggesting that its enrichment at the front requires proper membrane organization. Finally, we show that catalytic activity of calpain is required to limit pseudopod formation in the direction of chemoattractant and for efficient chemotaxis. Together, our findings identify calpain 2 as a novel component of the frontness signal that promotes polarization during chemotaxis.     

Spatial compartmentalization of tumor necrosis factor (TNF) receptor 1-dependent signaling pathways in human airway smooth muscle cells. Lipid rafts are essential for TNF-alpha-mediated activation of RhoA but dispensable for the activation of the NF-kappaB and MAPK pathways

Tumor necrosis factor (TNF)-alpha-induced activation of RhoA, mediated by TNF receptor 1 (TNFR1), is a prerequisite step in a pathway that leads to increased 20-kDa light chain of myosin (MLC20) phosphorylation and airway smooth muscle contraction. In this study, we have investigated the proximal events in TNF-alpha-induced RhoA activation. TNFR1 is localized to both lipid raft and nonraft regions of the plasma membrane in primary human airway smooth muscle cells. TNF-alpha engagement of TNFR1 recruited the adaptor proteins TRADD, TRAF-2, and RIP into lipid rafts and activated RhoA, NF-kappaB, and MAPK pathways. Depletion of cholesterol from rafts with methyl-beta-cyclodextrin caused a redistribution of TNFR1 to nonraft plasma membrane and prevented ligand-induced RhoA activation. By contrast, TNF-alpha-induced activation of NF-kappaB and MAPKs was unaffected by methyl-beta-cyclodextrin indicating that, in airway smooth muscle cells, activation of these pathways occurred independently of lipid rafts. Targeted knockdown of caveolin-1 completely abrogated TNF-alpha-induced RhoA activation, identifying this raft-resident protein as a positive regulator of the activation process. The signaling adaptors TRADD and RIP were also found to be necessary for ligand-induced RhoA activation. Taken together, our results suggest that in airway smooth muscle cells, spatial compartmentalization of TNFR1 provides a mechanism for generating distinct signaling outcomes in response to ligand engagement and define a mechanistic role for lipid rafts and caveolin-1 in TNF-alpha-induced activation of RhoA.     

Integrin-mediated adhesion regulates cell polarity and membrane protrusion through the Rho family of GTPases

Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through alpha 5 beta 1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.     

Hax1 regulates neutrophil adhesion and motility through RhoA

Kostmann disease is an inherited severe congenital neutropenia syndrome associated with loss-of-function mutations in an adaptor protein HS1-associated protein X-1 (Hax1). How Hax1 regulates neutrophil function remains largely unknown. In this paper, we use ribonucleic acid interference to deplete Hax1 in the neutrophil-like cell line PLB-985 and identify Hax1 as a negative regulator of integrin-mediated adhesion and chemotaxis. Using microfluidics, we show that depletion of Hax1 impairs neutrophil uropod detachment and directed migration. Hax1-deficient cells also display increased integrin-mediated adhesion and reduced RhoA activity. Moreover, depletion of RhoA induces increased neutrophil adhesion and impaired migration, suggesting that Hax1 regulates neutrophil adhesion and chemotaxis through RhoA. Accordingly, activation of RhoA is sufficient to rescue adhesion of Hax1-deficient neutrophils. Together, our findings identify Hax1 as a novel regulator of neutrophil uropod detachment and chemotaxis through RhoA.     

RhoA and ROCK promote migration by limiting membrane protrusions

Previously, we and others have shown that RhoA and ROCK signaling are required for negatively regulating integrin-mediated adhesion and for tail retraction of migrating leukocytes. This study continues our investigation into the molecular mechanisms underlying RhoA/ROCK-regulated integrin adhesion. We show that inhibition of ROCK up-regulates integrin-mediated adhesion, which is accompanied by both increased phosphotyrosine signaling through Pyk-2 and paxillin and inappropriate membrane protrusions. We provide evidence that inhibition of ROCK induces integrin adhesion by promoting remodeling of the actin cytoskeleton. Furthermore, we find that ROCK regulates membrane activity through a pathway involving cofilin. Inhibition of RhoA signaling allows the formation of multiple competing lamellipodia that disrupt productive migration of monocytes. Together, our results show that RhoA/ROCK signaling promotes migration by restricting integrin activity and membrane protrusions to the leading edge.     

The headpiece domain of dematin regulates cell shape, motility, and wound healing by modulating RhoA activation

RhoA is known to participate in cytoskeletal remodeling events through several signaling pathways, yet the precise mechanism of its activation remains unknown. Here, we provide the first evidence that dematin functions upstream of RhoA and regulates its activation. Primary mouse embryonic fibroblasts were generated from a dematin headpiece domain null (HPKO) mouse, and the visualization of the actin morphology revealed a time-dependent defect in stress fiber formation, membrane protrusions, cell motility, and cell adhesion. Rescue experiments using RNA interference and transfection assays revealed that the observed phenotypes are due to a null effect and not a gain of function in the mutant fibroblasts. In vivo wounding of adult HPKO mouse skin showed a decrease in wound healing (reepithelialization and granulation) compared to the wild-type control. Biochemical analysis of the HPKO fibroblasts revealed a sustained hyperphosphorylation of focal adhesion kinase (FAK) at tyrosine 397 as well as a twofold increase in RhoA activation. Inhibition of both RhoA and FAK signaling using C3 toxin and FRNK (focal adhesion kinase nonrelated kinase), respectively, revealed that dematin acts upstream of RhoA. Together, these results unveil a new function of dematin as a negative regulator of the RhoA activation pathway with physiological implications for normal and pathogenic signaling pathways.     

Requirement for Tec kinases in chemokine-induced migration and activation of Cdc42 and Rac

Cell polarization and migration in response to chemokines is essential for proper development of the immune system and activation of immune responses. Recent studies of chemokine signaling have revealed a critical role for PI3-Kinase, which is required for polarized membrane association of pleckstrin homology (PH) domain-containing proteins and activation of Rho family GTPases that are essential for cell polarization and actin reorganization. Additional data argue that tyrosine kinases are also important for chemokine-induced Rac activation. However, how and which kinases participate in these pathways remain unclear. We demonstrate here that the Tec kinases Itk and Rlk play an important role in chemokine signaling in T lymphocytes. Chemokine stimulation induced transient membrane association of Itk and phosphorylation of both Itk and Rlk, and purified T cells from Rlk(-/-)Itk(-/-) mice exhibited defective migration to multiple chemokines in vitro and decreased homing to lymph nodes upon transfer to wt mice. Expression of a dominant-negative Itk impaired SDF-1alpha-induced migration, cell polarization, and activation of Rac and Cdc42. Thus, Tec kinases are critical components of signaling pathways required for actin polarization downstream from both antigen and chemokine receptors in T cells.     

Intercellular adhesion molecule-1 (ICAM-1) regulates endothelial cell motility through a nitric oxide-dependent pathway

Coordinated regulation of endothelial cell migration is an integral process during angiogenesis. However, molecular mechanisms regulating endothelial cell migration remain largely unknown. Increased expression of cell adhesion molecules has been implicated during angiogenesis, yet the precise role of these molecules is unclear. Here, we examined the hypothesis that intercellular adhesion molecule-1 (ICAM-1) is important for endothelial cell migration. Total cell displacement and directional migration were significantly attenuated in ICAM-1-deficient endothelium. Closer examination of ICAM-1-deficient cells revealed decreased Akt Thr(308) and endothelial nitric-oxide synthase Ser(1177) phosphorylation and NO bioavailability, increased actin stress fiber formation, and a lack of distinct cell polarity compared with wild-type endothelium. Supplementation of ICAM-1 mutant cells with the NO donor DETA NONOate (0.1 microM) corrected the migration defect, diminished stress fiber formation, and enhanced pseudopod and uropod formation. These data demonstrate that ICAM-1 facilitates the development of cell polarity and modulates endothelial cell migration through a pathway regulating endothelial nitric-oxide synthase activation and organization of the actin cytoskeleton.     

Invasion of T-lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling.

Rho-like GTPases orchestrate distinct cytoskeletal changes in response to receptor stimulation. Invasion of T-lymphoma cells into a fibroblast monolayer is induced by Tiam1, an activator of the Rho-like GTPase Rac, and by constitutively active V12Rac1. Here we show that activated V12Cdc42 can also induce invasion of T-lymphoma cells. Activated RhoA potentiates invasion, but fails by itself to mimic Rac and Cdc42. However, invasion is inhibited by the Rho-inactivating C3 transferase. Thus, RhoA is required but not sufficient for invasion. Invasion of T-lymphoma cells is critically dependent on the presence of serum. Serum can be replaced by the serum-borne lipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) (10(-7)-10(-6) M), which act on distinct G protein-linked receptors to activate RhoA and phospholipase C (PLC)-Ca2+ signaling. LPA- and S1P-induced invasion is preceded by Rho-dependent F-actin redistribution and pseudopodia formation. However, expression of both V14RhoA and V12Rac1 does not bypass the LPA/S1P requirement for invasion, indicating involvement of an additional signaling pathway independent of RhoA. The PLC inhibitor U-73122, but not the inactive analog U-73343, abolishes invasion. Our results indicate that T-lymphoma invasion is driven by Tiam1/Rac or Cdc42 activation, and is dependent on LPA/S1P receptor-mediated RhoA and PLC signaling pathways which lead to pseudopod formation and enhanced infiltration.     

A proteomic and cellular analysis of uropods in the pathogen Entamoeba histolytica

Exposure of Entamoeba histolytica to specific ligands induces cell polarization via the activation of signalling pathways and cytoskeletal elements. The process leads to formation of a protruding pseudopod at the front of the cell and a retracting uropod at the rear. In the present study, we show that the uropod forms during the exposure of trophozoites to serum isolated from humans suffering of amoebiasis. To investigate uropod assembly, we used LC-MS/MS technology to identify protein components in isolated uropod fractions. The galactose/N-acetylgalactosamine lectin, the immunodominant antigen M17 (which is specifically recognized by serum from amoeba-infected persons) and a few other cells adhesion-related molecules were primarily involved. Actin-rich cytoskeleton components, GTPases from the Rac and Rab families, filamin, α-actinin and a newly identified ezrin-moesin-radixin protein were the main factors found to potentially interact with capped receptors. A set of specific cysteine proteases and a serine protease were enriched in isolated uropod fractions. However, biological assays indicated that cysteine proteases are not involved in uropod formation in E. histolytica, a fact in contrast to the situation in human motile immune cells. The surface proteins identified here are testable biomarkers which may be either recognized by the immune system and/or released into the circulation during amoebiasis.     

Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration

Transendothelial migration (TEM) is a tightly regulated process whereby leukocytes migrate from the vasculature into tissues. Rho guanosine triphosphatases (GTPases) are implicated in TEM, but the contributions of individual Rho family members are not known. In this study, we use an RNA interference screen to identify which Rho GTPases affect T cell TEM and demonstrate that RhoA is critical for this process. RhoA depletion leads to loss of migratory polarity; cells lack both leading edge and uropod structures and, instead, have stable narrow protrusions with delocalized protrusions and contractions. By imaging a RhoA activity biosensor in transmigrating T cells, we find that RhoA is locally and dynamically activated at the leading edge, where its activation precedes both extension and retraction events, and in the uropod, where it is associated with ROCK-mediated contraction. The Rho guanine nucleotide exchange factor (GEF) GEF-H1 contributes to uropod contraction but does not affect the leading edge. Our data indicate that RhoA activity is dynamically regulated at the front and back of T cells to coordinate TEM.     

STIM1调控细胞运动促进骨肉瘤转移的研究

目的:明确STIM1是否参与调控细胞运动促进骨肉瘤的转移。方法:应用靶向STIM1的si RNA沉默MG-63骨肉瘤细胞中STIM1的表达,然后用侵袭实验、迁移实验以及黏附实验检测骨肉瘤细胞侵袭、迁移与黏附能力的变化,采用Western Blot检测细胞FAK和paxillin的表达及Rac1和RhoA信号通路的活性。结果:转染靶向STIM1的si RNA后,MG-63骨肉瘤细胞中STIM1的蛋白表达和m RNA表达均明显降低(P0.05),细胞的侵袭、迁移与黏附能力均显著下降(P0.05),细胞伪足与细胞骨架的重要组分FAK和paxillin的表达及调控细胞运动的Rac1和RhoA信号通路活性均显著降低(P0.05)。结论:STIM1可能通过激活RhoA和Rac1的信号通路,增加FAK和paxillin的表达,从而调控骨肉瘤细胞运动,促进骨肉瘤转移。     

Cholesterol is obligatory for polarization and chemotaxis but not for endocytosis and associated signaling from chemoattractant receptors in human neutrophils

Plasma membrane cholesterol is critical for neutrophil chemotaxis, although how cholesterol affects chemotactic signaling pathway has not been clearly delineated. Here we demonstrate that cholesterol was absolutely required for polarized redistribution of key chemotactic mediators in human neutrophils in response to all chemoattractants tested (fMet-Leu-Phe, and the chemokines CXCL1, CXCL8 and CXCL12). In particular, PI3K and phosphatidylinositol-3,4,5 triphosphate (PIP₃) failed to accumulate at the front and phosphatase and tensin homolog (PTEN) at the back of chemoattractant-stimulated neutrophils after cholesterol depletion. Cholesterol depletion did not affect early chemoattractant signaling events such as G-protein activation, intracellular calcium flux or G-protein-independent endocytosis-linked signaling, including the activation of mitogen-activated protein kinase (MAPK), Hck and Fgr transduced by β-arrestin. During cell polarization, F-actin assemblies redistributed the cholesterol-rich microdomains and cytoskeleton-anchored proteins, including CD16 and CD44 from the leading edge. These data suggest that spatial polarization of chemotactic mediators is orchestrated by protein:protein interactions that organize cholesterol-rich domains of the plasma membrane.