HIV-1 Clade B Tat, but Not Clade C Tat, Increases X4 HIV-1 Entry into Resting but Not Activated CD4+ T Cells

CXCR4-using human immunodeficiency virus, type 1 (HIV-1) variants emerge late in the course of infection in >40% of individuals infected with clade B HIV-1 but are described less commonly with clade C isolates. Tat is secreted by HIV-1-infected cells where it acts on both uninfected bystander cells and infected cells. In this study, we show that clade B Tat, but not clade C Tat, increases CXCR4 surface expression on resting CD4⁺ T cells through a CCR2b-dependent mechanism that does not involve de novo protein synthesis. The expression of plectin, a cytolinker protein that plays an important role as a scaffolding platform for proteins involved in cellular signaling including CXCR4 signaling and trafficking, was found to be significantly increased following B Tat but not C Tat treatment. Knockdown of plectin using RNA interference showed that plectin is essential for the B Tat-induced translocation of CXCR4 to the surface of resting CD4⁺ T cells. The increased surface CXCR4 expression following B Tat treatment led to increased function of CXCR4 including increased chemoattraction toward CXCR4-using-gp120. Moreover, increased CXCR4 surface expression rendered resting CD4⁺ T cells more permissive to X4 but not R5 HIV-1 infection. However, neither B Tat nor C Tat was able to up-regulate surface expression of CXCR4 on activated CD4⁺ T cells, and both proteins inhibited the infection of activated CD4⁺ T cells with X4 but not R5 HIV-1. Thus, B Tat, but not C Tat, has the capacity to render resting, but not activated, CD4⁺ T cells more susceptible to X4 HIV-1 infection.     

Tumor Necrosis Factor Receptor Expression in HIV-1-Infected CD4+ T Cells

We investigated whether HIV-1 can regulate tumor necrosis factor receptor (TNFR) expression in SupT-1, a CD4 + T-cell line. The cells were infected with HIV-1 containing 1,000 cpm RT activity, as early as day 3 after infection and all along the culture the supernatant level of core protein p24 was >250 pg/ml, and on days 6 and 9 after infection, p24 was found in 10 % of the cells as determined by indirect immunofluorescence assay. The cells were growing without loss of viability. The study of TNFR expression was based on a microassay for measurement of binding of ¹²⁵I-TNFα to cells, in which free and cell-bound ligand separation was performed by centrifugation through oil. Scatchard analysis of TNFα binding on days 6 and 9 after infection revealed a 90 % increase in the expression of high-affinity membrane receptors in HIV + SupT-1 culture compared with uninfected cells (mean +/-S.D. = 501 +/-148.5 vs. 263 +/-77.8 receptors/cell, n = 9, P< 0.001) with no change in dissociation constants (mean +/? S.D. = 4.36 +/?1.06 vs. 4.00 +/?1.12 × 10^?10 m ).     

A Dichotomy in Cortical Actin and Chemotactic Actin Activity between Human Memory and Naive T Cells Contributes to Their Differential Susceptibility to HIV-1 Infection

Human memory and naive CD4 T cells can mainly be identified by the reciprocal expression of the CD45RO or CD45RA isoforms. In HIV-1 infection, blood CD45RO memory CD4 T cells are preferentially infected and serve as a major viral reservoir. The molecular mechanism dictating this differential susceptibility to HIV-1 remains largely obscure. Here, we report that the different susceptibility of memory and naive T cells to HIV is not determined by restriction factors such as Apobec3G or BST2. However, we observed a phenotypic distinction between human CD45RO and CD45RA resting CD4 T cells in their cortical actin density and actin dynamics. CD45RO CD4 T cells possess a higher cortical actin density and can be distinguished as CD45RO⁺Actin^high. In contrast, CD45RA T cells are phenotypically CD45RA⁺Actin^low. In addition, the cortical actin in CD45RO memory CD4 T cells is more dynamic and can respond to low dosages of chemotactic induction by SDF-1, whereas that of naive cells cannot, despite a similar level of the chemokine receptor CXCR4 present on both cells. We further demonstrate that this difference in the cortical actin contributes to their differential susceptibility to HIV-1; resting memory but not naive T cells are highly responsive to HIV-mediated actin dynamics that promote higher levels of viral entry and early DNA synthesis in resting memory CD4 T cells. Furthermore, transient induction of actin dynamics in resting naive T cells rescues HIV latent infection following CD3/CD28 stimulation. These results suggest a key role of chemotactic actin activity in facilitating HIV-1 latent infection of these T cell subsets.     

Collapsin Response Mediator Protein-1 Regulates Arp2/3-dependent Actin Assembly

Listeria monocytogenes is a bacterial parasite that uses host proteins to assemble an Arp2/3-dependent actin comet tail to power its movement through the host cell. Initiation of comet tail assembly is more efficient in cytosol than it is under defined conditions, indicating that unknown factors contribute to the reaction. We therefore fractionated cytosol and identified CRMP-1 as a factor that facilitates Arp2/3-dependent Listeria actin cloud formation in the presence of Arp2/3 and actin alone. It also scored as an important factor for Listeria actin comet tail formation in brain cytosol. CRMP-1 does not nucleate actin assembly on its own, nor does it directly activate the Arp2/3 complex. Rather, CRMP-1 scored as an auxiliary factor that promoted the ability of Listeria ActA protein to activate the Arp2/3 complex to trigger actin assembly. CRMP-1 is one member of a family of five related proteins that modulate cell motility in response to extracellular signals. Our results demonstrate an important role for CRMP-1 in Listeria actin comet tail formation and open the possibility that CRMP-1 controls cell motility by modulating Arp2/3 activation.     

Role of glycosphingolipid microdomains in CD4-dependent HIV-1 fusion

The fusion of HIV-1 with the plasma membrane of CD4⁺ cells is triggered by the interaction of HIV-1 surface envelope glycoprotein gp120 with the CD4 receptor, and requires coreceptors (CCR5 and CXCR4). Recent advances in the study of HIV-1 entry into CD4⁺ cells suggest that glycosphingolipids (GSL) may also participate in the fusion process. GSL are organized in functional microdomains which are associated with specific membrane proteins such as CD4. GSL-enriched microdomains were purified from human lymphocytes and reconstituted as a monomolecular film at the air–water interface of a Langmuir film balance. Surface pressure measurements allowed to characterize the sequential interaction of GSL with CD4 and with gp120. Using this approach, we identified globotriaosylceramide (Gb3) and ganglioside GM3 as the main lymphocyte GSL recognized by gp120. In both cases, the interaction was saturable and dramatically increased by CD4. We propose that GSL microdomains behave as moving platforms allowing the recruitment of HIV-1 coreceptors after the initial interaction between the viral particle and CD4. According to this model, the GSL microdomain may : i) stabilize the attachment of the virus with the cell surface through multiple low affinity interactions between the V3 domain of gp120 and the carbohydrate moiety of GSL, and ii) convey the virus to an appropriate coreceptor by moving freely in the outer leaflet of the plasma membrane. This model can be extrapolated to all envelope viruses (e.g. influenza virus) that use cell surface GSL of the host cells as receptors or coreceptors.     

Involvement of Arp2/3 complex in MCP-1-induced chemotaxis

The migrating monocyte shows dynamic actin polymerization in response to MCP-1. We investigated the involvement of the actin-related protein 2 and 3 complex (Arp2/3 complex) during chemotaxis of a human monocyte cell line (THP-1). To clarify whether the Arp2/3 complex directly polymerizes actin in response to MCP-1 stimulation, THP-1 cells were transfected with complementary DNA constructs encoding ScarWA. In ScarWA-transfected cells, neither recruitment of Arp2/3 complex at the leading edge nor actin polymerization was detected. Indeed, migration induced by MCP-1 was almost completely blocked. At the same time, transfection also interfered with the recruitment of integrin beta-1 at the leading edge and reduced affinity binding to fibronectin. Immunoprecipitation with an anti-Arp2 antibody showed that integrin beta-1 and WASP were co-precipitated under the condition of MCP-1 stimulation. These results indicate that interaction between the Arp2/3 complex and WASP stimulates actin polymerization and integrin beta-1-mediated adhesion during MCP-1-induced chemotaxis of THP-1 cells.     

Cortactin Scaffolds Arp2/3 and WAVE2 at the Epithelial Zonula Adherens

Cadherin junctions arise from the integrated action of cell adhesion, signaling, and the cytoskeleton. At the zonula adherens (ZA), a WAVE2-Arp2/3 actin nucleation apparatus is necessary for junctional tension and integrity. But how this is coordinated with cadherin adhesion is not known. We now identify cortactin as a key scaffold for actin regulation at the ZA, which localizes to the ZA through influences from both E-cadherin and N-WASP. Using cell-free protein expression and fluorescent single molecule coincidence assays, we demonstrate that cortactin binds directly to the cadherin cytoplasmic tail. However, its concentration with cadherin at the apical ZA also requires N-WASP. Cortactin is known to bind Arp2/3 directly (Weed, S. A., Karginov, A. V., Schafer, D. A., Weaver, A. M., Kinley, A. W., Cooper, J. A., and Parsons, J. T. (2000) J. Cell Biol. 151, 29–40). We further show that cortactin can directly bind WAVE2, as well as Arp2/3, and both these interactions are necessary for actin assembly at the ZA. We propose that cortactin serves as a platform that integrates regulators of junctional actin assembly at the ZA.     

Role of Glycosphingolipids in HIV-1 Entry: Requirement of Globotriosylceramide (Gb3) in CD4/CXCR4-dependent Fusion

We have recently shown that addition of human erythrocyte glycosphingolipids (GSL) to non-human CD4⁺ or GSL-depleted human CD4⁺ cells rendered those cells susceptible to gp120-gp41-mediated cell fusion (Puri et al., BBRC, 1998). One GSL fraction (Fraction 3) isolated from human erythrocyte GSL mixture exhibited the highest recovery of fusion following incorporation into CD4⁺ non-human and GSL-depleted HeLa-CD4 cells (HeLa-CD4/GSL^–). Structural analysis of Fraction 3 showed that this GSL had identical head group as the known GSL, Gal(14)Gal(1 4)Glc-Ceramide (Gb3) (Puri et al., PNAS, 1998). Here we report that presence of Gb3 in CD4⁺/CXCR4⁺ cells but not CD4⁺/CXCR4^– cells allows fusion with HIV-1_Lai-envelope glycoprotein expressing cells (TF228). Therefore, Gb3 functions in conjunction with HIV-1 co-receptor, CXCR4 to promote fusion. We propose that Gb3 functions by recruiting CD4 and/or CXCR4 at the fusion site through structurally specific interactions.     

Genetic and biochemical interactions between the Arp2/3 complex, Cmd1p, casein kinase II, and Tub4p in yeast

Arc35p, a component of the Arp2/3 complex, plays at least two distinct roles, regulating the actin cytoskeleton, but also microtubule function during cell division. Both functions involve calmodulin (CMD1). To investigate the pathway affecting microtubule function, we identified genes that are able to suppress the temperature-sensitive growth defect of the arc35-1 strain. Genes encoding gamma-tubulin (TUB4) or any subunit of casein kinase II (CKII) suppressed this growth defect, but did not suppress the growth defect of a mutant in another subunit of the Arp2/3 complex, arp2-1. We could also show a physical association of Arc35p with subunits of CKII, Cmd1p, and Tub4p. Based on the exclusive localization of Arc35p to the cytosolic Arp2/3 complex and on mutant phenotypes, we propose that the role of the Arc35p/CKII interaction might be to activate a cytosolic pool of gamma-tubulin, likely via calmodulin, for its nuclear and/or cytoplasmic functions.     

Ⅲ型纤连蛋白组件包含蛋白5通过抑制ERK1/2磷酸化抑制C3H10T1/2细胞成脂分化

旨在探究Ⅲ型纤连蛋白组件包含蛋白5(type Ⅲ domain-containing protein5,FNDC5)对C3H10T1/2细胞成脂分化的调控作用.利用qRT-PCR和Western印迹检测FNDC5在C3H10T1/2细胞成脂分化过程中的时序性表达规律;构建慢病毒包被的过表达/干扰FNDC5载体,转染C3...     

SCAR is a primary regulator of Arp2/3-dependent morphological events in Drosophila

The Arp2/3 complex and its activators, Scar/WAVE and Wiskott-Aldrich Syndrome protein (WASp), promote actin polymerization in vitro and have been proposed to influence cell shape and motility in vivo. We demonstrate that the Drosophila Scar homologue, SCAR, localizes to actin-rich structures and is required for normal cell morphology in multiple cell types throughout development. In particular, SCAR function is essential for cytoplasmic organization in the blastoderm, axon development in the central nervous system, egg chamber structure during oogenesis, and adult eye morphology. Highly similar developmental requirements are found for subunits of the Arp2/3 complex. In the blastoderm, SCAR and Arp2/3 mutations result in a reduction in the amount of cortical filamentous actin and the disruption of dynamically regulated actin structures. Remarkably, the single Drosophila WASp homologue, Wasp, is largely dispensable for these numerous Arp2/3-dependent functions, whereas SCAR does not contribute to cell fate decisions in which Wasp and Arp2/3 play an essential role. These results identify SCAR as a major component of Arp2/3-dependent cell morphology during Drosophila development and demonstrate that the Arp2/3 complex can govern distinct cell biological events in response to SCAR and Wasp regulation.     

Ionic interaction of the HIV-1 V3 domain with CCR5 and deregulation of T lymphocyte function

We have reported that the principal neutralizing domain of V3 of the HIV-1 gp120 induces an antigen-specific activation apoptosis of responding effector CD4+ T lymphocytes, a phenomenon inhibited by RANTES, an agonist of CCR5. Here, addressing the question of how a hypervariable region could induce such a selective reaction, we demonstrated that the magnitude of the activation phase was dependent on the number of basic amino acids present in the V3 peptide, an observation confirmed by using V3 peptides with appropriate basic amino acid substitutions. The relative position of the amino acids in the V3 peptide did not affect the biological phenomenon. Using surface plasmon resonance biosensor analysis, we also provided direct evidence of the influence of basic amino acids in the interaction between V3 and the amino terminal domain of CCR5. Sulphation of tyrosines in the CCR5 peptide was essential. Our results confirm gp120 modelling predictions and demonstrate simple molecular ionic interactions as capable of affecting key cell events, the wider biological implications of which need to be further explored.     

Arp2/3 complex interactions and actin network turnover in lamellipodia

Cell migration is initiated by lamellipodia-membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin-another prominent Arp2/3 complex regulator-and ADF/cofilin-previously implicated in driving both filament nucleation and disassembly-were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh.     

A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex

Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly.     

The actin regulatory protein HS1 interacts with Arp2/3 and mediates efficient neutrophil chemotaxis

HS1 is an actin regulatory protein and cortactin homolog that is expressed in hematopoietic cells. Antigen receptor stimulation induces HS1 phosphorylation, and HS1 is essential for T cell activation. HS1 is also expressed in neutrophils; however, the function of HS1 in neutrophils is not known. Here we show that HS1 localizes to the neutrophil leading edge, and is phosphorylated in response to the chemoattractant formyl-Met-Leu-Phe (fMLP) in adherent cells. Using live imaging in microchannels, we show that depletion of endogenous HS1 in the neutrophil-like PLB-985 cell line impairs chemotaxis. We also find that HS1 is necessary for chemoattractant-induced activation of Rac GTPase signaling and Vav1 phosphorylation, suggesting that HS1-mediated Rac activation is necessary for efficient neutrophil chemotaxis. We identify specific phosphorylation sites that mediate HS1-dependent neutrophil motility. Expression of HS1 Y378F, Y397F is sufficient to rescue migration of HS1-deficient neutrophils, however, a triple phospho-mutant Y222F, Y378F, Y397F did not rescue migration of HS1-deficient neutrophils. Moreover, HS1 phosphorylation on Y222, Y378, and Y397 regulates its interaction with Arp2/3. Collectively, our findings identify a novel role for HS1 and its phosphorylation during neutrophil directed migration.     

HIV-1 and SIV Infection Are Associated with Early Loss of Lung Interstitial CD4+ T Cells and Dissemination of Pulmonary Tuberculosis

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GRAIL (gene related to anergy in lymphocytes) regulates cytoskeletal reorganization through ubiquitination and degradation of Arp2/3 subunit 5 and coronin 1A

Anergy is an important mechanism for the maintenance of peripheral tolerance and avoidance of autoimmunity. The up-regulation of E3 ubiqitin ligases, including GRAIL (gene related to anergy in lymphocytes), is a key event in the induction and preservation of anergy in T cells. However, the mechanisms of GRAIL-mediated anergy induction are still not completely understood. We examined which proteins serve as substrates for GRAIL in anergic T cells. Arp2/3-5 (actin-related protein 2/3 subunit 5) and coronin 1A were polyubiquitinated by GRAIL via Lys-48 and Lys-63 linkages. In anergic T cells and GRAIL-overexpressed T cells, the expression of Arp2/3-5 and coronin 1A was reduced. Furthermore, we demonstrated that GRAIL impaired lamellipodium formation and reduced the accumulation of F-actin at the immunological synapse. GRAIL functions via the ubiquitination and degradation of actin cytoskeleton-associated proteins, in particular Arp2/3-5 and coronin 1A. These data reveal that GRAIL regulates proteins involved in the actin cytoskeletal organization, thereby maintaining the unresponsive state of anergic T cells.     

Actin and Arp2/3 localize at the centrosome of interphase cells

Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel (“lower dimer”) actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.