Establishment and maintenance of cell polarity during leukocyte chemotaxis.

The term polarity refers to the differential distribution of the macromolecular elements of a cell, resulting in its asymmetry in function, shape and/or content. Polarity is a fundamental property of all metazoan cells in at least some stages, and is pivotal to processes such as epithelial differentiation (apical/basal polarity), coordinated cell activity within the plane of a tissue (planar cell polarity), asymmetric cell division, and cell migration. In the last case, an apparently symmetric cell responds to directional cues provided by chemoattractants, creating a polarity axis that runs from the cell anterior, or leading edge, in which actin polymerization takes place, to the cell posterior (termed uropod in leukocytes), in which acto-myosin contraction occurs.

T cell costimulation by chemokine receptors.

Signals mediated by chemokine receptors may compete with T cell receptor stop signals and determine the duration of T cell-antigen-presenting cell interactions. Here we show that during T cell stimulation by antigen-presenting cells, T cell chemokine receptors coupled to G(q) and/or G(11) protein were recruited to the immunological synapse by a G(i)-independent mechanism. When chemokine receptors were sequestered at the immunological synapse, T cells became insensitive to chemotactic gradients, formed more stable conjugates and finally responded with enhanced proliferation and cytokine production.

Mastering time and space: immune cell polarization and chemotaxis.

Many immune cells can detect the direction and intensity of an extracellular chemical gradient, and migrate toward the source of stimulus. This process, called chemotaxis, is essential for immune system function and homeostasis, and its deregulation is associated with serious diseases. Chemotaxis is initiated by chemoattractant binding to heterotrimeric G protein-coupled receptors, which translate the gradients into accurate directional migration.

PTEN regulates motility but not directionality during leukocyte chemotaxis.

The localization at opposite cell poles of phosphatidylinositol-3 kinases and PTEN (phosphatase and tensin homolog on chromosome 10) governs Dictyostelium chemotaxis. To study this model in mammalian cells, we analyzed the dynamic redistribution of green fluorescent protein (GFP)-tagged PTEN chimeras during chemotaxis. N- or C-terminus GFP-tagged PTEN was distributed homogeneously in the cytoplasm of chemotaxing PTEN-negative Jurkat cells and PTEN-positive HL60 cells.

Statins inhibit HIV-1 infection by down-regulating Rho activity.

Human immunodeficiency virus (HIV)-1 infectivity requires actin-dependent clustering of host lipid raft-associated receptors, a process that might be linked to Rho guanosine triphosphatase (GTPase) activation. Rho GTPase activity can be negatively regulated by statins, a family of drugs used to treat hypercholesterolemia in man. Statins mediate inhibition of Rho GTPases by impeding prenylation of small G proteins through blockade of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Secreted MMP9 promotes angiogenesis more efficiently than constitutive active MMP9 bound to the tumor cell surface.

Association of matrix metalloprotease 9 (MMP9) to the cell membrane is considered important in tumor growth and angiogenesis. To dissect this regulatory mechanism, we generated raft and non-raft MMP9 chimeras to force membrane expression in the MCF-7 human breast carcinoma cell line. MMP9 targeting to non-raft cell surface domains rendered a constitutive active membrane MMP9 form, suggesting a contribution by the lipid environment in MMP activation.

Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis.

Spatially restricted activation of signaling molecules governs critical aspects of cell migration; the mechanism by which this is achieved nonetheless remains unknown. Using time-lapse confocal microscopy, we analyzed dynamic redistribution of lipid rafts in chemoattractant-stimulated leukocytes expressing glycosyl phosphatidylinositol-anchored green fluorescent protein (GFP-GPI). Chemoattractants induced persistent GFP-GPI redistribution to the leading edge raft (L raft) and uropod rafts of Jurkat, HL60, and dimethyl sulfoxide-differentiated HL60 cells in a pertussis toxin-sensitive, actin-dependent manner.

CCR5 expression influences the progression of human breast cancer in a p53-dependent manner.

Chemokines are implicated in tumor pathogenesis, although it is unclear whether they affect human cancer progression positively or negatively. We found that activation of the chemokine receptor CCR5 regulates p53 transcriptional activity in breast cancer cells through pertussis toxin-, JAK2-, and p38 mitogen-activated protein kinase-dependent mechanisms. CCR5 blockade significantly enhanced proliferation of xenografts from tumor cells bearing wild-type p53, but did not affect proliferation of tumor xenografts bearing a p53 mutation.

From rafts to crafts: membrane asymmetry in moving cells.

Many important biological events, including the leukocyte-mediated immune response, wound repair, axon guidance and developmental patterning, involve persistent cell movement towards a directional signal, a process termed chemotaxis. Establishment of functional and spatial cell polarity is an absolute requirement for this response. We propose that redistribution of specific membrane microdomains, termed rafts, during cell migration is a pivotal step in achieving polarity.

Blocking of HIV-1 infection by targeting CD4 to nonraft membrane domains.

Human immunodeficiency virus (HIV)-1 infection depends on multiple lateral interactions between the viral envelope and host cell receptors. Previous studies have suggested that these interactions are possible because HIV-1 receptors CD4, CXCR4, and CCR5 partition in cholesterol-enriched membrane raft domains. We generated CD4 partitioning mutants by substituting or deleting CD4 transmembrane and cytoplasmic domains and the CD4 ectodomain was unaltered.

Quantitative determination of tumor cell intravasation in a real-time polymerase chain reaction-based assay.

Tumor cells acquire the ability to enter blood vessels surrounding the primary tumor, endowing them with the capacity to disseminate and become established in distant sites, originating a metastasis. Determination of the intravasation ability of tumor cells is thus important, as it can be correlated with their potential malignancy. To analyze the intravasation phenotype of human tumor cells in vivo, we performed chick embryo chorioallantoic membrane (CAM) assays.

Specific SHP-2 partitioning in raft domains triggers integrin-mediated signaling via Rho activation.

Cell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts. By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling. Serum-depleted, nonattached cells expressing the raft SHP-2 form, but not non-raft SHP-2, display signaling events resembling those observed after fibronectin attachment, such as beta1 integrin clustering, 397Y-FAK phosphorylation, and ERK activation, and also increases Rho-GTP levels.

Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization.

Redistribution of specialized molecules in migrating cells develops asymmetry between two opposite cell poles, the leading edge and the uropod. We show that acquisition of a motile phenotype in T lymphocytes results in the asymmetric redistribution of ganglioside GM3- and GM1-enriched raft domains to the leading edge and to the uropod, respectively. This segregation to each cell pole parallels the specific redistribution of membrane proteins associated to each raft subfraction.

Membrane raft microdomains in chemokine receptor function.

Cell chemotaxis requires the acquisition and maintenance of both spatial and functional asymmetry between initially equivalent cell parts. In leukocytes one becomes the leading edge and the other, the rear edge or uropod. The acquisition of this cell polarity is controlled by an array of chemoattractants, including those of the chemokine family.

Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection.

HIV-1 infection triggers lateral membrane diffusion following interaction of the viral envelope with cell surface receptors. We show that these membrane changes are necessary for infection, as initial gp120-CD4 engagement leads to redistribution and clustering of membrane microdomains, enabling subsequent interaction of this complex with HIV-1 co-receptors. Disruption of cell membrane rafts by cholesterol depletion before viral exposure inhibits entry by both X4 and R5 strains of HIV-1, although viral replication in infected cells is unaffected by this treatment.

A role for chemokine receptor transactivation in growth factor signaling.

Complex cell responses require the integration of signals delivered through different pathways. We show that insulin-like growth factor (IGF)-I induces specific transactivation of the Gi-coupled chemokine receptor CCR5, triggering its tyrosine phosphorylation and Galpha recruitment. This transactivation occurs via a mechanism involving transcriptional upregulation and secretion of RANTES, the natural CCR5 ligand.

Cells on the move: a dialogue between polarization and motility.

Throughout evolution, both prokaryotic and eukaryotic cells have developed a variety of biochemical mechanisms to define the direction and proximity of extracellular stimuli. This process is essential for the cell to reply properly to the environmental cues that determine cell migration, proliferation, and differentiation. Chemotaxis is the cellular response to chemical attractants that direct cell migration, a process that plays a central role in many physiological situations, such as host immune responses, angiogenesis, wound healing, embryogenesis, and neuronal patterning, among others.

Membrane raft microdomains mediate front-rear polarity in migrating cells.

The acquisition of spatial and functional asymmetry between the rear and the front of the cell is a necessary step for cell chemotaxis. Insulin-like growth factor-I (IGF-I) stimulation of the human adenocarcinoma MCF-7 induces a polarized phenotype characterized by asymmetrical CCR5 chemokine receptor redistribution to the leading cell edge. CCR5 associates with membrane raft microdomains, and its polarization parallels redistribution of raft molecules, including the raft-associated ganglioside GM1, glycosylphosphatidylinositol-anchored green fluorescent protein and ephrinB1, to the leading edge.

Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility.

The coordinated interplay of substrate adhesion and deadhesion is necessary for cell motility. Using MCF-7 cells, we found that insulin-like growth factor I (IGF-I) induces the adhesion of MCF-7 to vitronectin and collagen in a dose- and time-dependent manner, suggesting that IGF-I triggers the activation of different integrins. On the other hand, IGF-I promotes the association of insulin receptor substrate 1 with the focal adhesion kinase (FAK), paxillin, and the tyrosine phosphatase SHP-2, resulting in FAK and paxillin dephosphorylation.

The matrix metalloproteinase-9 regulates the insulin-like growth factor-triggered autocrine response in DU-145 carcinoma cells.

The androgen-independent human prostate adenocarcinoma cell line DU-145 proliferates in serum-free medium and produces insulin-like growth factors (IGF)-I, IGF-II, and the IGF type-1 receptor (IGF-1R). They also secrete three IGF-binding proteins (IGFBP), IGFBP-2, -3, and -4. Of these, immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of 31 and 19 kDa.

Physical mapping of human insulin-like growth factor-I using specific monoclonal antibodies.

The primary structure of recombinant human (h) insulin-like growth factor-I (IGF-I) epitopes recognized by a panel of 28 monoclonal antibodies (mAbs) is characterized. Pairwise mAb epitope mapping defines eight 'epitopic clusters' (I-VIII) which cover nearly the entire solvent-exposed IGF-I surface. Monoclonal antibody reactivity with 32 overlapping synthetic peptides and with IGF-I mutants is used to associate these epitopic clusters with the probable primary IGF-I sequences recognized.