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1.
J Cell Sci ; 125(Pt 6): 1478-87, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22328509

RESUMO

Laminin-binding integrins (α3ß1, α6ß1, α6ß4, α7ß1) are almost always expressed together with tetraspanin CD151. In every coexpressing cell analyzed to date, CD151 makes a fundamental contribution to integrin-dependent motility, invasion, morphology, adhesion and/or signaling. However, there has been minimal mechanistic insight into how CD151 affects integrin functions. In MDA-MB-231 mammary cells, tetraspanin CD151 knockdown impairs α6 integrin clustering and functions without decreasing α6 integrin expression or activation. Furthermore, CD151 knockdown minimally affects the magnitude of α6 integrin diffusion, as measured using single particle tracking. Instead, CD151 knockdown has a novel and unexpected dysregulating effect on the mode of α6 integrin diffusion. In control cells α6 integrin shows mostly random-confined diffusion (RCD) and some directed motion (DMO). In sharp contrast, in CD151-knockdown cells α6 integrin shows mostly DMO. In control cells α6 diffusion mode is sensitive to actin disruption, talin knockdown and phorbol ester stimulation. By contrast, CD151 knockdown cell α6 integrin is sensitive to actin disruption but desensitized to talin knockdown or phorbol ester stimulation, indicating dysregulation. Both phorbol ester and EGF stimulate cell spreading and promote α6 RCD in control cells. By contrast, CD151-ablated cells retain EGF effects but lose phorbol-ester-stimulated spreading and α6 RCD. For α6 integrins, physical association with CD151 promotes α6 RCD, in support of α6-mediated cable formation and adhesion. By comparison, for integrins not associated with CD151 (e.g. αv integrins), CD151 affects neither diffusion mode nor αv function. Hence, CD151 support of α6 RCD is specific and functionally relevant, and probably underlies diverse CD151 functions in skin, kidney and cancer cells.


Assuntos
Integrina alfa6/metabolismo , Tetraspanina 24/fisiologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Feminino , Humanos , Integrina alfa6/genética , Distribuição Aleatória , Tetraspanina 24/genética
2.
Blood ; 112(7): 2770-9, 2008 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-18641366

RESUMO

Vascular endothelial-cadherin (VE-cad) is localized to adherens junctions at endothelial cell borders and forms a complex with alpha-, beta-, gamma-, and p120-catenins (p120). We previously showed that the VE-cad complex disassociates to form short-lived "gaps" during leukocyte transendothelial migration (TEM); however, whether these gaps are required for leukocyte TEM is not clear. Recently p120 has been shown to control VE-cad surface expression through endocytosis. We hypothesized that p120 regulates VE-cad surface expression, which would in turn have functional consequences for leukocyte transmigration. Here we show that endothelial cells transduced with an adenovirus expressing p120GFP fusion protein significantly increase VE-cad expression. Moreover, endothelial junctions with high p120GFP expression largely prevent VE-cad gap formation and neutrophil leukocyte TEM; if TEM occurs, the length of time required is prolonged. We find no evidence that VE-cad endocytosis plays a role in VE-cad gap formation and instead show that this process is regulated by changes in VE-cad phosphorylation. In fact, a nonphosphorylatable VE-cad mutant prevented TEM. In summary, our studies provide compelling evidence that VE-cad gap formation is required for leukocyte transmigration and identify p120 as a critical intracellular mediator of this process through its regulation of VE-cad expression at junctions.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Quimiotaxia de Leucócito , Leucócitos/citologia , Leucócitos/metabolismo , Fosfoproteínas/metabolismo , Cateninas , Células Cultivadas , Endocitose , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio/metabolismo , Junções Comunicantes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Meia-Vida , Humanos , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , delta Catenina
3.
J Immunol ; 177(9): 6440-9, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17056576

RESUMO

Endothelial cell ICAM-1 interacts with leukocyte beta(2) integrins to mediate adhesion and transmit outside-in signals that facilitate leukocyte transmigration. ICAM-1 redistribution and clustering appear necessary for leukocyte transmigration, but the mechanisms controlling ICAM-1 redistribution and clustering have not been identified. We recently reported that Src kinase phosphorylation of endothelial cortactin regulates polymorphonuclear cell (PMN) transmigration. In this study, we tested the hypotheses that the Src family kinase-cortactin pathway mediates association of ICAM-1 with the actin cytoskeleton and that this association is required for ICAM-1 clustering and leukocyte transmigration. Cross-linking ICAM-1 induced cytoskeletal remodeling and a decrease in ICAM-1 lateral mobility, as assessed by fluorescence recovery after photobleaching. Cytoskeletal remodeling after ICAM-1 cross-linking was reduced by knockdown of cortactin by small interfering RNA, by expression of a cortactin mutant deficient in Src phosphorylation sites (cortactin3F), and by the Src kinase inhibitor PP2. Pretreatment of cytokine-activated human endothelial monolayers with cortactin small interfering RNA significantly decreased both actin and ICAM-1 clustering around adherent PMN and the formation of actin-ICAM-1 clusters required for PMN transmigration. Our data suggest a model in which tyrosine phosphorylation of cortactin dynamically links ICAM-1 to the actin cytoskeleton, enabling ICAM-1 to form clusters and facilitate leukocyte transmigration.


Assuntos
Citoesqueleto de Actina/metabolismo , Adesão Celular , Movimento Celular , Cortactina/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Neutrófilos/imunologia , Actinas/metabolismo , Adesão Celular/genética , Movimento Celular/genética , Células Cultivadas , Cortactina/antagonistas & inibidores , Cortactina/genética , Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Fosforilação , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Tirosina , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
4.
J Cell Biol ; 169(6): 871-84, 2005 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-15967811

RESUMO

Stress granules (SGs) are cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress. GW bodies/processing bodies (PBs) are distinct cytoplasmic sites of mRNA degradation. In this study, we show that SGs and PBs are spatially, compositionally, and functionally linked. SGs and PBs are induced by stress, but SG assembly requires eIF2alpha phosphorylation, whereas PB assembly does not. They are also dispersed by inhibitors of translational elongation and share several protein components, including Fas-activated serine/threonine phosphoprotein, XRN1, eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, eIF4G, and PABP-1 are restricted to SGs, whereas DCP1a and 2 are confined to PBs. SGs and PBs also can harbor the same species of mRNA and physically associate with one another in vivo, an interaction that is promoted by the related mRNA decay factors TTP and BRF1. We propose that mRNA released from disassembled polysomes is sorted and remodeled at SGs, from which selected transcripts are delivered to PBs for degradation.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Estresse Fisiológico/metabolismo , Animais , Células COS , Chlorocebus aethiops , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/ultraestrutura , Fator de Iniciação 2 em Eucariotos/genética , Células HeLa , Humanos , Polirribossomos/genética , Polirribossomos/metabolismo , Biossíntese de Proteínas/genética , Transporte Proteico/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Proc Natl Acad Sci U S A ; 101(24): 8969-74, 2004 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-15184684

RESUMO

The development of more selective immunosuppressive agents to mitigate transplant rejection and autoimmune diseases requires effective strategies of blocking signaling pathways in T cells. Current immunosuppressive strategies use cyclosporin A (CsA) or FK506 to inhibit calcineurin, which dephosphorylates and promotes the nuclear import of nuclear factor of activated T cells (NFAT) transcription factors. These nuclear NFATs then transactivate cytokine genes that regulate proliferative responses of T cells. Both CsA and FK506 have debilitating side effects, including nephrotoxicity, hypertension, diabetes, and seizures, that argue for the development of alternative or complementary agents. To this end, we developed cell-based assays for monitoring NFAT dynamics in nonlymphoid cells to identify small molecules that inhibit NFAT nuclear import. Interestingly, we found that the majority of these small molecules suppress NFAT signaling by interfering with "capacitative" or "store-operated" calcium mobilization, thus raising the possibility that such mobilization processes are relevant targets in immunosuppression therapy. Further, these small molecules also show dose-dependent suppression of cytokine gene expression in T cells. Significantly, the IC(50) of CsA in primary T cells was reduced by the addition of suboptimal concentrations of these compounds, suggesting the possibility that such small molecules, in combination with CsA, offer safer means of immunosuppression.


Assuntos
Cálcio/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Imunossupressores/farmacologia , Proteínas Nucleares , Fatores de Transcrição/antagonistas & inibidores , Animais , Calcineurina/metabolismo , Cálcio/química , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Linhagem Celular , Núcleo Celular/metabolismo , Técnicas de Química Combinatória/métodos , Cricetinae , Ciclosporina/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Concentração Inibidora 50 , Interleucina-2/biossíntese , Camundongos , Fatores de Transcrição NFATC , Compostos Orgânicos/química , Compostos Orgânicos/farmacologia , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Biochemistry ; 42(40): 11716-25, 2003 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-14529282

RESUMO

The endothelial isoform of nitric oxide synthase (eNOS), a key regulator of vascular tone, is activated in endothelial cells by diverse Ca(2+)-mobilizing agonists, including vascular endothelial growth factor (VEGF). Although the activation state of eNOS and the subcellular localization of the enzyme are both highly regulated, the relationship between enzyme activity and subcellular targeting remains obscure. We aim here to elucidate this relationship by direct dynamic imaging analysis of Ca(2+)/CaM-dependent eNOS activation in living endothelial cells, using high-resolution confocal microscopy and donor dequenching fluorescence resonance energy transfer (FRET) techniques. Confocal images show a complex pattern of eNOS subcellular distribution; the enzyme is concentrated in both the plasma membrane and internal membranes, with robust expression in the perinuclear region. We construct a fusion protein between eNOS and the FRET-based calcium sensor cameleon, and analyze the temporal and spatial pattern of VEGF-mediated calcium mobilization using donor dequenching FRET methods. We find that VEGF promotes rapid mobilization of intracellular calcium throughout the regions of the cell in which eNOS is distributed. We further create a series of fusion proteins and use FRET imaging methods to study the interactions between eNOS and its obligate allosteric activator protein calmodulin. We clone the FRET acceptor EYFP (enhanced yellow fluorescent protein) at the C-terminus of calmodulin, and the FRET donor ECFP (enhanced cyan fluorescent protein) into eNOS at a site adjacent to its calmodulin-binding domain. FRET imaging analysis of individual endothelial cells cotransfected with eNOS-ECFP and calmodulin-EYFP shows that VEGF induces interactions between eNOS and calmodulin wherever both are present in the cell. Our studies provide evidence that the pool of rapidly responsive receptor-activated eNOS is distributed throughout endothelial cells in both plasma membrane and internal membrane structures, and that this distribution parallels the localization of agonist-induced intracellular Ca(2+) changes in the vicinity of eNOS.


Assuntos
Calmodulina/metabolismo , Endotélio Vascular/enzimologia , Óxido Nítrico Sintase/metabolismo , Receptores de Superfície Celular/fisiologia , Animais , Proteínas de Bactérias/genética , Células COS , Sinalização do Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Calmodulina/genética , Bovinos , Linhagem Celular , Chlorocebus aethiops , Proteínas de Drosophila/genética , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde , Líquido Intracelular/enzimologia , Líquido Intracelular/metabolismo , Proteínas Luminescentes/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo III , Proteínas Recombinantes de Fusão/genética , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismo , Transfecção , Fator A de Crescimento do Endotélio Vascular/fisiologia
7.
Proc Natl Acad Sci U S A ; 99(10): 6907-12, 2002 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-11997458

RESUMO

Immune cells are activated during cellular responses to antigen by two described mechanisms: (i) direct uptake of antigen and (ii) extraction and internalization of membrane components from antigen-presenting cells. Although endocytosis of microbial antigens by pattern recognition molecules (PRM) also activates innate immunity, it is not known whether this involves extraction and internalization of microbial surface components. Epithelial cells on mucosal surfaces use a variety of receptors that are distinct from the classical endocytic PRM to bind and internalize intact microorganisms. Nonclassical receptor molecules theoretically could act as a type of endocytic PRM if these molecules could recognize, bind, extract, and internalize a pathogen-associated molecule and initiate cell signaling. We report here that the interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the outer core oligosaccharide of the lipopolysaccharide (LPS) in the outer membrane of Pseudomonas aeruginosa satisfies all of these conditions. P. aeruginosa LPS was specifically recognized and bound by CFTR, extracted from the organism's surface, and endocytosed by epithelial cells, leading to a rapid (5- to 15-min) and dynamic translocation of nuclear transcription factor NF-kappa B. Inhibition of epithelial cell internalization of P. aeruginosa LPS prevented NF-kappa B activation. Cellular activation depended on expression of wild-type CFTR, because both cultured Delta F508 CFTR human airway epithelial cells and lung epithelial cells of transgenic-CF mice failed to endocytose LPS and translocate NF-kappa B. CFTR serves as a critical endocytic PRM in the lung epithelium, coordinating the effective innate immune response to P. aeruginosa infection.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Lipopolissacarídeos/imunologia , NF-kappa B/imunologia , Pseudomonas aeruginosa/imunologia , Mucosa Respiratória/imunologia , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular/imunologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Humanos , Bicamadas Lipídicas , Camundongos , Camundongos Endogâmicos C3H , Mucosa Respiratória/citologia
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