RESUMO
Antibody production depends on B cell internalization and presentation of antigens to helper T cells. To acquire antigens displayed by antigen-presenting cells, B cells form immune synapses and extract antigens by the mechanical activity of the acto-myosin cytoskeleton. While cytoskeleton organization driving the initial formation of the B cell synapse has been studied, how the cytoskeleton supports antigen extraction remains poorly understood. Here we show that after initial cell spreading, F-actin in synapses of primary mouse B cells and human B cell lines forms a highly dynamic pattern composed of actin foci interspersed with linear filaments and myosin IIa. The foci are generated by Arp2/3-mediated branched-actin polymerization and stochastically associate with antigen clusters to mediate internalization. However, antigen extraction also requires the activity of formins, which reside near the foci and produce the interspersed filaments. Thus, a cooperation of branched-actin foci supported by linear filaments underlies B cell mechanics during antigen extraction.
Assuntos
Citoesqueleto de Actina/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Actinas/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Formação de Anticorpos/imunologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos/metabolismo , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Sinapses Imunológicas/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
αMß2 integrin (complement receptor 3) is a major receptor for phagocytosis in macrophages. In other contexts, integrins' activities and functions are mechanically linked to actin dynamics through focal adhesions. We asked whether mechanical coupling of αMß2 integrin to the actin cytoskeleton mediates phagocytosis. We found that particle internalization was driven by formation of Arp2/3 and formin-dependent actin protrusions that wrapped around the particle. Focal complex-like adhesions formed in the phagocytic cup that contained ß2 integrins, focal adhesion proteins and tyrosine kinases. Perturbation of talin and Syk demonstrated that a talin-dependent link between integrin and actin and Syk-mediated recruitment of vinculin enable force transmission to target particles and promote phagocytosis. Altering target mechanical properties demonstrated more efficient phagocytosis of stiffer targets. Thus, macrophages use tyrosine kinase signalling to build a mechanosensitive, talin- and vinculin-mediated, focal adhesion-like molecular clutch, which couples integrins to cytoskeletal forces to drive particle engulfment.
Assuntos
Macrófagos/imunologia , Mecanotransdução Celular , Fagocitose/imunologia , Quinase Syk/genética , Talina/genética , Vinculina/genética , Citoesqueleto de Actina/imunologia , Citoesqueleto de Actina/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Actinas/genética , Actinas/imunologia , Animais , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Adesões Focais/imunologia , Adesões Focais/ultraestrutura , Forminas/genética , Forminas/imunologia , Regulação da Expressão Gênica , Humanos , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/imunologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Fagossomos/imunologia , Fagossomos/ultraestrutura , Poliestirenos , Cultura Primária de Células , Células RAW 264.7 , Quinase Syk/imunologia , Células THP-1 , Talina/imunologia , Vinculina/imunologiaRESUMO
Leukocytes are constantly produced in the bone marrow and released into the circulation. Many different leukocyte subpopulations exist that exert distinct functions. Leukocytes are recruited to sites of inflammation and combat the cause of inflammation via many different effector functions. Virtually all of these processes depend on dynamic actin remodeling allowing leukocytes to adhere, migrate, phagocytose, and release granules. However, actin dynamics are not possible without actin-binding proteins (ABP) that orchestrate the balance between actin polymerization, branching, and depolymerization. The homologue of the ubiquitous ABP cortactin in hematopoietic cells is hematopoietic cell-specific lyn substrate-1, often called hematopoietic cell-specific protein-1 (HCLS1 or HS1). HS1 has been reported in different leukocytes to regulate Arp2/3-dependent migration. However, more evidence is emerging that HS1 functions go far beyond just being a direct actin modulator. For example, HS1 is important for the activation of GTPases and integrins, and mediates signaling downstream of many receptors including BCR, TCR, and CXCR4. In this review, we summarize current knowledge on HS1 functions and discuss them in a pathophysiologic context.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/genética , Leucemia/genética , Leucócitos/imunologia , Quinases da Família src/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Actinas/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Adesão Celular , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Movimento Celular , Proliferação de Células , Cortactina/genética , Cortactina/imunologia , Regulação da Expressão Gênica , Humanos , Leucemia/imunologia , Leucemia/patologia , Leucócitos/classificação , Leucócitos/patologia , Fagocitose , Ligação Proteica , Transdução de Sinais , Quinases da Família src/imunologiaRESUMO
Cell-mediated transmission and dissemination of sexually-acquired human immunodeficiency virus 1 (HIV-1) in the host involves the migration of immature dendritic cells (iDCs). iDCs migrate in response to the HIV-1 envelope protein, gp120, and inhibiting such migration may limit the mucosal transmission of HIV-1. In this study, we elucidated the mechanism of HIV-1-gp120-induced transendothelial migration of iDCs. We found that gp120 enhanced the binding of Wiskott-Aldrich Syndrome protein (WASp) and the Actin-Related Protein 2/3 (Arp2/3) complex with ß-actin, an interaction essential for the proper formation of podosomes, specialized adhesion structures required for the migration of iDCs through different tissues. We further identified Leukocyte-Specific Protein 1 (LSP1) as a novel component of the WASp-Arp2/3-ß-actin complex. Pretreating iDCs with an active fragment of the secretory glycoprotein Slit2 (Slit2N) inhibited HIV-1-gp120-mediated migration and podosome formation, by inducing the cognate receptor Roundabout 1 (Robo1) to bind to and sequester WASp and LSP1 from ß-actin. Slit2N treatment also inhibited Src signaling and the activation of several downstream molecules, including Rac1, Pyk2, paxillin, and CDC42, a major regulator of podosome formation. Taken together, our results support a novel mechanism by which Slit2/Robo1 may inhibit the HIV-1-gp120-induced migration of iDCs, thereby restricting dissemination of HIV-1 from mucosal surfaces in the host.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Proteínas dos Microfilamentos/imunologia , Proteínas do Tecido Nervoso/imunologia , Receptores Imunológicos/imunologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/imunologia , Actinas/metabolismo , Western Blotting , Células Cultivadas , Células Dendríticas/metabolismo , Quinase 2 de Adesão Focal/imunologia , Quinase 2 de Adesão Focal/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Proteínas do Tecido Nervoso/metabolismo , Paxilina/imunologia , Paxilina/metabolismo , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas pp60(c-src)/imunologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Pseudópodes/imunologia , Interferência de RNA , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/imunologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas RoundaboutRESUMO
WASP, the product of the gene mutated in Wiskott-Aldrich syndrome, is expressed only in hematopoietic cells and is the archetype of a family of proteins that include N-WASP and Scar/WAVE. WASP plays a critical role in T cell activation and actin reorganization. WASP has multiple protein-interacting domains. Through its N-terminal EVH1 domain WASP binds to its partner WASP interacting protein (WIP) and through its C-terminal end it interacts with and activates the Arp2/3 complex. In lymphocytes, most of WASP is sequestered with WIP and binding to WIP is essential for the stability of WASP. The central proline-rich region of WASP serves as docking site to several adaptor proteins. Through these multiple interactions WASP integrates many cellular signals to actin cytoskeleton remodeling. In this review, we have summarized recent developments in the biology of WASP and the role of WIP in regulating WASP function. We also discuss WASP-independent functions of WIP.