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
Cytotoxic T lymphocytes (CTLs) kill by forming immunological synapses with target cells and secreting toxic proteases and the pore-forming protein perforin into the intercellular space. Immunological synapses are highly dynamic structures that boost perforin activity by applying mechanical force against the target cell. Here, we used high-resolution imaging and microfabrication to investigate how CTLs exert synaptic forces and coordinate their mechanical output with perforin secretion. Using micropatterned stimulatory substrates that enable synapse growth in three dimensions, we found that perforin release occurs at the base of actin-rich protrusions that extend from central and intermediate locations within the synapse. These protrusions, which depended on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, were required for synaptic force exertion and efficient killing. They also mediated physical deformation of the target cell surface during CTL-target cell interactions. Our results reveal the mechanical basis of cellular cytotoxicity and highlight the functional importance of dynamic, three-dimensional architecture in immune cell-cell interfaces.
Assuntos
Sinapses Imunológicas/imunologia , Perforina/imunologia , Linfócitos T Citotóxicos/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Actinas/imunologia , Animais , Camundongos , Proteína da Síndrome de Wiskott-Aldrich/imunologiaRESUMO
T cell antigen receptor (TCR) signaling triggers selective cytokine expression to drive T cell proliferation and differentiation required for immune defense and surveillance. The nuclear signaling events responsible for specificity in cytokine gene expression upon T cell activation are largely unknown. Here, we uncover formation of a dynamic actin filament network in the nucleus that regulates cytokine expression for effector functions of CD4+ T lymphocytes. TCR engagement triggers the rapid and transient formation of a nuclear actin filament network via nuclear Arp2/3 complex, induced by elevated nuclear Ca2+ levels and regulated via N-Wasp and NIK. Specific interference with TCR-induced formation of nuclear actin filaments impairs production of effector cytokines and prevents generation of antigen-specific antibodies but does not interfere with immune synapse formation and cell proliferation. Ca2+-regulated actin polymerization in the nucleus allows CD4+ T cells the rapid conversion of TCR signals into effector functions required for T cell help.
Assuntos
Actinas/imunologia , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Transferência Adotiva , Animais , Linhagem Celular , Humanos , CamundongosRESUMO
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
Migration is crucial for the function of dendritic cells (DCs), which act as outposts of the immune system. Upon detection of pathogens, skin- and mucosa-resident DCs migrate to secondary lymphoid organs where they activate T cells. DC motility relies critically on the actin cytoskeleton, which is regulated by the actin-related protein 2/3 (ARP2/3) complex, a nucleator of branched actin networks. Consequently, loss of ARP2/3 stimulators and upstream Rho family GTPases dramatically impairs DC migration. However, nothing is known yet about the relevance of ARP2/3 inhibitors for DC migration. We previously demonstrated that the AP-1-associated adaptor protein Gadkin inhibits ARP2/3 by sequestering it on intracellular vesicles. Consistent with a role of Gadkin in DC physiology, we here report Gadkin expression in bone marrow-derived DCs and show that its protein level and posttranslational modification are regulated upon LPS-induced DC maturation. DCs derived from Gadkin-deficient mice were normal with regards to differentiation and maturation, but displayed increased actin polymerization. While the actin-dependent processes of macropinocytosis and cell spreading were not affected, loss of Gadkin significantly impaired DC migration in vitro, however, in vivo DC migration was unperturbed suggesting the presence of compensatory mechanisms.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Proteínas de Membrana/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/imunologia , Actinas/metabolismo , Animais , Comunicação Celular/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/metabolismo , Fenômenos do Sistema Imunitário/imunologia , Ativação Linfocitária/imunologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Transcrição AP-1/imunologia , Fator de Transcrição AP-1/metabolismo , Proteínas rho de Ligação ao GTP/imunologia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
T lymphocytes are highly motile cells that decelerate upon antigen recognition. These cells can either completely stop or maintain a low level of motility, forming contacts referred to as synapses or kinapses, respectively. Whether similar or distinct molecular mechanisms regulate T-cell deceleration during synapses or kinapses is unclear. Here, we used microfabricated channels and intravital imaging to observe and manipulate T-cell kinapses and synapses. We report that high-affinity antigen induced a pronounced deceleration selectively dependent on Ca(2+) signals and actin-related protein 2/3 complex (Arp2/3) activity. In contrast, low-affinity antigens induced a switch of migration mode that promotes T-cell exploratory behavior, characterized by partial deceleration and frequent direction changes. This switch depended on T-cell receptor binding but was largely independent of downstream signaling. We propose that distinct mechanisms of T-cell deceleration can be triggered during antigenic recognition to favor local exploration and signal integration upon suboptimal stimulus and complete arrest on the best antigen-presenting cells.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Movimento Celular/imunologia , Microambiente Celular/imunologia , Imunidade Celular/imunologia , Linfócitos T/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Análise de Variância , Animais , Cálcio/metabolismo , Citometria de Fluxo , Sinapses Imunológicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estatísticas não ParamétricasRESUMO
Defects in Wiskott-Aldrich Syndrome protein (WASp) underlie development of WAS, an X-linked immunodeficiency and autoimmunity disorder of childhood. Nucleation-promoting factors (NPFs) of the WASp family generate F-actin in the cytosol via the VCA (verprolin-homology, cofilin-homology, and acidic) domain and support RNA polymerase II-dependent transcription in the nucleus. Whether nuclear-WASp requires the integration of its actin-related protein (ARP)2/3-dependent cytoplasmic function to reprogram gene transcription, however, remains unresolved. Using the model of human TH cell differentiation, we find that WASp has a functional nuclear localizing and nuclear exit sequences, and accordingly, its effects on transcription are controlled mainly at the level of its nuclear entry and exit via the nuclear pore. Human WASp does not use its VCA-dependent, ARP2/3-driven, cytoplasmic effector mechanisms to support histone H3K4 methyltransferase activity in the nucleus of TH1-skewed cells. Accordingly, an isolated deficiency of nuclear-WASp is sufficient to impair the transcriptional reprogramming of TBX21 and IFNG promoters in TH1-skewed cells, whereas an isolated deficiency of cytosolic-WASp does not impair this process. In contrast, nuclear presence of WASp in TH2-skewed cells is small, and its loss does not impair transcriptional reprogramming of GATA3 and IL4 promoters. Our study unveils an ARP2/3:VCA-independent function of nuclear-WASp in TH1 gene activation that is uncoupled from its cytoplasmic role in actin polymerization.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Actinas/imunologia , Núcleo Celular/imunologia , Células Th1/imunologia , Células Th2/imunologia , Transcrição Gênica/imunologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/genética , Núcleo Celular/genética , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/imunologia , Células HeLa , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Células Jurkat , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologia , Transcrição Gênica/genética , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
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.
Assuntos
Linfócitos B/imunologia , Proteínas do Citoesqueleto/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Linfócitos T Reguladores/imunologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia , 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 , Animais , Linfócitos B/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Estrutura Terciária de Proteína/fisiologia , Linfócitos T Reguladores/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/imunologia , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
T cell receptor (TCR)-mediated cytoskeletal reorganization is considered to be actin-related protein (Arp) 2/3 complex dependent. We therefore examined the requirement for Arp2/3- and formin-dependent F-actin nucleation during T cell activation. We demonstrated that without Arp2/3-mediated actin nucleation, stimulated T cells could not form an F-actin-rich lamellipod, but instead produced polarized filopodia-like structures. Moreover, the microtubule-organizing center (MTOC, or centrosome), which rapidly reorients to the immunological synapse through an unknown mechanism, polarized in the absence of Arp2/3. Conversely, the actin-nucleating formins, Diaphanous-1 (DIA1) and Formin-like-1 (FMNL1), did not affect TCR-stimulated F-actin-rich structures, but instead displayed unique patterns of centrosome colocalization and controlled TCR-mediated centrosome polarization. Depletion of FMNL1 or DIA1 in cytotoxic lymphocytes abrogated cell-mediated killing. Altogether, our results have identified Arp2/3 complex-independent cytoskeletal reorganization events in T lymphocytes and indicate that formins are essential cytoskeletal regulators of centrosome polarity in T cells.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Polaridade Celular/imunologia , Centrossomo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Actinas/imunologia , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Centrossomo/imunologia , Centrossomo/ultraestrutura , Proteínas do Citoesqueleto/imunologia , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Imunofluorescência , Forminas , Humanos , Immunoblotting , Imunoprecipitação , Células Jurkat , Microscopia Eletrônica de Varredura , Linfócitos T/imunologia , Linfócitos T/ultraestrutura , TransfecçãoRESUMO
Reorganization of the actin cytoskeleton requires nucleating factors, which initiate actin fiber formation. In this issue of Immunity, the functions of two classes of actin nucleators are examined in T lymphocytes, giving insight into the regulation of T cell polarity (Gomez et al., 2007).