RESUMO
Although RNA aptamers can show comparable or better specificity and affinity to antibodies and have the advantage of being able to access different live cell compartments, they are often much less stable in vivo. We report here the first aptamer that binds human retinoblastoma protein (RB) and is stable in live cells. RB is both a key protein in cell cycle control and also a tumour suppressor. The aptamer was selected from an RNA library against a unique 12-residue helical peptide derived from RB rather than the whole protein molecule. It binds RB with high affinity (K d = 5.1 ± 0.1 nM) and is a putative RNA G-quadruplex structure formed by an 18-nucleotide sequence (18E16 - GGA GGG UGG AGG GAA GGG), which may account for its high stability. Confocal fluorescence microscopy of live cells transfected with the aptamer shows it is stable intracellularly and efficient in entering the nucleus where an analogous antibody was inaccessible. The findings demonstrate this aptamer is an advanced probe for RB in live cell applications.
RESUMO
In order to mount high-affinity antibody responses, B cells internalise specific antigens and process them into peptides loaded onto MHCII for presentation to T helper cells (TH cells). While the biochemical principles of antigen processing and MHCII loading have been well dissected, how the endosomal vesicle system is wired to enable these specific functions remains much less studied. Here, we performed a systematic microscopy-based analysis of antigen trafficking in B cells to reveal its route to the MHCII peptide-loading compartment (MIIC). Surprisingly, we detected fast targeting of internalised antigen into peripheral acidic compartments that possessed the hallmarks of the MIIC and also showed degradative capacity. In these vesicles, internalised antigen converged rapidly with membrane-derived MHCII and partially overlapped with cathepsin-S and H2-M, both required for peptide loading. These early compartments appeared heterogenous and atypical as they contained a mixture of both early and late endosomal markers, indicating a specialized endosomal route. Together, our data suggest that, in addition to in the previously reported perinuclear late endosomal MIICs, antigen processing and peptide loading could have already started in these specialized early peripheral acidic vesicles (eMIIC) to support fast peptide-MHCII presentation.
Assuntos
Apresentação de Antígeno , Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Transferência Adotiva , Animais , Linfócitos B/citologia , Endossomos/metabolismo , Feminino , Humanos , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transporte Proteico , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
During B-cell activation, the dynamic reorganisation of the cytoskeleton is crucial for multiple cellular responses, such as receptor signalling, cell spreading, antigen internalisation, intracellular trafficking, and antigen presentation. However, the role of intermediate filaments (IFs), which represent a major component of the mammalian cytoskeleton, is not well defined. Here, by using multiple super-resolution microscopy techniques, including direct stochastic optical reconstruction microscopy, we show that IFs in B cells undergo drastic reorganisation immediately upon antigen stimulation and that this reorganisation requires actin and microtubules. Although the loss of vimentin in B cells did not impair B-cell development, receptor signalling, and differentiation, vimentin-deficient B cells exhibit altered positioning of antigen-containing and lysosomal associated membrane protein 1 (LAMP1+) compartments, implying that vimentin may play a role in the fine-tuning of intracellular trafficking. Indeed, vimentin-deficient B cells exhibit impaired antigen presentation and delayed antibody responses in vivo. Thus, our study presents a new perspective on the role of IFs in B-cell activation.
RESUMO
Wiskott-Aldrich syndrome protein (WASp) is a main cytoskeletal regulator in B cells. WASp-interacting protein (WIP) binds to and stabilizes WASp but also interacts with actin. Using mice with a mutated actin binding domain of WIP (WIPΔABD), we here investigated the role of WIP binding to actin during B cell activation. We found an altered differentiation of WIPΔABD B cells and diminished antibody affinity maturation after immunization. Mechanistically, WIPΔABD B cells showed impaired B cell receptor (BCR)-induced PI3K signaling and actin reorganization, likely caused by diminished CD81 expression and altered CD19 dynamics on the B cell surface. WIPΔABD B cells displayed reduced in vivo motility, concomitantly with impaired chemotaxis and defective F-actin polarization, HS1 phosphorylation, and polarization of HS1 to F-actin-rich structures after CXCL12 stimulation in vitro. We thus concluded that WIP binding to actin, independent of its binding to WASp, is critical for actin cytoskeleton plasticity in B cells.
Assuntos
Actinas/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Movimento Celular , Imunidade Humoral , Animais , Afinidade de Anticorpos , Antígenos CD/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Polaridade Celular , Quimiotaxia , Proteínas do Citoesqueleto , Difusão , Centro Germinativo/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de SinaisRESUMO
PKCß-null (Prkcb-/-) mice are severely immunodeficient. Here we show that mice whose B cells lack PKCß failed to form germinal centers and plasma cells, which undermined affinity maturation and antibody production in response to immunization. Moreover, these mice failed to develop plasma cells in response to viral infection. At the cellular level, we have shown that Prkcb-/- B cells exhibited defective antigen polarization and mTORC1 signaling. While altered antigen polarization impaired antigen presentation and likely restricted the potential of GC development, defective mTORC1 signaling impaired metabolic reprogramming, mitochondrial remodeling, and heme biosynthesis in these cells, which altogether overwhelmingly opposed plasma cell differentiation. Taken together, our study reveals mechanistic insights into the function of PKCß as a key regulator of B cell polarity and metabolic reprogramming that instructs B cell fate.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Ativação Linfocitária/imunologia , Plasmócitos/imunologia , Proteína Quinase C beta/imunologia , Animais , Heme/biossíntese , Camundongos , Camundongos Knockout , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Plasmócitos/citologiaRESUMO
Wiskott-Aldrich syndrome (WAS) is an immune pathology associated with mutations in WAS protein (WASp) or in WASp interacting protein (WIP). Together with the small GTPase Cdc42 and other effectors, these proteins participate in the remodelling of the actin network downstream of BCR engagement. Here we show that mice lacking the adaptor protein ITSN2, a G-nucleotide exchange factor (GEF) for Cdc42 that also interacts with WASp and WIP, exhibited increased mortality during primary infection, incomplete protection after Flu vaccination, reduced germinal centre formation and impaired antibody responses to vaccination. These defects were found, at least in part, to be intrinsic to the B cell compartment. In vivo, ITSN2 deficient B cells show a reduction in the expression of SLAM, CD84 or ICOSL that correlates with a diminished ability to form long term conjugates with T cells, to proliferate in vivo, and to differentiate into germinal centre cells. In conclusion, our study not only revealed a key role for ITSN2 as an important regulator of adaptive immune-response during vaccination and viral infection but it is also likely to contribute to a better understanding of human immune pathologies.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Linfócitos B/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/patologia , Orthomyxoviridae/imunologia , Linfócitos T/imunologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Adesão Celular , Proliferação de Células , Vacinas contra Influenza/administração & dosagem , Camundongos , Análise de SobrevidaRESUMO
B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunidade Inata , Influenza Humana/imunologia , Interleucina-4/genética , Células Matadoras Naturais/imunologia , Infecção por Zika virus/imunologia , Animais , Galinhas , Cães , Centro Germinativo/citologia , Humanos , Interleucina-4/metabolismo , Macaca , Macrófagos/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Autophagy is important in a variety of cellular and pathophysiological situations; however, its role in immune responses remains elusive. Here, we show that among B cells, germinal center (GC) cells exhibited the highest rate of autophagy during viral infection. In contrast to mechanistic target of rapamycin complex 1-dependent canonical autophagy, GC B cell autophagy occurred predominantly through a noncanonical pathway. B cell stimulation was sufficient to down-regulate canonical autophagy transiently while triggering noncanonical autophagy. Genetic ablation of WD repeat domain, phosphoinositide-interacting protein 2 in B cells alone enhanced this noncanonical autophagy, resulting in changes of mitochondrial homeostasis and alterations in GC and antibody-secreting cells. Thus, B cell activation prompts a temporal switch from canonical to noncanonical autophagy that is important in controlling B cell differentiation and fate.
Assuntos
Autofagia/imunologia , Linfócitos B/imunologia , Linfócitos B/virologia , Viroses/imunologia , Animais , Regulação para Baixo , Centro Germinativo/imunologia , Centro Germinativo/virologia , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Repetições WD40/genéticaRESUMO
Receptor organization and dynamics at the cell membrane are important factors of signal transduction regulation. Using super-resolution microscopy and single-particle tracking, we show how the negative coreceptor CD22 works with the cortical cytoskeleton in restraining BCR signalling. In naïve B cells, we found endogenous CD22 to be highly mobile and organized into nanodomains. The landscape of CD22 and its lateral diffusion were perturbed either in the absence of CD45 or when the CD22 lectin domain was mutated. To understand how a relatively low number of CD22 molecules can keep BCR signalling in check, we generated Brownian dynamic simulations and supported them with ex vivo experiments. This combined approach suggests that the inhibitory function of CD22 is influenced by its nanoscale organization and is ensured by its fast diffusion enabling a "global BCR surveillance" at the plasma membrane.
Assuntos
Linfócitos B/fisiologia , Citoesqueleto/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais , Animais , Linfócitos B/citologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de FluorescênciaRESUMO
Cancer-associated fibroblasts (CAFs) are non-cancerous cells found in solid tumors that remodel the tumor matrix and promote cancer invasion and angiogenesis. Here, we demonstrate that Cdc42EP3/BORG2 is required for the matrix remodeling, invasion, angiogenesis, and tumor-growth-promoting abilities of CAFs. Cdc42EP3 functions by coordinating the actin and septin networks. Furthermore, depletion of SEPT2 has similar effects to those of loss of Cdc42EP3, indicating a role for the septin network in the tumor stroma. Cdc42EP3 is upregulated early in fibroblast activation and precedes the emergence of the highly contractile phenotype characteristic of CAFs. Depletion of Cdc42EP3 in normal fibroblasts prevents their activation by cancer cells. We propose that Cdc42EP3 sensitizes fibroblasts to further cues-in particular, those activating actomyosin contractility-and thereby enables the generation of the pathological activated fibroblast state.
Assuntos
Fibroblastos/metabolismo , Fibroblastos/patologia , Reguladores de Proteínas de Ligação ao GTP/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Septinas/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Regulação para CimaRESUMO
Humans with Wiskott-Aldrich syndrome display a progressive immunological disorder associated with compromised Wiskott-Aldrich Syndrome Interacting Protein (WIP) function. Mice deficient in WIP recapitulate such an immunodeficiency that has been attributed to T cell dysfunction; however, any contribution of B cells is as yet undefined. Here we have shown that WIP deficiency resulted in defects in B cell homing, chemotaxis, survival, and differentiation, ultimately leading to diminished germinal center formation and antibody production. Furthermore, in the absence of WIP, several receptors, namely the BCR, BAFFR, CXCR4, CXCR5, CD40, and TLR4, were impaired in promoting CD19 co-receptor activation and subsequent PI3 kinase (PI3K) signaling. The underlying mechanism was due to a distortion in the actin and tetraspanin networks that lead to altered CD19 cell surface dynamics. In conclusion, our findings suggest that, by regulating the cortical actin cytoskeleton, WIP influences the function of CD19 as a general hub for PI3K signaling.
Assuntos
Antígenos CD19/fisiologia , Linfócitos B/imunologia , Proteínas de Transporte/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Transdução de Sinais/imunologia , Citoesqueleto de Actina/ultraestrutura , Actinas/análise , Animais , Formação de Anticorpos , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Linfócitos B/ultraestrutura , Proteínas de Transporte/genética , Células Cultivadas , Quimiocinas/farmacologia , Quimiocinas/fisiologia , Quimiotaxia/efeitos dos fármacos , Proteínas do Citoesqueleto , Centro Germinativo/imunologia , Centro Germinativo/patologia , Haptenos , Hemocianinas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfopoese , Proteínas de Membrana/imunologia , Camundongos , Fosforilação , Plasmócitos/imunologia , Processamento de Proteína Pós-Traducional , Quimera por Radiação , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Quimiocinas/fisiologia , Tetraspaninas/análise , Vacínia/imunologia , Vacínia/patologiaRESUMO
The layer of macrophages at the subcapsular sinus (SCS) captures pathogens entering the lymph node, preventing their global dissemination and triggering an immune response. However, how infection affects SCS macrophages remains largely unexplored. Here we show that infection and inflammation disrupt the organization of SCS macrophages in a manner that involves the migration of mature dendritic cells to the lymph node. This disrupted organization reduces the capacity of SCS macrophages to retain and present antigen in a subsequent secondary infection, resulting in diminished B cell responses. Thus, the SCS macrophage layer may act as a sensor or valve during infection to temporarily shut down the lymph node to further antigenic challenge. This shutdown may increase an organism's susceptibility to secondary infections.
Assuntos
Linfócitos B/imunologia , Movimento Celular/imunologia , Coinfecção/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Infecções Cutâneas Estafilocócicas/imunologia , Staphylococcus aureus , Animais , Antígenos/imunologia , Linfócitos B/patologia , Células Dendríticas/imunologia , Linfonodos/imunologia , Linfonodos/patologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The contractile actomyosin cytoskeleton and its connection to the plasma membrane are critical for control of cell shape and migration. We identify three STRIPAK complex components, FAM40A, FAM40B and STRN3, as regulators of the actomyosin cortex. We show that FAM40A negatively regulates the MST3 and MST4 kinases, which promote the co-localization of the contractile actomyosin machinery with the Ezrin/Radixin/Moesin family proteins by phosphorylating the inhibitors of PPP1CB, PPP1R14A-D. Using computational modelling, in vitro cell migration assays and in vivo breast cancer metastasis assays we demonstrate that co-localization of contractile activity and actin-plasma membrane linkage reduces cell speed on planar surfaces, but favours migration in confined environments similar to those observed in vivo. We further show that FAM40B mutations found in human tumours uncouple it from PP2A and enable it to drive a contractile phenotype, which may underlie its role in human cancer.
Assuntos
Autoantígenos/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Transporte/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Autoantígenos/genética , Neoplasias da Mama/genética , Proteínas de Ligação a Calmodulina/genética , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Biologia Computacional , Proteínas do Citoesqueleto/metabolismo , Drosophila melanogaster , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares , Metástase Neoplásica , Proteínas de Ligação a Fosfato , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Quinases Associadas a rho/metabolismoRESUMO
The small Rho GTPase Cdc42, known to interact with Wiskott-Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell-intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Imunidade Humoral/imunologia , Infecções por Orthomyxoviridae/imunologia , Proteína cdc42 de Ligação ao GTP/imunologia , Animais , Formação de Anticorpos/imunologia , Linfócitos B/metabolismo , Linfócitos B/ultraestrutura , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Citometria de Fluxo , Expressão Gênica/imunologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Imunidade Humoral/genética , Vírus da Influenza A/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Bacterial enhancer-dependent transcription systems support major adaptive responses and offer a singular paradigm in gene control analogous to complex eukaryotic systems. Here we report new mechanistic insights into the control of one-membrane stress-responsive bacterial enhancer-dependent system. Using millisecond single-molecule fluorescence microscopy of live cells we determine the localizations, two-dimensional diffusion dynamics and stoichiometries of complexes of the bacterial enhancer-binding ATPase PspF during its action at promoters as regulated by inner membrane interacting negative controller PspA. We establish that a stable repressive PspF-PspA complex is located in the nucleoid, transiently communicating with the inner membrane via PspA. The PspF as a hexamer stably binds only one of the two psp promoters at a time, suggesting that psp promoters will fire asynchronously and cooperative interactions of PspF with the basal transcription complex influence dynamics of the PspF hexamer-DNA complex and regulation of the psp promoters.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Viabilidade Microbiana , Transativadores/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase Sigma 54/metabolismo , Estresse Fisiológico , Transcrição GênicaRESUMO
A key role is emerging for the cytoskeleton in coordinating receptor signaling, although the underlying molecular requirements remain unclear. Here we show that cytoskeleton disruption triggered signaling requiring not only the B cell receptor (BCR), but also the coreceptor CD19 and tetraspanin CD81, thus providing a mechanism for signal amplification upon surface-bound antigen stimulation. By using superresolution microscopy, we demonstrated that endogenous IgM, IgD, and CD19 exhibited distinct nanoscale organization within the plasma membrane of primary B cells. Upon stimulation, we detect a local convergence of receptors, although their global organization was not dramatically altered. Thus, we postulate that cytoskeleton reorganization releases BCR nanoclusters, which can interact with CD19 held in place by the tetraspanin network. These results not only suggest that receptor compartmentalization regulates antigen-induced activation but also imply a potential role for CD19 in mediating ligand-independent "tonic" BCR signaling necessary for B cell survival.
Assuntos
Actinas/imunologia , Antígenos CD19/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Tetraspanina 28/imunologia , Actinas/metabolismo , Animais , Antígenos CD19/genética , Antígenos CD19/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Membrana Celular/imunologia , Membrana Celular/metabolismo , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Citometria de Fluxo , Immunoblotting , Imunoglobulina D/imunologia , Imunoglobulina D/metabolismo , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Modelos Imunológicos , Nanoestruturas , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Tetraspanina 28/genética , Tetraspanina 28/metabolismoRESUMO
During the activation of humoral immune responses, B cells acquire antigen for subsequent presentation to cognate T cells. Here we show that after mouse B cells accumulate antigen, it is maintained in a polarized distribution for extended periods in vivo. Using high-throughput imaging flow cytometry, we observed that this polarization is preserved during B cell division, promoting asymmetric antigen segregation among progeny. Antigen inheritance correlates with the ability of progeny to activate T cells: Daughter cells receiving larger antigen stores exhibit a prolonged capacity to present antigen, which renders them more effective in competing for T cell help. The generation of progeny with differential capacities for antigen presentation may have implications for somatic hypermutation and class switching during affinity maturation and as B cells commit to effector cell fates.
Assuntos
Apresentação de Antígeno , Antígenos/análise , Antígenos/imunologia , Linfócitos B/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Linfócitos B/citologia , Divisão Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Simulação por Computador , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Imunológicos , Muramidase/análise , Muramidase/imunologiaRESUMO
The B cell receptor (BCR) mediates B cell antigen gathering and acquisition for presentation to T cells. Although the amount of antigen presentation to T cells determines the extent of B cell activation, the molecular mechanisms underlying antigen gathering remain unexplored. Here, through a combination of high-resolution imaging, genetics and quantitative mass spectrometry, we demonstrate that adaptors Grb2 and Dok-3, and ubiquitin ligase Cbl in signaling BCR microclusters mediate association with the microtubule motor dynein. Furthermore, we visualize the localization and movement of these microclusters on the underlying microtubule network. Importantly, disruption of this network or diminished dynein recruitment in Grb2-, Dok-3-, or Cbl-deficient B cells, does not influence microcluster formation or actin-dependent spreading, but abrogates directed movement of microclusters and antigen accumulation. Thus we identify a surprising but pivotal role for dynein and the microtubule network alongside Grb2, Dok-3, and Cbl in antigen gathering during B cell activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Antígenos/imunologia , Dineínas/imunologia , Proteína Adaptadora GRB2/imunologia , Proteínas Proto-Oncogênicas c-cbl/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Dineínas/metabolismo , Proteína Adaptadora GRB2/metabolismo , Camundongos , Microtúbulos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Signaling microclusters are a common feature of lymphocyte activation. However, the mechanisms controlling the size and organization of these discrete structures are poorly understood. The Ezrin-Radixin-Moesin (ERM) proteins, which link plasma membrane proteins with the actin cytoskeleton and regulate the steady-state diffusion dynamics of the B cell receptor (BCR), are transiently dephosphorylated upon antigen receptor stimulation. In this study, we show that the ERM proteins ezrin and moesin influence the organization and integrity of BCR microclusters. BCR-driven inactivation of ERM proteins is accompanied by a temporary increase in BCR diffusion, followed by BCR immobilization. Disruption of ERM protein function using dominant-negative or constitutively active ezrin constructs or knockdown of ezrin and moesin expression quantitatively and qualitatively alters BCR microcluster formation, antigen aggregation, and downstream BCR signal transduction. Chemical inhibition of actin polymerization also altered the structure and integrity of BCR microclusters. Together, these findings highlight a crucial role for the cortical actin cytoskeleton during B cell spreading and microcluster formation and function.
Assuntos
Actinas/imunologia , Linfócitos B/imunologia , Proteínas do Citoesqueleto/imunologia , Ativação Linfocitária/imunologia , Proteínas dos Microfilamentos/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Actinas/genética , Actinas/metabolismo , Animais , Antígenos/genética , Antígenos/imunologia , Linfócitos B/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/genética , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosforilação/genética , Fosforilação/imunologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/genéticaRESUMO
We have investigated exchange of molecules between different membrane domains on a highly compartmentalized cell, the spermatozoon. Using Alexa Fluor 555-cholera toxin B-subunit we have observed clustering of preexisting GM1 gangliosides which diffused across the anterior acrosome-equatorial segment interface but did not access the postacrosome. By contrast, single lipid and protein molecules readily exchanged between all three domains, although they diffused more slowly on nearing and crossing to the postacrosome. Thus, two types of diffusion interfaces are present on sperm heads, an "open" interface and a "mass filter" interface. The latter seems to be due to a protein-cytoskeleton network.