RESUMO
AIMS: To characterize the specificity and effect of pH and ionic strength on the kinetics of virus binding to histo-blood group antigens (HBGA)-conjugated magnetic beads. METHODS AND RESULTS: HBGAs from porcine gastric mucin (PGM) have been conjugated to magnetic beads (PGM-MB) for concentration of NoV. A GII.4 virus was used for the detailed binding kinetics study and a panel of genogroup I (GI) NoVs, genogroup II (GII) NoVs and recombinant NoVs (rNoVs) were used for specificity and binding efficiency assays. We determined that NoV can be captured after 15min of incubation with PGM-MB, and virus recovery efficiency is decreased after extended incubation times. rNoV binding as measured by ELISA and NoV recovery as measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), were both enhanced significantly at acidic pH conditions. rNoV binding to PGM as measured by ELISA was increased up to 66%. While real-time RT-PCR analyses suggest that NoV could be concentrated as much as 1000-fold at neutral pH, up to 3·4-fold further increase of NoV recovery was achieved by adjusting the pH of the sample to 3·0-4·2. Variation between GI and GII viral binding to the PGM-MB at basic pH was observed. All five GI rNoVs tested and 6 of 9 GII rNoVs were captured by PGM. All eight GI strains tested were concentrated by PGM-MB, ranging from 28-fold (GI.4) to 1502-fold (GI.1). Eleven of 13 GII strains were concentrated from 30-fold (GII.5) to 1014-fold (GII.4, lab strain) by PGM-MB. GI and GII rNoVs viral capsid proteins were recovered with high salt conditions, but results were inconsistent for whole virus recovery. CONCLUSIONS: All GI and 85% of GII NoVs tested could be captured and concentrated by PGM-MB method. The binding occurred rapidly and was enhanced at low pH. SIGNIFICANCE AND IMPACT OF THE STUDY: These results facilitated development of a prototype method for sensitive detection of NoV in samples requiring larger volumes.
Assuntos
Separação Imunomagnética , Norovirus/metabolismo , Virologia/métodos , Ligação Viral , Animais , Ensaio de Imunoadsorção Enzimática , Mucinas Gástricas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Norovirus/genética , RNA/isolamento & purificação , Sensibilidade e Especificidade , SuínosRESUMO
The class IA phosphatidylinositol 3-kinases (PI3K) is involved in controlling changes in cell morphology, which is a highly coordinated cellular event. This event is powered by actin filament polymerization and remodeling. The gain-of-function mutations in the catalytic subunit of p110α of class IA PI3K, which occur in up to one-third of human colorectal cancers (CRCs), are capable of causing dysregulation of cell signaling and thus may result in the alteration in cell morphology and motility and in turn cause cancer metastasis. In vivo studies have demonstrated that cell lines bearing the H1047R point mutation, the most frequent cancer-specific mutation in the kinase domain of p110α, are more metastatic than cells carrying wild-type p110α. In the current study, we show that the H1047R in p110α of PI3K decreases F-actin polymerization, increases the formation of filopodia and significantly changes the cell morphology in HCT116 cancer cells. The anti-apoptotic protein B-cell lymphoma 2 (Bcl-2), which is also involved in actin polymerization and cell migration, is downregulated by the H1047R mutation in p110α. Our data suggest that the H1047R mutation in PI3K is responsible for the rearrangement of the cytoskeleton, alteration in cell morphology and enhancing cell motility, and that Bcl-2 may be involved in the H1047R mutation-mediated morphological changes and increased migratory capability.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/fisiologia , Sistema Nervoso Central/patologia , Guanilato Ciclase/fisiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Criança , Xenoenxertos , Humanos , Camundongos , Invasividade Neoplásica , Transdução GenéticaRESUMO
Cell polarization is a key feature of cell motility, driving cell migration to tissues. CD43 is an abundantly expressed molecule on the T-cell surface that shows distinct localization to the migrating T-cell uropod and the distal pole complex (DPC) opposite the immunological synapse via association with the ezrin-radixin-moesin (ERM) family of actin regulatory proteins. CD43 regulates multiple T-cell functions, including T-cell activation, proliferation, apoptosis, and migration. We recently demonstrated that CD43 regulates T-cell trafficking through a phosphorylation site at Ser-76 (S76) within its cytoplasmic tail. Using a phosphorylation-specific antibody, we now find that CD43 phosphorylation at S76 is enhanced by migration signals. We further show that CD43 phosphorylation and normal T-cell trafficking depend on CD43 association with ERM proteins. Interestingly, mutation of S76 to mimic phosphorylation enhances T-cell migration and CD43 movement to the DPC while blocking ERM association, showing that CD43 movement can occur in the absence of ERM binding. We also find that protein kinase CΘ can phosphorylate CD43. These results show that while CD43 binding to ERM proteins is crucial for S76 phosphorylation, CD43 movement and regulation of T-cell migration can occur through an ERM-independent, phosphorylation-dependent mechanism.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Leucossialina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Linfócitos T/fisiologia , Animais , Movimento Celular/fisiologia , Células Cultivadas , Sinapses Imunológicas/metabolismo , Isoenzimas/metabolismo , Leucossialina/genética , Camundongos , Camundongos Transgênicos , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/citologiaRESUMO
T cells interacting with APCs undergo rearrangement of surface receptors and cytoskeletal elements to face the zone of contact with the APC. This polarization process is thought to affect T cell signaling by organizing a specialized domain on the T cell surface and to direct T cell effector function toward the appropriate APC. We have investigated the contribution of TCR, CD28, and LFA-1 signaling to T cell cytoskeletal polarization by assaying the response of an Ag-specific Th1 clone toward a panel of transfected APCs expressing MHC class II alone or in combination with ICAM-1 or B7-1. We show that polarization of talin, an actin-binding protein, occurs in response to integrin engagement. In contrast, reorientation of the T cell microtubule-organizing center (MTOC) is dependent on and directed toward the site of TCR signaling, regardless of whether integrins or costimulatory molecules are engaged. MTOC reorientation in response to peptide-MHC complexes is sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. CD28 coengagement overcomes this sensitivity, as does activation via Ab cross-linking of the TCR or via covalent peptide-MHC complexes, suggesting that phosphatidylinositol 3-kinase is not required per se but rather plays a role in signal amplification. Engagement of TCR in trans with LFA-1 results in separation of MTOC reorientation and cortical cytoskeletal polarization events, indicating that the two processes are not directly mechanistically linked. These studies show that T cells mobilize individual cytoskeletal components in response to distinct and specific cell surface interactions.
Assuntos
Antígenos CD28/metabolismo , Citoesqueleto/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Actinas/metabolismo , Androstadienos/farmacologia , Células Apresentadoras de Antígenos/imunologia , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Polaridade Celular , Citoesqueleto/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/imunologia , Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/ultraestrutura , Talina/metabolismo , Células Th1/imunologia , Células Th1/ultraestrutura , Transfecção , WortmaninaRESUMO
Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Junções Intercelulares/metabolismo , Proteínas/metabolismo , Linfócitos T/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Compartimento Celular , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Modelos Biológicos , Prolina , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Proteínas/isolamento & purificação , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteína da Síndrome de Wiskott-Aldrich , Proteína-Tirosina Quinase ZAP-70 , Proteína cdc42 de Ligação ao GTP/isolamento & purificaçãoRESUMO
The large mucin CD43 is actively excluded from T cell/APC interaction sites, concentrating in a membrane domain distal to the site of TCR engagement. The cytoplasmic region of CD43 was necessary and sufficient for this antipodal movement. ERM cytoskeletal adaptor proteins colocalized with CD43 in this domain. An ERM dominant-negative mutant blocked the distal accumulation of CD43 and another known ERM binding protein, Rho-GDI. Inhibition of ERM function decreased the production of IL-2 and IFNgamma, without affecting PKC(theta) focusing or CD69 upregulation. These results indicate that ERM proteins organize a complex distal to the T cell/APC interaction site and provide evidence that full T cell activation may involve removal of inhibitory proteins from the immunological synapse.