RESUMEN
The hematopoietic actin regulatory protein hematopoietic lineage cell-specific protein 1 (HS1) is required for cell spreading and signaling in lymphocytes, but the scope of HS1 function in Ag presentation has not been addressed. We show that dendritic cells (DCs) from HS1(-/-) mice differentiate normally and display normal LPS-induced upregulation of surface markers and cytokines. Consistent with their normal expression of MHC and costimulatory molecules, HS1(-/-) DCs present OVA peptide efficiently to CD4(+) T cells. However, presentation of OVA protein is defective. Similarly, MHC class I-dependent presentation of VSV8 peptide to CD8(+) T cells occurs normally, but cross-presentation of GRP94/VSV8 complexes is defective. Analysis of Ag uptake pathways shows that HS1 is required for receptor-mediated endocytosis, but not for phagocytosis or macropinocytosis. HS1 interacts with dynamin 2, a protein involved in scission of endocytic vesicles. However, HS1(-/-) DCs showed decreased numbers of endocytic invaginations, whereas dynamin-inhibited cells showed accumulation of these endocytic intermediates. Taken together, these studies show that HS1 promotes an early step in the endocytic pathway that is required for efficient Ag presentation of exogenous Ag by DCs.
Asunto(s)
Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Endocitosis/inmunología , Factor Estimulante de Colonias de Granulocitos/inmunología , Animales , Western Blotting , Separación Celular , Células Dendríticas/metabolismo , Células Dendríticas/ultraestructura , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Ovalbúmina/inmunologíaRESUMEN
Dendritic cells (DCs) are professional APCs that reside in peripheral tissues and survey the body for pathogens. Upon activation by inflammatory signals, DCs undergo a maturation process and migrate to lymphoid organs, where they present pathogen-derived Ags to T cells. DC migration depends on tight regulation of the actin cytoskeleton to permit rapid adaptation to environmental cues. We investigated the role of hematopoietic lineage cell-specific protein 1 (HS1), the hematopoietic homolog of cortactin, in regulating the actin cytoskeleton of murine DCs. HS1 localized to lamellipodial protrusions and podosomes, actin-rich structures associated with adhesion and migration. DCs from HS1(-/-) mice showed aberrant lamellipodial dynamics. Moreover, although these cells formed recognizable podosomes, their podosome arrays were loosely packed and improperly localized within the cell. HS1 interacts with Wiskott-Aldrich syndrome protein (WASp), another key actin-regulatory protein, through mutual binding to WASp-interacting protein. Comparative analysis of DCs deficient for HS1, WASp or both proteins revealed unique roles for these proteins in regulating podosomes with WASp being essential for podosome formation and with HS1 ensuring efficient array organization. WASp recruitment to podosome cores was independent of HS1, whereas HS1 recruitment required Src homology 3 domain-dependent interactions with the WASp/WASp-interacting protein heterodimer. In migration assays, the phenotypes of HS1- and WASp-deficient DCs were related, but distinct. WASp(-/y) DCs migrating in a chemokine gradient showed a large decrease in velocity and diminished directional persistence. In contrast, HS1(-/-) DCs migrated faster than wild-type cells, but directional persistence was significantly reduced. These studies show that HS1 functions in concert with WASp to fine-tune DC cytoarchitecture and direct cell migration.
Asunto(s)
Quimiotaxis/inmunología , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos/inmunología , Proteína del Síndrome de Wiskott-Aldrich/inmunología , Actinas/genética , Actinas/metabolismo , Animales , Presentación de Antígeno/inmunología , Western Blotting , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Movimiento Celular/inmunología , Células Cultivadas , Citoesqueleto/inmunología , Citoesqueleto/metabolismo , Células Dendríticas/metabolismo , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Unión Proteica , Seudópodos/inmunología , Seudópodos/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
The Cancer Genome Atlas study in thyroid cancer exposed the genomic landscape of ~500 PTCs and revealed BRAFV600E-mutant tumors as having different prognosis, contrasting indolent cases and those with more invasive disease. Here, we describe the generation and characterization of six novel BRAFV600E-driven papillary thyroid cancer (PTC) cell lines established from a BrafV600E+/-/Pten+/-/TPO-Cre mouse model that spontaneously develop thyroid tumors. The novel cell lines were obtained from animals representing a range of developmental stages and both sexes, with the goal of establishing a heterogeneous panel of PTC cell lines sharing a common driver mutation. These cell lines recapitulate the genetics and diverse histopathological features of BRAFV600E-driven PTC, exhibiting differing degrees of growth, differentiation, and invasive potential that may help define mechanisms of pathogenesis underlying the heterogeneity present in the patient population. We demonstrate that these cell lines can be used for a variety of in vitro applications and can maintain the potential for in vivo transplantation into immunocompetent hosts. We believe that these novel cell lines will provide powerful tools for investigating the molecular basis of thyroid cancer progression and will lead to the development of more personalized diagnostic and treatment strategies for BRAFV600E-driven PTC.
RESUMEN
Dendritic cell (DC) migration is required for efficient presentation of antigen to T cells and the initiation of an adaptive immune response. In spite of its importance, many aspects of DC migration have not been characterized. DCs encounter a variety of environments with different stiffness and geometry, but the effect of these parameters on DC migration has not yet been determined. We addressed this question by comparing DC motility on standard migration surfaces (polydimethylsiloxane (PDMS)-coated coverslips) and micropost array detectors (mPADs). These two surfaces differ in both stiffness and geometry. We found that DC migration was affected by substrate type, with significant increases in speed and significant decreases in persistence time on mPADs made of PDMS as compared to spin-coated PDMS coverslips. To determine whether the geometry or compliance of the post arrays was responsible for these changes in DC migration, we quantified DC motility on mPADs of identical geometry but different stiffness. Migration was indistinguishable on these mPADs, suggesting that DCs are responsive to geometry of ligand presentation and not stiffness. Further, by micropatterning ligands on flat PDMS surfaces in similar geometries to the mPAD arrays, we determined that DCs respond to the geometry of printed ligand. Finally, we used a variety of small molecule inhibitors to identify pathways involved in geometry sensing. We saw a significant role for myosin contractility and α5ß1 integrin engagement. We also noted significant reorganization of the actin cytoskeleton into dynamic actin rings when DCs were motile on posts. From these experiments, we conclude that DCs are insensitive to substrate compliance in the range tested but respond to changes in geometry via a mechanism that involves integrin function, myosin contractility, and remodeling of the actin cytoskeleton. As a possible explanation, we postulate a consistent role for filopodial extension and contraction as the driver of DC motility.
Asunto(s)
Movimiento Celular , Células Dendríticas/fisiología , Citoesqueleto de Actina , Animales , Fenómenos Biomecánicos , Adhesión Celular , Dimetilpolisiloxanos , Integrinas/fisiología , Ratones Endogámicos C57BL , Miosinas/fisiologíaRESUMEN
Full T cell activation requires coordination of signals from multiple receptor-ligand pairs that interact in parallel at a specialized cell-cell contact site termed the immunological synapse (IS). Signaling at the IS is intimately associated with actin dynamics; T cell receptor (TCR) engagement induces centripetal flow of the T cell actin network, which in turn enhances the function of ligand-bound integrins by promoting conformational change. Here, we have investigated the effects of integrin engagement on actin flow, and on associated signaling events downstream of the TCR. We show that integrin engagement significantly decelerates centripetal flow of the actin network. In primary CD4+ T cells, engagement of either LFA-1 or VLA-4 by their respective ligands ICAM-1 and VCAM-1 slows actin flow. Slowing is greatest when T cells interact with low mobility integrin ligands, supporting a predominately drag-based mechanism. Using integrin ligands presented on patterned surfaces, we demonstrate that the effects of localized integrin engagement are distributed across the actin network, and that focal adhesion proteins, such as talin, vinculin, and paxillin, are recruited to sites of integrin engagement. Further analysis shows that talin and vinculin are interdependent upon one another for recruitment, and that ongoing actin flow is required. Suppression of vinculin or talin partially relieves integrin-dependent slowing of actin flow, indicating that these proteins serve as molecular clutches that couple engaged integrins to the dynamic actin network. Finally, we found that integrin-dependent slowing of actin flow is associated with reduction in tyrosine phosphorylation downstream of the TCR, and that this modulation of TCR signaling depends on expression of talin and vinculin. More generally, we found that integrin-dependent effects on actin retrograde flow were strongly correlated with effects on TCR signaling. Taken together, these studies support a model in which ligand-bound integrins engage the actin cytoskeletal network via talin and vinculin, and tune TCR signaling events by modulating actin dynamics at the IS.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Sinapsis Inmunológicas/inmunología , Integrina alfa4beta1/metabolismo , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Talina/metabolismo , Vinculina/metabolismo , Actinas/metabolismo , Línea Celular Tumoral , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Células Jurkat , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Paxillin/metabolismo , Fosforilación , Transducción de Señal/inmunología , Tirosina/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Dendritic cell migration to the T-cell-rich areas of the lymph node is essential for their ability to initiate the adaptive immune response. While it has been shown that the actin cytoskeleton is required for normal DC migration, the role of many of the individual cytoskeletal molecules is poorly understood. In this study, we investigated the contribution of the Arp2/3 complex binding protein, haematopoietic lineage cell-specific protein 1 (HS1), to DC migration and force generation. We quantified the random migration of HS1-/- DCs on 2D micro-contact printed surfaces and found that in the absence of HS1, DCs have greatly reduced motility and speed. This same reduction in motility was recapitulated when adding Arp2/3 complex inhibitor to WT DCs or using DCs deficient in WASP, an activator of Arp2/3 complex-dependent actin polymerization. We further investigated the importance of HS1 by measuring the traction forces of HS1-/- DCs on micropost array detectors (mPADs). In HS1 deficient DCs, there was a significant reduction in force generation (3.96 ± 0.40 nN per cell) compared to WT DCs (13.76 ± 0.84 nN per cell). Interestingly, the forces generated in DCs lacking WASP were only slightly reduced compared to WT DCs. Taken together, these findings show that HS1 and Arp2/3 complex-mediated actin polymerization are essential for the most efficient DC random migration and force generation.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/fisiología , Células Dendríticas/fisiología , Factor Estimulante de Colonias de Granulocitos/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/antagonistas & inhibidores , Actinas/metabolismo , Animales , Bioingeniería , Fenómenos Biofísicos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos/deficiencia , Factor Estimulante de Colonias de Granulocitos/genética , Indoles/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/fisiologíaRESUMEN
Effector T cell migration into inflamed sites greatly exacerbates tissue destruction and disease severity in inflammatory diseases, including graft-versus-host disease (GVHD). T cell migration into such sites depends heavily on regulated adhesion and migration, but the signaling pathways that coordinate these functions downstream of chemokine receptors are largely unknown. Using conditional knockout mice, we found that T cells lacking the adaptor proteins CRK and CRK-like (CRKL) exhibit reduced integrin-dependent adhesion, chemotaxis, and diapedesis. Moreover, these two closely related proteins exhibited substantial functional redundancy, as ectopic expression of either protein rescued defects in T cells lacking both CRK and CRKL. We determined that CRK proteins coordinate with the RAP guanine nucleotide exchange factor C3G and the adhesion docking molecule CASL to activate the integrin regulatory GTPase RAP1. CRK proteins were required for effector T cell trafficking into sites of inflammation, but not for migration to lymphoid organs. In a murine bone marrow transplantation model, the differential migration of CRK/CRKL-deficient T cells resulted in efficient graft-versus-leukemia responses with minimal GVHD. Together, the results from our studies show that CRK family proteins selectively regulate T cell adhesion and migration at effector sites and suggest that these proteins have potential as therapeutic targets for preventing GVHD.
Asunto(s)
Quimiotaxis , Proteínas Proto-Oncogénicas c-crk/fisiología , Linfocitos T/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Trasplante de Médula Ósea , Adhesión Celular , Polaridad Celular , Células Cultivadas , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Inflamación/metabolismo , Inflamación/patología , Tejido Linfoide/inmunología , Tejido Linfoide/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/fisiología , Transducción de Señal , Linfocitos T/trasplante , Migración Transendotelial y Transepitelial , Proteína de Unión al GTP rac1/metabolismoRESUMEN
A mutant human connexin50 (hCx50), hCx50P88S, has been linked to cataracts inherited as an autosomal dominant trait. The functional, biochemical and cellular behavior of wild-type and mutant hCx50 were examined in transfected cells. hCx50P88S was unable to induce gap junctional currents by itself, and it abolished gap junctional currents when co-expressed with wild-type (wt) hCx50. Cells transfected with hCx50P88S showed cytoplasmic accumulations of Cx50 immunoreactivity in addition to staining at appositional membranes; these accumulations did not significantly co-localize with markers for the endoplasmic reticulum, Golgi apparatus, lysosomes, endosomes or vimentin filaments. Immunoelectron microscopy studies localized hCx50P88S to cytoplasmic membrane stacks in close vicinity to the endoplasmic reticulum. In contrast, aggresome-like accumulations were induced by treatment of wt hCx50-transfected cells with proteasomal inhibitors. The formation of hCx50P88S accumulations in transiently transfected cells was not blocked by treatment with Brefeldin A suggesting that they form before Cx50 transits through the Golgi apparatus to the plasma membrane. Treatment of HeLa-hCx50P88S cells with cycloheximide demonstrated the presence of a very stable pool of hCx50P88S. Taken together, these results suggest that the P to S mutation at amino acid residue 88 causes a defect that leads to decreased degradation and subsequent accumulation of hCx50P88S in a cellular structure different from aggresomes.
Asunto(s)
Catarata/genética , Proteínas del Ojo/genética , Animales , Línea Celular , Conexinas , Electrofisiología , Proteínas del Ojo/metabolismo , Células HeLa/ultraestructura , Humanos , Immunoblotting , Microscopía Electrónica , Mutación , Oligopéptidos/farmacología , Fenilbutiratos/farmacología , Transporte de Proteínas/efectos de los fármacos , TransfecciónRESUMEN
T cell trafficking between the blood and lymphoid organs is a complex, multistep process that requires several highly dynamic and coordinated changes in cyto-architecture. Members of the ezrin, radixin and moesin (ERM) family of actin-binding proteins have been implicated in several aspects of this process, but studies have yielded conflicting results. Using mice with a conditional deletion of ezrin in CD4+ cells and moesin-specific siRNA, we generated T cells lacking ERM proteins, and investigated the effect on specific events required for T cell trafficking. ERM-deficient T cells migrated normally in multiple in vitro and in vivo assays, and could undergo efficient diapedesis in vitro. However, these cells were impaired in their ability to adhere to the ß1 integrin ligand fibronectin, and to polarize appropriately in response to fibronectin and VCAM-1 binding. This defect was specific for ß1 integrins, as adhesion and polarization in response to ICAM-1 were normal. In vivo, ERM-deficient T cells showed defects in homing to lymphoid organs. Taken together, these results show that ERM proteins are largely dispensable for T cell chemotaxis, but are important for ß1 integrin function and homing to lymphoid organs.
Asunto(s)
Proteínas del Citoesqueleto/genética , Tejido Linfoide/metabolismo , Proteínas de Microfilamentos/genética , Linfocitos T/metabolismo , Animales , Adhesión Celular/genética , Quimiotaxis/genética , Quimiotaxis/inmunología , Proteínas del Citoesqueleto/metabolismo , Integrina beta1/metabolismo , Ligandos , Tejido Linfoide/inmunología , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/metabolismo , Receptores CCR7/metabolismo , Linfocitos T/inmunología , Migración Transendotelial y Transepitelial/ética , Migración Transendotelial y Transepitelial/genéticaRESUMEN
Pleckstrin-2 is composed of 2 pleckstrin homology (PH) domains and a disheveled-Egl-10-pleckstrin (DEP) domain. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to D3 and D5 phosphoinositides. Pleckstrin-2 expressed in Jurkat T cells bound to the cellular membrane and enhanced actin-dependent spreading only after stimulation of the T-cell antigen receptor or the integrin alpha4beta1. A pleckstrin-2 variant containing point mutations in both PH domains failed to associate with the Jurkat membrane and had no effect on spreading under the same conditions. Although still membrane bound, a pleckstrin-2 variant containing point mutations in the DEP domain demonstrated a decreased ability to induce membrane ruffles and spread. Pleckstrin-2 also colocalized with actin at the immune synapse and integrin clusters via its PH domains. Although pleckstrin-2 can bind to purified D3 and D5 phosphoinositides, the intracellular membrane association of pleckstrin-2 and cell spreading are dependent on D3 phosphoinositides, because these effects were disrupted by pharmacologic inhibition of phosphatidylinositol 3-kinase (PI3K). Our results indicate that pleckstrin-2 uses its modular domains to bind to membrane-associated phosphatidylinositols generated by PI3K, whereby it coordinates with the actin cytoskeleton in lymphocyte spreading and immune synapse formation.