RESUMEN
Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the alpha subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.
Asunto(s)
Proteínas del Citoesqueleto , Vesículas Secretoras/metabolismo , Serina Endopeptidasas/metabolismo , Linfocitos T Citotóxicos/inmunología , beta-N-Acetilhexosaminidasas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Actinas/aislamiento & purificación , Animales , Catepsina D , Membrana Celular/ultraestructura , Polaridad Celular , Gránulos Citoplasmáticos/ultraestructura , Aparato de Golgi/ultraestructura , Granzimas , Síndrome de Hermanski-Pudlak , Hipopigmentación , Síndromes de Inmunodeficiencia , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Mutantes , Vesículas Secretoras/ultraestructura , Linfocitos T Citotóxicos/ultraestructura , Talina/aislamiento & purificación , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTPRESUMEN
Specific T cell responses to a variety of self and microbial lipids depend on proper assembly and intracellular trafficking of CD 1 molecules that intersect with and load processed lipid antigens. These pathways involve unique membrane trafficking and chaperones that are distinct from those utilized for major histocompatibility complex (MHC)-mediated presentation of peptide antigens, and thus define unique lipid antigen presentation pathways. Furthermore, recent studies have identified components of lipid metabolism that participate in lipid delivery, uptake, processing and loading onto CD1 molecules. Defects in these pathways result in impaired T cell development and function, underscoring their critical role in the lipid-specific T cell immune responses.