RESUMEN
HIV-1 Nef is a key factor in AIDS pathogenesis. Here, we report that Nef potently inhibits motility of fibroblasts and chemotaxis of HIV-1-infected primary human T lymphocytes toward the chemokines SDF-1alpha, CCL-19, and CCL-21 ex vivo. Furthermore, Nef inhibits guided motility of zebrafish primordial germ cells toward endogenous SDF-1a in vivo. These migration defects result from Nef-mediated inhibition of the actin remodeling normally triggered by migratory stimuli. Nef strongly induces phosphorylation of cofilin, inactivating this evolutionarily conserved actin-depolymerizing factor that promotes cell motility when unphosphorylated. Nef-dependent cofilin deregulation requires association of Nef with the cellular kinase Pak2. Disruption of Nef-Pak2 association restores the cofilin phosphorylation levels and actin remodeling that facilitate cell motility. We conclude that HIV-1 Nef alters Pak2 function, which directly or indirectly inactivates cofilin, thereby restricting migration of infected T lymphocytes as part of a strategy to optimize immune evasion and HIV-1 replication.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Movimiento Celular , VIH-1/patogenicidad , Factores de Virulencia/fisiología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/fisiología , Línea Celular , Células Cultivadas , Fibroblastos/virología , Humanos , Linfocitos T/virología , Quinasas p21 Activadas/metabolismoRESUMEN
Regulation of cell polarity is a process observed in all cells. During directed migration, cells orientate their microtubule cytoskeleton and the microtubule-organizing-center (MTOC), which involves integrins and downstream Cdc42 and glycogen synthase kinase-3beta activity. However, the contribution of G protein-coupled receptor signal transduction for MTOC polarity is less well understood. Here, we report that the heterotrimeric Galpha(12) and Galpha(13) proteins are necessary for MTOC polarity and microtubule dynamics based on studies using Galpha(12/13)-deficient mouse embryonic fibroblasts. Cell polarization involves the Galpha(12/13)-interacting leukemia-associated RhoGEF (LARG) and the actin-nucleating diaphanous formin mDia1. Interestingly, LARG associates with pericentrin and localizes to the MTOC and along microtubule tracks. We propose that Galpha(12/13) proteins exert essential functions linking extracellular signals to microtubule dynamics and cell polarity via RhoGEF and formin activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Polaridad Celular , Fibroblastos/citología , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Microtúbulos/metabolismo , Animales , Antígenos/metabolismo , Movimiento Celular , Fibroblastos/enzimología , Forminas , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Ratones , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Unión Proteica , Factores de Intercambio de Guanina Nucleótido Rho , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Scratch-wound assays are frequently used to study directed cell migration, a process critical for embryogenesis, invasion, and tissue repair. The function and identity of trimeric G-proteins in cell behavior during wound healing is not known. Here we show that Galpha12/13, but not Galphaq/11 or Galphai, is indispensable for coordinated and directed cell migration. In mouse embryonic fibroblasts endogenous Rho activity is present at the rear of migrating cells but also at the leading edge, whereas it is undetectable at the cell front of Galpha12/13-deficient mouse embryonic fibroblasts. Spatial activation of Rho at the wound edge can be stimulated by lysophosphatidic acid. Active Rho colocalizes with the diaphanous-related formin Dia1 at the cell front. Galpha12/13-deficient cells lack Dia1 localization to the wound edge and are unable to form orientated, stable microtubules during wound healing. Knock down of Dia1 reveals its requirement for microtubule stabilization as well as polarized cell migration. Thus, we identified Galpha12/13-proteins as essential components linking extracellular signals to localized Rho-Dia1 function during directed cell movement.