RESUMEN
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
The RhoA-effector Dia1 controls actin-dependent processes such as cytokinesis, SRF transcriptional activity, and cell motility. Dia1 polymerizes actin through its formin homology (FH) 2 domain. Here we show that Dia1 acts upstream of RhoA independently of its effects on actin assembly. Dia1 binds to the leukemia-associated Rho-GEF (LARG) through RhoA-dependent release of Dia1 autoinhibition. The FH2 domain stimulates the guanine nucleotide exchange activity of LARG in vitro. Our results reveal that Dia1 is necessary for LPA-stimulated Rho/ROCK signaling and bleb-associated cancer cell invasion. Thus, Dia1-dependent RhoA activation constitutes a positive feedback mechanism to modulate cell behavior.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Línea Celular Tumoral , Retroalimentación , Forminas , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Modelos Biológicos , Mutación , Invasividad Neoplásica , ARN Interferente Pequeño/genética , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Proteína de Unión al GTP rhoA/genéticaRESUMEN
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.