RESUMEN
The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization.
Asunto(s)
Ciona intestinalis/citología , Regulación del Desarrollo de la Expresión Génica , Miosinas/genética , Notocorda/citología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Polaridad Celular/efectos de los fármacos , Ciona intestinalis/efectos de los fármacos , Ciona intestinalis/embriología , Ciona intestinalis/metabolismo , Citocalasina B/farmacología , Embrión no Mamífero , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Expresión Génica , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Miosinas/metabolismo , Notocorda/efectos de los fármacos , Notocorda/embriología , Notocorda/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/genética , Factores de Intercambio de Guanina Nucleótido ras/metabolismoRESUMEN
Although biochemical and genetic methods have detected many activator-transcription factor interactions, the direct functional targets of most activators remain undetermined. For this study, photo-cross-linkers positioned within the Gal4 C-terminal acidic activating region were used to identify polypeptides in close physical proximity to Gal4 during transcription activation in vitro. Of six specifically cross-linked polypeptides, three (Tra1, Taf12, and Gal11) are subunits of four complexes (SAGA, Mediator, NuA4, and TFIID) known to play a role in gene regulation. These cross-linking targets had differential effects on activation. SAGA was critical for activation by Gal4, Gal11 contributed modestly to activation, and TFIID and NuA4 were not important for activation under our conditions. Tra1, Taf12, and Gal11 have also been identified as cross-linking targets of the Gcn4 acidic central activating region. Our results demonstrate that two unrelated acidic activators converge on the same set of functional targets.