RESUMEN
We previously identified Waf1 Cip1 stabilizing protein 39 (WISp39) as a binding partner for heat shock protein 90 (Hsp90). We now report that WISp39 has an essential function in the control of directed cell migration, which requires WISp39 interaction with Hsp90. WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge. WISp39 binds Coronin 1B, known to regulate the Arp2/3 complex and Cofilin at the leading edge. WISp39 preferentially interacts with phosphorylated Coronin 1B, allowing it to complex with Slingshot phosphatase (SSH) to dephosphorylate and activate Cofilin. WISp39 also regulates Arp2/3 complex localization at the leading edge. WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant. We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Inmunofilinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Factores Despolimerizantes de la Actina/genética , Línea Celular Tumoral , Movimiento Celular/genética , Activación Enzimática/genética , Células HEK293 , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Inmunofilinas/genética , Proteínas de Microfilamentos/biosíntesis , Fosfoproteínas Fosfatasas , Fosforilación , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño , Proteínas de Unión a TacrolimusRESUMEN
Capping protein (CP) binds to barbed ends of growing actin filaments and inhibits elongation. CP is essential for actin-based motility in cell-free systems and in Dictyostelium. Even though CP is believed to be critical for creating the lamellipodial actin structure necessary for protrusion and migration, CP's role in mammalian cell migration has not been directly tested. Moreover, recent studies have suggested that structures besides lamellipodia, including lamella and filopodia, may have unappreciated roles in cell migration. CP has been postulated to be absent from filopodia, and thus its role in filopodial activity has remained unexplored. We report that silencing CP in both cultured mammalian B16F10 cells and in neurons of developing neocortex impaired cell migration. Moreover, we unexpectedly observed that low levels of CP were detectable in the majority of filopodia. CP depletion decreased filopodial length, altered filopodial shape, and reduced filopodial dynamics. Our results support an expansion of the potential roles that CP plays in cell motility by implicating CP in filopodia as well as in lamellipodia, both of which are important for locomotion in many types of migrating cells.
Asunto(s)
Proteína CapZ/fisiología , Movimiento Celular , Seudópodos/ultraestructura , Actinas/metabolismo , Animales , Línea Celular Tumoral , Forma de la Célula , Técnicas de Silenciamiento del Gen , Ratones , Seudópodos/metabolismoRESUMEN
A current model posits that cofilin-dependent actin severing negatively impacts dendritic spine volume. Studies suggested that increased cofilin activity underlies activity-dependent spine shrinkage, and that reduced cofilin activity induces activity-dependent spine growth. We suggest instead that both types of structural plasticity correlate with decreased cofilin activity. However, the mechanism of inhibition determines the outcome for spine morphology. RNAi in rat hippocampal cultures demonstrates that cofilin is essential for normal spine maintenance. Cofilin-F-actin binding and filament barbed-end production decrease during the early phase of activity-dependent spine shrinkage; cofilin concentration also decreases. Inhibition of the cathepsin B/L family of proteases prevents both cofilin loss and spine shrinkage. Conversely, during activity-dependent spine growth, LIM kinase stimulates cofilin phosphorylation, which activates phospholipase D-1 to promote actin polymerization. These results implicate novel molecular mechanisms and prompt a revision of the current model for how cofilin functions in activity-dependent structural plasticity.
Asunto(s)
Cofilina 1/metabolismo , Espinas Dendríticas/fisiología , Regulación hacia Abajo , Neuronas/fisiología , Actinas/metabolismo , Animales , Células Cultivadas , Cofilina 1/genética , Espinas Dendríticas/efectos de los fármacos , Femenino , Hipocampo/citología , Inmunohistoquímica , Quinasas Lim/metabolismo , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Microscopía Confocal , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfolipasa D/metabolismo , Fosforilación , Unión Proteica , Interferencia de ARN , Ratas , Tacrolimus/farmacología , Imagen de Lapso de TiempoRESUMEN
We have identified a unique human microtubule-associated protein (MAP) named ASAP for ASter-Associated Protein. ASAP localizes to microtubules in interphase, associates with the mitotic spindle during mitosis, localizes to the central body during cytokinesis and directly binds to purified microtubules by its COOH-terminal domain. Overexpression of ASAP induces profound bundling of cytoplasmic microtubules in interphase cells and aberrant monopolar spindles in mitosis. Depletion of ASAP by RNA interference results in severe mitotic defects: it provokes aberrant mitotic spindle, delays mitotic progression, and leads to defective cytokinesis or cell death. These results suggest a crucial role for ASAP in the organization of the bipolar mitotic spindle, mitosis progression, and cytokinesis and define ASAP as a key factor for proper spindle assembly.
