RESUMEN
Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) is a new member of the gelsolin family of actin regulatory proteins [Klaavuniemi, T., Yamashiro, S., and Ono, S. (2008) J. Biol. Chem. 283, 26071-26080]. It is an unconventional gelsolin-related protein with four gelsolin-like (G) domains (G1-G4), unlike typical gelsolin-related proteins with three or six G domains. GSNL-1 severs actin filaments and caps the barbed end in a calcium-dependent manner similar to that of gelsolin. In contrast, GSNL-1 has properties different from those of gelsolin in that it remains bound to F-actin and does not nucleate actin polymerization. To understand the mechanism by which GSNL-1 regulates actin dynamics, we investigated the domain-function relationship of GSNL-1 by analyzing activities of truncated forms of GSNL-1. G1 and the linker between G1 and G2 were sufficient for actin filament severing, whereas G1 and G2 were required for barbed end capping. The actin severing activity of GSNL-1 was inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and a PIP2-sensitive domain was mapped to G1 and G2. At least two actin-binding sites were detected: a calcium-dependent G-actin-binding site in G1 and a calcium-independent G- and F-actin-binding site in G3 and G4. These results reveal both conserved and different utilization of G domains between C. elegans GSNL-1 and mammalian gelsolin for actin regulatory functions.
Asunto(s)
Proteínas de Capping de la Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Sensoras del Calcio Intracelular/química , Proteínas Sensoras del Calcio Intracelular/metabolismo , Fosfatidilinositoles/metabolismo , Proteínas de Capping de la Actina/química , Proteínas de Capping de la Actina/fisiología , Factores Despolimerizantes de la Actina/química , Factores Despolimerizantes de la Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Factores Despolimerizantes de la Actina/fisiología , Actinas/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Gelsolina/química , Gelsolina/metabolismo , Gelsolina/fisiología , Proteínas Sensoras del Calcio Intracelular/genética , Proteínas Sensoras del Calcio Intracelular/fisiología , Modelos Biológicos , Peso Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica/fisiología , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína/fisiologíaRESUMEN
Recent live cell image analysis of actin dynamics in lamellipodia of motile cells has shown that regulated treadmilling, which supports actin-based propulsion of functionalized particles in biomimetic reconstituted motility assays, is also responsible for lamellipodia extension. In both cases, filaments are created by branching with Arp2/3 complex only at the membrane or particle surface, grow transiently and are capped; ADF/cofilin enhances the treadmilling but does not sever filaments in the body of the meshwork. Differences between the cellular and biomimetic systems suggest that additional regulatory mechanisms take place in lamellipodia.