RESUMO
Fesselin or avian synaptopodin 2 is a member of the synaptopodin family of actin binding proteins. Fesselin promotes G-actin polymerization and the formation of large actin complexes that can be collected by low-speed centrifugation. Because of the potential role of fesselin in some cancers and its effects on actin, we further investigated the effect of fesselin on actin. Fesselin initiated actin polymerization under a variety of conditions, including the virtual absence of salt. Actin filaments formed at low salt concentrations in the presence of fesselin were similar to filaments polymerized in the presence of 100 mM KCl. In both cases, the filaments were long and straight with a common orientation. Highly ordered actin bundles formed with increasing times of incubation. Blockers of actin growth at the barbed end (cytochalasin D and CapZ) did not prevent fesselin from polymerizing actin. Low concentrations of fesselin increased the critical concentration of actin. Both observations are consistent with preferential growth at the pointed end of actin filaments. These results indicate a role of fesselin in organizing cellular actin. These and other results indicate that fesselin is part of a cellular actin organizing center.
Assuntos
Actinas/química , Proteínas de Membrana/química , Proteínas dos Microfilamentos/química , Animais , Aves , Microscopia Eletrônica , Conformação ProteicaRESUMO
An analysis of the primary structure of the actin-binding protein fesselin revealed it to be the avian homologue of mammalian synaptopodin 2 [Schroeter, Beall, Heid, and Chalovich (2008) Biochem. Biophys. Res. Commun. 371, 582-586]. We isolated two synaptopodin 2 isoforms from rabbit stomach that corresponded to known types of human synaptopodin 2. The purification scheme used was that developed for avian fesselin. These synaptopodin 2 forms shared several key functions with fesselin. Both avian fesselin and mammalian synaptopodin 2 bound to Ca(2+)-calmodulin, alpha-actinin and smooth-muscle myosin. In addition, both proteins stimulated the polymerization of actin in a Ca(2+)-calmodulin-dependent manner. Synaptopodin 2 has never before been shown to polymerize actin in the absence of alpha-actinin, to polymerize actin in a Ca(2+)-calmodulin-dependent manner, or to bind to Ca(2+)-calmodulin or myosin. These properties are consistent with the proposed function of synaptopodin 2 in organizing the cytoskeleton.
Assuntos
Proteínas Musculares/química , Proteínas Musculares/isolamento & purificação , Proteínas Musculares/metabolismo , Actinina/metabolismo , Animais , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/isolamento & purificação , Proteínas de Ligação a Calmodulina/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/isolamento & purificação , Proteínas dos Microfilamentos/metabolismo , Músculo Liso/química , Músculo Liso/metabolismo , Miosinas/metabolismo , CoelhosRESUMO
Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and alpha-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins.