Participation of LIMK1 and cofilin in the regulation of Golgi organization and dynamics.

JAUSORO I; BOLLATI F; PAGLINI G; CACERES A

Casapueblo, Maldonado

Simposio; Calcium Signalling, with special attention to cell motility and the cytoskeleton; 2005

During the last decade, an increasing number of experimental evidences have suggested that the cytoskeleton has a crucial role in axon formation. During recent years, a growing body of evidence have accumulated suggesting that the actin cytoskeleton has a key role in regulating axon formation. In particular, signaling pathways, that include the smallGTPases of the Rho/Rac/Cdc42 superfamily, have been shown to be important for actin reorganization and dynamics during neuronal polarization. Among the downstream effectors of these GTPases are proteins that directly regulate actin turnover, such as the actin-depolymerizing factor (ADF) and cofilin. They are hightly expressed in neuronal growth cones where the regulation of actin dynamics is essential for axon outgrowth. The overexpression of ADF-cofilin enhances neurite outgrowth and growth cone motility in developing neurons. Recently, in vivo and in vitro studies have demonstrated that ADF-cofilin is a substrate of a novel family of kinases, designated as LIMKs. Phosphorylation of ADF-cofilin at a single site (ser 3) by LIMKs inhibits its binding to actin monomers and its actin-depolymerizing activity. In cultured neurons, the proteins levels increases 24 h after plating, just at the time in which cells are beginning to extend axons. After that time point, LIMK1 protein levels remain unchanged until the end of the second week in vitro when an additional and significant increase is detected. A downstream target of Rac and cdc42 is PAK1, a direct activator of LIMK1 through phosphorylation of Thr508. In our laboratory we have demostrated that during the establishment of neuronal polarity the active form of LIMK1 is localized in vesicles distributed along shaft and growth cone of neurites that will became an axon. Besides, overexpression of wt-LIMK1 dramatically reduces the formation of tubules that emerge from the Golgi, whereas that of LIMK1-kd or S3A-cofilin has the opposite effect. This also suggests the involvement of LIMK1 and actin dynamics in the regulation of Golgi morphology and tubules formation. The analysis of its subcelular distribution reveales that LIMK1 is enriched in the Golgi apparatus and growth cones, and that LIM domains are required for LIMK1 localization to the Golgi apparatus, whereas a region containing the PDZ domain is required for its localization at growth cones, and its exclusion from the cell nucleus.The presence of LIMK1 and cofilin in the golgi apparatus is consistent with recent studies showing that members of RHO-GTPasas family are localized in this organel. Our observations also raise the interesting possibility of LIMK1 regulating the organization and trafficking of Golgi-derived vesicles.