Naslov
Dual role of a Rac GTPase in the regulation of cell motility

Autori
Weber, Igor ; Faix, J. ; Filić, Vedrana ; Marinović, Maja

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
From Physics To Life Sciences / Forro, Laszlo ; Pavuna, Davor - Lausanne : Ecole Polytechnique Federale de Lausanne, 2010, 15-15

Skup
From Solid State To BioPhysics V

Mjesto i datum
Cavtat, Hrvatska, 12.06.2010. - 19.06.2010

Vrsta sudjelovanja
Pozvano predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Rho GTPases; actin cytoskeleton; Dictyostelium

Sažetak
Rac proteins are members of a broad family of monomeric Rho GTPases that act as key regulators of the actin cytoskeleton. Rac1A from highly motile Dictyostelium cells binds to the IQGAP-related protein DGAP1, which forms a cortical complex with actin-binding proteins cortexillins. This complex localizes to the rear end of moving cells and to the cleavage furrow of dividing cells, where it supports efficient cytokinesis. To gain further insight into the role of Rac1A in Dictyostelium cells, we created a fluorescent probe that specifically binds its GTP-bound form. A screen based on yeast two-hybrid and GST pull-down assays resulted in the selection of an interaction partner specific for the active form of Rac1A, a GBD (GTPase-binding domain) from rat PAK1 kinase. PAK1_GBD was fused N-terminally to YFP and expressed in Dictyostelium cells. In non-motile cells, our probe was strongly enriched throughout the cortex, while in motile cells it always localized to the leading edge (Fig. 1). During phagocytosis and macropinocytosis, the probe localized to endocytotic cups. During cytokinesis and chemotaxis the probe didn’t show any prominent localization. In order to demonstrate an interaction between PAK1_GBD and GTP-Rac1A in living cells, we employed fluorescence resonance energy transfer (FRET) approach. A unimolecular probe was constructed, where PAK1_GBD and Rac1A were sandwiched between florescent proteins YFP and mRFP. This FRET probe has a prominent cortical localization and measurements by sensitized emission of the acceptor indicate that PAK1_GBD and GTP-Rac1A interact in the cortex of living cells. Altogether, our results demonstrate that localization of active Rac1A, as reported by our probe PAK1_GBD-YFP, does not correspond to localization of GFP-DGAP1 and GFP-cortexillin, the other components of the aforementioned cortical complex that contains Rac1A. Based on these results, we propose that Rac1A has a dual role in regulation of the actin cytoskeleton. In addition to its established role in recruitment of the DGAP1-cortexillin complex to the rear parts of a polarized cell, it also participates in signaling pathways that control de novo actin polymerization at the protruding regions of the cell.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA