Naslov
Rac1 GTPases play a dual role in the regulation of cell motility

Autori
Filić, Vedrana ; Marinović, Maja ; Weber, Igor

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

Izvornik
Hrvatski mikroskopijski simpozij 2012, Knjiga sažetaka / Gajović, A. ; Tomašić, N. - Zagreb : Hrvatsko mikroskopijsko društvo, 2012, 16-17

ISBN
978-953-57138-1-4

Skup
Hrvatski mikroskopijski simpozij 2012

Mjesto i datum
Pula, Hrvatska, 16.11.2012. - 17.11.2012

Vrsta sudjelovanja
Pozvano predavanje

Vrsta recenzije
Domaća recenzija

Ključne riječi
confocal microscopy; live cell imaging; FRET; Dictyostelium; actin cytoskeleton

Sažetak
Dictyostelium Rac1A is a member of a broad family of monomeric Rho GTPases that are involved in regulation of the actin cytoskeleton. It was previously shown that Rac1A binds the IQGAP- related protein DGAP1, which forms a cortical complex with cortexillin heterodimer. To gain more insight into its role in living cells, we decided to create a fluorescent probe that specifically binds the active form of Rac1A. As a first step in the selection of an appropriate GTPase-binding domain to be used in construction of the probe, we performed a Y2H screen using four candidate domains as baits. Only GBD from rat PAK1 interacted with active forms of Rac1A, Rac1C and RacC, but not with their inactive forms. After this interaction was confirmed by GST pull-down assay, PAK1_GBD was N-terminally fused to YFP and expressed in wild-type cells. In non-motile cells, our probe was strongly enriched throughout the cortex, while in motile cells it always localized to the leading edge. In order to demonstrate an interaction between GTP-Rac1A and PAK1_GBD in vivo, we constructed a unimolecular probe for intramolecular FRET (fluorescence resonance energy transfer) (Figure 1a), which had a prominent cortical localization (Figure 1b). Using the method of sensitized emission of the acceptor, we were able to detect significant FRET effect in cortical regions of living cells with the quadruple construct (Figure 1b, 1c), and no FRET in a similar triple construct that lacks Rac1A (Figure 1d). We analyzed FRET efficiency for five different constructs that clearly segregate into a positive and a negative group (Figure 1e). Results of uniform chemoattractant stimulation experiments show a transient recruitment of PAK1_GBD-YFP to the cell cortex. This effect was stronger in Rac1A- overexpressing cells and in DGAP1-null cells, whereas in DGAP1-overexpressing cells it was weaker than in wild-type cells. Spatial and temporal dynamics of PAK1_GBD-YFP probe during random motility and endocytosis strongly resembles localization of other components involved in signalling pathways leading to actin polymerization. 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, components of a quartenary cortical complex that also contains activated Rac1A. Based on these results, we propose that Rac1A has a dual role in regulation of the actin cytoskeleton in Dictyostelium [1]. 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 signalling pathways that control de novo actin polymerization at the protruding regions of the cell.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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