Naslov
Visualization and analysis of small GTPases dynamics in highly motile cells

Autori
Marinović, Maja ; Šoštar, Marko ; Filić Mileta, Vedrana ; Antolović, Vlatka ; Weber, Igor

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

Izvornik
17th International European Light Microscopy Initiative Meeting (ELMI) : Program and Abstract book / Weber, Igor ; Tolić, Iva ; Kovačević, Goran ; Vidoš, Ana - Zagreb : Institut Ruđer Bošković ; Hrvatsko mikroskopijsko društvo, 2017, 152-152

ISBN
978-953-7941-16-1

Skup
17th International European Light Microscopy Initiative Meeting (ELMI)

Mjesto i datum
Dubrovnik, Hrvatska, 23.05.2017. - 26.05.2017

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Image analysis ; Dictyostelium ; Rac1

Sažetak
Small Rho GTP-ases play various important roles in the regulation of the actin cytoskeleton dynamics in eukaryotic cells, including rapidly moving Dictyostelium amoebae. In contrast to other cell types, appropriate probes for monitoring the activity of many Rho GTPases in Dictyostelim are still lacking. Here we describe the use of a newly developed fluorescent probe for visualization of the active form of Rac1 GTPases in live Dictyostelium cells. The probe is based on the GTPase-binding domain (GBD) from DPAKa kinase fused with the yellow fluorescent protein. The probe was selected on the basis of the yeast two-hybrid screen, GST pull-down assay, and FRET measurements in live cells by fluorescence lifetime imaging microscopy (FLIM). Images of the cells expressing the DPAKa(GBD)-DYFP probe obtained with the confocal microscope exhibit a high membrane-to-cytoplasm intensity ratio, which enables discerning fine details of Rac1 activity within the cell membrane. Importantly, due to its high contrast, the probe enables prolonged imaging of live cells with low-intensity excitation light, thus contributing to low levels of photobleaching and phototoxicity. Additionally, overexpression of DPAKa(GBD)-DYFP probe doesn’t have any effects on cell motility, cytokinesis and cell growth. The detailed information about the active Rac1 membrane distribution obtained by imaging of DPAKa(GBD)-DYFP will facilitate the quantitative analysis of the mechanisms that regulate the Rac1 dynamics.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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