Naslov
Image processing for the analysis of Rac1 dynamics in Dictyostelium amoebae

Autori
Šoštar, Marko ; Marinović, Maja ; Filić, Vedrana ; Pavin, Nenad ; Weber, Igor

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

Izvornik
NEUBIAS Bioimage analysis symposium : Booklet / - , 2018, 137-137

Skup
NEUBIAS, Bioimage Analysis Symposium

Mjesto i datum
Szeged, Mađarska, 2018

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Image analysis ; Dictyostelium ; Rac1

Sažetak
Rho family small GTPases are a group of signaling proteins that regulate and coordinate many aspects of cellular activities driven by the actin cytoskeleton. We are especially interested in understanding their function in random cell migration. Among eukaryotic cells, Dictyostelium amoebae are capable of the fastest re- polarization, sometimes taking only 30 seconds to completely reverse their polarity. As such, they represent a convenient testing ground for conceptual models that link small GTPase signaling with cell morphodynamics during migration. Here, we present initial experimental and modeling results regarding the dynamics of Rac1 GTPases and their effector molecules in Dictyostelium cells. Prerequisite for monitoring the Rac1 activity and dynamics is a biosensor able to match these fast dynamics and suitable for sustained imaging at high recording rates. We briefly describe the characteristics of a fluorescent probe developed for this purpose based on the GTPase-binding domain of DPAKa kinase. This probe is highly specific since it binds only to the active form of Rac1. Furthermore, it has a low cytoplasmic background, thus enabling to resolve small variations of the Rac1 activity in the cell membrane. Since Dictyostelium cells change shape very quickly, using efficient methods to measure temporally resolved distributions of fluorescently labeled proteins in living cells is crucial. Therefore, we rely on specific image analysis tools and procedures for quantifying spatio- temporal patterns of fluorescently labeled biomolecules in the cortex of moving cells. We primarily use QuimP, a set of plugins for ImageJ, which utilizes an active contour algorithm to separate cells from the image background, so even lengthy time series of amoeboid cells can be effectively and quickly analyzed. Obtained information include centroid displacement, distance traveled, speed, elongation and circularity of cells, cell area, cytoplasmic and cortical fluorescence distributions etc. The obtained data ara easily exportable and can be additionally processed if needed. For example, quantitative features of emerging patterns in the cortex can be extracted. Also, we can visualize the distributions of active Rac1 and its effectors in the form of kymographs and relate their dynamics by performing wavelet or cross- correlation analysis. Finally, we present comparative analysis of the spatio-temporal distributions of active Rac1 and its effectors obtained by confocal microscopy and a reaction- diffusion model.

Izvorni jezik
Engleski

Znanstvena područja
Fizika, Biologija

POVEZANOST RADA