Biophotonics approach to regulation of the actin cytoskeleton dynamics by small GTPase proteins
Actin cytoskeleton, a dynamic filamentous meshwork composed of protein actin and numerous associated proteins, enables cells to maintain or change shape, to move, divide and internalize nutrients. In order to perform such vitally important functions, actin cytoskeleton undergoes fast rearrangements and forms local, short-living functional domains in the cell. Precise control of the cytoskeleton remodeling is guaranteed by multiple intracellular signaling pathways and important roles in this process are played by small GTPase proteins from the Rho family. Analysis by standard biochemical assays alone can provide only limited information about spatio-temporal dynamics of the Rho GTPase activity. In order to capture these dynamics in real time, we plan to use the tools of biophotonics to image and analyze signaling events and correlate them with remodeling of the actin cytoskeleton. Probes that incorporate fluorescent proteins will be used to non-invasively report the changing subcellular localization and activity of the small Rho-GTPase Rac1A in living cells. Confocal laser scanning microscopy will be utilized to gain information about local recruitment of proteins and their interactions with high spatial and temporal resolution. As the model organism we use amoeboid Dictyostelium cells which are amenable to elaborate genetic engineering and biochemical analyses. These free-living cells are highly motile and display a rich repertoire of activities that involve rapid formation, transformation and deconstruction of specialized cytoskeletal structures. Basic principles that govern remodeling of the cytoskeleton and the molecules involved are similar in animal cells and Dictyostelium, which established this organism as an important model in cytoskeleton and signaling research. Dictyostelium cells are active phagocytes, perform chemotaxis and aggregate to form a motile multicellular assembly, making this research relevant for understanding of such fundamental processes as cellular immune response, cancer metastasis, and embryonic development. Besides providing new biological insights, introduction of experimental tools of cellular biophotonics and training of young scientists to develop and use these tools will provide significant benefit to scientific community in Croatia. While advanced light microscopy is finding a widespread and ever expanding use in biosciences worldwide, it is still underrepresented in Croatian scientific milieu. This project is envisaged to set up a site of expertise and a reference laboratory for application of biophotonics methods in Croatia. Our intention is to raise awareness about potential of such methods in life sciences, to offer advice and opportunity for collaboration to interested colleagues, and to make available advanced courses including hands-on experience to students.