Oscillations in the cytoskeleton are of general importance in cell biology as a means of spatial and temporal regulation of the cellular organization, cell division and motility. The project OSCITON focuses on two oscillatory processes. First, we will investigate the mechanism of microtubule-driven kinetochore oscillations during mitosis, and the role of kinesin-8 motors in this process.
These oscillations generate tension between sister kinetochores, which is required to pass the spindle checkpoint. We will use HeLa cells expressing tubulin-GFP and mRFP-CENP-B, a kinetochore protein, in combination with advanced light microscopy and depletion or overexpression of kinesin-8 motors. To study the forces acting on kinetochores, we will perturb the force balance by performing laser microsurgery of the microtubules attached to kinetochores.
The experiments will be accompanied by the development of a theoretical model, which will allow us to explore the role of kinesin-8 in the oscillations. Second, we will study the oscillatory repolarization of Dictyostelium cells during random migration. We propose that the competition of DGAP1/cortexillin and SCAR/Wave complexes for the common activator Rac1 plays a central role in this process. To test this hypothesis, we will use a novel fluorescent probe for active Rac1 GTPases, generate cells with disturbed balance of Rac1 effectors, and cells deficient in functional Rac1. Dynamics of the probes for Rac1, its effectors, and F-actin will be studied experimentally in live cells and theoretically.
This interdisciplinary project is designed to optimize the use of resources and bring together researchers with the expertise in a range of disciplines, including molecular and cellular biology, biochemistry, microscopy, and theoretical biophysics. Through joining forces of all investigators of the cytoskeletal dynamics in Croatia, we intend to assemble a team that will play a prominent role in this field of research internationally.