Agenda

The actin cytoskeleton is a complex network of filaments distributed throughout the cell cytoplasm. This cytoskeleton mediates essential cellular functions such as cell migration, endocytosis, and cell division. Its efficiency relies on rapid renewal of actin filaments in a cyclic process involving their polymerization, disassembly, and recycling of actin subunits into polymerizable monomers. In cells, this turnover can occur on timescales of few seconds to few minutes and is tightly regulated by the coordinated action of multiple actin binding proteins (ABPs). Our understanding of these mechanisms is incomplete, limiting our ability to reconstitute various actin-based cellular processes, particularly when encapsulating these biomimetic systems into cell-like microenvironments. A better understanding of these molecular mechanisms requires identifying the optimal combinations and concentrations of ABPs capable of efficiently catalyzing the various steps of actin turnover. This is possible if an efficient and high-throughput strategy is implemented to characterize and compare the individual and combinatorial effects of ABPs. During my PhD, I developed novel in vitro assays based of the exchange dynamics of actin-bound nucleotides, or on measurement of actin ATP consumption. Using these sensitive assays, I screened ABPs involved in actin disassembly and recycling, determined their activity as a function of concentration, and identified optimal conditions for rapid filament turnover. In addition, I compared the efficiency of these proteins at high F-actin concentration, close to cellular levels. Finally, I demonstrated that rapid turnover is maintained under confined conditions by encapsulating an optimized actin system into cell-sized vesicles. Overall, this work establishes a quantitative framework for reconstituting rapid actin turnover in confined, cell-like environments and provides a foundation for reconstituting various actin-based dynamic processes, such as endocytosis and cell division, in vesicles with deformable membranes.