Optimizing actin turnover in cell-like conditions

Abstract A dynamic actin cytoskeleton, characterized by rapid filament turnover, is essential for driving intracellular transport and cellular movement. Despite extensive study, the contributions of many actin-binding proteins (ABPs) that regulate the different steps of the actin turnover cycle (filament assembly, disassembly and recycling into polymerizable actin monomers) remain ill-defined. Here, we introduce novel sensitive in vitro assays to quantitatively assess how ABPs catalyze actin turnover. By accurately measuring nucleotide exchange dynamics and ATP consumption, these assays enable robust characterization of ABP activity across broad concentration ranges. Using these methods and modeling of these reactions, we systematically examined the contributions of five conserved regulators, both individually and in combination, and identified conditions that maximize turnover efficiency. We also determined that increasing F-actin concentration to cellular levels affects ABP activity. Finally, we demonstrated that rapid actin turnover is preserved during encapsulation in cell-sized vesicles using the cDICE method. Together, these advances provide versatile tools and new insights into actin cytoskeletal dynamics under physiologically relevant conditions. Competing Interest Statement The authors have declared no competing interest.