Single-molecule turnover dynamics of actin and membrane coat proteins in clathrin-mediated endocytosis

Actin is required for clathrin-mediated endocytosis (CME) in yeast. Experimental observations indicate that this actin assembly generates force to deform the membrane and overcome the cell’s high turgor pressure, but the precise molecular details remain unresolved. Based on previous models, we predicted that actin at endocytic sites continually polymerize and disassemble, turning over multiple times during an endocytic event. Here we applied single-molecule speckle tracking in live fission yeast to directly measure this predicted turnover within the CME assembly for the first time. In contrast with the overall ~20-sec lifetimes of actin and actin-associated proteins in endocytic patches, we detected single-molecule residence times around 1 to 2 sec, and high turnover rates of membrane-associated proteins in CME. Furthermore, we find heterogeneous behaviors in many proteins’ motions. These results indicate that rapid and continuous turnover is a key feature of the endocytic machinery and suggest revising quantitative models of force production.