Running Against Time - A Kinetic Limit for Endophilin Action during Rapid Endocytosis

Abstract

Time is a limiting factor for many cellular processes. Neurons are particularly sensitive to time as they are specialized for rapid communication. For instance, synaptic vesicle endocytosis could occur within 50 milliseconds to recycle vesicle membranes from the plasma membrane. By contrast, endocytosis in non-neuronal cells occurs slowly, often taking minutes to complete. While a similar set of protein machinery is involved in both slow and rapid endocytosis, it is unknown whether the difference in time duration impacts the action of these proteins. To address this issue, we have investigated the dynamic action of endophilin. Endophilin is a critical protein for both rapid and slow endocytosis. Biochemical studies have shown that endophilin converts flat membranes into bilayer tubules and vesicles in vitro. To examine how endophilin acts within physiological time windows to remodel membranes, we have developed kinetic assays to monitor how endophilin binds membranes, induces local membrane deformation, and produces large-scale changes of membrane morphology. We find that endophilin undergoes a sensing-to-bending transition, which takes about 30 milliseconds. These results show that endophilin does not immediately deform membranes upon its arrival. The bending function is activated only after two amphipathic helices become ready for membrane insertion. Surprisingly, we find that endophilin is fairly slow in producing large-scale membrane changes. While local membrane deformation occurs rapidly, it takes minutes for endophilin to induce the extensive morphological changes for membrane tubulation and vesiculation. Therefore, we propose that endophilin's action depends on how much time it has on endocytic membranes. In other words, it is the kinetic property, rather than the thermodynamic equilibrium, that dominates endophilin action during rapid endocytosis.