Clathrin‐mediated endocytosis: Dynamics and Dynamin

Dynamin is essential for clathrin‐mediated endocytosis (CME), but its exact function and mechanism of action remain unknown. Using TIR‐FM together with new particle tracking software and statistical analyses we identified 3 kinetically‐distinct subpopulations of eGFP‐labeled clathrin coated pits (CCPs): 2 short‐lived abortive species and 1 longer‐lived productive species. siRNA‐mediated knock‐down of dynamin‐2 and reintroduction of WT or mutant dynamin‐1 showed that it plays an early regulatory role and controls the rate of CCV formation. We have also developed fluid sup ported bilayers with e xcess membrane r eservoir, (SUPER) templates, to assay vesicle formation and membrane fission. Under physiological conditions in the constant presence of GTP, dynamin cooperatively organizes into self‐limited assemblies at the necks of emergent vesicles and is sufficient to mediate membrane fission leading to continuous vesicle release. Together, these in vivo and in vitro approaches provide evidence that dynamin plays a dual role in CME, functioning at early stages to regulate CCV formation and at later stages to directly mediate membrane fission. Supported by NIH GM42455, GM73165, MH61345.