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Dynamin is a mechanochemical GTPase essential for membrane fission during clathrimmediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynaminamphiphysin complexes during the membrane fission using electron microscopy (EM) and highspeed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynaminamphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission. [ABSTRACT FROM AUTHOR]