Abstract
Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated 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 dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin 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.
Highlights
Clathrin mediated endocytosis (CME) is the best characterized endocytic pathway by which cells incorporate extracellular molecules into cells with the aid of clathrin coat (Kirchhausen, Owen, & Harrison, 2014; McMahon & Boucrot, 2011)
We tried to clarify how the membrane constriction and fission by dynamin-amphiphysin ring complexes are correlated with guanine nucleotide conditions during GTP hydrolysis
high-speed atomic force microscopy (HS-AFM) analyses directly demonstrated that GTP hydrolysis induces dynamic longitudinal movement of the dynamin-amphiphysin rings as well as constriction during cluster formation (Figure 3)
Summary
Clathrin mediated endocytosis (CME) is the best characterized endocytic pathway by which cells incorporate extracellular molecules into cells with the aid of clathrin coat (Kirchhausen, Owen, & Harrison, 2014; McMahon & Boucrot, 2011). CME is required for various essential processes including neuronal transmission, signal transduction and other cell membrane activities such as cell adhesion and migration. For precise progression of membrane invagination and fission during CME, various proteins need to be assembled in a temporally and spatially coordinated manner at the site of endocytosis. One of those endocytic proteins, dynamin, is a GTPase essential for membrane fission in CME (Antonny et al, 2016; Ferguson & De Camilli, 2012; Schmid & Frolov, 2011). Cook, Mesa, & Urrutia, 1996; T. A. Cook, Urrutia, & McNiven, 1994)
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