Practice question

Formation of endocytic vesicle demands energetically unfavorable fission of neck connecting bud to parent membrane against line tension and bending resistance. In clathrin-mediated pathway adaptor AP2 and accessory proteins FCHo, epsin together with clathrin lattice driving polymerization stabilize deeply invaginated bud with omega shape. Constriction of neck performed by dynamin large 96 kDa GTPase assembling into helical oligomer about two turns around neck 50 nm diameter PH domain binding PI(4,5)P2. Polymerization aligns G domains enhancing GTPase 100 fold. Upon concerted GTP binding and cooperative hydrolysis helix compacts from 20 nm to 4 nm radius and twist extends producing high curvature shear stress breaking membrane continuum executing hemi-fission then full fission releasing coated vesicle. Actin polymerization via Arp2/3 and cortactin assists under high tension at apical membranes. SNARE interactions drive downstream fusion with early endosome after uncoating, NSF ATPase disassembles cis-SNAREs post-fusion, Rab5 GTP hydrolysis coordinates early tethering but not scission. Dominant negative dynamin K44A unable to bind GTP arrests pits as elongated tubules. Therefore vesicle scission uniquely depends dynamin-mediated GTP hydrolysis providing mechanoenzyme activity essential for endocytosis.