Fig. 3.

Dynamin-lipid tubes generated with extruded liposomes constrict and fragment upon addition of GTP. (A) To create extruded liposomes, lipid in chloroform solvent is dried under nitrogen gas in a glass tube and stored under vacuum overnight. The lipid is then resuspended in buffer and extruded through a 1 µm polycarbonate membrane (Avanti). (B) Liposomes are added to dynamin in HCB100 and incubated for ~2 h to generate dynamin–lipid tubes, which are observed using negative stain TEM (panels C and D). GTP is added either directly to the sample in the tube or by placing the grid with sample adhered to its surface on a drop of GTP in solution. (C, D) Negative stain EM of dynamin–lipid tubes prior to GTP addition are 50 nm in diameter. (E–G) In the presence of GTP, dynamin–lipid tubes fragment and constrict to 40 nm in diameter. Scale bar, 100 nm.