A schematic alignment of mammalian and yeast dynamins is presented and highlights the domain organization for each protein. The GTPase, middle, and GED topology is conserved among all family members. GED, GTPase effector domain; PH, pleckstrin homology; PRD, proline-rich domain; MTS, mitochondria targeting sequence; TM, transmembrane. (For Arabidopsis dynamins see Hong et al., 2003).

Oligomeric structures of dynamins are visualized using negative stain TEM. (A) Oligomers are generated under low salt conditions or in the presence of nonhydrolyzable nucleotides. (B) Dynamin spiral structures are shown that were dialyzed in the presence of GDP/BeF. (C) Larger spiral structures are observed for Dnm1 in the presence of GMP-PCP. (D) MxA curved filaments and occasional rings are presented after incubation with GMP-PCP. All protein structures were generated in HCB100. Scale bar, 100 nm.

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.

Biochemical assays quantify dynamin assembly and conformational changes upon addition of GTP. (A) The sup/pellet assay quantifies dynamin assembly under different conditions by centrifuging the sample at 100,000g and running the supernatant and pellet fractions on a gel (top). Imaging software quantifies the relative amount of protein in the pellet (bottom). (B) 90° light scattering measures the relative change in tube conformation. Addition of GTP (indicated with an arrow) leads to a rapid and dramatic decrease in scattering.

Dnm1–lipid tubes are imaged using negative stain and cryo-EM. (A) Negative stain imaging of Dnm1–lipid tubes suggests flattening occurs due to stain and sample dehydration (B) Corresponding Fourier transform of image in A shows spots indicating a 2D lattice, indicative of tube flattening. (C) Cryo-EM image of Dnm1–lipid tube. (D) Fourier transform of image in (C) provides a regular helical pattern with layer lines suggesting a helical structure.

Cryo-EM imaging and reconstructions of dynamin are presented in the nonconstricted and constricted states. (A) Dynamin–lipid tubes formed as in Fig. 3 and imaged using cryo-EM. (B, C) When GTP is added, the tubes constrict the lipid bilayer, leading to bulges in regions of undecorated lipid. (D) ΔPRD dynamin forms well-ordered tubes in the presence of lipid and GMP-PCP, which diffract to ~20 Å resolution (Fourier transform in inset). Such images are averaged using helical reconstruction methods to generate a three-dimensional reconstruction (panel F). (E) ΔPRD dynamin–lipid tubes in both the nonconstricted and constricted states are boxed (blue and red boxes respectively) for image processing using the IHRSR reconstruction method (Egelman, 2006). (F) 3D map of dynamin–lipid tube solved by helical reconstruction methods (Zhang & Hinshaw, 2001) represented at both high (yellow mesh) and low (blue) thresholds. (G) IHRSR image reconstructions of the nonconstricted (left) and constricted (right) ΔPRD dynamin–lipid tubes (high-yellow mesh and low–blue thresholds) (Chen et al., 2004). Scale bar for A–E, 100 nm. Scale bar for F and G, 10 nm.

Dynamin examined by several diverse image techniques. (A) Rotary shadowing of dynamin–lipid tubes reveals the surface of the tube and subsequently the hand of the helices. (B) Fourier transform of the shadowed image confirm the long (gray) pitch helix is left-handed and the short (black) pitch helix is right-handed. (C) STEM analysis of a dynamin spiral (highlighted by arrowhead) provides an accurate measure of mass over a defined length when compared with a TMV particle (arrow). Scale bar, 50 nm. (D) AFM imaging of dynamin tubes was used to examine conformational changes triggered by GTP hydrolysis. A zoomed image of dynamin tubes is shown in the inset. Scale bar, 2 µm.