Molecular mechanism of DRP1 assembly studied in vitro by cryo-electron microscopy

Mitochondria are dynamic organelles that continually adapt their morphology by fusion andfission events. An imbalance between fusion and fission has been linked to major neurodegenerativediseases, including Huntington's, Alzheimer's, and Parkinson's diseases. Amember of the Dynamin superfamily, dynamin-related protein 1 (DRP1), a dynamin-relatedGTPase, is required for mitochondrial membrane fission. Self-assembly of DRP1 into oligomersin a GTP-dependent manner likely drives the division process. We show here thatDRP1 self-assembles in two ways: i) in the presence of the non-hydrolysable GTP analogGMP-PNP into spiral-like structures of ~36 nm diameter; and ii) in the presence of GTP intorings composed of 13−18 monomers. The most abundant rings were composed of 16 monomersand had an outer and inner ring diameter of ~30 nm and ~20 nm, respectively. Threedimensionalanalysis was performed with rings containing 16 monomers. The single-particlecryo-electron microscopy map of the 16 monomer DRP1 rings suggests a side-by-sideassembly of the monomer with the membrane in a parallel fashion. The inner ring diameterof 20 nm is insufficient to allow four membranes to exist as separate entities. Furthermore,we observed that mitochondria were tubulated upon incubation with DRP1 protein in vitro.The tubes had a diameter of ~ 30nm and were decorated with protein densities. [...