title: A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function.
abstract: Dynamin, the paradigmatic membrane fission catalyst, assembles as helical scaffolds that hydrolyse GTP to sever the tubular necks of clathrin-coated pits. Using a facile assay system of supported membrane tubes (SMrT) engineered to mimic the dimensions of necks of clathrin-coated pits, we monitor the dynamics of a dynamin-catalysed tube-severing reaction in real time using fluorescence microscopy. We find that GTP hydrolysis by an intact helical scaffold causes progressive constriction of the underlying membrane tube. On reaching a critical dimension of 7.3 nm in radius, the tube undergoes scission and concomitant splitting of the scaffold. In a constant GTP turnover scenario, scaffold assembly and GTP hydrolysis-induced tube constriction are kinetically inseparable events leading to tube-severing reactions occurring at timescales similar to the characteristic fission times seen in vivo. We anticipate SMrT templates to allow dynamic fluorescence-based detection of conformational changes occurring in self-assembling proteins that remodel membranes.
authors.affiliation: Indian Institute of Science Education and Research, Dr. Homi Bhabha Road Pashan, Pune 411008, Maharashtra, India.
authors.forename: Srishti
authors.initials: S
authors.lastname: Dar
authors.affiliation: Indian Institute of Science Education and Research, Dr. Homi Bhabha Road Pashan, Pune 411008, Maharashtra, India.
authors.forename: Sukrut C
authors.initials: SC
authors.lastname: Kamerkar
authors.affiliation: Indian Institute of Science Education and Research, Dr. Homi Bhabha Road Pashan, Pune 411008, Maharashtra, India.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Pucadyil
mesh_terms: D002384:Catalysis; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D003034:Coated Pits, Cell-Membrane / Q000737:chemistry / Q000378:metabolism; D034284:Dynamin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D059008:Time-Lapse Imaging / Q000379:methods*
chemical_list: D008563:Membrane Lipids; D019269:Phosphatidylinositol 4,5-Diphosphate; D006160:Guanosine Triphosphate; D034284:Dynamin I