Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

29-POS Board 29 SINGLE MOLECULE MASS PHOTOMETRY OF DYNAMIN AND MEMBRANE INTERACTION REVEALS ITS POLYDISPERSITY AND OLIGOMER-SPECIFIC INTERACTIONS Manish S Kushwah 1 ; Philipp Kukura 1 ; 1 University of Oxford, Department of Chemistry, Oxford, Oxfordshire, United Kingdom Dynamin, the prototypical member of dynamin superfamily, is a large multi-domain GTPase, and is essential for membrane fission during clathrin-mediated endocytosis. Electron and fluorescence microscopy studies along with conductance measurements have provided detailed preliminary insights into dynamin polymerization in both solution and on membranes, as well as effects of nucleotide binding, membrane constriction and membrane fission upon GTP hydrolysis. Furthermore, extensive x-ray crystallography and cryo-electron microscopy of dynamin in various nucleotide-bound states have provided a detailed picture of GTP-hydrolysis catalyzed membrane fission. A quantitative understanding of dynamin polymerization, the critical initial step in translating from a solubilised to a functional form, however, remains elusive and requires an approach enabling dynamic observation of the dynamin polymerization and its interactions at the molecular level. Here, we use mass photometry, a label-free, imaging- based method to accurately determine the molecular mass of single molecules (Young and Hundt et al., 2018), to measure oligomeric distributions of dynamin-1 over a range of concentrations and conditions. We find an equilibrium between monomer, dimer and tetramer at lower concentrations (<100nM), moving towards 2-8mers at higher concentrations (<500nM). Interestingly, dimers and tetramers dominate (>50nM) making them the most stable species and GTP binding shifts the equilibrium towards tetrameric species (200-500nM). The abundances of these species as a function of concentration reveals the energetics of oligomerization and effect of GTP binding on associated equilibria. We next compared binding of oligomers to lipid vesicles (~20nm diameter) and supported bilayers and found that the dimer shows preference to bilayers over vesicles, with the trend is reversed for the tetramer, indicating a curvature- dependent binding affinity of the oligomers. These results represent a first step towards a quantitative view of dynamin polymerization.

83