Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

55-POS Board 55 IN VITRO QUANTIFICATION OF THE STOICHIOMETRY OF DYNAMIN FISSION MACHINERY Javier Vera Lillo 1 ; Juan Manuel Martínez Gálvez 1 ; Julene Ormaetxea Gisasola 1 ; Anna V Shnyrova 1 ; Vadim A Frolov 1,2 ; 1 Biophysics Institute (CSIC, UPV/EHU) , Department of Biochemistry and Molecular Biology, University of the Basque Country, Leioa, Vizcaya, Spain 2 IKERBASQUE, Basque Foundation for Science, Bilbao, Vizcaya, Spain Dynamin 1 (Dyn1) is a large GTPase implicated in membrane fission during endocytosis. Dyn1 takes energy from GTP hydrolysis to severe the necks of endocytic vesicles. For that, Dyn1 oligomerizes around the necks into helical structures enabling cooperative GTP hydrolysis. The hydrolysis causes acute constriction of the helix, leading to membrane scission. The stoichiometry and pathways of self-assembly of this helical fission machinery remains a subject of debate. Here we used Dyn1-eGFP to determine the minimal number of dynamins needed to produce membrane fission in vitro. We found that eGFP addition had minor effect of on membrane-mediated self-assembly and GTPase activities of Dyn1. We further quantified the Dyn1-eGFP interaction with lipid membrane nanotubes at low protein/lipid ratios. We found that under such circumstances Dyn1-eGFP oligomers stably bound to membrane templates were generally smaller than a single helical rung. Yet they responded to GTP addition and produced stochastic membrane scission. We discuss possible mechanistic interpretations of these findings.

70