Understanding membrane fission through the study of dynamin's twisting activity – DYNTWIST

Eukaryotic cells are separated from their environment by a lipid membrane mostly impermeable to nutrients essential to their life. In order to incorporate them, proteins transiently interacting with the lipids are form membrane buds. To finalize internalization, buds need to be separated from the membrane, a step called fission. Lipid membranes are auto-sealable objects, making their fission not easy. In spite of physics describing well the elastic free energy associated with the deformation of membranes, the energy cost of fission is hard to estimate both experimentally and theoretically. However in cells, a constant number of fission events sustain the activity of the membrane traffic. Dynamin, a protein with unique mechanical properties has been implicated in the rupture of lipid bilayers. It is able to polymerize into a helix at the neck of nascent buds and strangle them by torsion until they break. As we have participated in the discovery of the twisting activity of dynamin, the first motor of this kind ever described, we propose to understand membrane fission by a detailed investigation of the mechanical gear of dynamin. To achieve this goal, we will measure the relevant parameters that describe the twisting activity of the dynamin polymer: the twisting speed, the torque applied to the membrane, the energetic yield and the total energy consumed by a fission event. These measurements will be used to build and optimize and theoretical model, which in turn, will lead us to important information on the kinetics and the energy cost of membrane fission. To do these measurements, we apply to this Appel à projets 'Jeunes Chercheurs' from the ANR, and ask for the funding of two adapted experimental set-ups. The first one, combining optical tweezers with micropipettes and imaging will allow us to quantify the elastic parameters of the dynamin-membrane tubule, and establish a realistic theoretical description of it. Then, it will be used to study the role of membrane tension in fission. The second set-up will be magnetic tweezers adapted to the measurement of the torque applied by dynamin to the membrane tubules. It will also be used to quantify energy consumption.