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BP: Fachverband Biologische Physik

BP 4: Membranes and Vesicles I

BP 4.1: Hauptvortrag

Montag, 31. März 2014, 15:00–15:30, HÜL 386

Role of membrane elasticity in clathrin-mediated endocytosis — Sandrine Morlot¹, Saleem Mohammed¹, Nicolas Chiaruttini¹, Valentina Galli¹, Marius Klein³, Luìs Dinis⁴, Martin Lenz⁵, Giovanni Cappello³, and •Aurélien Roux^{1, 2} — ¹Biochemistry Department, University of Geneva, CH-1211 Geneva, Switzerland — ²Swiss National Centre for Competence in Research Programme Chemical Biology, CH-1211 Geneva, Switzerland — ³Institut Curie, Centre de Recherche; CNRS, UMR 168, Physico-Chimie Curie; Université Pierre et Marie Curie, F-75248 Paris, France — ⁴Departamento de Fìsica Atòmica, Molecular y Nuclear, Facultad de Ciencas Fìsicas, Universidad Complutense de Madrid, ES-28040, Madrid, Spain — ⁵James Franck Institute, University of Chicago, IL-60637 Chicago, U.S.A.

In Clathrin-mediated endocytosis, Clathrin assembles into a soccerball-like structure at the plasma membrane that was proposed to deform the membrane by scaffolding. However, controversies in the community have appeared on the exact role of Clathrin: does its polymerization force is sufficient to curve the membrane, or deformation by other means (protein insertion) is required? We studied the formation of Clathrin buds from Giant Unilamellar Vesicles, and found that the pits can be flattened when membrane tension is increased. This suggested that the Clathrin polymerization force could be counteracted by membrane tension, which we further proved by directly measuring Clathrin polymerization force: by pulling a membrane tube out of a GUV aspirated in a micropipette, we can measure the force required to hold the tube through an optical tweezer system. When Clathrin is added, it polymerizes onto the GUV predominantly, and the force drops. From these measurements, we can deduce that the polymerization strength of Clathrin is in the range of a few hundred micronewtons per meter. This value confirms that clathrin polymerization can be counteracted efficiently by membrane tension. To finalize endocytosis, the clathrin-bud needs to be separated from the plasma membrane. Membrane fission requires the constriction and breakage of a transient neck, splitting one membrane compartment into two. The GTPase Dynamin forms a helical coat that constricts membrane necks of Clathrin-coated pits to promote their fission. Dynamin constriction is necessary but not sufficient, questioning the minimal requirements for fission. Here we show that fission occurs at the edge of the Dynamin coat, where it is connected to the uncoated membrane. At this location, the specific shape of the membrane increases locally its elastic energy, facilitating fission by reducing its energy barrier. We predict that fission kinetics should depend on tension, bending rigidity and the Dynamin constriction torque. We verify that fission times depend on membrane tension in controlled conditions in vitro and in Clathrin-mediated endocytosis in vivo. By numerically estimating the energy barrier from the increased elastic energy, and measuring the Dynamin torque, we show that: 1- Dynamin torque, about 1nN.nm, is huge but necessary to achieve constriction, and 2- Dynamin work sufficiently reduces the energy barrier to promote spontaneous fission.