Rôle de la tension et de la courbure membranaires de l'appareil de Golgi dans le transport intracellulaire – Golgi/Tension

Intracellular transport is mediated by small tubulo-vesicular carriers that concentrate cargo molecules and bud off the membrane of the donor compartment. Molecular sorting and membrane deformation are critical initial steps in intracellular transport. Although the molecular mechanisms leading to sorting and deformation are being clarified, very little is known about the corresponding biophysical mechanisms. The role of membrane physical parameters, such as membrane tension or membrane curvature, is particularly poorly understood. The COPI coat protein complex is involved in both lipid and protein sorting and membrane deformation at the level of the Golgi apparatus. Recruitment of coatomer by membrane bound Arf1-GTP leads to self-assembly of the COPI coat. COPI has been shown to regulate retrograde transport from the Golgi apparatus to the endoplasmic reticulum and intra-Golgi transport. We have recently succeeded in reconstituting the self-assembly of the COPI coat on model membranes (giant unilamellar vesicles, GUVs) and showed that 1) COPI specifically assembles on membranes in the liquid disordered state and 2) COPI-induced membrane deformation is facilitated by a low membrane tension (Manneville et al. PNAS 2008). Our results also suggest that the COPI-coat mediates lipid sorting. In the present proposal, we wish to investigate the interplay between COPI coat proteins and physical parameters during molecular sorting and membrane deformation. This issue will be addressed both by quantitative in vitro experiments and in vivo experiments. The in vitro part of the project will be built on our minimal COPI-coat reconstitution on GUV membranes (Manneville et al. PNAS 2008) and the development of a new microscope combining confocal fluorescence microscopy, force measurements by optical tweezers and control of membrane tension by micropipette aspiration. Membrane tubes will be pulled from GUVs at fixed tension using a streptavidin-coated bead coupled to biotinylated lipids in the GUV membrane and manipulated by the optical tweezers. Parameters such as the membrane tension, the membrane bending rigidity, and the force exerted on the bead can be measured and/or tuned with this assay. The new set-up will allow us to address questions related to membrane deformation by 1) quantifying the threshold tension below which deformation occurs upon COPI coat assembly; 2) measuring the force exerted by the COPI coat on the membrane during coat assembly and subsequent membrane deformation; 3) testing the mechanical role of membrane tension and the role of candidate proteins in membrane fission by the COPI coat. Experiments will also be designed to study molecular sorting. The diameter of the tube is set by membrane tension. This enables us to measure sorting as a function of membrane curvature. Sorting of COPI coat proteins will be investigated first. We will then focus on the role of COPI in curvature-induced lipid sorting. In a second part of the proposal we propose to test in vivo the conclusions drawn from the in vitro work. We will upgrade the experimental set-up in order to visualize live cells under the microscope. Streptavidin-coated beads will be incorporated into intact cells and manipulated by the optical tweezers to couple them to the membrane of the Golgi apparatus. The coupling will be achieved by expressing biotinylated Golgi proteins in vivo. Mechanical tension will be imposed on the Golgi membranes via bead displacement. Our experiments should provide the first measurement of the tension of the Golgi apparatus. We will investigate the role of membrane tension on COPI-mediated transport to test the idea along which a low tension facilitates membrane deformation. We will also try to understand how the tension of the Golgi apparatus is regulated and to identify tension regulators in vivo. Finally, by extracting membrane tubes from Golgi membranes, the role of membrane curvature in the sorting of COPI proteins will be addressed and the influence of COPI on lipid sorting will be tested. Taken together, our results should lead to a better understanding of the mechanics of membrane deformation by coat proteins and of their role in lipid and protein sorting. The tools and methodologies developed during the project should also be useful to tackle a wide variety of questions regarding intracellular transport at the interface between physics and biology.