Protein and lipid functions in membrane scission during clathrin-independent endocytosis – ProLipScis

Membrane scission is a critical step in the molecular chain of events that lead to the formation on intracellular transport intermediates. The GTPase dynamin plays an important role in scission. Yet, converging evidence from yeast, Drosophila and mammalian systems suggests that particularly in the case of clathrin-independent endocytosis, other mechanisms may also contribute to efficient scission ' either in synergy with dynamin, or on their own right. Our preliminary evidence on cells and model membrane systems suggests that tubular endocytic membrane invaginations that are induced in a coat-independent manner by the bacterial Shiga toxin ' a model cargo for clathrin-independent endocytosis ' undergo scission when they are transferred from physiological temperature to room temperature or below. Since cholesterol is essential for this process, we hypothesize that the membranes in these invaginations are near a miscibility critical point (phase boundary), and that an external cue may amplify dynamic, small-scale membrane heterogeneities resulting from critical fluctuations by changing the membrane's proximity to this point. Collective effects lead to force generation for scission if the process occurs in a tubular membrane, as predicted by theoretical considerations. Apart from changing our view on the scission reaction, this model has radical conceptual implications on how the membrane fabric is itself an integrate part of biologically relevant trafficking processes. We expect to obtain the following scientific results whose implications go beyond the immediate context of biological membrane scission: - Compositional information of membrane subdomains (Shiga toxin-induced tubules) that are formed by dynamic protein-lipid clustering and that undergo defined biological reactions (invaginations and scission). We expect to find that such subdomains are enriched in so-called raft lipids. - Physical information on the phase behavior of such membrane subdomains. We expect that these lipid compositions are in a state close to a phase boundary at physiological temperatures. - Identification of the protein machinery involved in inducing scission without dynamin (or in synergy with dynamin). We expect that actin will be a critical player, and that actin forces are crucial for vesicle scission. - Integrative view of protein and lipid function in membrane scission. We expect to find that membrane states as those found in STxB-induced tubules are prone for lipid repartition heterogeneity-driven membrane scission, induced by a specific protein machinery such as actin. - We also expect to show that the concepts derived from the work on Shiga toxin can be extended to other exogenous cargos that enter cells by clathrin-independent endocytosis. The conceptual challenges of our project are high numerous and represent not simply a logical development of existing models, but rather a radically new thinking about biologically relevant membrane organization at mesoscopic scales. The combination of solid preliminary data, dedicated experimentation, and a solid grasp on the physical and biological concepts should ensure us with a maximum of chances of success. Our proposal will also respond to the following technical challenges and thereby is expected to make contributions that will widely penetrate the cell biology community: - Secondary ion mass spectrometry (SIMS) for lipid imaging. We expect that the development of SIMS protocols for lipid imaging will be a break-through contribution in the field of lipid cell biology, in which the cellular localization of minimally-modified lipids is one of the biggest current challenges. - Reconstitution of membrane invagination and scission on pore suspending lipid bilayers. In current liposomal systems, it is very difficult and most of the time practically impossible to manipulate proteins machinery at both sides of the membrane. We expect that the development of pore-suspending bilayers will offer a technical solution to this challenge. Again, it's the combination of solid preliminary data with strong expertise that is expected to maximize chances of success. The project is positioned at the interface between biology and physics, and involves challenging chemical synthesis schemes for the generation of tailor-made lipid species. The primary scientific question ' membrane scission ' is motivated from a biology background. However, we are extremely aware that collective behavior of biomolecules needs to be taken into account when reasoning about membrane processes, and conventional cell biology concepts fail to take up this challenge. By creating a team of biologists, physicists, and chemists, we directly position our work within the framework of physical concepts on critical fluctuations near critical temperatures that are expected to become a rich ground for truly innovative scientific discovery in membrane biology.