Reconstitution of clathrin/AP-1 coated membrane microdomains on proteoliposomes

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A fundamental feature of eukaryotic cells is their compartmentalization into membrane-bound organelles organized in membrane microdomains. Vesicular transport along the organelles of the secretory and the endocytic pathways relies on the dynamic assembly/disassembly of sorting devices (coats) that are recruited onto defined membrane microdomains to form transport intermediates. Using liposome-based systems, we have reconstituted the selective assembly of the clathrin/AP-1 coat on synthetic membranes. This coat functions in protein sorting/transport between the secretory and the endocytic pathways of eukaryotic cells. Using mass spectrometry, we identified the protein networks associated with AP-1, an analysis which surprisingly revealed that AP-1 coat assembly drives the concomitant assembly of other cellular machines involved in actin polymerization and membrane fusion. In this proposal, we want to combine biochemical, molecular and biophysical methods (Fluorescence Cross Correlation Spectroscopy) to visualize and measure the dynamic assembly of these cellular machineries onto synthetic membrane microdomains. We want here to address four main biological questions: 1) what is the dynamic behavior of the three different machineries during the assembly process? 2) what is the on/of states of these machines during the overall assembly process leading to the formation of a stable structure in the plane of membranes, 3) What is the role of actin polymerization in this process and 4) does this protein-driven assembly process lead to a phase partition of membrane lipids (liquid ordered/disordered phases). Adapted biophysical methods measuring physical parameters of association/dissociation, diffusion of membrane components will help us to answer those questions. Our working hypothesis, partly based on previous and current results, is that AP-1 coat, taken as a paradigm, binds onto flat membranes, diffuses in the plane of membranes using forces generated by actin polymerization in order to form a stable patch, potentially stabilized by clathrin polymerization. Actin polymerization could subsequently help forming a tubular transport intermediate that would ultimately bud.