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

真核细胞的一个基本特征是它们被分隔成在膜微域中组织的膜结合细胞器。囊泡沿分泌和内吞途径细胞器的运输依赖于分类装置(外套)的动态组装/拆卸，这些分类装置被招募到特定的膜微域上以形成运输中间体。利用基于脂质体的系统，我们已经在合成膜上重建了笼蛋白/AP-1涂层的选择性组装。这一外壳在真核细胞的分泌和内吞途径之间的蛋白质分选/运输中起作用。利用质谱仪，我们确定了与AP-1相关的蛋白质网络，这一分析令人惊讶地揭示了AP-1涂层组装驱动参与肌动蛋白聚合和膜融合的其他细胞机器的伴随组装。在这个方案中，我们希望结合生物化学、分子和生物物理方法(荧光交叉相关光谱)来可视化和测量这些细胞机械在合成膜微域上的动态组装。我们想要解决四个主要的生物学问题：1)三种不同的机器在组装过程中的动态行为？2)这些机器在整个组装过程中的开启/变化状态导致在膜平面上形成稳定的结构，3)肌动蛋白聚合在这个过程中扮演什么角色，4)这种蛋白质驱动的组装过程是否导致膜脂的相分配(液体有序/无序相)。采用适当的生物物理方法测量膜组分的缔合/解离、扩散的物理参数将有助于我们回答这些问题。我们的工作假设部分基于以前和现在的结果，即AP-1涂层作为一个范例，结合到平坦的膜上，利用肌动蛋白聚合产生的力在膜平面上扩散，以便形成一个稳定的斑块，并有可能通过分子筛蛋白聚合稳定下来。肌动蛋白聚合随后可以帮助形成最终发芽的管状运输中间体。

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.