Dynamics of Clathrin Coat Formation in Cells

描述（由申请人提供）：细胞膜的重组和组分从一个脂质双层结合区室到另一个脂质双层结合区室的转运是细胞内部结构的基础。网格蛋白是许多此类过程的主要分子支架-最值得注意的是，受体介导的配体如转铁蛋白、LDL、生长因子和激素的内吞作用。病毒和其他病原体和致病毒素篡夺这一途径进入细胞。通过基因组编辑和单荧光团灵敏度成像的组合，现在可以直接观察活细胞中网格蛋白依赖性膜运输的生物化学。该组合相当于体内单分子生物化学，解决了关于基本成分及其作用时间点的模糊性，特别是当许多步骤具有随机性而不是完全确定性时。我们的工作在前一个补助金期间的分子机制的涂层坑启动说明了价值的单荧光团灵敏度成像方法。在这项研究中，我们将分析外套组装中过渡检查点的分子机制，开发和应用特定磷酸肌醇脂质在调节网格蛋白膜运输中的作用的探针，并将我们的“成像生物化学”扩展到多细胞组装和活组织的3D背景。我们将利用一种变革性的新成像技术，即点阵光片显微镜（LLSM），该技术可实现全细胞和多细胞标本（包括活组织）的快速、高分辨率、高灵敏度3D可视化。

DESCRIPTION (provided by applicant): Reorganization of cellular membranes and transport of components from one lipid-bilayer bounded compartment to another underlie much of the internal structure of a cell. Clathrin is the principal molecular scaffold for a number of such processes -- most notably, receptor-mediated endocytosis of ligands such as transferrin, LDL, growth factors, and hormones. Viruses and other pathogens and pathogenic toxins usurp this pathway to enter cells. Direct observation of the biochemistry of clathrin-dependent membrane traffic in living cells is now possible, through a combination of genome editing and single-fluorophore sensitivity imaging. The combination, which amounts to in vivo, single-molecule biochemistry, resolves ambiguities about essential components and the time points at which they function, particularly when many of the steps have a stochastic rather than fully deterministic character. Our work in the previous grant period on the molecular mechanism of coated-pit initiation illustrates the value of single-fluorophore-sensitivity imaging approaches. I the research described in this proposal, we will analyze the molecular mechanisms of transitional checkpoints in coat assembly, develop and apply probes for the roles of specific phosphoinositide lipids in regulating clathrin- based membrane traffic, and extend our "imaging biochemistry" to the 3D context of multicellular assemblies and living tissues. We will capitalize on a transformative new imaging technology, lattice light- sheet microscopy (LLSM), which enables rapid, high-resolution, high-sensitivity 3D visualization of whole cells and multicellular specimens, including living tissues.