Dynamics of Clathrin Coat Formation in Cells

描述(由申请人提供)：细胞膜的重组和成分从一个脂质双层结合的隔室到另一个隔间的运输是细胞内部结构的大部分基础。笼状蛋白是许多这类过程的主要分子支架，最显著的是受体介导的配体内吞作用，如转铁蛋白、低密度脂蛋白、生长因子和激素。病毒和其他病原体和致病毒素篡夺了进入细胞的这一途径。通过基因组编辑和单一荧光团敏感性成像的组合，直接观察活细胞中依赖于笼蛋白的膜运输的生物化学现在是可能的。这种结合相当于体内的单分子生物化学，解决了关于基本成分及其发挥作用的时间点的模棱两可的问题，特别是当许多步骤具有随机而不是完全确定性的特征时。我们在上一次赠款期间对涂层凹坑启动的分子机制所做的工作表明了单一荧光团敏感成像方法的价值。在这项研究中，我们将分析涂层组装中过渡性检查点的分子机制，开发和应用特定的肌醇磷脂在调节基于笼状蛋白的膜交通中的作用，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环境中。我们将利用一种变革性的新成像技术--晶格光片显微镜(LLSM)，它能够快速、高分辨率、高灵敏度地对整个细胞和多细胞标本(包括活组织)进行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.