Modeling the nucleation of clathrin coated vesicles at the cell membrane

这个K99/R 00的建议是集中在研究蛋白质的相互作用，驱动网格蛋白介导的内吞作用中使用的网格蛋白包被的囊泡的成核。该提案的主要目标是确定外壳成核的机制，并确定使网格蛋白外壳成核所必需且足够的蛋白质的最小核心集（在数十种涉及的蛋白质中）。这里开发的计算模型必须（1）确定涂层成核中许多参与者的功能，（2）再现建模系统的实验性质，以及（3）易于解释以指导进一步的实验和模型。三个具体目标中的每一个都有一个K99阶段组件，为研究R 00阶段的这三个要求提供直接培训和经验。在我的第一个目标的K99阶段，我开发了一个有限的反应扩散（RD）模型的网格蛋白外套成核，捕获强烈牵连的子集的五种蛋白质和一种脂质之间的相互作用。该模型包括空间和动态细节。第二个目标的K99阶段优化了模型的参数，以与相同蛋白质子集上的新脂质体结合实验一致。在第三个目标的K99阶段，我们将创建复杂的简化地图，高度详细的模拟（例如，生物过程如涂层成核的模拟）。作为该方法的测试案例，我们将研究β发夹蛋白的折叠机制。在R 00阶段，我将开发的反应扩散模型，以量化的序列的蛋白质协会，导致涂层成核取决于蛋白质和脂质的浓度，以及隔离独特的蛋白质子集与特定的跨膜货物耦合，形成囊泡。随着更多蛋白质被引入模型，模型将不断优化以匹配实验，并将使用Aim 3的主方程框架分析结果。K99项目将在一年结束前完成，该项目将与NIH的Gerhard Hummer博士和匹兹堡大学的林顿特劳布博士合作。网格蛋白介导的内吞作用的研究和研发软件包的开发将消耗独立职位的前三年。该建议将计算和理论生物物理学与系统级生物过程的研究相结合。

This K99/R00 proposal is focused on investigating the protein interactions driving the nucleation of the clathrin-coated vesicles used in clathrin-mediated endocytosis. The main goal of this proposal is to determine the mechanisms of coat nucleation and identify the minimum core set of proteins (out of dozens of implicated proteins) that is necessary and sufficient to nucleate a clathrin coat. The computational model developed here must (1) determine the functions of the many participants in coat nucleation, (2) reproduce experimental properties of the system modeled, and (3) be readily interpretable to guide further experiments and models. Each of the three specific aims has a K99 phase component that provides direct training and experience for these three requirements of the R00 phase of the research. In the K99 phase of my first aim, I develop a limited reaction-diffusion (RD) model of clathrin coat nucleation that captures the interactions between a strongly implicated subset of five proteins and one lipid. This model includes both spatial as well as dynamic detail. The K99 phase of the second aim optimizes the parameters of the model to agree with new liposome binding experiments on the same subset of proteins. In the K99 phase of the third aim, we will create simplified maps of complex, highly detailed simulations (e.g., simulations of biological processes like coat nucleation) using a coarse master equation framework. As a test case for the method, we will study the mechanisms of folding in a beta hairpin protein. In the R00 phase I will develop the reaction-diffusion model to quantify how the sequence of protein associations that lead to coat nucleation depends on the protein and lipid concentrations, as well as to isolate the unique protein subsets that couple with specific transmembrane cargoes to form vesicles. The model will be continually optimized to match experiment as more proteins are introduced to the model, and the results will be analyzed using the master-equation framework of Aim 3. The K99 projects will be completed before the end of one year working with Dr. Gerhard Hummer at the NIH and collaborating with Dr. Linton Traub at University of Pittsburgh. The study of clathrin-mediated endocytosis and the development of the RD software package will consume the first three years of the independent position. This proposal integrates computational and theoretical biophysics with studies of systems-level biological processes.