Modeling the nucleation of clathrin coated vesicles at the cell membrane

这项K99/R00提案的重点是研究在网格蛋白介导的内吞作用中驱动网格蛋白包被囊泡成核的蛋白质相互作用。本提案的主要目标是确定外壳成核的机制，并确定最小的核心蛋白集（从数十个相关蛋白中），这是形成网格蛋白外壳所必需的和足够的。这里开发的计算模型必须(1)确定包被成核中许多参与者的功能，(2)再现所建模系统的实验特性，(3)易于解释，以指导进一步的实验和模型。这三个具体目标中的每一个都有一个K99阶段的组成部分，它为研究的R00阶段的这三个需求提供了直接的培训和经验。在我的第一个目标的K99阶段，我开发了网格蛋白外壳成核的有限反应扩散（RD）模型，该模型捕获了五种蛋白质和一种脂质之间的强烈关联子集之间的相互作用。这个模型既包括空间细节，也包括动态细节。第二个目标的K99阶段优化了模型的参数，使其与新的脂质体结合实验在相同的蛋白质亚群上一致。在第三个目标的K99阶段，我们将使用粗糙的主方程框架创建复杂的、高度详细的模拟（例如，像外壳成核这样的生物过程的模拟）的简化图。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R00阶段，我将开发反应扩散模型，以量化导致外壳成核的蛋白质关联序列如何依赖于蛋白质和脂质浓度，以及分离与特定跨膜货物偶联形成囊泡的独特蛋白质亚群。随着更多蛋白质的引入，模型将不断优化以匹配实验，并使用Aim 3的主方程框架对结果进行分析。K99项目将在一年内完成，与NIH的Gerhard Hummer博士合作，并与匹兹堡大学的Linton Traub博士合作。网格蛋白介导内吞作用的研究和研发软件包将占用独立岗位的前三年时间。这一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.