Developing quantitative understanding of adaptor-clathrin coating at the trans-Golgi network

所有细胞都含有专门执行特定功能的细胞器。这些细胞器的功能取决于蛋白质进出每个细胞器的正确运输。这种运输是由小泡介导的，这些小泡在细胞周围以高度特异性的运动编排携带各种货物。因此，囊泡的形成将特异性地将选择的蛋白质传递到特定的细胞器，对细胞的正常功能至关重要。该项目旨在了解含有特定蛋白质的特定囊泡如何在细胞中形成。目的是将分子/生化实验与计算/算术建模结合起来，以了解囊泡交通，并实现捕获这一基本细胞过程的潜在生物物理原理的长期目标。更广泛的影响活动包括研究本身的内在价值，例如，缺乏向各种细胞器输送酶会导致严重疾病。该项目将提供跨学科培训机会，并举办讲习班和课程，以有效地弥合生物和物理领域之间的差距。该项目的另一个目标是通过积极招募和指导代表性不足的群体，参与旨在增加少数民族代表性的项目，促进和发展社区外展项目，以提高科学意识和素养，从而促进科学公平。分泌途径和内体途径间的蛋白质运输对生长、分裂和分化等活动至关重要。运输是由囊泡介导的，囊泡从一个舱中选择货物，然后将货物运送到下一个舱。货物选择是由包衣晶格介导的，包衣晶格聚集在初芽的细胞质方面，这一过程涉及多层次的子过程，这些子过程在时间和空间上因果或功能上系统地相互联系。目标是确定控制涂层的基本原理，并对促进涂层的调节网络的紧急特性进行预测性理解。该项目将侧重于在跨高尔基网络（TGN）上组装用于分拣货物的涂层网格的子过程。TGN的晶格形成是通过一个复杂的机制进行的，在这个机制中，适配器的内核首先组装，然后是网格蛋白的外层。该项目旨在了解由四聚AP1复合物或三个单体高尔基定位-adaptin ARF-binding （GGA1-3）适配器和网格蛋白组成的适配器-网格蛋白（AC）涂层模块形成的动力学和生物物理参数。接头通过与活化的Arf gtpase相互作用组装在膜上，这些Arfs在TGN上的活化是由BIG1和BIG2鸟嘌呤核苷酸交换因子介导的。该项目将使用CRISPR/ cas9修饰的敲除（KO）细胞系来鉴定介导每个AC涂层模块募集的特定Arfs和BIGs。适配器-网格蛋白（AC）涂层将通过数学描述关键组分的行为来建模：BIG1， BIG2, Arf1-3, AP1， GGA1-3和网格蛋白。整个涂覆过程是多步骤、多组分和可变的。单独的实验无法处理这种复杂性，需要计算建模来阐明过程的时空参数。此外，AC囊泡形成的复杂性需要发展复杂的数学方法来模拟系统的行为。该项目结合了不同的专业知识，最终寻求破译涂层模块组装的一般原理，这将定义生命规则之一。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

All cells contain organelles that are specialized to perform a specific function. The function of these organelles depends on the correct transport of proteins into and out of each organelle. This transport is mediated by small vesicles that carry various cargos around the cell in a highly specific orchestration of movement. Thus, the formation of vesicles that will specifically deliver select proteins, to a specific organelle, is critically important to the normal function of cells. The project seeks to understand how specific vesicles containing specific proteins form in the cells. The aim is to unite molecular/biochemical experiments with computational/arithmetic modeling to understand vesicular traffic with the long-term goal of capturing the underlying biophysical principles of this essential cellular process. Broader Impact activities include the intrinsic merit of the research itself, e.g., the lack of delivery of enzymes to various organelles causes severe diseases. The project will provide cross-discipline training opportunities and develop workshops and courses to effectively bridge the gap between biological and physical fields. Another goal of the project is to advance scientific equity by actively recruiting and mentoring under-represented groups and by participating in programs aimed at increasing representation of minorities and promoting and developing community outreach programs to increase science awareness and literacy.Protein transport between the compartments of the secretory and endosomal pathways is essential for activities such as growth, division and differentiation. Transport is mediated by vesicles that select cargo from one compartment, and then deliver the cargo to the next compartment. Cargo selection is mediated by coating lattices that assemble on the cytoplasmic aspect of nascent buds in a process involving a hierarchy of subprocesses systematically linked to one another causally or functionally in time and space. The goal is to identify underlying principles that govern coating and to develop a predictive understanding of the emergent properties of the regulatory networks that facilitate coating. The project will focus on the subprocesses that assemble coating lattices for sorting cargo at the trans-Golgi network (TGN). Lattice formation at the TGN proceeds via a complex mechanism, in which an inner core of adaptors assembles first, followed by an outer layer of clathrin. The project seeks to understand the dynamics and the biophysical parameters regulating the formation of adaptor-clathrin (AC) coating modules composed of the tetrameric AP1 complex or three monomeric Golgi-localized -adaptin ARF-binding (GGA1-3) adaptors and clathrin. The adaptors assemble on the membrane by interacting with activated Arf GTPases and the activation of such Arfs at the TGN is mediated by the BIG1 and the BIG2 guanine nucleotide exchange factors. The project will use CRISPR/Cas9-modified knock-out (KO) cell lines to identify the specific Arfs and BIGs that mediate the recruitment of each AC coating module. Adaptor-clathrin (AC) coating will be modeled by mathematically describing the behavior of key components: BIG1, BIG2, Arf1-3, AP1, GGA1-3 and clathrin. The overall coating process is multi-step, multi-component, and variable. Experiments alone cannot deal with this complexity, and computational modeling is needed to illuminate the spatio-temporal parameters of the process. Furthermore, the complexity of AC vesicle formation requires the development of sophisticated mathematical methods to model the behavior of the system. This project combines diverse expertise and ultimately seeks to decipher the general principles of coating module assembly which will define one of the Rules of Life.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.