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

所有细胞都含有专门用于执行特定功能的细胞器。这些细胞器的功能取决于蛋白质正确地进出每个细胞器。这种运输是由小囊泡介导的，这些小泡以高度特定的运动编排在细胞周围携带各种货物。因此，囊泡的形成对细胞的正常功能至关重要，这些囊泡将特定的蛋白质运送到特定的细胞器。该项目试图了解含有特定蛋白质的特定囊泡是如何在细胞中形成的。其目的是将分子/生化实验与计算/算术建模相结合，以了解囊泡运输，长期目标是捕捉这一基本细胞过程的潜在生物物理原理。更广泛的影响活动包括研究本身的内在价值，例如，缺乏向各种细胞器提供酶会导致严重疾病。该项目将提供跨学科培训机会，并开发讲习班和课程，以有效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，通过参与旨在增加少数群体代表性的计划，以及促进和发展社区外联计划来提高科学意识和识字能力，以促进科学公平。运输是由小囊调节的，小囊从一个车厢选择货物，然后将货物运送到下一个车厢。货物选择是通过包裹晶格来调节的，这些晶格聚集在新芽的细胞质方面，在一个过程中涉及在时间和空间上相互系统地相互因果或功能联系的子过程层次。目标是确定管理涂层的基本原则，并对促进涂层的监管网络的紧急性质发展一种预测性的理解。该项目将重点放在组装涂层格子以在跨高尔基网络(TGN)分拣货物的子工艺上。TGN的晶格形成是通过一个复杂的机制进行的，其中首先组装适配器的内核，然后组装外层的笼状蛋白。该项目旨在了解由四聚体AP1复合体或三个单体组成的适配器-网状蛋白(AC)涂层模块的形成的动力学和生物物理参数，该模块由高尔基定位的-Adaptin ARF结合(GGA1-3)适配器和网状蛋白组成。接头通过与激活的Arf GTP酶相互作用组装在膜上，这种ARF在TGN的激活是由BIG1和BIG2鸟核苷酸交换因子介导的。该项目将使用CRISPR/Cas9修饰的敲除(KO)细胞系来确定调节每个AC涂层模块招募的特定ARF和BIG。适配器-网状蛋白(AC)涂层将通过数学描述关键成分BIG1、BIG2、Arf1-3、AP1、GGA1-3和网状蛋白的行为来建模。整个涂布过程是多步骤、多组分、多变的。仅靠实验不能处理这种复杂性，需要计算模型来阐明这一过程的时空参数。此外，交流泡形成的复杂性要求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则，这将定义生活规则之一。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.