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，小泡携带各种货物在细胞周围以高度特异性的运动编排。因此，将特定蛋白质递送到特定细胞器的囊泡的形成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。其目的是将分子/生物化学实验与计算/算术建模相结合，以了解囊泡交通的长期目标是捕获这一基本细胞过程的基本生物物理原理。更广泛的影响活动包括研究本身的内在价值，例如，缺乏酶对各种细胞器的递送导致严重的疾病。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，并通过参与旨在增加少数群体代表性的计划，促进和发展社区外展计划，以提高科学意识和识字率，促进科学公平。分泌和内体途径之间的蛋白质运输对于生长，分裂和分化等活动至关重要。转运是由囊泡介导的，囊泡从一个隔室选择货物，然后将货物递送到下一个隔室。货物选择介导的涂层晶格组装在细胞质方面的新生芽在一个过程中，涉及层次的子过程系统地相互联系的因果关系或功能在时间和空间。我们的目标是确定管理涂层的基本原则，并制定一个预测性的理解，促进涂层的监管网络的新兴属性。该项目将重点关注在trans-Golgi网络（TGN）上组装用于分类货物的涂层网格的子过程。在TGN的晶格形成通过复杂的机制进行，其中适配器的内核首先组装，然后是网格蛋白的外层。该项目旨在了解调节由四聚体AP 1复合物或三个单体高尔基体定位的ARF结合（GGA 1 -3）衔接子和网格蛋白组成的衔接子-网格蛋白（AC）涂层模块形成的动力学和生物物理参数。衔接子通过与活化的Arf GTP酶相互作用在膜上组装，并且TGN处的此类Arf的活化由BIG 1和BIG 2鸟嘌呤核苷酸交换因子介导。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包被模块募集的特定Arfs和BIG。将通过数学描述关键组分（BIG 1、BIG 2、Arf 1 -3、AP 1、GGA 1 -3和网格蛋白）的行为对衔接子-网格蛋白（AC）涂层进行建模。整个涂层过程是多步骤、多组分和可变的。单靠实验无法处理这种复杂性，需要计算建模来阐明该过程的时空参数。此外，AC囊泡形成的复杂性需要开发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，并最终寻求破译涂层模块组装的一般原则，这将定义生活的规则之一。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.