Development of biophysical principles of vehicle coating

车辆涂料生物物理原理的发展

• 批准号: 1615607
• 负责人: Elizabeth Sztul
• 金额: $ 98.9万
• 依托单位: University of Alabama at Birmingham
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1615607&HistoricalAwards=false
• 关键词: Development; biophysical; principles; vehicle; coating

All mammalian cells transport proteins via the secretory pathway to sustain indispensable cell activities such as growth, division and differentiation. Transport is mediated by small vesicles that bud from one compartment, move towards the next compartment, and then fuse in order to deliver their cargo. Vesicular traffic involves a hierarchy of molecular subprocesses linked to one another in time and space. The goal of this project is to model vesicle traffic with the long-term goal of capturing the underlying biophysical principles of this essential cellular process. In addition, this project will support interdisciplinary synergy by providing cross-discipline training opportunities and developing workshops and courses to effectively bridge the gap between biological and physical fields. Importantly, a focus 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. This project focuses on the formation of vesicles that transport proteins at the ER-Golgi interface. Such vesicles form through the recruitment of the coatomer complex (also called COPI complex) to the nascent bud. The recruitment is mediated by a small GTPase of the ARF (ADP-ribosylation factor) family. Like all GTPases, ARF acts as a molecular switch by cycling between an inactive state when it is bound to a GDP, and an activated state when it is bound to GTP. Only the active form of ARF can recruit coatomer and initiate vesicle formation. The activation of ARF is mediated by a nucleotide exchange factor (GEF) called GBF1. Hence, GBF1 activates ARF, which then recruits coatomer to make COPI vesicle. In addition to coat recruitment, ARF also regulates the sorting of cargo proteins into the nascent bud, and this process requires that ARF continuously cycle from between the GDP and the GTP-bound state. To facilitate the GTP to GDP conversion, a GTPase activating protein (GAP) called ArfGAP1 is required. Hence, the process of vesicle formation requires, at the very minimum, 4 key components: GBF1, ARF, coatomer and ArfGAP1. While we have descriptive knowledge of each molecular subprocess mediated by each component, we lack the knowledge of how these processes are connected in time and space to result in vesicle formation. We also lack even basic understanding of the governing biophysical principles. Thus, the aim of this project is to elucidate this enigma. We propose four Specific Aims: (1) Determine diffusion type and parameters of the 4 key components in cytosol; (2) Determine membrane association processes for the 4 key components; (3) Determine volume and surface densities and define binding constants and reaction rates for the 4 key components; and (4) Develop mathematical models describing the formation of the ternary coating complex. We will measure the dynamic properties of the 4 key components by fluorescence recovery after photobleaching (FRAP) under various experimentally induced conditions to mathematically describe their behavior. The resulting models will be refined through targeted perturbations of the system. Our models will provide the framework for future integration of other participants such as cargo proteins, lipids and a multitude of known regulatory factors that together ensure specificity to vesicular traffic. We stress that the machineries and the mechanisms we will model are highly conserved, and that analogous processes mediate coating of other types of vesicles for transport between different cellular compartments. Thus, our models will be generally applicable and will form an obligatory foundation for understanding the biophysical processes that govern vesicle traffic. A systems understanding of vesicle coating based on the laws of physics is crucial for future manipulation in vivo and for building synthetic organelles and cells.The funding for this project comes from both the Division of Molecular and Cell Biology and the Division of Mathematical Sciences

所有哺乳动物细胞都通过分泌途径转运蛋白质，以维持细胞生长、分裂和分化等必不可少的细胞活动。运输是由小泡介导的，这些小泡从一个隔室萌发，向下一个隔室移动，然后融合以运送货物。水疱交通涉及在时间和空间上相互联系的分子亚过程的层次结构。这个项目的目标是模拟囊泡交通，长期目标是捕捉这一基本细胞过程的潜在生物物理原理。此外，该项目将通过提供跨学科培训机会和举办讲习班和课程来有效地弥合生物和物理领域之间的差距，从而支持跨学科的协同作用。重要的是，一个重点是通过积极招募和指导代表性不足的群体，参与旨在增加少数群体代表性的项目，促进和发展社区外展项目来提高科学意识和素养，从而促进科学公平。这个项目的重点是在er -高尔基界面上运输蛋白质的囊泡的形成。这些囊泡是通过向初生芽招募涂层复合体（也称为COPI复合体）而形成的。募集是由ARF （adp -核糖化因子）家族的一个小GTPase介导的。像所有的GTPases一样，ARF作为一个分子开关，当它与GTP结合时处于无活性状态，当它与GTP结合时处于激活状态。只有活性形式的ARF才能招募涂层并引发囊泡形成。ARF的激活由一种名为GBF1的核苷酸交换因子（GEF）介导。因此，GBF1激活ARF， ARF随后招募涂层制造COPI囊泡。除了外膜招募，ARF还调节货物蛋白进入初芽的分选，这一过程需要ARF在GDP和gtp结合状态之间不断循环。为了促进GTP到GDP的转化，需要一种叫做ArfGAP1的GTP酶激活蛋白（GAP）。因此，囊泡形成过程至少需要4个关键组分：GBF1、ARF、coatomer和ArfGAP1。虽然我们对每个成分介导的每个分子亚过程有描述性的了解，但我们缺乏这些过程如何在时间和空间上连接以导致囊泡形成的知识。我们甚至对支配生物物理的原理也缺乏基本的了解。因此，这个项目的目的是阐明这个谜。我们提出了四个具体目标：(1)确定细胞质溶胶中4种关键组分的扩散类型和参数；(2)确定4个关键组分的膜关联工艺；(3)确定4种关键组分的体积和表面密度，确定结合常数和反应速率；(4)建立描述三元涂层配合物形成的数学模型。我们将在不同的实验诱导条件下，通过光漂白后荧光恢复（FRAP）来测量4种关键组分的动态特性，以数学方式描述它们的行为。所得到的模型将通过对系统进行有针对性的扰动而得到改进。我们的模型将为未来整合其他参与者提供框架，如货物蛋白、脂质和众多已知的调节因子，共同确保对囊泡交通的特异性。我们强调，我们将建模的机械和机制是高度保守的，并且类似的过程介导其他类型的囊泡在不同细胞间运输的涂层。因此，我们的模型将是普遍适用的，并将形成理解控制囊泡交通的生物物理过程的必要基础。基于物理定律的囊泡包覆系统理解对于未来体内操作和构建合成细胞器和细胞至关重要。这个项目的资金来自分子和细胞生物学部和数学科学学部