Probing the Energetic Cost of Cargo Encapsulation in Coated Vesicles

探讨包被囊泡中货物封装的能量成本

• 批准号: 9314585
• 负责人: Jeanne Casstevens Stachowiak
• 金额: $ 40.35万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9314585
• 关键词: Adaptor Signaling Protein; Address; Automobile Driving; Binding; Biological Assay; Biological Models; Caliber; Capsid Proteins; Cell physiology; Cell surface; Cells; Clathrin; Clinical; Coated vesicle; Crowding; Cystic Fibrosis; Defect; Disease; Encapsulated; Equilibrium; Evaluation; Familial Hypercholesterolemia; Health; Human; Huntington Disease; Hydrophobicity; In Vitro; Knowledge; Lead; Measures; Membrane; Methodology; Mission; Molecular; Molecular Weight; Mutate; Mutation; Optics; Pathogenicity; Pathology; Patients; Physiological; Play; Process; Proteins; Public Health; Research; Role; Shapes; Side; Signal Transduction; Structure; Surface; Synaptic Transmission; System; Testing; Therapeutic; United States National Institutes of Health; Vesicle; Viral; Virus; Work; cell motility; coated pit; cost; density; design; disability; experimental study; extracellular; human disease; innovation; intercellular communication; membrane assembly; molecular mass; novel strategies; pathogen; physical model; polymerization; pressure; prevent; public health relevance; tool

DESCRIPTION (provided by applicant): Assembling coated membrane vesicles (CVs) during cellular processes such as synaptic transmission and protein traffic requires coordinated interactions between transmembrane cargo molecules and coat proteins, rapidly shaping the membrane into a highly curved CV that encapsulates a specific cargo. Defects in CV assembly and exploitation of CVs by pathogens lead to devastating diseases that cumulatively impact hundreds of millions of patients each year. While the molecular components and structures of CVs have been largely identified, critical physiological questions remain unanswered. Specifically, determining how the coat physically senses and adapts to cargo and elucidating the criteria that determine the size and cargo content of CVs are key remaining steps toward understanding the role of CV assembly in human disease. To address these questions, the objective of the proposed work is to quantify and compare the energetic costs of cargo encapsulation with the energetic contributions of coat assembly during CV formation. Recent work in our lab has demonstrated that the energetic cost of encapsulating cargo molecules increases exponentially with their concentration on membrane surfaces, a consequence of increased steric pressure among them. Similarly, our work has shown that concentrating coat components to the levels found in CVs creates a substantial steric pressure on the opposite membrane surface that drives membrane curvature, in opposition to pressure from cargo molecules. In contrast to current understanding, these results suggest that concentrating cargo molecules, rather than bending membranes, represents the major physical barrier to forming CVs. These observations lead to the central hypothesis that assembly of the coat lattice sterically confines molecular components on the cargo and coat sides of the membrane, setting up a competition between opposing membrane surface pressures that collectively shape nascent CVs. Using assembly of clathrin-coated pits as a model system, experiments in three aims will test this hypothesis. Using minimal membrane systems and quantitative optical assays, experiments in Aim 1 will measure the energetic cost of cargo encapsulation as a function of cargo concentration and molecular mass. In contrast, experiments in Aim 2 will use minimal systems to quantify and compare the energetic drivers of cargo encapsulation, including coat polymerization, steric pressure among coat components, and hydrophobic insertion. Finally, Aim 3 will probe the physiological balance between the costs and drivers of cargo encapsulation in living cells. These experiments will determine the impact of cargo concentration and molecular weight on the size and coat composition of CVs. Using innovative methodologies to quantify the energetics of CV formation, the significance of the proposed work will be a critical evaluation of the extent to which an energetic competition between cargo and coat components determines the size and molecular content of CVs, a key step toward understanding and addressing pathologies arising from misregulation, mutation, and pathogenic exploitation of CV assembly.

描述（由申请人提供）：在突触传递和蛋白质运输等细胞过程中组装包被膜囊泡（CV）需要跨膜货物分子和外壳蛋白之间的协调相互作用，快速将膜塑造成封装特定货物的高度弯曲的CV。 CV 组装缺陷和病原体对 CV 的利用会导致毁灭性的疾病，每年累计影响数亿患者。虽然CV的分子成分和结构已基本被确定，但关键的生理问题仍未得到解答。具体来说，确定外套如何物理感知和适应货物，并阐明确定 CV 大小和货物内容的标准，是了解 CV 组装在人类疾病中的作用的关键剩余步骤。为了解决这些问题，拟议工作的目标是量化并比较货物封装的能量成本与 CV 形成过程中涂层组装的能量贡献。我们实验室最近的工作表明，封装货物分子的能量成本随着它们在膜表面的浓度呈指数增加，这是它们之间的空间压力增加的结果。同样，我们的工作表明，将涂层成分浓缩到 CV 中的水平会在相对的膜表面上产生很大的空间压力，从而驱动膜曲率，与来自货物分子的压力相反。与目前的理解相反，这些结果表明，浓缩货物分子，而不是弯曲膜，代表了形成 CV 的主要物理障碍。这些观察结果得出了一个中心假设，即涂层晶格的组装在空间上将分子成分限制在膜的货物侧和涂层侧，从而在共同塑造新生CV的相反膜表面压力之间建立竞争。使用网格蛋白包被的凹坑的组装作为模型系统，三个目标的实验将检验这一假设。目标 1 中的实验将使用最小的膜系统和定量光学测定来测量货物封装的能量成本，作为货物浓度和分子质量的函数。相比之下，目标 2 中的实验将使用最小的系统来量化和比较货物封装的能量驱动因素，包括涂层聚合、涂层成分之间的空间压力和疏水插入。最后，目标 3 将探讨活细胞中货物封装的成本和驱动因素之间的生理平衡。这些实验将确定货物浓度和分子量对 CV 尺寸和涂层组成的影响。使用创新方法来量化 CV 形成的能量，拟议工作的意义将是对货物和外套成分之间的能量竞争在多大程度上决定 CV 的大小和分子含量进行批判性评估，这是理解和理解 CV 的关键一步。解决因 CV 组装的失调、突变和致病利用而引起的病理学。