Probing the Energetic Cost of Cargo Encapsulation in Coated Vesicles

描述（由申请人提供）：在细胞过程（如突触传递和蛋白质运输）期间组装包被膜囊泡（CV）需要跨膜货物分子和外壳蛋白之间的协调相互作用，将膜快速成形为封装特定货物的高度弯曲CV。CV组装缺陷和病原体对CV的利用导致每年累积影响数亿患者的毁灭性疾病。虽然CV的分子组成和结构已经在很大程度上得到了鉴定，但关键的生理问题仍然没有答案。具体而言，确定外套如何物理感知和适应货物，并阐明确定CV的大小和货物含量的标准是理解CV组装在人类疾病中的作用的关键步骤。为了解决这些问题，建议的工作的目标是量化和比较货物封装的能量成本与CV形成过程中的涂层组件的能量贡献。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上的浓度呈指数级增加，这是它们之间空间压力增加的结果。类似地，我们的工作已经表明，将涂层组分浓缩到CV中发现的水平在相反的膜表面上产生了大量的空间压力，该空间压力驱动膜弯曲，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，集中货物分子，而不是弯曲膜，代表了形成CV的主要物理障碍。这些观察结果导致了中心假设，即涂层晶格的组装在空间上限制了膜的货物侧和涂层侧上的分子组分，从而在共同形成新生CV的相对膜表面压力之间建立了竞争。使用网格蛋白涂层的凹坑组装作为模型系统，在三个目标的实验将验证这一假设。使用最小的膜系统和定量光学测定，目标1中的实验将测量作为货物浓度和分子量的函数的货物封装的能量成本。相比之下，目标2中的实验将使用最小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、涂层组分之间的空间压力和疏水插入。最后，目标3将探索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的大小和涂层组成的影响。使用创新的方法来量化CV形成的能量学，所提出的工作的意义将是对货物和涂层组分之间的能量竞争在多大程度上决定CV的大小和分子含量的关键评估，这是理解和解决CV组装的失调，突变和致病性利用所引起的病理的关键一步。

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.