Mechanism of fenestrae assembly in mammalian endothelial cells

哺乳动物内皮细胞窗孔组装机制

• 批准号: 9166840
• 负责人: RADU VIRGIL STAN
• 金额: $ 32.4万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9166840
• 关键词: Actins; Address; Apical; Biochemistry; Biogenesis; Biological; Biological Assay; Blood; Blood Vessels; Caliber; Capillary Endothelial Cell; Caveolae; Cell Culture System; Cell Line; Cell membrane; Cell surface; Cells; Cellular Structures; Complex; Cytoplasm; Cytoskeleton; Data; Defect; Diabetes Mellitus; Disease; Electron Microscopy; Endocrine Glands; Endocytosis; Endothelial Cells; Event; Exocytosis; Fluorescence Microscopy; Homeostasis; Human; Inflammation; Investigation; Kidney Failure; Knowledge; Lead; Leukocytes; Life; Maintenance; Malignant Neoplasms; Measures; Membrane; Membrane Protein Traffic; Modeling; Molecular; Morphogenesis; Mus; Nutrient; Organ; Oxygen; Pathogenesis; Play; Positioning Attribute; Pre-Eclampsia; Property; Protein Biochemistry; Proteins; Reagent; Respiratory Diaphragm; Role; Subcellular structure; Testing; Time; Tissues; Vascular Endothelial Cell; Vascular Endothelial Growth Factors; Vesicle; Visceral; Work; attenuation; base; caveolin 1; cofilin; depolymerization; human disease; insight; live cell microscopy; postnatal; tool; tumor growth; wasting

FENESTRAE are circular transcellular pores in vascular endothelial cells playing critical roles in the maintenance of normal endothelial barrier function, blood homeostasis and ultimately survival. Fenestrae are spanned in most cases by a protein barrier called a fenestral diaphragm (FD). There is a significant knowledge gap in our understanding of the basic cell biological mechanism of fenestrae assembly, which is the subject of this proposal. Recently, our group demonstrated that Plasmalemma Vesicle Associated Protein (PLVAP or PV1) is critical for FD assembly, and that absence of PV1 in mice and humans causes abnormal fenestrae and early postnatal lethality due to multiple vascular defects. Critically for this proposal, PV1 is the only known marker for fenestrae, and we will exploit this property in our investigation of fenestrae assembly mechanisms. Our central hypothesis is that fenestrae assembly follows a three-step model: In Step 1, controlled actin depolymerization leads to cell thinning and apposition of apical and basal plasma membranes to within 50 nm. In Step 2, fenestrae pores form by fusion of exocytic vesicles with apical and basal membranes. In Step 3, the FD assembles using exocytosed PV1 and its interacting partners. This hypothesis is based on our key new observations including: 1) Arp2/3 complex inhibition induces fenestrae assembly; 2) de novo formation of the FD requires exocytosis of PV1 from an internal pool; 3) fenestrae assembly does not require caveolin 1, caveolae or PV1 endocytosis. We will critically test specific aspects of this model, using a functionally validated endothelial cell culture system. In Aim 1 we will define the role and mechanism of actin depolymerization in fenestrae formation. Using our fenestrae morphogenesis assay, we will determine the spatio-temporal relationship between actin disassembly and cell thinning, and will elucidate the roles of actin nucleators and actin depolymerizers. In Aim 2 we will define the mechanism of fenestrae pore formation. We will use live-cell microscopy to measure the timing of PV1 delivery to the cell surface, and identify the intracellular compartment(s) from which exocytosed PV1 is derived. In Aim 3 we will define the determinants of fenestrae diaphragm assembly. We hypothesize that PV1 is the major FD structural component, but requires additional proteins for functional FD assembly. We will use a combination of biochemistry and cellular complementation to test PV1 oligomerization structurally and functionally, and will test five PV1 interacting proteins we recently identified for roles in FD assembly. We are uniquely positioned both in terms of key and necessary expertise to complete this work. These investigations will define the assembly mechanism for an intricate and physiologically relevant cellular structure, providing valuable cell biological insight that is currently lacking.

FENESTRAE 是血管内皮细胞中的圆形跨细胞孔，在 维持正常的内皮屏障功能、血液稳态和最终生存。窗孔是 在大多数情况下，由称为窗孔隔膜（FD）的蛋白质屏障跨越。有一个重要的知识 我们对窗孔组装的基本细胞生物学机制的理解存在差距，这是 这个建议。最近，我们的团队证明了质膜囊泡相关蛋白（PLVAP 或 PV1) 对于 FD 组装至关重要，小鼠和人类中 PV1 的缺失会导致窗孔异常和 由于多发性血管缺陷导致产后早期死亡。对于该提案至关重要的是，PV1 是唯一已知的 窗孔的标记，我们将在研究窗孔组装机制时利用这一特性。 我们的中心假设是窗孔组装遵循三步模型：在第一步中，受控肌动蛋白 解聚导致细胞变薄以及顶端和基底质膜的并置到 50 nm 以内。 在步骤 2 中，胞吐囊泡与顶膜和基底膜融合形成窗孔。在步骤 3 中， FD 使用胞吐的 PV1 及其相互作用伙伴进行组装。这个假设是基于我们的关键新 观察结果包括：1) Arp2/3 复合物抑制诱导窗孔组装； 2）从头形成 FD 需要 PV1 从内部池中胞吐； 3) 窗孔组装不需要caveolin 1， 小凹或 PV1 内吞作用。我们将使用经过功能验证的模型严格测试该模型的特定方面 内皮细胞培养系统。在目标 1 中，我们将定义肌动蛋白解聚的作用和机制 窗孔的形成。使用我们的窗孔形态发生测定，我们将确定时空 肌动蛋白分解和细胞变薄之间的关系，并将阐明肌动蛋白成核剂和 肌动蛋白解聚剂。在目标 2 中，我们将定义窗孔形成的机制。我们将使用活细胞 显微镜测量 PV1 递送到细胞表面的时间，并识别细胞内 从中衍生出胞吐的 PV1 的区室。在目标 3 中，我们将定义窗孔的决定因素 隔膜组件。我们假设 PV1 是主要的 FD 结构组件，但需要额外的 用于功能性 FD 组装的蛋白质。我们将结合使用生物化学和细胞互补 从结构和功能上测试 PV1 寡聚化，并将测试我们最近的五种 PV1 相互作用蛋白 确定了 FD 组装中的角色。我们在关键和必要的专业知识方面都处于独特的地位 完成这项工作。这些研究将定义复杂且复杂的组装机制。 生理相关的细胞结构，提供目前缺乏的有价值的细胞生物学见解。