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是血管内皮细胞中圆形的跨细胞孔，在 维持正常的内皮屏障功能，血液稳态和最终生存。 Fenestrae是 在大多数情况下，由称为烟膜膜片（FD）的蛋白质屏障跨越。有重要的知识 在我们对fenestrae组装基本细胞生物学机制的理解中的差距，这是 这个建议。最近，我们的小组表明浆膜囊泡相关蛋白（PLVAP或 PV1）对于FD组件至关重要，并且在小鼠和人类中缺乏PV1会导致异常的Fenestrae和 由于多个血管缺陷而引起的早期产后致死性。对于此提案，PV1是唯一已知的 Fenestrae的标记，我们将在调查Fenestrae组装机制中利用这一财产。 我们的中心假设是芬斯特雷集会遵循三步模型：在步骤1中，受控肌动蛋白 解聚会导致顶端和基底质膜的细胞变薄和对50 nm以内的细胞变薄。 在步骤2中，通过与顶膜和基底膜融合的外囊囊泡形成fenestrae毛孔。在步骤3中， FD使用胞外PV1及其相互作用伙伴组装。该假设基于我们的关键新知识 观察结果包括：1）ARP2/3复合抑制会导致吞ans体组装； 2）从头形成 FD需要内部池中的PV1胞吐作用； 3）Fenestrae组装不需要小窝蛋白1， 小窝或PV1内吞作用。我们将使用经过功能验证的批判性测试该模型的特定方面 内皮细胞培养系统。在AIM 1中，我们将定义肌动蛋白解聚的作用和机制 Fenestrae组。使用我们的Fenestrae形态发生测定法，我们将确定时空 肌动蛋白分解与细胞稀疏之间的关系，并将阐明肌动蛋白成核的作用和 肌动蛋白去聚合物。在AIM 2中，我们将定义Fenestrae孔形成的机制。我们将使用活细胞 显微镜测量PV1向细胞表面递送的时间，并识别细胞内 从中得出外旋转PV1的隔室。在AIM 3中，我们将定义Fenestrae的决定因素 膜片组件。我们假设PV1是主要的FD结构组件，但需要额外 功能FD组件的蛋白质。我们将结合生物化学和细胞互补 为了在结构和功能上测试PV1低聚，并将测试五个PV1相互作用蛋白 确定在FD组件中的角色。我们在关键和必要的专业知识方面都有独特的位置 完成这项工作。这些调查将定义复杂和 生理上相关的细胞结构，提供了当前缺乏的有价值的细胞生物学见解。