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中文摘要
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窗孔是血管内皮细胞中的圆形跨细胞孔,在血管内皮细胞的增殖和分化中起关键作用。 维持正常内皮屏障功能、血液稳态和最终存活。窗孔是 在大多数情况下,由称为窗隔(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 这个提议。最近,我们的小组证明质膜囊泡相关蛋白(PLVAP或 PV 1)对于FD组装是关键的,并且在小鼠和人类中PV 1的缺乏导致异常的窗孔, 由于多处血管缺陷导致的出生后早期死亡。对于该提案至关重要的是,PV 1是唯一已知的 标记的窗,我们将利用这个属性在我们的调查窗组装机制。 我们的中心假设是,窗孔组装遵循三步模型:在第一步,控制肌动蛋白 解聚导致细胞变薄和顶端和基底质膜的并置在50 nm以内。 在步骤2中,通过胞吐囊泡与顶膜和基膜融合形成窗孔。在步骤3中, FD使用胞吐的PV 1及其相互作用伴侣组装。这一假设是基于我们的关键新的 观察结果包括:1)Arp 2/3复合物抑制诱导窗孔组装; 2)新形成的 FD需要从内部池胞吐PV 1; 3)窗孔组装不需要小窝蛋白1, 小窝或PV 1内吞作用。我们将严格测试该模型的特定方面,使用功能验证的 内皮细胞培养系统。在目标1中,我们将定义肌动蛋白解聚在 窗孔形成使用我们的窗孔形态发生试验,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 显微镜下测量PV 1递送到细胞表面的时间,并鉴定细胞内的 细胞外切的PV 1来源于的隔室。在目标3中,我们将定义窗的决定因素 隔膜组件我们假设PV 1是FD的主要结构成分,但需要额外的 用于功能性FD组装的蛋白质。我们将结合生物化学和细胞互补 测试PV 1寡聚化的结构和功能,并将测试五个PV 1相互作用蛋白,我们最近 在FD装配中的角色。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。这些调查将确定一个复杂的组装机制, 生理相关的细胞结构,提供了目前缺乏的有价值的细胞生物学见解。
英文摘要
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.
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Novel regulators, components and function of endothelial diaphragms
  • 批准号:
    10339380
  • 项目类别:
  • 资助金额:
    $41.0万
  • 财政年份:
    2021
  • 负责人:
    RADU VIRGIL STAN
  • 依托单位:
Novel regulators, components and function of endothelial diaphragms
  • 批准号:
    10560620
  • 项目类别:
  • 资助金额:
    $41.0万
  • 财政年份:
    2021
  • 负责人:
    RADU VIRGIL STAN
  • 依托单位:
Olympus TIRF Microscope for Simultaneous Multichannel Live cell Imaging
  • 批准号:
    8447267
  • 项目类别:
  • 资助金额:
    $17.96万
  • 财政年份:
    2013
  • 负责人:
    RADU VIRGIL STAN
  • 依托单位:
Plasmalemma vesicle-associated protein (PV-1) as a novel target in cancer immunot
  • 批准号:
    8491338
  • 项目类别:
  • 资助金额:
    $21.11万
  • 财政年份:
    2013
  • 负责人:
    RADU VIRGIL STAN
  • 依托单位:
海外基金