Mechanism of fenestrae assembly in mammalian endothelial cells

窗孔是血管内皮细胞中的圆形跨细胞孔，在血管内皮细胞的生长发育过程中起着至关重要的作用。 维持正常的内皮屏障功能，血液动态平衡，最终存活。Fenestrae是 在大多数情况下，被一种称为窗孔隔膜(FD)的蛋白质屏障跨越。有一项重要的知识 在我们对窗孔组装的基本细胞生物学机制的理解上存在差距，这是 这项提议。最近，本课题组证明了质膜泡相关蛋白(PLVAP或PLVAP) PV1)对FD组装至关重要，而在小鼠和人类中缺乏PV1会导致异常的窗孔和 多发性血管缺陷所致的早期产后死亡。对这一提议至关重要的是，PV1是唯一已知的 我们将在研究窗帘组装机制时利用这一特性。 我们的中心假设是，Fenestrae组装遵循一个三步模型：在第一步，受控肌动蛋白 解聚导致细胞变薄，顶端和基底部质膜贴合到50 nm以内。 在步骤2中，窗孔是由胞外小泡与顶膜和基膜融合形成的。在步骤3中， FD利用排出的PV1及其相互作用的伙伴进行组装。这一假设是基于我们的关键新发现 观察包括：1)Arp2/3复合体抑制诱导窗孔组装；2)从头形成 FD需要从内部池吐出PV1；3)隔板组件不需要小窝1， 小凹或PV1内吞。我们将严格测试此模型的特定方面，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的作用和机制 栅栏队形。使用我们的窗孔形态发生分析，我们将确定时空 肌动蛋白分解和细胞变薄之间的关系，并将阐明肌动蛋白核因子和 肌动蛋白解聚体。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传递到细胞表面的时间，并识别细胞内 (S)从其衍生出胞外PV1的隔室。在目标3中，我们将定义围栏的决定因素 隔膜组件。我们假设PV1是主要的FD结构组件，但需要额外的 功能性FD组装所需的蛋白质。我们将使用生物化学和细胞互补的组合 为了从结构和功能上测试PV1齐聚，并将测试我们最近开发的五种PV1相互作用蛋白 为FD程序集中的角色标识。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。这些研究将定义一种复杂和 生理上相关的细胞结构，提供了目前所缺乏的有价值的细胞生物学见解。

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.