Role of AJC in umbrella cell function and dysfunction

摘要：顶端连接复合体(AJC)是伞状细胞屏障的重要组成部分。 多部分带状结构，由紧密连接、附着连接、桥粒和 相关的细胞骨架。AJC的功能包括调节细胞旁流量、细胞间黏附和 机械转导。尽管有证据表明伞状细胞AJC对尿路上皮功能和 在几种下尿路疾病中被破坏，我们对伞状细胞的关键方面了解有限 AJC生物学和病理生物学包括：(I)AJC如何在周期性膀胱面前保持其连续性 填充和排空；(Ii)AJC如何组织以经历这些过渡以及AJC的职能 这些事件中的细胞骨架；以及(Iii)伞状细胞AJC如何感知张力以及是否处于病理状态 高膀胱内压刺激AJC相关的机械转导通路。我们的初步研究 包括新的发现，在膀胱充盈期间，AJC周长显著扩大，这一过程 取决于肌动蛋白细胞骨架和囊泡交通的变化，可能是针对AJC的。相比之下， AJC在膀胱排尿后不久收缩，这一事件由非肌肉肌球蛋白II引发的收缩驱动 肌动蛋白细胞骨架，RhoA，以及内吞作用。根据现有的数据，我们假设关键 伞状细胞AJC的功能是通过动态扩张来维持尿路上皮屏障功能 并在正常和病理条件下作为机械转导的场所。至 为了检验这一全局假设，我们提出了以下实验。在目标1中，我们将使用一种新开发的 双向拉伸装置，结合活细胞图像分析，以确定增加的应变是否触发 连接相关蛋白向AJC的胞吐作用，如果菌株的释放刺激了它们的 内吞作用。我们还将评估阻断AJC扩张是否扰乱尿路上皮屏障功能。在目标2中，我们 将重点破译与AJC相关的伞状细胞细胞骨架的功能和组织。我们 将使用超分辨率共聚焦成像以及电子显微镜来重建伞状细胞AJC 在3D中。此外，我们将确定福尔马林是否会驱动肌动蛋白聚合，以响应填充。在《目标3》中，我们将 使用张力传感器确定与伞状细胞AJC相关的跨膜蛋白是否 力量，并评估连接相关的信号通路是否被激活以响应部分膀胱 出口梗阻(PBOO)。这些研究完成后，我们将对伞状细胞如何 AJC动力学有助于尿路上皮屏障功能、AJC的组织及其功能 相关的细胞骨架，以及有关AJC如何感知和响应的重要新信息 机械环境中的扰动，包括对多溴联苯的反应。

Abstract: A critical component of the umbrella cell barrier is the apical junctional complex (AJC), a multipartite, belt-like structure comprised of the tight junction, the adherens junction, desmosomes, and an associated cytoskeleton. Functions of the AJC include regulation of paracellular flux, cell-cell adhesion, and mechanotransduction. Despite evidence that the umbrella cell AJC is integral to urothelial function and disrupted in several lower urinary tract disorders, we have limited understanding of key aspects of umbrella cell AJC biology and pathobiology including: (i) how the AJC maintains its continuity in the face of cyclical bladder filling and voiding; (ii) how the AJC is organized to undergo these transitions and the function of the cytoskeleton in these events; and (iii) how the umbrella cell AJC senses tension and whether pathologically high intravesical pressures stimulate AJC-associated mechanotransduction pathways. Our preliminary studies include the novel findings that during bladder filling the AJC perimeter expands dramatically, a process that depends on changes in the actin cytoskeleton and vesicular traffic, likely directed toward the AJC. In contrast, the AJC contracts soon after bladder voiding, events driven by the non-muscle myosin II-triggered contraction of the actin cytoskeleton, RhoA, as well as endocytosis. Based on available data, we hypothesize that critical functions of the umbrella cell AJC are to maintain urothelial barrier function by undergoing dynamic expansion and contraction and to serve as a site of mechanotransduction under normal and pathological conditions. To test this global hypothesis, we propose the following experiments. In Aim 1, we will use a newly developed biaxial stretching device, coupled with live-cell image analysis, to determine if increased strain triggers exocytosis of junction-associated proteins directed toward the AJC, and if release of strain stimulates their endocytosis. We will also assess if blocking AJC expansion perturbs urothelial barrier function. In Aim 2, we will focus on deciphering the function and organization of the umbrella cell AJC-associated cytoskeleton. We will use super-resolution confocal imaging, as well as electron microscopy to reconstruct the umbrella cell AJC in 3D. In addition, we will determine if formins drive actin polymerization in response to filling. In Aim 3, we will use tension sensors to determine if transmembrane proteins associated with the umbrella cell AJC sense force, and assess whether junction-associated signaling pathways are activated in response to partial bladder outlet obstruction (PBOO). Upon completion of these studies we will have new insights into how umbrella cell AJC dynamics contribute to urothelial barrier function, the organization of the AJC and the function of its associated cytoskeleton, and important new information about how the AJC senses and responds to perturbations in its mechanical milieu, including in response to PBOO.