Role of AJC in umbrella cell function and dysfunction

摘要： 伞状细胞屏障的关键组成部分是顶端连接复合体（AJC）， 多部分带状结构，由紧密连接、粘附连接、桥粒和 相关的细胞骨架。 AJC 的功能包括调节细胞旁通量、细胞间粘附和 力传导。尽管有证据表明伞细胞 AJC 是尿路上皮功能不可或缺的一部分， 在几种下尿路疾病中受到破坏，我们对伞细胞关键方面的了解有限 AJC 生物学和病理学包括：(i) AJC 如何在面对周期性膀胱时保持其连续性 填充和排空； (ii) AJC 的组织方式如何进行这些转变以及 AJC 的职能 这些事件中的细胞骨架； (iii) 伞细胞 AJC 如何感知张力以及是否病理性地 高膀胱内压力刺激 AJC 相关的机械传导途径。我们的初步研究 包括以下新发现：在膀胱充盈期间，AJC 周长急剧扩大，这一过程 取决于肌动蛋白细胞骨架和囊泡交通的变化，可能针对 AJC。相比之下， AJC 在膀胱排尿后不久就会收缩，这是由非肌肉肌球蛋白 II 触发的收缩驱动的事件 肌动蛋白细胞骨架、RhoA 以及内吞作用。根据现有数据，我们假设关键 伞细胞 AJC 的功能是通过动态扩张来维持尿路上皮屏障功能 和收缩，并作为正常和病理条件下的机械传导部位。至 测试这个全局假设，我们提出以下实验。在目标 1 中，我们将使用新开发的 双轴拉伸装置，结合活细胞图像分析，以确定增加的应变是否会触发 连接相关蛋白的胞吐作用定向到 AJC，并且如果应变的释放刺激了它们 内吞作用。我们还将评估阻断 AJC 扩张是否会扰乱尿路上皮屏障功能。在目标 2 中，我们 将重点破译伞细胞 AJC 相关细胞骨架的功能和组织。我们 将使用超分辨率共焦成像以及电子显微镜来重建伞状细胞 AJC 在 3D 中。此外，我们将确定福尔明是否会响应填充而驱动肌动蛋白聚合。在目标 3 中，我们将 使用张力传感器确定与伞细胞 AJC 相关的跨膜蛋白是否有感觉 力，并评估连接相关信号通路是否响应部分膀胱而被激活 出口阻塞（PBOO）。完成这些研究后，我们将对伞细胞如何 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 PBOO 的响应。

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.