Role of AJC in umbrella cell function and dysfunction

AJC 在伞细胞功能和功能障碍中的作用

基本信息

批准号：
10655616
负责人：
Gerard L Apodaca
金额：
$ 63.61万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-06 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10655616
关键词：
3-Dimensional Actins Actomyosin Acute Adherens Junction Adhesions Affect Animal Model Apical Benign Prostatic Hypertrophy Binding Biology Bladder Bladder Dysfunction Cell Line Cell physiology Cell-Cell Adhesion Cells Complex Contracts Coupled Cystitis Cytoskeleton Data Desmosomes Devices Electron Microscopy Endocytosis Endothelial Cells Enterocytes Environment Epithelium Event Exocytosis Functional disorder Gene Expression Global Change Image Image Analysis Integral Membrane Protein Interstitial Cystitis Length Lower urinary tract Mechanics Membrane Membrane Proteins Microscopy Myosin Type II Obstruction Organ of Corti Pathologic Pathology Pathway interactions Patients Polymers Process Proliferating Proteins Regulation Renal tubule structure Reporting Role Sarcomeres Signal Pathway Site Spinal cord injury Stomach Stretching Structure Testing Tight Junctions Time Tissues Urine Urologic Diseases Urothelial Cell Urothelium Variant confocal imaging experimental study force sensor genetic regulatory protein insight intravesical live cell imaging mechanotransduction non-muscle myosin novel pneumocyte polymerization preservation pressure response sensor trafficking ultra high resolution

项目摘要

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.

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 周长急剧扩张，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。到测试这个全局假设，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。