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：
10277473
负责人：
Gerard L Apodaca
金额：
$ 66.08万
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/10277473
关键词：
3-Dimensional Actins Actomyosin Acute Adherens Junction Adhesions Affect Animal Model Apical Benign Prostatic Hypertrophy 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 Environment Epithelial Event Exocytosis Functional disorder Gene Expression Global Change Image Image Analysis Integral Membrane Protein Interstitial Cystitis Length Lower urinary tract Mechanics Membrane Microscopy Myosin Type II Obstruction Organ of Corti Pathologic Pathology Pathway interactions Patients Periodicity Process Proliferating Proteins Regulation Renal tubule structure Reporting Resolution Role Sarcomeres Signal Pathway Site Spinal cord injury Stomach Stretching Structure Testing Tight Junctions Time Tissues Urine Urologic Diseases Urothelial Cell Urothelium Variant base 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

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成分是顶端连接络合物（AJC），A 多部分的皮带状结构由紧密连接，粘附连接，脱染色和一个组成相关的细胞骨架。 AJC的功能包括调节细胞细胞通量，细胞 - 细胞粘附和机械转导。尽管有证据表明伞状细胞AJC是尿路上皮功能不可或缺的在几个较低的尿路疾病中破坏了，我们对伞状细胞的关键方面的理解有限 AJC生物学和病理生物学包括：（i）面对周期性膀胱，AJC如何保持其连续性填充和排尿；（ii）如何组织AJC进行这些过渡和功能这些事件中的细胞骨架；（iii）伞状细胞AJC如何感应张力以及病理学上是否有高静脉压力刺激与AJC相关的机械转导途径。我们的初步研究包括新发现的结果，即膀胱填充AJC外围会大大扩展，这一过程是取决于肌动蛋白细胞骨架和囊泡流量的变化，可能针对AJC。相比之下，膀胱后不久，AJC收缩，由非肌肉肌球蛋白II触发收缩驱动的事件肌动蛋白细胞骨架，RhoA以及内吞作用。基于可用数据，我们假设这一关键伞电池AJC的功能是通过动态扩展来维持尿路上皮屏障功能和收缩，并在正常和病理条件下充当机械转移的位置。到检验该全球假设，我们提出以下实验。在AIM 1中，我们将使用新开发的双轴拉伸装置，再加上活细胞图像分析，以确定增加的应变触发因素是否触发针对AJC的连接相关蛋白的胞吐作用，如果释放的释放会刺激其刺激内吞作用。我们还将评估阻止AJC扩展的尿路上皮屏障功能。在AIM 2中，我们将重点放在解密伞状细胞AJC相关的细胞骨架的功能和组织上。我们将使用超分辨率共聚焦成像以及电子显微镜来重建雨伞池AJC 在3D中。此外，我们将确定formins是否响应填充而驱动肌动蛋白聚合。在AIM 3中，我们将使用张力传感器来确定跨膜蛋白是否与雨伞细胞AJC感觉相关力，并评估是否响应部分膀胱激活与连接相关的信号通路出口阻塞（PBOO）。完成这些研究后，我们将对雨伞细胞的方式有新的见解 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感应和响应的重要新信息机械环境中的扰动，包括响应PBOO。