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

• 批准号: 10482413
• 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
• 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/10482413
• 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

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.

翻译后摘要：伞细胞屏障的一个关键组成部分是顶端连接复合体（AJC）， 由紧密连接、粘附连接、桥粒和桥粒组成的多分裂的带状结构 相关的细胞骨架AJC的功能包括调节细胞旁流、细胞-细胞粘附和细胞间粘附。 机械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， 在几种下尿路疾病中，我们对伞细胞的关键方面的了解有限， AJC生物学和病理学包括：（i）AJC如何在周期性膀胱炎中保持其连续性 （二）如何组织AJC进行这些过渡和功能的 细胞骨架在这些事件中的作用;以及（iii）伞细胞AJC如何感知张力以及是否在病理上 较高膀胱内压刺激AJC相关机械转导途径。我们的初步研究 包括新的发现，即在膀胱充盈期间，AJC周长急剧扩张，这一过程 依赖于肌动蛋白细胞骨架和囊泡交通的变化，可能是针对AJC的。与此相反， 膀胱排尿后AJC立即收缩，这是由非肌肉肌球蛋白II触发的收缩引起的 肌动蛋白细胞骨架，RhoA，以及内吞。根据现有数据，我们假设， 伞细胞AJC的功能是通过动态扩张维持尿路上皮屏障功能 和收缩，并在正常和病理条件下作为机械传导的位点。到 为了检验这个全局假设，我们提出了以下实验。在目标1中，我们将使用新开发的 双轴拉伸设备，结合活细胞图像分析，以确定增加的应变是否触发 连接相关蛋白的胞吐作用直接作用于AJC，如果应变的释放刺激它们的 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， 将集中在破译伞细胞AJC相关的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细胞AJC 在3D中。此外，我们将确定是否formins驱动肌动蛋白聚合反应填充。在目标3中，我们 使用张力传感器来确定是否与伞细胞AJC有义相关的跨膜蛋白 力，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）。在完成这些研究后，我们将有新的见解如何伞细胞 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，以及关于AJC如何感知和响应的重要新信息。 在其机械环境中的扰动，包括响应PBOO。