Molecular basis of brush border assembly

描述（由申请方提供）：本项目的长期目标是阐明肠上皮细胞刷状缘组装的潜在机制：营养吸收的唯一部位以及与肠腔中积累的细菌和细菌产物相互作用的主要表面。刷状缘位于肠上皮细胞的顶端，包含多达1000个紧密排列的、由肌动蛋白支撑的膜突起，称为微绒毛，它们从细胞表面延伸到几乎相同的长度。功能 这种排列的结果是容纳维持肠道内稳态所需的膜相关营养加工和宿主防御机制的巨大能力。尽管微绒毛位于胃肠道中的关键生理界面，但关于微绒毛肌动蛋白束如何成核、微绒毛长度如何控制或微绒毛如何在肠上皮细胞分化过程中实现完美紧密包装的信息很少。使用CACO-2BBE细胞培养模型来探索分化过程中肠上皮细胞顶面的物理重塑，我们的实验室取得了一系列令人兴奋的发现，为刷状缘形成的基本机制提供了见解。在刷状缘组装的早期阶段，我们观察到微绒毛聚集在一起，并在其尖端相互作用， 形成“圆锥形”结构。随着分化的进行，观察到的“圆锥形”通过并入更多的微绒毛而变大。这些结构的电子显微镜显示，第一次，在这些圆锥形的相邻微绒毛是物理连接到彼此的线状链接。这些观察结果表明，刷状缘组装过程中微绒毛的紧密包装可能是由这些突起固有的粘附复合物驱动的。我们还确定了钙粘蛋白超家族的成员，原钙粘蛋白-24（PCDH 24），它可能在形成微绒毛间粘附连接中发挥作用。PCDH 24是一种新的微绒毛组分，其在微绒毛尖端表现出显著的富集（用超分辨率光学显微镜和免疫EM观察到），并且该分子的shRNA介导的敲低显著损害了CACO- 2BBE分化期间的微绒毛堆积。基于这些和其他初步发现，我们提出PCDH 24在微绒毛尖端处产生微绒毛间粘附链接，这是在刷状缘组装期间这些突起的紧密包装所需的。本文提出的目的将开始通过研究以下内容来检验这一假设：（1）在刷状缘组装和肠道病原体消除过程中PCDH 24的靶向和需求，（2）PCDH 24的粘附能力，以及（3）PCDH 24微绒毛尖端定位的潜在机制。鉴于我们在定义刷状缘功能的生物学和物理基础方面的专业知识，我们的研究小组能够很好地测试这一假设，并对GI上皮生物学的这一基本方面产生新的见解。

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate mechanisms underlying assembly of the enterocyte brush border: the sole site of nutrient absorption and the primary surface of interaction with bacteria and bacterial products that accumulate in the intestinal lumen. Found at the apex of the enterocyte, the brush border contains up to one thousand tightly packed, actin bundle-supported membrane protrusions known as microvilli, which extend off the cell surface to a nearly identical length. The functional consequence of this arrangement is an immense capacity for housing membrane-associated nutrient processing and host defense machinery that is required for maintaining gut homeostasis. Despite being positioned at a critical physiological interface in the GI tract, there s little information on how microvillar actin bundles are nucleated, how microvillar length is controlled, or how microvilli achieve perfectly tight packing during enterocyte differentiation. Using the CACO-2BBE cell culture model to explore the physical remodeling of the enterocyte apical surface during differentiation, our laboratory made a series of exciting discoveries that provide insight on fundamental mechanisms of brush border formation. During the early stages of brush border assembly, we observe that microvilli cluster together and interact at their tips to form 'tepee' shaped structures. As differentiation proceeds, the observed 'tepees' grow larger by incorporating more microvilli. Electron microscopy of these structures revealed, for the first time that adjacent microvilli in these tepees are physically connected to each other by thread-like links. These observations suggest that the tight packing of microvilli during brush border assembly may be driven by adhesion complexes that are inherent to these protrusions. We also identified a member of the cadherin superfamily, protocadherin-24 (PCDH24), which could play a role in forming inter-microvillar adhesion links. PCDH24 is a novel microvillar component that exhibits striking enrichment at microvillar tips (observed with super-resolution light microscopy and immuno- EM) and shRNA-mediated knockdown of this molecule significantly impairs microvillar packing during CACO- 2BBE differentiation. Based on these and other preliminary findings, we propose that PCDH24 creates inter- microvillar adhesion links at microvillar tips, which are required for the tight packing of these protrusions during brush border assembly. The Aims proposed herein will begin to test this hypothesis by investigating: (1) the targeting and requirement for PCDH24 during brush border assembly and effacement by enteric pathogens, (2) the adhesion capacity of PCDH24, and (3) the mechanism underlying the microvillar tip localization of PCDH24. Given our expertise in defining the biological and physical underpinnings of brush border function, our group is well positioned to test this hypothesis and generate novel insight on this fundamental aspect of GI epithelial biology.