Molecular basis of brush border assembly

描述(由申请人提供)：该项目的长期目标是阐明肠细胞刷状边界组装的潜在机制，肠细胞刷状边界是营养吸收的唯一部位，也是与细菌和积聚在肠腔中的细菌产物相互作用的主要表面。发现于肠细胞的顶端，刷状边缘包含多达1000个紧密排列的微绒毛：肌动蛋白束支持的膜突起，从细胞表面延伸到几乎相同的长度。这种安排的功能结果是具有巨大的容量来容纳与膜相关的营养处理和宿主防御机械，这是维持肠道内环境稳定所必需的。尽管S位于胃肠道的关键生理界面，但对于微绒毛肌动蛋白束是如何成核的，微绒毛长度是如何控制的，或者微绒毛如何在肠细胞分化过程中实现完美的紧密堆积，我们几乎没有什么信息。使用CACO-2BBE细胞培养模型来探索分化过程中肠细胞顶端表面的物理重塑，我们的实验室取得了一系列令人兴奋的发现，为深入了解刷状边缘形成的基本机制提供了深入的了解。在刷子边缘组装的早期阶段，我们观察到微绒毛聚集在一起，并在它们的尖端相互作用 形成“圆锥形”的结构。随着分化的进行，观察到的“帐篷”通过合并更多的微绒毛而变得更大。对这些结构的电子显微镜首次显示，这些帐篷中相邻的微绒毛通过线状连接物理上相互连接。这些观察表明，刷状边缘组装过程中微绒毛的紧密堆积可能是由这些突起固有的黏附复合体驱动的。我们还鉴定了钙粘蛋白超家族的一个成员，原钙粘蛋白-24(PCDH24)，它在微绒毛的顶端表现出显著的富集性。有趣的是，PCDH24基因敲除也会导致微绒毛聚集缺陷。基于这些和其他初步发现，我们认为PCDH24在微绒毛顶端创建了微绒毛之间的粘附链，这是在刷子边缘组装过程中这些突起紧密填充所必需的。本文提出的目标将通过调查：(1)刷子边缘组装过程中PCDH24的靶向和要求，(2)PCDH24的黏附能力，以及(3)PCDH24微绒毛尖端定位的机制，开始检验这一假说。鉴于我们在定义刷子边缘功能的生物学和物理基础方面的专业知识，我们的团队处于有利地位，可以测试这一假设，并对胃肠道上皮生物学的这一基本方面产生洞察力。

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 microvilli: actin bundle-supported membrane protrusions that 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 exhibits striking enrichment at the tips of microvilli. Interestingly, knockdown of PCDH24 also gives rise to defects in microvillar clustering. 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, (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 insight on this fundamental aspect of GI epithelial biology.