Function of Myosin-X in Intestinal Apical Domain Assembly

描述（由申请方提供）：肠顶端结构域提供了允许营养吸收的选择性屏障，同时也形成了防止肠腔内容物移位的保护性屏障。顶端结构域的选择性屏障功能由微绒毛和顶端连接调节。在肠道疾病中，顶端结构域的功能障碍改变了其选择性屏障功能：微绒毛吸收不良在乳糜泻和微绒毛包涵体疾病中是明显的，顶端连接屏障的破坏有助于克罗恩病的发病机制和肠道病原体的感染。本研究的长期目标是了解肠道疾病中顶端结构域组装的过程。到目前为止，还没有直接靶向顶端结构域组装或拆卸的疗法。 丝状伪足是肌动蛋白为基础的结构，理论上在微绒毛和顶端连接的组装中起作用。肌球蛋白-X是丝状伪足形成所需的分子马达，从而导致肌球蛋白-X在顶端结构域组装中起作用的假设。初步慢病毒RNA干扰研究强烈表明肌球蛋白X对于微绒毛的形成是必需的，初步观察可能表明肌球蛋白X在连接蛋白定位中发挥作用。这个建议的具体目标是双重的：以确定肌球蛋白-X和丝状伪足在微绒毛的形成中的作用;并确定肌球蛋白-X在顶端连接复合物（AJC）组装中的作用。 使用Caco-2细胞作为模型系统，肌球蛋白-X在顶端结构域结构中的定位和动力学将通过共聚焦显微镜和活细胞成像来确定。将在稳定的肌球蛋白-X敲低/拯救系统中测试肌球蛋白-X对微绒毛形成是必要的这一假设。为了测试AJC形成是否需要肌球蛋白-X，将通过肌球蛋白-X敲低细胞中的钙开关诱导AJC组装。 这项研究将确定肌球蛋白-X在肠细胞顶端结构域的功能作用，并揭示顶端结构域形成的新分子机制。拟议的实验将进一步我们的理解顶端域组装，这是一个必要的第一步，以解决顶端域功能障碍的肠道疾病。 公共卫生相关性：肠细胞功能障碍导致吸收不良和上皮屏障破坏。这种功能障碍导致肠道疾病，如乳糜泻和炎症性肠病（IBD）。这项研究调查了肠道细胞中的关键结构如何组装，这一过程可能在乳糜泻和IBD等疾病中受损。

DESCRIPTION (provided by applicant): Intestinal apical domain provides a selective barrier that allows for absorption of nutrients, while it also forms a protective barrier against translocation of gut lumenal contents. The selective barrier function of the apical domain is modulated by microvilli and apical junctions. In intestinal disease, dysfunction of the apical domain alters its selective barrier function: malabsorption by microvilli is evident in celiac disease and microvillous inclusion disease, and disruption of the apical junction barrier contributes to the pathogenesis of Crohn's disease and infection by intestinal pathogens. The long-term objective of this research is to understand the process of apical domain assembly in intestinal disease. Thus far, there are no therapies that directly target apical domain assembly or disassembly. Filopodia are actin-based structures theorized to function in the assembly of microvilli and apical junctions. Myosin-X is a molecular motor that is required for filopodial formation, thus leading to the hypothesis that myosin-X functions in apical domain assembly. Preliminary lentivirus RNA interference studies strongly suggest myosin-X is necessary for microvillar formation, and preliminary observations may suggest a role for myosin-X in junctional protein localization. The specific aims of this proposal are two-fold: to determine the role of myosin-X and filopodia in the formation of microvilli; and to determine the role of myosin-X in apical junctional complex (AJC) assembly. Using Caco-2 cells as a model system, the localization and dynamics of myosin-X in apical domain structures will be determined by confocal microscopy and live-cell imaging. The hypothesis that myosin-X is necessary for microvillar formation will be tested in a stable myosin-X knockdown/rescue system. To test whether myosin-X is needed for AJC formation, AJC assembly will be induced by calcium switch in myosin-X knockdown cells. This study will determine the functional roles of myosin-X at the apical domain of intestinal cells and will reveal a novel molecular mechanism of apical domain formation. The proposed experiments will further our understanding of apical domain assembly, which is a necessary first step to address apical domain dysfunction in intestinal disease. PUBLIC HEALTH RELEVANCE: Intestinal cell dysfunction leads to malabsorption and breach of the epithelial barrier. Such dysfunctions contribute to intestinal diseases such as celiac disease and inflammatory bowel disease (IBD). This study investigates how key structures in intestinal cells assemble, a process that can be impaired in diseases like celiac disease and IBD.