Function of Myosin-X in Intestinal Apical Domain Assembly

描述(申请人提供)：肠道顶端区域提供了一种选择性屏障，允许吸收营养物质，同时也形成了一种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受微绒毛和顶端连接的调节。在肠道疾病中，顶区的功能障碍改变了其选择性屏障功能：在乳糜泻和微绒毛包涵体病中，微绒毛明显吸收不良，顶端连接屏障的破坏导致克罗恩病的发病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的组装过程。到目前为止，还没有直接针对根尖区域组装或拆卸的治疗方法。丝状伪足是以肌动蛋白为基础的结构，理论上认为它在微绒毛和顶端连接的组装中发挥作用。肌球蛋白-X是一种分子马达，是形成丝状孔所必需的，因此导致了肌球蛋白-X在顶端结构域组装中的功能的假说。初步的慢病毒RNA干扰研究强烈表明肌球蛋白-X对微绒毛的形成是必需的，初步观察可能提示肌球蛋白-X在结合蛋白定位中的作用。这项建议的具体目的有两个：确定肌球蛋白-X和丝状伪足在微绒毛形成中的作用；以及确定肌球蛋白-X在顶端连接复合体(AJC)组装中的作用。以Caco-2细胞为模型系统，利用共聚焦显微镜和活细胞成像技术研究肌球蛋白X在顶端结构中的定位和动态变化。肌球蛋白-X是微绒毛形成所必需的这一假说将在稳定的肌球蛋白-X击倒/挽救系统中得到检验。为了测试肌球蛋白-X是否需要AJC的形成，AJC的组装将由肌球蛋白-X基因敲除细胞中的钙开关诱导。这项研究将确定肌球蛋白-X在肠道细胞顶端区域的功能作用，并将揭示一种新的顶端区域形成的分子机制。拟议的实验将加深我们对顶域组装的理解，这是解决肠道疾病顶域功能障碍的必要的第一步。 与公共卫生相关：肠道细胞功能障碍会导致吸收不良和上皮屏障的破坏。这种功能障碍会导致肠道疾病，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是如何组装的，这一过程在乳糜泻和IBD等疾病中可能会受到损害。

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.