Function of Myosin-X in Intestinal Apical Domain Assembly

描述（由申请人提供）：肠顶端结构域提供了一个选择性屏障，允许营养物质的吸收，同时它也形成了一个防止肠管内容物移位的保护屏障。顶端结构域的选择性屏障功能是由微绒毛和顶端连接调节的。在肠道疾病中，根尖结构域的功能障碍改变了其选择性屏障功能：乳糜泻和微绒毛包涵性疾病中明显存在微绒毛吸收不良，根尖连接屏障的破坏有助于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的顶端结构域组装过程。到目前为止，还没有直接针对根尖结构域组装或拆卸的治疗方法。丝状足是基于肌动蛋白的结构，理论上在微绒毛和根尖连接的组装中起作用。肌球蛋白- x是丝状面形成所必需的分子马达，因此导致假设肌球蛋白- x在顶端结构域组装中起作用。慢病毒RNA干扰的初步研究强烈表明，肌球蛋白- x是微绒毛形成所必需的，初步观察可能表明肌球蛋白- x在连接蛋白定位中的作用。该提案的具体目的有两个：确定肌球蛋白- x和丝状足在微绒毛形成中的作用；并确定肌球蛋白- x在根尖连接复合体（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确定肌球蛋白- x在细胞顶端结构域的定位和动态。肌球蛋白- x是微绒毛形成所必需的假设将在稳定的肌球蛋白- x敲除/拯救系统中得到验证。为了检验AJC的形成是否需要myosin-X，在myosin-X敲低的细胞中，钙开关会诱导AJC组装。本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一种新的分子机制。本实验将进一步加深我们对根尖结构域组装的理解，这是解决肠道疾病根尖结构域功能障碍的必要的第一步。

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.