Function of Myosin-X in Intestinal Apical Domain Assembly

肌球蛋白-X 在肠顶端域组装中的功能

• 批准号: 8113136
• 负责人: Katy C Liu
• 金额: $ 3.38万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8113136
• 关键词: Actins; Address; Apical; Atrophic; Biological Assay; Biological Models; Caco-2 Cells; Calcium; Celiac Disease; Cell Line; Cells; Complex; Confocal Microscopy; Crohn' s disease; Cytomegalovirus Infections; Data; Defect; Development; Disease; E-Cadherin; Epithelial; Epithelial Cells; Fibroblasts; Filopodia; Functional disorder; Image; Immunofluorescence Immunologic; Infection; Inflammatory Bowel Diseases; Intestinal Diseases; Intestines; Life; Malabsorption Syndromes; Measures; Molecular; Molecular Motors; Movement; Myosin ATPase; Nutrient; Pathogenesis; Process; Proteins; RNA Interference; Research; Resistance; Role; Scanning Electron Microscopy; Small Intestines; Staging; Structure; Subfamily lentivirinae; System; Testing; Tight Junctions; Transmission Electron Microscopy; Work; absorption; base; cellular imaging; cellular microvillus; novel; pathogen; protein complex; public health relevance; research study; vasodilator-stimulated phosphoprotein; zonula occludens-1 protein

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.

描述（由申请人提供）：肠顶端区域提供了允许吸收营养物质的选择性屏障，同时它还形成了防止肠腔内容物易位的保护性屏障。顶端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病中很明显，顶端连接屏障的破坏导致克罗恩病和肠道病原体感染的发病机制。这项研究的长期目标是了解肠道疾病中顶端域组装的过程。迄今为止，还没有直接针对顶端域组装或拆卸的疗法。 丝状伪足是基于肌动蛋白的结构，理论上在微绒毛和顶端连接的组装中发挥作用。肌球蛋白-X 是丝状伪足形成所需的分子马达，因此推测肌球蛋白-X 在顶端域组装中发挥作用。初步的慢病毒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，并且初步观察可能表明肌球蛋白-X在连接蛋白定位中的作用。该提案的具体目标有两个：确定肌球蛋白-X 和丝状伪足在微绒毛形成中的作用；并确定肌球蛋白-X 在顶端连接复合体 (AJC) 组装中的作用。 使用 Caco-2 细胞作为模型系统，肌球蛋白-X 在顶端域结构中的定位和动态将通过共焦显微镜和活细胞成像来确定。肌球蛋白-X 对于微绒毛形成是必需的这一假设将在稳定的肌球蛋白-X 敲低/救援系统中进行测试。为了测试 AJC 形成是否需要肌球蛋白-X，将通过肌球蛋白-X 敲低细胞中的钙转换来诱导 AJC 组装。 这项研究将确定肌球蛋白-X 在肠细胞顶端域的功能作用，并将揭示顶端域形成的新分子机制。拟议的实验将进一步加深我们对顶端域组装的理解，这是解决肠道疾病中顶端域功能障碍的必要的第一步。 公众健康相关性：肠细胞功能障碍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，例如乳糜泻和炎症性肠病（IBD）。这项研究调查了肠道细胞的关键结构如何组装，这一过程在乳糜泻和炎症性肠病等疾病中可能会受到损害。