Investigating the role of EPS8 in microvillar growth

研究 EPS8 在微绒毛生长中的作用

• 批准号: 10164775
• 负责人: Isabella M Gaeta
• 金额: $ 3.07万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10164775
• 关键词: Actins; Address; Affect; Apical; Architecture; Area; Bacterial Infections; Binding; Biological Assay; Biotin; Brush Border; Bundling; C-terminal; Celiac Disease; Cell Culture Techniques; Cell membrane; Cells; Chimera organism; Complex; Conflict (Psychology); Confocal Microscopy; Disease; Distal; EPS8 gene; Electron Microscopy; Electrons; Elongation Factor; Epidermal Growth Factor Receptor Pathway Substrate 8; Epithelial Cells; Event; Fibrinogen; Filament; Fimbrin; Growth; Health; Human; Image; Intestines; Knockout Mice; LS174T colon cancer cell line; Label; Length; Ligase; Literature; Mass Spectrum Analysis; Methods; Microfilaments; Microscopy; Microvillus inclusion disease; Modeling; Molecular; Molecular Biology Techniques; Morphogenesis; Morphology; Mus; Organism; Phenotype; Physiological; Physiology; Play; Plus End of the Actin Filament; Positioning Attribute; Process; Proteins; Proteome; Proteomics; Resolution; Role; Signal Transduction; Site; Specific qualifier value; Specificity; Structure; Surface; Testing; Tissue Stains; Tissues; Transmission Electron Microscopy; Villus; apical membrane; base; cellular microvillus; crosslink; in vitro activity; in vivo; insight; intestinal crypt; intestinal epithelium; knock-down; live cell imaging; loss of function; monomer; mouse model; nutrient absorption; pathogen; protein complex; tool; villin

PROJECT SUMMARY/ABSTRACT Microvilli are actin-based protrusions located at the apical surface of transporting epithelial cells. In the context of the mammalian intestine, microvilli collectively comprise the intestinal brush border, which acts as a first line of defense against pathogens and increases the surface are for nutrient absorption. Despite the importance of microvilli for intestinal health and human viability, little is known about the molecular events that underlie microvillar assembly. Early ultrastructural analysis by transmission electron microscopy revealed that the distal tips of microvilli are occupied by an electron dense plaque known as the “distal tip complex”, which embeds the barbed ends of actin filaments. As the barbed ends of actin filaments are the preferred site of actin monomer addition, proteins that regulate actin filament length typically target to these ends. One candidate distal tip complex protein is epidermal growth factor receptor pathway substrate 8 (EPS8). Intestinal tissue staining of EPS8 revealed striking distal tip localization along the length of the intestinal crypt-villus. Additionally, the domain architecture of EPS8 lends the potential to bind plasma membrane, actin, and other signaling factors, features that position it as an attractive candidate for orchestrating microvillar assembly. Moreover, studies have shown that loss of EPS8 in both cell culture and mouse knockout models results in shortened microvilli. Despite these findings, how EPS8 promotes microvillar growth remains unknown. Thus, this proposal seeks to define the mechanism by which EPS8 promotes microvillar growth using live cell, super resolution, and transmission electron microscopy in combination with molecular biology techniques. Moreover, as EPS8 is remarkably specific to the distal tips of microvilli, this specificity can be harnessed to define other distal tip complex proteins with a biotin proximity labeling approach. By creating a chimeric fusion with the biotin ligase BioID2 (EPS8-BioID2), proteins residing in the distal tip complex can be biotinylated, isolated, and identified by mass spectrometry. As such, revealing the identity of distal tip complex proteins would provide insight into the general mechanism of microvillar growth, a process essential for normal brush border physiology. Thus, understanding mechanisms underlying microvillar growth can provide insight into diseases where microvillar morphology is compromised, such as microvillus inclusion disease, celiac disease, and Chron’s disease.

项目概要/摘要 微绒毛是位于运输上皮细胞顶端表面的基于肌动蛋白的突起。 在哺乳动物肠道中，微绒毛共同组成肠刷状缘，充当第一线 尽管很重要，但防御病原体和增加表面是为了营养吸收。 微绒毛对于肠道健康和人类生存能力至关重要，但对其背后的分子事件知之甚少 透射电子显微镜的早期超微结构分析表明，远端微绒毛组装。 微绒毛的尖端被称为“远端尖端复合体”的电子致密斑块占据，该斑块嵌入 肌动蛋白丝的倒刺末端是肌动蛋白单体的优选位点。 此外，调节肌动蛋白丝长度的蛋白质通常靶向这些末端。 复合蛋白是表皮生长因子受体途径底物8 (EPS8)的肠组织染色。 EPS8揭示了沿肠隐窝绒毛长度的显着远端定位。 EPS8 的结构具有结合质膜、肌动蛋白和其他信号因子、特征的潜力 此外，研究表明，它是组织微别墅组装的一个有吸引力的候选者。 尽管如此，细胞培养物和小鼠敲除模型中 EPS8 的缺失都会导致微绒毛缩短。 研究结果表明，EPS8 如何促进微绒毛生长仍然未知，因此，该提案旨在定义 EPS8 利用活细胞、超分辨率和传输促进微绒毛生长的机制 电子显微镜与分子生物学技术相结合此外，由于 EPS8 具有异常特异性。 对于微绒毛的远端，可以利用这种特异性来定义其他远端复合蛋白 通过与生物素连接酶 BioID2 (EPS8-BioID2) 建立嵌合融合， 存在于远端复合物中的蛋白质可以通过质谱法进行生物素化、分离和鉴定。 这样，揭示远端复合蛋白的身份将提供对一般机制的深入了解。 微绒毛生长，这是正常刷状缘生理学所必需的过程，因此，了解其机制。 潜在的微绒毛生长可以深入了解微绒毛形态受损的疾病， 例如微绒毛包涵体病、乳糜泻和慢性病。