Investigating the role of EPS8 in microvillar growth

项目摘要/摘要 微绒毛是以肌动蛋白为基础的突起，位于运输上皮细胞的顶面。在上下文中 在哺乳动物的肠道中，微绒毛共同构成肠道刷状缘，起着第一线的作用。 对病原体的防御和增加表面是为了吸收养分。尽管重要的是 微绒毛对肠道健康和人类生存能力的影响，对其背后的分子事件知之甚少。 微绒毛组装。早期的透射电子显微镜超微结构分析显示，远端 微绒毛的尖端被一种电子密度高的斑块占据，这种斑块被称为“远端尖端复合体”，它嵌入了 肌动蛋白细丝的带刺末端。由于肌动蛋白细丝的带刺末端是肌动蛋白单体的首选位置 此外，调节肌动蛋白细丝长度的蛋白质通常针对这些末端。1个候选远端 复合蛋白是表皮生长因子受体途径底物8(Eps8)。肠组织染色 EPS8显示沿着肠腺绒毛长度的末端有明显的定位。此外，域 Eps8的结构提供了结合质膜、肌动蛋白和其他信号因子、功能的可能性 这使其成为策划微绒毛组装的有吸引力的候选者。此外，研究表明， 在细胞培养和小鼠基因敲除模型中，Eps8的丢失都会导致微绒毛变短。尽管如此 研究发现，Eps8是如何促进微绒毛生长的尚不清楚。因此，本提案试图界定 Eps8通过活细胞、超分辨和传递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非常具体 对于微绒毛的末端，这种特异性可以用来定义其他末端复合体蛋白。 生物素亲和标记法。通过创建与生物素连接酶BioID2(Eps8-BioID2)的嵌合融合， 驻留在远端末端复合体中的蛋白质可以被生物素化，分离，并通过质谱学进行鉴定。AS 因此，揭示远端末端复合体蛋白的特性将为深入了解 微绒毛生长，这是一个正常的刷子边缘生理必不可少的过程。因此，理解机制 潜在的微绒毛生长可以提供对微绒毛形态受损的疾病的洞察， 如微绒毛包涵体病、乳糜泻和慢性疾病。

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.