The role of TTC7A in apical lumen formation and polarized trafficking in the intestinal epithelium

TTC7A 在肠上皮顶腔形成和极化运输中的作用

基本信息

批准号：
10464502
负责人：
Katlynn Bugda Gwilt
金额：
$ 7.5万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-01 至 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10464502
关键词：
1-Phosphatidylinositol 4-Kinase 3-Dimensional Address Affect Age Apical Apoptotic Applications Grants Architecture Automobile Driving Biological Assay Biology Caco-2 Cells Cell Line Cell Maintenance Cell Polarity Cell membrane Cells Cellular Membrane Cholera Toxin Cyst Defect Development Development Polarity Developmental Biology Diarrhea Dimerization Disease Endoplasmic Reticulum Endosomes Enterocolitis Enterocytes Epithelial Epithelial Cells Etiology Fc Receptor Functional disorder Gastrointestinal Diseases Gastrointestinal tract structure Generations Genes Gland Goals Golgi Apparatus Image Immune Immunoglobulin G Inflammatory Bowel Diseases Intestinal Atresia Intestines Knowledge Label Lead Ligands Lipids Location Maps Mediating Membrane Membrane Proteins Modeling Molecular Chaperones Monitor Movement Mutation Organ Organoids Pathology Pathway interactions Patients Phenotype Phosphatidylinositols Phosphotransferases Process Production Proteins Recycling Resolution Role SERPINA4 gene Scaffolding Protein Site Sorting - Cell Movement Source Space Perception Specialized Epithelial Cell Symptoms Testing Therapeutic Tissues Training Translating apical membrane base basolateral membrane disease-causing mutation experimental study gastrointestinal epithelium gut inflammation human disease in vivo infancy inorganic phosphate intestinal epithelium knock-down loss of function loss of function mutation monolayer mutant neonatal Fc receptor new technology novel polarized cell protein complex scaffold spatiotemporal trafficking transcytosis

项目摘要

PROJECT SUMMARY The goal of this grant application is to understand how mutations in tetratricopeptide repeat domain 7a (TTC7A) affect formation of the polarized apical membrane; and how these mutations cause human disease. TTC7A loss of function mutations result in severe infantile-onset gastrointestinal disease, with phenotypes related to both gut epithelial and immune cell dysfunction. Previous studies suggest that loss of function in TTC7A results in altered apico-basolateral polarity and lumen formation in intestinal epithelial cells, although how this occurs remains unclear. Prevailing models suggest that initial organization of the apical membrane occurs at an initiation site (AMIS), which is enriched with distinct phosphoinositides compared to the basolateral membrane. The putative function of TTC7A is to serve as a chaperone and scaffolding protein for the phosphoinositide (PI) kinase – PI4KIIIα - at the plasma membrane. This kinase is principally responsible for the generation phosphoinositide PI4-phosphate (PI4P), which is one of the major precursors to the apically enriched PI(4,5P)2 and basolaterally enriched PI(3,4,5)P3. The role of PIs in specialized compartments is thought to partly direct vesicular cargo to the correct subcellular compartment – also responsible for proper polarity and lumenogenesis. Given the known function of TTC7A in coordinating PI4KIIIα localization, we hypothesize that TTC7A mutations perturb PI4KIIIαs normal functionality in cells, resulting in disordered spatiotemporal production of PI(4,5)P2 and PI(3,4,5)P3, thus driving improper cellular polarity, lumenogenesis and endosomal trafficking. To test this idea, we investigate the role of TTC7A in cell polarization, epithelial lumen formation, and endosomal trafficking. We propose studies using patient-derived enteroids, intestinal epithelial cell (Caco2) monolayers and Caco2 cyst cultures to determine the contribution of TTC7A to cell polarity and lumenogenesis. In Aim 1 of this proposal, we will further develop novel technology to monitor the formation of the early apical membrane, protein and lipid movement during apical membrane formation (and lumenogenesis), and how the proper subcellular localization of TTC7A contributes to these processes. We will carry out high resolution confocal and lightsheet imaging of phosphoinositide and apical and basolateral proteins localization in the polarization and lumen formation of Caco2- cysts and enteroids. In Aim 2 of this proposal we further translate our recently established high- throughput, quantitative endosomal trafficking assays for studies on primary patient derived enteroids. Further, we develop novel imaging to spatially probe endosomal trafficking in live enteroids. Our studies seek to identify if the membrane scaffolding function of TTC7A is important for correct spatial orientation of epithelial cells, and how the loss of TTC7A induces abnormal intestinal lumen formation. The results of these experiments may plausibly reveal general rules underlying epithelial development and suggest therapeutic approaches for TTC7A deficiency as well as other more common GI pathologies. Further, successful completion of these aims will provide extraordinary training towards my goal in becoming an epithelial biologist.

项目概要本拨款申请的目标是了解四肽重复结构域 7a (TTC7A) 中的突变是如何发生的影响极化顶膜的形成；以及这些突变如何导致人类疾病。的功能突变导致严重的婴儿期胃肠道疾病，其表型与肠道和肠道相关先前的研究表明，TTC7A 功能的丧失会导致上皮细胞和免疫细胞功能的改变。肠上皮细胞的顶端-基底外侧极性和管腔形成，尽管其发生方式仍然存在流行的模型表明顶膜的初始组织发生在起始位点。（AMIS），与基底外侧膜相比，其富含不同的磷酸肌醇。 TTC7A 的功能是充当磷酸肌醇 (PI) 激酶的伴侣和支架蛋白 – PI4KIIIα - 在质膜上，该激酶主要负责生成磷酸肌醇。 PI4-磷酸盐 (PI4P)，是顶部富集 PI(4,5P)2 和基底外侧的主要前体之一富集的 PI(3,4,5)P3 PI 在特殊隔室中的作用被认为部分引导囊泡货物。正确的亚细胞区室 - 也负责正确的极性和发光。通过研究 TTC7A 在协调 PI4KIIIα 定位中的功能，我们发现 TTC7A 突变会扰乱 PI4KIIIα 细胞的正常功能，导致 PI(4,5)P2 和 PI(3,4,5)P3 的时空产生紊乱，从而驱动不正确的细胞极性、腔发生和内体运输为了检验这个想法，我们研究了。我们的研究提出了 TTC7A 在细胞极化、上皮腔形成和内体运输中的作用。使用患者来源的肠类、肠上皮细胞 (Caco2) 单层和 Caco2 囊肿培养物确定 TTC7A 对细胞极性和发光发生的贡献在本提案的目标 1 中，我们将进一步进行。开发新技术来监测早期顶膜的形成、蛋白质和脂质运动在顶膜形成（和管腔生成）过程中，以及 TTC7A 如何正确进行亚细胞定位我们将为这些过程做出贡献。磷酸肌醇以及顶端和基底外侧蛋白在极化和管腔形成中的定位 Caco2- 囊肿和肠样蛋白在本提案的目标 2 中，我们进一步翻译了我们最近建立的高- 用于研究原发性患者来源的肠样蛋白的通量、定量内体运输测定。我们开发了新颖的成像技术来空间探测活肠类体内的内体运输。如果 TTC7A 的膜支架功能对于上皮细胞的正确空间定向很重要，并且 TTC7A 的缺失如何导致肠腔形成异常？这些实验的结果可能是这样的。合理地揭示了上皮发育的一般规则并提出了 TTC7A 的治疗方法此外，成功完成这些目标将有助于消除缺乏症以及其他更常见的胃肠道疾病。为我成为上皮生物学家的目标提供非凡的培训。