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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.

项目总结