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

项目摘要 这项资助申请的目的是了解三联肽重复结构域7a（TTC 7A）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。TTC 7A损失 功能突变导致严重的消化道疾病，表型与两个肠道 上皮和免疫细胞功能障碍。以前的研究表明，TTC 7A功能的丧失导致了 肠上皮细胞的顶端-基底侧极性和管腔形成，尽管这是如何发生的仍然存在。 不清楚流行的模型表明，顶端膜的初始组织发生在起始位点 （AMIS），其与基底外侧膜相比富含不同的磷酸肌醇。推定的 TTC 7A的功能是作为磷脂酰肌醇（PI）激酶的伴侣蛋白和支架蛋白。 PI 4KIII α -在质膜上。这种激酶主要负责产生磷酸肌醇 PI 4-磷酸盐（PI 4P），是顶部富集PI（4，5 P）2和基底外侧富集PI（4，5 P）2的主要前体之一。 富集PI（3，4，5）P3。PI在特化隔室中的作用被认为是部分引导囊泡货物， 正确的亚细胞区室-也负责适当的极性和管腔形成。鉴于已知 TTC 7A在协调PI 4KIII α定位中的功能，我们假设TTC 7A突变扰乱PI 4KIII α定位， 细胞中的正常功能，导致PI（4，5）P2和PI（3，4，5）P3的无序时空产生，因此 驱动不适当的细胞极性、内腔形成和内体运输。为了验证这个想法，我们调查了 TTC 7A在细胞极化、上皮管腔形成和内体运输中的作用。我们建议研究 使用患者来源的类肠、肠上皮细胞（Caco 2）单层和Caco 2囊肿培养物， 确定TTC 7A对细胞极性和管腔形成的贡献。在本建议的目标1中，我们将进一步 开发新技术来监测早期根尖膜的形成、蛋白质和脂质的运动 在顶端膜形成（和管腔形成），以及如何正确的亚细胞定位TTC 7A 有助于这些进程。我们将进行高分辨率的共焦和光片成像， 磷脂酰肌醇和顶端和基底外侧蛋白定位在极化和管腔形成的 Caco 2-囊肿和肠样囊肿。在本提案的目标2中，我们进一步将我们最近建立的高- 用于研究原发性患者来源的类肠病的通量、定量内体运输测定。此外，本发明还 我们开发了新的成像技术来空间探测活的类肠细胞中的内体运输。我们的研究试图找出 如果TTC 7A的膜支架功能对于上皮细胞的正确空间取向是重要的，以及 TTC 7A的缺失如何诱导异常肠腔形成。这些实验的结果可能 揭示了上皮细胞发育的一般规律，并提出了TTC 7A的治疗方法 缺乏症以及其他更常见的胃肠道疾病。此外，成功实现这些目标将 为我成为一名上皮生物学家的目标提供了非凡的训练。

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.