Role of Membrane Trafficking in Epithelial Homeostasis

膜运输在上皮稳态中的作用

• 批准号: 10365484
• 负责人: HEIKE FOLSCH
• 金额: $ 35.48万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10365484
• 关键词: Amino Acids; Apical; Area; Biochemical; Biochemistry; Biological; Biological Assay; Cell Adhesion Molecules; Cell Polarity; Cell membrane; Cell physiology; Cells; Chronic; Clathrin Adaptors; Complement; Complex; Crohn' s disease; Development; Disease; Disseminated Malignant Neoplasm; Endocytosis; Endosomes; Epithelial; Epithelial Cells; Exocytosis; Focal Adhesions; Growth Hormone Receptor; Health; Homeostasis; Human; Human body; Imaging Techniques; Inflammation; Integrins; Kidney Diseases; Laboratories; Lipids; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Microscopy; Molecular; Monomeric GTP-Binding Proteins; Nature; Nutrient; Organ; Organizational Change; Phosphotransferases; Play; Polycystic Kidney Diseases; Post-Translational Protein Processing; Process; Proteins; Receptor Signaling; Recycling; Research; Role; Sorting - Cell Movement; Speed; Surface; TLR3 gene; Testing; Tissues; Vesicle; Waste Products; Work; apical membrane; basolateral membrane; cell motility; experimental study; human disease; hypercholesterolemia; innovation; insight; intestinal epithelium; migration; monolayer; novel; novel strategies; phosphoinositide-3,4,5-triphosphate; photoactivation; prevent; receptor; recruit; trafficking; trans-Golgi Network; uptake; wound; wound healing

Epithelial cells play critical roles in many organs and are responsible for elemental processes such as nutrient uptake and waste product secretion. To fulfill these functions, they polarize their plasma membrane into apical and basolateral domains. To maintain tissue homeostasis, epithelial cells must continuously sort newly synthesized and internalized surface receptors to the correct target domain. And after tissue damage, epithelial cells must correctly migrate into the wound and re-colonize the wound area. Loss of polarity is associated with numerous diseases including metastatic cancer, polycystic kidney disease, and Crohn’s disease. How epithelial cell polarity is regulated is thus an important cell biological question with profound implications for human health. Our work identified the epithelial cell-specific clathrin adaptor complex AP-1B as crucial for polarized recycling of cargos to the basolateral domain. Cargos that depend on AP-1B for basolateral localization include important signaling receptors such as epidermal growth hormone receptor whose missorting to the apical domain has been implicated in cancer and polycystic kidney disease, and toll-like receptor 3 whose missorting results in chronic inflammation. A major unresolved question in the field is how AP-1B uniquely functions in epithelial cells, and why its close cousin AP-1A mostly fails to substitute. Our previous work suggested that AP-1B changes the organization of recycling endosomes (REs) to accommodate AP-1B’s function in basolateral sorting. Only AP- 1B but not AP-1A localizes in REs where it triggers the formation of a lipid domain enriched in PI(3,4,5)P3, and facilitates the recruitment of accessory factors including a vesicle-tethering complex (the exocyst) and a lipid kinase (PIPKIg-90). We recently showed that AP-1B expression reduced the speed of collective cell migration after monolayer wounding, a novel function independent of basolateral sorting. We will test the central hypothesis that AP-1B plays dual roles in establishment and maintenance of epithelial monolayers by 1) directing cargos to the basolateral membrane by generating a sorting platform in REs at steady-state and 2) during cell migration by modulating the availability of focal adhesion molecules at the plasma membrane. We will test this hypothesis in our proposed experiments by using state-of-the-art imaging techniques including live TIRF microscopy in combination with photoactivation and unbiased screens including BioID and mass spectrometry to complement innovative cell biological and biochemistry approaches to: 1. Determine how AP-1B generates a basolateral sorting platform, and 2. Determine how AP-1B controls cell migration. Our studies will define new mechanisms governing the organization of polarized epithelial cells at steady-state and during cell migration.

上皮细胞在许多器官中发挥着关键作用，负责营养等基本过程 吸收和废物分泌。为了实现这些功能，它们将质膜极化到顶端 和基底外侧域。为了维持组织稳态，上皮细胞必须不断地进行新的分类 合成并内化表面受体到正确的目标域。而组织损伤后，上皮 细胞必须正确迁移到伤口并重新定植伤口区域。极性丧失与 许多疾病，包括转移性癌症、多囊肾病和克罗恩病。如何上皮 因此，细胞极性的调节是一个重要的细胞生物学问题，对人类健康具有深远的影响。 我们的工作确定了上皮细胞特异性网格蛋白接头复合物 AP-1B 对于极化回收至关重要 货物到基底外侧域。依赖 AP-1B 进行基底外侧定位的货物包括重要的 信号受体，例如表皮生长激素受体，其顶端结构域的错配已被证实 与癌症和多囊肾病有关，以及 Toll 样受体 3 的错配导致慢性肾病 炎。该领域尚未解决的一个主要问题是 AP-1B 如何在上皮细胞中发挥独特的作用，以及 为什么它的近亲 AP-1A 大多无法替代。我们之前的工作表明 AP-1B 改变了 组织回收内体 (RE) 以适应 AP-1B 在基底外侧分选中的功能。仅 AP- 1B 但 AP-1A 不定位于 RE 中，在 RE 中触发富含 PI(3,4,5)P3 的脂质结构域的形成，并且 促进辅助因子的募集，包括囊泡束缚复合物（外囊）和脂质 激酶（PIPKIg-90）。我们最近发现 AP-1B 表达降低了集体细胞迁移的速度 单层损伤后，一种独立于基底外侧排序的新功能。我们将检验中心假设 AP-1B 在上皮单层的建立和维持中发挥双重作用，1) 将货物引导至 通过在稳态和 2) 细胞迁移过程中在 RE 中生成分选平台来对基底外侧膜进行分选 通过调节质膜上粘着斑分子的可用性。我们将检验这个假设 在我们提出的实验中，使用最先进的成像技术，包括实时 TIRF 显微镜 与光活化和无偏筛选（包括 BioID 和质谱）相结合以补充 创新的细胞生物学和生物化学方法： 1. 确定 AP-1B 如何产生基底外侧 分选平台，以及 2. 确定 AP-1B 如何控制细胞迁移。我们的研究将定义新的机制 控制稳态和细胞迁移过程中极化上皮细胞的组织。