Role of Membrane Trafficking in Epithelial Homeostasis

上皮细胞在许多器官中起着关键作用，并负责营养等基本过程 摄取和废物产物的分泌。为了完成这些功能，它们将质膜极化成顶端 和基底侧域。为了维持组织的动态平衡，上皮细胞必须不断地进行新的分类 合成表面受体并将其内化到正确的靶域。在组织损伤后，上皮细胞 细胞必须正确地迁移到伤口，并在伤口区域重新定植。极性的丧失与 多种疾病，包括转移性癌症、多囊肾病和克罗恩病。如何上皮化 因此，细胞极性的调控是一个重要的细胞生物学问题，对人类健康有着深远的影响。 我们的工作发现上皮细胞特异性的网状蛋白适配器复合体AP-1B对极化循环至关重要 把货物运到基边域。依赖AP-1B进行基侧定位的货物包括重要的 信号受体，如表皮生长激素受体，其错误分类到顶端区域 与癌症和多囊肾病有关，以及Toll样受体3，其错误分类导致慢性 发炎。该领域的一个主要悬而未决的问题是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。我们先前的工作表明，AP-1B改变了 循环内小体(RES)的组织，以适应AP-1B在基侧分选中的功能。只有AP- 1B而不是AP-1A定位于RES，在那里它触发了富含PI(3，4，5)P3的脂类结构域的形成，以及 促进辅助因子的募集，包括囊泡系留复合体(外囊)和脂类 蛋白激酶(PIPKIg-90)。我们最近发现AP-1B的表达降低了细胞的集体迁移速度 在单层损伤后，一种不依赖于基侧分类的新功能。我们将检验中心假说 AP-1B在上皮单层的建立和维持中起双重作用：1)引导货物 在稳态和2)细胞迁移过程中，通过在RES中生成分选平台来获得基侧膜 通过调节细胞膜上焦点黏附分子的可用性。我们将检验这一假设 在我们提出的实验中，使用了最先进的成像技术，包括实时TIRF显微镜 结合光活化和包括BioID和质谱学在内的无偏屏幕来补充 创新的细胞生物学和生物化学方法：1.确定AP-1B如何产生基底侧向 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 控制极化上皮细胞在稳态和细胞迁移过程中的组织。

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.