Molecular Mechanisms of Basolateral Targeting in Polarized Epithelial Cells

 描述(申请人提供)：上皮细胞在许多器官中扮演关键角色，并负责营养吸收或废物分泌等基本过程。无论是在正常活动期间还是在伤口愈合过程中，对于上皮细胞如何在其整个生命周期中保持其固有的细胞极性，存在着根本的差距。为了维持细胞的极性，上皮细胞必须不断地将新合成和内化的跨膜蛋白分类到正确的膜域(基侧或顶端)。在大多数上皮细胞中，胞质适配器复合体AP-1B是这一过程的关键，它将蛋白质输送到运输囊泡的基侧膜上。AP-1B囊泡是如何形成的，以及囊泡形成是如何调节的，这些都是悬而未决的问题。长期目标是了解AP-1B是如何在分子水平上被调控的，以及AP-1B的放松调控如何可能有助于疾病的发展。中心假设是，AP-1B在结构性基侧分选过程中控制的网络也可能被AP-1B用来控制上皮细胞的迁移。此外，我们假设只有AP-1B中的几个关键氨基酸是赋予这些过程特异性所必需的。这些假设背后的理论基础是，我们发现的在碱侧分选中与AP-1B一起工作的大多数蛋白质也是已知的，它们也驱动细胞迁移和癌变。此外，虽然AP-1B在基侧分选过程中控制着这个细胞迁移网络，但与之密切相关的AP-1A不能。在强大的初步数据的指导下，这些假设将通过追求两个具体目标来检验：1)AP-1B如何在囊泡形成过程中促进外囊招募；2)AP-1B如何控制细胞迁移。在第一个目标下，我们将使用定点突变来确定AP-1B中的关键氨基酸残基，以揭示AP-1B是如何控制碱侧排序的。突变的复合体将使用既定的方案结合最先进的共聚焦和电子显微镜进行分析。在第二个目标下，我们将使用最先进的成像技术，包括活细胞成像和超分辨率免疫荧光显微镜，结合使用基因敲除方法耗尽AP-1B和其他候选蛋白质，来研究AP-1B在细胞迁移中的功能。该提议具有创新性，因为它是第一个分析AP-1B在细胞迁移中的作用，这将潜在地扩大其在上皮细胞动态平衡中的功能，从完全极化的细胞的基侧分选到损伤后的细胞迁移。这项拟议的研究具有重要意义，因为它将导致对AP-1B功能在健康和疾病中的贡献的新见解，并将导致对AP-1B在正常伤口愈合和疾病修复过程中结构性基侧分选和上皮细胞迁移过程中的调控有更深入的理解。这有可能为开发新的药物筛选和未来的翻译研究提供新的目标蛋白。

 DESCRIPTION (provided by applicant): Epithelial cells play critical roles in many organs and are responsible for elemental processes like nutrient uptake or waste product secretion. There is a fundamental gap in understanding how epithelial cells maintain their intrinsic cell polarity throughout their lifetime, both during normal activity and during wound healing processes. To maintain cell polarity, epithelial cells must continuously sort newly synthesized and internalized transmembrane proteins to the correct membrane domains (basolateral or apical). Essential to this process in most epithelia is the cytosolic adaptor complex AP-1B that delivers proteins to the basolateral membrane in transport vesicles. How AP-1B vesicles form and how vesicle formation is regulated are unresolved questions. The long-term goals are to understand how AP-1B is regulated on a molecular level and how deregulation of AP-1B may contribute to disease development. The central hypotheses are that a network controlled by AP-1B during constitutive basolateral sorting may also be used by AP-1B to control epithelial cell migration. Furthermore, we hypothesize that only a few critical amino acids in AP-1B are necessary to confer specificity to these processes. The rationale behind these hypotheses is that most of the proteins that we discovered to work with AP-1B in basolateral sorting are also known for driving cell migration and cancerous transformations. Moreover, while AP-1B controls this cell migration network during basolateral sorting, the closely related AP-1A does not. Guided by strong preliminary data, these hypotheses will be tested by pursuing two specific aims: 1) How does AP-1B facilitate exocyst recruitment during vesicle formation; and 2) How does AP-1B control cell migration. Under the first aim, we will use site-directed mutagenesis to determine critical amino acid residues in AP- 1B to unravel how AP-1B controls basolateral sorting. Mutant complexes will be analyzed using established protocols in combination with state-of-the-art confocal and electron microscopy. Under the second aim, we will examine a function for AP-1B in cell migration using state-of-the-art imaging techniques including live-cell imaging and super-resolution immunofluorescence microscopy in combination with depletion of AP-1B and other candidate proteins using gene knock down approaches. The proposal is innovative, because it is the first one to analyze AP-1B in cell migration, which will potentially broaden its functions i epithelial cell homeostasis from basolateral sorting in fully polarized cells to cell migration aftr wounding. The proposed research is significant, because it will result in novel insights regarding the contribution of AP-1B function in health and disease, and will lead to an advanced understanding of AP-1B regulation during constitutive basolateral sorting and epithelial cell migration during normal wound healing and in diseases. This has the potential to inform the development of new drug screens toward novel target proteins and future translational research.