Molecular Mechanisms of Basolateral Targeting in Polarized Epithelial Cells

极化上皮细胞基底外侧靶向的分子机制

• 批准号: 9135451
• 负责人: HEIKE FOLSCH
• 金额: $ 33.27万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9135451
• 关键词: Amino Acids; Apical; Automobile Driving; Binding; Cancer Model; Cancerous; Carcinoma; Cell Line; Cell Polarity; Cell physiology; Cell-Matrix Junction; Cells; Chimeric Proteins; Clathrin Adaptors; Clathrin-Coated Vesicles; Colon; Complex; Confocal Microscopy; Crohn' s disease; Data; Development; Disease; Disseminated Malignant Neoplasm; Electron Microscopy; Endosomes; Epithelial; Epithelial Cells; Epithelium; Excision; Future; Genes; Goals; Growth; Health; Homeostasis; Human; Human body; Image; Imaging Techniques; Immunofluorescence Microscopy; Individual; Integral Membrane Protein; Integrins; Knowledge; Laboratories; Lead; Life; Lipids; Malignant Neoplasms; Mediating; Membrane; Membrane Fusion; Molecular; Mutate; Nutrient; Organ; Outcome; Patients; Phosphotransferases; Play; Preclinical Drug Evaluation; Process; Proteins; Protocols documentation; Recruitment Activity; Recycling; Regulation; Research; Resolution; Role; Signal Transduction; Site-Directed Mutagenesis; Sorting - Cell Movement; Specificity; Speed; Surface; Testing; Tissues; Translational Research; Transport Vesicles; Tyrosine; Vesicle; Waste Products; Work; Wound Healing; apical membrane; base; basolateral membrane; cancer cell; cell motility; human disease; hypercholesterolemia; innovation; insight; knock-down; live cell imaging; migration; monolayer; mutant; new therapeutic target; novel; novel strategies; novel therapeutics; overexpression; phosphoinositide-3,4,5-triphosphate; polarized cell; prevent; protein protein interaction; receptor; uptake; wound

 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.

 描述（由申请人提供）：上皮细胞在许多器官中发挥关键作用，并负责营养摄取或废物分泌等基本过程。在理解上皮细胞如何在其一生中保持其固有的细胞极性方面存在根本性的差距，无论是在正常活动期间还是在伤口愈合过程中。为了维持细胞极性，上皮细胞必须不断地将新合成和内化的跨膜蛋白分选到正确的膜结构域（基底侧或顶端）。在大多数上皮细胞中，对该过程至关重要的是胞质适配器复合物AP-1B，其在运输囊泡中将蛋白质递送至基底外侧膜。AP-1B囊泡如何形成以及囊泡的形成是如何被调节的是尚未解决的问题。长期目标是了解AP-1B在分子水平上是如何调节的，以及AP-1B的失调如何有助于疾病的发展。中心假设是，网络控制的AP-1B在组成性基底外侧分选也可以使用AP-1B控制上皮细胞迁移。此外，我们假设AP-1B中只有少数关键氨基酸是赋予这些过程特异性所必需的。这些假设背后的基本原理是，我们发现在基底外侧分选中与AP-1B一起工作的大多数蛋白质也被认为是驱动细胞迁移和癌性转化的蛋白质。此外，虽然AP-1B在基底外侧分选过程中控制着这种细胞迁移网络，但密切相关的AP-1A却没有。在强有力的初步数据的指导下，这些假设将通过追求两个特定的目标进行测试：1）AP-1B如何促进囊泡形成过程中的外囊募集; 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如何控制基底外侧分选。将使用已建立的方案结合最先进的共聚焦和电子显微镜分析突变复合物。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消除AP-1B和其他候选蛋白质，研究AP-1B在细胞迁移中的功能。该提案是创新的，因为它是第一个分析AP-1B在细胞迁移中的作用，这将潜在地将其在上皮细胞稳态中的功能从完全极化细胞中的基底外侧分选扩展到创伤后的细胞迁移。拟议的研究是重要的，因为它将导致关于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中的组成性基底外侧分选和上皮细胞迁移过程中的调节的深入理解。这有可能为开发新的靶蛋白和未来的转化研究提供信息。