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

基本信息

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
项目类别：
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&apos 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

项目摘要

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（由适用提供）：上皮细胞在许多器官中起关键作用，并负责营养摄取或废物产品分泌等元素过程。了解上皮细胞在正常活动和伤口愈合过程中如何保持其内在细胞极性的基本差距。为了维持细胞极性，上皮细胞必须连续分类新合成和内部化的跨膜蛋白到正确的膜结构域（basolatellar或顶端）。在大多数上皮的过程中，这一过程至关重要的是胞质适配器复合物AP-1B，该复合物AP-1B将蛋白质传递到运输蔬菜中的底层膜。 AP-1B蔬菜如何形成以及如何调节蔬菜形成是未解决的问题。长期目标是了解如何根据分子水平调节AP-1B，以及AP-1B的放松管制如何有助于疾病的发展。中心假设是，AP-1B也可以使用由AP-1B控制的网络来控制上皮细胞迁移。此外，我们假设AP-1B中只有几个关键的氨基酸对于将这些过程的特异性汇总是必要的。这些假设背后的理由是，我们发现与AP-1B一起在基底外侧分类中使用的大多数蛋白质也以驱动细胞迁移和取消转化而闻名。此外，虽然AP-1B在基底外侧分类过程中控制此细胞迁移网络，但与密切相关的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在场地组建过程中如何封装外循环招募； 2）AP-1B如何控制细胞迁移。在第一个目的下，我们将使用定位的诱变来确定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与最先进的共聚焦和电子显微镜结合使用建立的方案来分析突变复合物。在第二个目标下，我们将使用最先进的成像技术在细胞迁移中检查AP-1B的功能，包括活细胞成像和超分辨率免疫荧光显微镜，并使用AP-1B的耗竭和其他候选蛋白使用基因敲除方法结合使用。该提案具有创新性，因为它是第一个在细胞迁移中分析AP-1B的提案，这可能会扩大其功能I的上皮细胞稳态，从完全极化细胞中的基底外侧分类到获得后细胞迁移。拟议的研究很重要，因为它将导致有关AP-1B功能在健康和疾病中的贡献的新见解，并将在正常伤口愈合期间和疾病中对AP-1B调节的高度了解。这有可能告知新药物筛查对新型靶蛋白和未来翻译研究的开发。