LIVE-CELL IMAGING OF CFTR OLIGOMERIZATION, TRAFFICKING AND INTERACTIONS

CFTR 寡聚化、运输和相互作用的活细胞成像

基本信息

批准号：
7844880
负责人：
Peter Michael Haggie
金额：
$ 30.82万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-01 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7844880
关键词：
Address Adrenergic Agents Adrenergic Receptor Apical Area Binding Biochemical Biology Cell Fractionation Cell Surface Proteins Cell membrane Cell surface Cells Cessation of life Chloride Channels Chloride Ion Chlorides Cholera Clathrin Color Complex Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Diarrhea Disease Electrophysiology (science)Endocytosis Enterotoxins Epithelium Event Fluorescence Fluorescence Microscopy Gastrointestinal tract structure Genes Goals Image Immunoprecipitation Individual Infection Ion Channel Kidney Label Life Longevity Macromolecular Complexes Mediating Methods Microscopy Mutagenesis Mutation Pancreas Pathology Pathway interactions Phosphorylation Photobleaching Polycystic Kidney Diseases Process Protein Kinase Recurrence Recycling Regulation Regulator Genes Reporting Research Resolution Respiratory physiology Role Scaffolding Protein Small Interfering RNA Sorting - Cell Movement Spottings Structural Protein Structure Surface Sweat Glands Techniques Testing Tissues adrenergic apical membrane cellular imaging coated pit ezrin fluorescence imaging imaging modality insight novel particle patch clamp polypeptide premature protein complex public health relevance research study single molecule trafficking

项目摘要

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated ion channel present in the apical surface of several epithelia including the airways, pancreas, kidney, sweat glands and gastrointestinal tract. CFTR is central to several disease pathologies including cystic fibrosis, polycystic kidney disease and enterotoxin-induced secretory diarrheas such as cholera. The long-term objective of this proposal is to use live cell fluorescence imaging to address key unresolved issues in the CFTR field. Specific Aim 1 investigates the structure of CFTR channels in live cells using single molecule fluorescence techniques. The hypotheses will be tested that PDZ domain interactions at the C-terminus of CFTR and activation of protein kinases modulate the oligomeric state of CFTR. These studies will yield insight into the structure of CFTR and possible mechanisms that regulate channel activity. Specific Aim 2 investigates CFTR trafficking at the apical plasma membrane of cells. CFTR will be directly labeled to visualize individual endocytic and recycling events with excellent spatial and temporal resolution. The role of protein kinase A and CFTR domain interactions in the itinerary of CFTR will be determined. Regulation of CFTR trafficking is one of the most controversial areas in the field and resolution of this issue is warranted. Specific Aim 3 will investigate whether CFTR is integrated into a cell surface protein complex with regulatory and structural proteins. The dynamics of interactions between CFTR and 22-adrenergic receptor (22AR), a key regulator of CFTR activity, will be probed by two color single particle tracking techniques. The hypothesis will be tested that CFTR and 22AR form transient, as opposed to stable, complexes to modulate CFTR activity. The proposed experiments will provide new insights into the structure, activity and regulation of CFTR activity that are not possible with alternative techniques. PUBLIC HEALTH RELEVANCE: The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel present in many tissues including the airways and digestive tract. CFTR activity is important in several diseases including cystic fibrosis, polycystic kidney disease and secretory diarrheas (e.g., cholera). The goal of this proposal is to use live cell microscopy to investigate unresolved issues in the field of CFTR research.

描述（由申请人提供）：囊性纤维化跨膜电导调节剂（CFTR）是一个cAMP调节的离子通道，存在于几个上皮的顶部表面，包括气道，胰腺，胰腺，肾脏，汗腺和胃肠道。 CFTR是几种疾病病理学的核心，包括囊性纤维化，多囊性肾脏疾病和肠毒素诱导的分泌性腹泻，例如霍乱。该建议的长期目标是使用活细胞荧光成像来解决CFTR领域中的关键未解决问题。特定的目标1使用单分子荧光技术研究了活细胞中CFTR通道的结构。这些假设将测试CFTR的C末端的PDZ结构域相互作用以及蛋白激酶的激活调节CFTR的寡聚状态。这些研究将深入了解CFTR的结构以及调节通道活性的可能机制。具体目标2研究了细胞顶端质膜的CFTR运输。 CFTR将直接标记以可视化具有出色的空间和时间分辨率的单个内吞和回收事件。将确定蛋白激酶A和CFTR结构域相互作用在CFTR行程中的作用。 CFTR贩运的监管是该领域最有争议的领域之一，因此有必要解决该问题。具体目标3将研究CFTR是否与调节蛋白和结构蛋白的细胞表面蛋白质复合物集成在一起。 CFTR和22-肾上腺素受体之间的相互作用动力学（22AR）是CFTR活性的关键调节剂，将通过两种颜色的单个粒子跟踪技术进行探测。该假设将被检验，即CFTR和22AR形成瞬态，而不是稳定的复合物来调节CFTR活性。提出的实验将为CFTR活性的结构，活性和调节提供新的见解，而替代技术是不可能的。公共卫生相关性：囊性纤维化跨膜电导调节剂（CFTR）是包括气道和消化道在内的许多组织中的一个离子通道。 CFTR活性在几种疾病中很重要，包括囊性纤维化，多囊性肾脏疾病和分泌性腹泻（例如霍乱）。该建议的目的是使用活细胞显微镜研究CFTR研究领域的未解决问题。