ASL PHYSIOLOGY STUDIED BY NOVEL FLUORESCENCE METHODS

通过新颖的荧光方法研究 ASL 生理学

• 批准号: 6202600
• 负责人: ALAN S VERKMAN
• 金额: $ 18.09万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202600
• 关键词: biophysics; body fluid viscosity; body fluids; chloride channels; cystic fibrosis; fluorescent dye /probe; membrane fusion; method development; photochemistry; pulmonary surfactants; respiratory epithelium; syntaxin; time resolved data; tissue /cell culture; trachea; vesicle /vacuole

The application remains focused on the mechanisms by which the Cystic Fibrosis genotype leads to clinical abnormalities. Aim 1 is a direct extension of recent work indicating that CFTR is a modulator of intracellular vesicle fusion. Aims 2 and 3 concern the biophysics of the airspace surface liquid (ASL) layer. The proposed studies utilize novel optical methods including fluorescent indicators of [Cl/-] and vesicle fusion, photobleaching and time-resolved fluorescence measurements of viscosity, and 3-d tracking. Specific Aim 1. To investigate the mechanisms and significance of the CFTR-dependent stimulation of intracellular vesicle fusion. We reported that expression of cAMP activation of CFTR in stably transfected 3T3 fibroblasts increases endosome fusion by greater than 2-fold. New technical developments will be used to determine whether CFTR regulates endosome fusion in native CFTR-expressing versus 'CF' epithelial cells and whether vesicle fusion in the secretory pathway is regulated by CFTR. The hypothesis will be tested that syntaxins are involved in the CFTR- regulation of endosome fusion. Specific Aim 2: To develop quantitative fluorescence methods to measure ASL fluid thickness, ionic composition and viscosity. Novel Cl/- and Na+- sensitive indicators will be used to measure ASL fluid composition in situ without fluid sampling and other perturbations. Photobleaching recovery and time-resolved fluorescence anisotrophy will be used to measure ASL fluid viscosity. Measurements will be carried out on primary cultures of human and bovine tracheal epithelia for comparison with intact bovine and monkey trachea. The role of submucosal glands in ASL fluid properties will be studied. Specific Aim 3. To study the regulation of ASL fluid thickness, composition and viscosity, and to characterize their abnormality in Cystic Fibrosis. The methods developed in Aim 2 will be used to compare CFTR- expressing versus CF-epithelia. The hypotheses will be tested that ASL salt concentration and viscosity are elevated in CF, epithelial, and that UTP normalizes ASL fluid properties.

该应用程序仍集中在囊性的机制上 纤维化基因型导致临床异常。目标1是直接 最近的工作的扩展，表明CFTR是 细胞内囊泡融合。目标2和3涉及的生物物理学 空域表面液体（ASL）层。拟议的研究利用了新颖 光学方法包括[Cl/ - ]和囊泡的荧光指标 融合，光漂白和时间分辨的荧光测量 粘度和3D跟踪。 具体目的1。研究 CFTR依赖于细胞内囊泡融合的刺激。我们报道 CFTR在稳定转染的3T3中表达CFTR的表达 成纤维细胞可将内体融合增加超过2倍。新的 技术发展将用于确定CFTR是否对 天然CFTR表达与“ CF”上皮细胞的内体融合 分泌途径中的囊泡融合是否由CFTR调节。这 假设将检验，即语法素参与CFTR- 调节内体融合。 特定目的2：开发定量荧光方法以测量 ASL流体厚度，离子成分和粘度。新颖的Cl/ - 和Na+ - 敏感指标将用于测量原位ASL流体组成 没有流体采样和其他扰动。光漂白恢复 时间分辨荧光各向异性将用于测量ASL 流体粘度。测量将在原始培养物上进行 人类和牛气管上皮，可与完整的牛和 猴子气管。粘膜腺在ASL流体特性中的作用将 被研究。 特定目的3。研究ASL流体厚度的调节， 成分和粘度，并表征其囊性异常 纤维化。 AIM 2中开发的方法将用于比较CFTR- 表达与CF-上皮。假设将测试ASL 盐浓度和粘度在CF，上皮中升高，并且 UTP归一化ASL流体特性。