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.