Mapping CFTR Clusters in 3D via Single Molecule Step Photobleaching Analysis in Epithelial Cells

Abstract

Protein oligomerization is crucial for cellular regulation. Various methods have been used to study the oligomerization of membrane proteins and multimeric channels including single molecule step photobleaching subunit counting. However, the membrane proteins function in a quasi 2D environment within a larger 3D cellular context encompassing membrane delivery and recycling via exocytosis and endocytosis and scaffolding and functional interactions within multiprotein complexes. We present PISA (photobleaching intensity step analysis), a new single molecule approach to map oligomerization states of fluorescently tagged proteins in 3D in cells where the membrane interface is imaged via TIRF microscopy. PISA counts oligomer subunits via sequential photobleaching steps, and additionally maps these distributions with nm precision in z due to magnitude differences of the intensity steps within the TIRF evanescent field. We applied PISA to map distributions of clusters of cystic fibrosis transmembrane conductance regulator (CFTR) in primary human airway epithelial cells. CFTR is an anion channel which conducts chloride and bicarbonate ions through the apical plasma membrane of epithelial cells and is known to interact with other proteins which form a macromolecular complex that regulates its activity at the cell surface. We measured membrane distributions of CFTR-eGFP as well as intracellular clusters of CFTR that were distributed at distances over 100 nm from the membrane. Treatment of the cells with vasoactive intestinal peptide (VIP) and cholesterol oxidase (COase) both resulted in a shift of CFTR clusters towards the membrane. VIP treatment resulted in larger CFTR cluster size (mean 2.9 subunits/cluster) and population (mean 28 units/ROI) as compared to untreated cells (mean 2.1 subunits/cluster) and cluster population (mean 22 units/ROI). COase treatment resulted only in lower cluster population (mean 9 units/ROI). Results on untreated cells were corroborated by TEM.