Tracking of CFTR diffusion in cell membranes. (A, upper) Schematic of CFTR in the plasma membrane showing its C-terminal PDZ binding domain, the PDZ-domain protein EBP50, the actin-binding protein ezrin, and the actin cytoskeleton. The location of the epitope (3HA) in the fourth extracllular loop is also shown. (A, lower) Fluorescence micrographs of MDCK cells stably expressing CFTR-3HA (left) and nontransfected control cells (right), labeled with anti-HA antibody, secondary biotinylated Fab fragment, and streptavidin-conjugated Qdots. Image acquisition time was 15 ms, as used in SPT measurements. (B) Representative trajectories for CFTR-3HA diffusion in the plasma membrane of MDCK cells (upper) and COS7 fibroblasts (middle). Total trajectory time >12 s. For comparison, trajectories of immobilized Qdots and for parformaldehyde-fixed cells are shown (lower), acquired using the same parameters as for live cells. Scale bar applies to all trajectories. (C) MSD versus time plots for CFTR-3HA in MDCK cells (solid line), immobilized Qdots (dotted line), and CFTR mutant CFTR-Δ26 that lacks its C-terminal PDZ binding domain (dashed line). Plots are averaged for 16–38 individual trajectories.