PCL is required for effective mucociliary and cough clearance. (a) Schema of microanatomy of normal ASL. Note mixing of bacteria in “turbulent” mucus. (b) Schema depicting hypothetical volume depletion of ASL covering CF airway epithelial surfaces. Note that the volume depletion is reflected in both the generation of a more concentrated mucus layer and the depletion of the PCL. PCL depletion allows interactions to occur between the tethered mucins of the glycocalyx and the mucus layer. Note motile bacteria penetrating into thickened, stationary mucus. (c) Evidence for ASL volume depletion in CF airway epithelia. ASL height was measured immediately, 12 hours, and 24 hours after deposition of PBS containing Texas red dextran on the epithelial surface of the cell (pseudocolored green). Left: Representative confocal microscopy images. Right: Mean data for normal (circles) and CF (squares) ASL heights. *CF ASL is significantly shallower than normal (P < 0.05; n = 6 per group). (d) Mucus (bead) rotational velocity 24 hours after administration of PBS containing fluorescent markers. At t = 0 hours, both normal and CF cultures exhibited rotational velocities of about 45 μm/s. (*P < 0.05, CF vs. normal; n = 6 per group). (e) Low-power electron micrograph of perfluorocarbon/osmium–fixed CF airway culture 24 hours after volume addition and with rotational mucus transport abolished. Note close apposition of mucus layer and the glycocalyx covering flattened cilia and the cell surface. (f) Light micrograph of freshly excised CF bronchus stained with Alcin blue periodic acid-Schiffs for mucus. As in the in vitro model, note close apposition (annealing) between secreted mucins and the cell surface (indicated by white arrow). NL, normal.