Mucus layer and PCL transport by human airway epithelia. (a) Mucus transport in WD airway epithelial cultures was identified by the rotational movement of 1-μm fluorescent microspheres in the mucus layer. The image shown was acquired as a single 5-second exposure; the streaks represent the paths of individual microspheres (field diameter ∼2 mm). (b) Mucus transport rates were calculated as the linear velocities of fluorescent microspheres (a) and plotted against the distance from the center of the rotation (radial). (c) Transport of PCL revealed by photoactivation of caged fluorescein-conjugated dextrans. The ASL was labeled with caged fluorescein-conjugated dextran (10,000 Da) and the fate of photoreleased dextran fluorescence was determined. Conventional fluorescence microscopy at low power was used (bars = 0.2 mm). Left: Migration of the released fluorescent dextran during the 20-second period of observation. Right: After removing the mucus layer (+DTT), the migration of released fluorescent dextran was minimal. Both the mucus layer and the PCL are labeled by photoactivation. The absence of a “smear” during movement implies that both the mucus layer and the PCL move at the same velocity. The absence of PCL movement after mucus removal suggests that mucus movement is critical for PCL movement. (d) Models of ASL transport. Lateral fluid velocity profiles in ASL predicted for three different considerations, with the ordinate aligned to the diagram of the ciliary beat cycle at the left. Curve A approximates velocity profiles in the PCL predicted from theoretical considerations of ciliary propulsion of water (60, 61). Note nominally zero velocities at 70–75% of the ciliary length, below the level of the return stroke. Curve B depicts the velocity profile predicted for PCL flow driven solely by frictional interactions with the mucus layer. Curve C depicts the velocity profile for the PCL from the observations in this work that the flows of the PCL and mucus layer are essentially indistinguishable.