(A) Schematic representation of the Gel-on-Brush hypothesis of the periciliary layer: tethered macromolecules, such as membrane-bound mucins, form a brush-like structure of the PCL. (B, C) Morphological evidence for the Gel-on-Brush model is revealed by rapid freeze imaging of HBE cultures exhibiting extensive mesh-like structure with mesh (depicted by the arrow in (C)) on the order of ~ 20–40 nm in the PCL. Immunological evidence showing the presence of tethered mucins on freshly excised human airway tissue: (D) MUC1 (red) is located at the bottom of the PCL; (E) MUC4 (green) spans the whole PCL. Scale bars in (B, D, E) = 7 μm, bar in (C) = 100 nm, double-head arrow in (C) = 30 nm. White box in (B) denotes area of magnification.