Fig. 3

Mucus threads from surface goblet cells were thinner than mucus bundles. A Image sequence from a low-resolution time-lapse where threads collected beads on an explanted WT piglet trachea. Speed of corresponding movie (Additional file 4) increased 16×. Mucus bundle coming out of a gland opening indicated by light blue arrowhead. Dark blue arrow: immobile bundle. Yellow, green and purple arrows indicate different moving mucus assemblies. B Low resolution image of bundles (LTL, blue arrow) and beads gathered into threads (yellow arrow) on WT piglet tracheas (arrowhead: gland opening). C Low-resolution image of explanted WT piglet trachea with bundle from a submucosal gland, LTL (green) and threads, UEA1 (red). D Airyscan high-resolution Z-stack of LTL-stained bundles (green) from submucosal glands and UEA1-stained threads (red) in a live explanted WT piglet trachea. E Thickness analysis of mucus bundles (LTL, green) and mucus threads (UEA1, red) in the image in D. Inset: zoom of the threads in the image center. F Mucus bundle thickness was calculated as a mean of 10 measurements close to the gland opening (1A, 2A) and further along the bundle (1B, 2B), as indicated in (E). Bundles were thicker close to the gland opening, Mann–Whitney test: 1A vs. 1B P < 0.0001 ****, 2A vs. 2B P = 0.0052 **. Thread thickness was calculated in the same way, as indicated in (E). Based on bundle thickness in live explants compared to fixed scanning electron micrographs, the maximum mucus thread thickness was determined to 2 µm (dashed line). Thread T3 was thicker than T4, Mann–Whitney test P < 0.0001 ****. Bundles were thicker than threads, Mann–Whitney test P = 0.0095 **. Data presented as median with interquartile range. G Scanning electron micrograph from a WT piglet trachea with mucus bundle from a submucosal gland (blue arrowhead). H Scanning electron micrograph of WT piglet trachea mucus thread (yellow arrows), GC: goblet cell, sGC: secreting goblet cell