Figure 4

Microrheological analysis of CYS domain-enriched mucus scraped from mouse colons. (a) Study design for the analysis of PEG-bead diffusion. After checking their PEG coating, fluorescent beads were loaded in mucus scraped from the colons of wild-type and transgenic mice. Tracking and analysis of bead diffusion were conducted using Fiji and Icy, respectively. (b) Coating of 200 nm diameter YG beads (green fluorescence) with low-molecular-weight PEG. Effective coating was checked by incubation with avidin–rhodamine (red fluorescence). The avidin adsorbs onto uncoated beads (yellow fluorescence on merged pictures), but not beads coated with PEG (see the x3 enlargements). Scale bar = 50 µm. (c) MSD ± sem as a function of time for 200 nm PEG-coated beads embedded in mucus from wild-type (in blue) or transgenic (in purple) mice. (d) Diffusion coefficients distribution (t = 1 s) of 200 nm fluorescent beads embedded in colonic mucus from wild-type (in blue) or transgenic (in purple) mice. The average diffusion coefficient is represented by the vertical dashed line. (c,d) Data are averaged from 5 independent experiments. (e) Mucus mesh-size distribution calculated with the obstruction-scaling model in mucus from wild-type and transgenic mice. Data were pooled from 5 independent experiments. (f) MSD ± sem as a function of time for 1 µm PEG-coated beads embedded in mucus from wild-type (in blue) or transgenic (in purple) mice. (g) Diffusion coefficients distribution (t = 1 s) of 1 µm fluorescent beads embedded in colonic mucus from wild-type (in blue) or transgenic (in purple) mice. The average diffusion coefficient is represented by the vertical dashed line. (f,g) Data are averaged from 3 independent experiments. (h) MSD of 200 nm (filled circle) and 1 µm (empty square) particles in mucus of wild-type (WT, blue) and transgenic (Tg, purple) mice scaled by their radius R. (d,g) Diffusion coefficients lower than the uncertainty were excluded from the analysis.