Fig. 3: Bap1 possesses a unique 57-amino acid loop with broad, nonspecific adhesion.
a Biofilm adhesion assay for different Bap1 mutants (structure shown schematically) in a ΔrbmC strain background. BSA was used during biofilm growth at increasing concentrations as a non-specific competitor for glass surface adhesion. ΔBC denotes the Δbap1ΔrbmC double mutant. All data are depicted as the mean ± SD (n = 3 biologically independent samples). b, c In situ staining and associated quantification of biofilms formed by different Bap1 mutants, tagged by 3×FLAG at the C-terminus and labeled using anti-FLAG-Cy3. Note a functional copy of RbmC is present in this assay to anchor the biofilms to the substrate regardless of whether the mutant Bap1 is functional. b Representative cross-sectional images of the bottom layer and side views of biofilms formed by cells expressing WT Bap1 (Left), Bap1Δ57aa (Middle), and Bap1Δβ-prismB+57aa (Right). c Quantification of Bap1 localization within the biofilm for different mutants (structure shown schematically). Shown are the ratios between the immunosignals of 3×FLAG-tagged Bap1 at the biofilm-glass interface and the total signal integrated over the entire biofilm cluster, for each indicated strain. Different colors and symbols correspond to different biological replicates (n = 4). Statistical analysis was performed using unpaired, two-tailed t-test with Welch’s correction. ns stands for not significant; ***p < 0.001. Exact p values from left to right: 0.0007, 0.7163, 0.0003, 0.0665. d Peptide sequence of the 57aa loop. Blue dash = aromatic residues, red dot = positively charged residues. e Microbead adsorption assay for quantifying adhesive properties of the 57aa peptide. Top: a representative image of 5 μm silica bead (Left, bright field) and FITC-labeled 57aa peptide adsorbed on the bead (Right). Bottom: a representative image of 5 μm silica beads coated with lipids, labeled with RhPE (Left) for lipids and the FITC-labeled 57aa peptide adsorbed on lipid layer (Right). f Excess fluorescence signal on the surface of the beads compared to the solution signal, on silica surface (Top) and supported lipid layer (Bottom), respectively. FITC was used at the same molecular concentration (1.5 µM) as a control. g Confocal images of human jejunum tissue slices stained with 1 μM FITC-labeled 57aa peptide and 300 nm DAPI.
