Fig. 4: The jordan mutation does not disrupt barbed-end capping in stereocilia, but does alter MYO15A trafficking on cellular actin filaments.
A, B Actin barbed-end assay in detergent-permeabilized inner hair cells from mouse cochlear explants acutely isolated at P7. TMR-labelled G-actin (green) was added prior to fixation to identify uncapped barbed ends. Phalloidin labelling of F-actin (magenta) is overlaid. In both Myo15ajd/jd and littermate Myo15a+/jd controls, barbed-ends were detected at row 2 stereocilia tips, and at the tips of all stereocilia rows in Myo15ash2/sh2 hair cells. C Quantification of TMR-barbed end incorporation in row 1 and 2 stereocilia (from left to right columns, n = 75, 81, 98, 90, 102, 99, 119, 102 stereocilia respectively, 9 hair cells from 3 independent mice per genotype), ****, P < 0.0001, one-way ANOVA with Å Ãdák’s multiple comparisons test. D HeLa cells were transfected with EGFP-tagged Myo15a-2 expression constructs or EGFP alone (green) as indicated, fixed and probed with phalloidin (magenta) and Hoechst (blue). Wild-type protein trafficked to filopodia tips (red arrowheads), while jordan and shaker-2 mutants did not. Boxed regions are magnified (inverted grayscale). E LLC-PK1-CL4 cells were transfected with EGFP-tagged Myo15a-2 (green), fixed and labelled with phalloidin (magenta) and Hoechst (blue). The jordan mutant concentrated at microvillar tips, similar to wild-type, whereas the shaker-2 mutant did not. Orthogonal projections are shown (inverted grayscale). Images are representative from at least three independent experiments. Data are mean ± SD. Scale bars, 5 µm (A, B); 20 µm (D, E).
