Figure 5. Photoconversion of dendra2-β-actin reveals stable actin in stereocilia cores.
(a) Schematic of the experimental design, possible outcomes of the experiment and theoretical data of each predicted model. Cultured utricles biolistically transfected with pDendra2-β-actin were mounted onto a spinning-disk microscope for live imaging. The distal tip compartments of stereocilia were targeted with a 405 nm laser to photoconvert dendra2 from green to red. Dashed lines indicate the tip of the stereocilium and the lower boundary of the photoconverted region. (b) Time-lapse images from a representative cell before photoconversion, immediately after photoconversion and at 10 and 20 h post-photoconversion. Photoconverted regions of interest are denoted by a yellow dashed circles. A 488 nm laser and 561 nm laser were used to excite non-photoconverted dendra2-β-actin (green) and photoconverted dendra2-β-actin (red), respectively. During photoconversion, dendra2-β-actin in the cuticular plate was also converted and can be seen moving into the shafts of stereocilia (Supplementary Movie 7). (c) The distance from the boundary of the photoconverted dendra2-β-actin (red) and the distance from the distal tip of the stereocilium (green) were measured every 8 h and plotted for stereocilia tips 1 and 2 indicated by the yellow arrow and arrowheads, respectively, in panel b. Measurements were made with both the green and red channels displayed to determine the insertion point of the photoconverted stereocilium into the cuticular plate. The distance from the lower boundary of the photoconverted dendra2-β-actin to point of insertion of the stereocilium into the apical surface of the hair cell does not change, consistent with steady-state tip localization of photoconverted dendra2-β-actin. In contrast, the distance from the distal tip to the apical surface increases over time, consistent with stereocilia elongation. (d) Automated measurements were made using Volocity software to plot the distance from the centroid of the photoconverted dendra2-β-actin to the edge of the non-photoconverted cuticular plate (green). Scale bar, 5 μm.