Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin

Abstract

Stereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips. The onset of mechanotransduction during mouse postnatal development refines stereocilia length and width. However, it is unclear how actin is differentially regulated between stereocilia in the tallest row of the bundle and the shorter, mechanotransducing rows. Here, we show actin turnover is increased at the tips of mechanotransducing stereocilia during bundle maturation. Correspondingly, from birth to postnatal day 6, these stereocilia had increasing amounts of available actin barbed ends, where monomers can be added or lost readily, as compared with the non-mechanotransducing stereocilia in the tallest row. The increase in available barbed ends depended on both mechanotransduction and MYO15 or EPS8, which are required for the normal specification and elongation of the tallest row of stereocilia. We also found that loss of the F-actin-severing proteins ADF and cofilin-1 decreased barbed end availability at stereocilia tips. These proteins enriched at mechanotransducing stereocilia tips, and their localization was perturbed by the loss of mechanotransduction, MYO15, or EPS8. Finally, stereocilia lengths and widths were dysregulated in Adf and Cfl1 mutants. Together, these data show that actin is remodeled, likely by a severing mechanism, in response to mechanotransduction.

Keywords: ADF; actin; cofilin; development; mechanotransduction; morphogenesis; morphology; protrusion; stereocilia.

Figure 1.. Postnatal actin incorporation patterns.

(A) Actin-EGFP expression was induced at P6 and the organ of Corti was harvested at P28. Actin-EGFP in inner hair cells (IHCs) is shown in grey or green and phalloidin in red. Arrows indicate row 1 stereocilia and arrowheads row 2 stereocilia. Scale bar is 5 μm. (B) Fluorescence recovery after photobleaching of EGFP-actin in P3-P4 IHC stereocilia. Images on the left are maximum intensity projections of an example cell at various timepoints. Grey and orange arrowheads track row 1 and row 2 tips, respectively, through the time series. Scale bar is 1 μm. (C) The fraction of normalized fluorescence recovery relative to row 2 is plotted (19 cells from 3 mice, mean ± SD). Cells are from the middle turn of the organ of Corti. **** denotes significance (p < 0.0001) and n.s. denotes no significance (p > 0.05).

Figure 2.. Rhodamine-labeled actin incorporation during postnatal development of auditory hair cells.

(A) A diagram outlining the assay where hair cells are permeabilized to allow purified proteins to access and bind to the underlying actin cytoskeleton. F-actin is stained with phalloidin. (B) Exogenous, purified Flag-FSCN1 was incubated with intact or permeabilized tissue at P5 and detected by an anti-Flag antibody (green). Endogenous F-actin was stained with phalloidin (red) to show inner hair cell (IHC) stereocilia. (C-D) Rhodamine labeled actin (red, grey) incorporation in permeabilized IHCs (C) at P0, P1, P2, and P6 or (D) P6 outer hair cells (OHCs). Endogenous F-actin was labeled with phalloidin (green). Regions marked by boxes are magnified to the right, where the approximate onset of mechanotransduction (MET) is indicated. (E) Permeabilized P6 IHCs were labeled with exogenous, purified His-CAPZα1/β and detected by anti-His immunostaining (green, grey). Endogenous F-actin was stained with phalloidin (red). Scale bars in large rectangular images equal 5 μm and in small square images 1 μm. (F-G) Quantification of relative rhodamine-actin incorporation at the tips of rows 1, 2, and 3 (n = 35 to 42 cells from control mice). (F) Rhodamine-actin intensity at stereocilia tips normalized to the average intensity at row 2 tips. (G) The values of the graph in (F), normalized to the ratio of cross-sectional area between rows (0.85:1.00:0.43 for IHCs and 0.73:1.00:0.57 for OHCs; measured from scanning electron micrographs). Black bars in all graphs indicate mean ± SD. **** denotes significance (p < 0.0001) and n.s. denotes no significance (p > 0.05).

Figure 3.. Rhodamine-labeled actin incorporation when mechantransduction is disrupted.

(A-B) Rhodamine-actin (red, grey) incorporation in permeabilized inner and outer hair cells of P6 Tmie^+/− control and Tmie^−/− mutant mice. Phalloidin stained F-actin is green. Boxed regions are magnified in (B). (C) The graph shows the normalized fluorescence intensity of rhodamine-actin at row 1 or row 2 stereocilia tips in Tmie^+/− controls (n = 61 cells from 3 mice) and Tmie^−/− mutants (n = 58 cells from 3 mice). Each data point represents the average tip intensity for each row of a cell normalized to the average intensity at cell borders. (D-G) P4 explants were incubated without (control) or with 100 μM tubocurarine (Tubo) for 18 hours. (D) Rhodamine-actin incorporation (red, grey) and phalloidin stained F-actin (green). (E) EPS8 immunostaining (red, grey) and phalloidin stained F-actin (green). (F) Ratio of rhodamine-actin fluorescence intensities of row 2 to row 1 stereocilia tips from control (n = 25 cells from 3 mice) and Tubo treated explants (n = 22 cells from 3 mice). (G) Ratio of EPS8 fluorescence intensities of row 1 to row 2 stereocilia tips from control (n = 25 cells from 3 mice) and Tubo treated explants (n = 30 cells from 3 mice). Black bars in all graphs indicate mean ± SD. Each data point represents a cell. **** denotes significance (p < 0.0001); *** denotes significance (p < 0.001); n.s. denotes no significance (p > 0.05). Scale bars equal 5 μm in large squares and 1 μm in insets. All images are of hair cells from the middle turn. See also Figure S1.

Figure 4.. ADF/CFL1 localization in auditory hair cells.

(A-D) Immunolocalization of ADF/CFL1 (red or grey) and β-actin (green) during postnatal development. (A) Top panels, IHCs at P0, P1, P2, and P6. Bottom panels show ADF/CFL1 immunolocalization in the organ of Corti including both IHCs and OHCs. (B) P6 IHC stereocilia imaged by Airyscan microscopy. (C) P2 OHC stereocilia. (D) P4 Tmie^+/− control and Tmie^−/− mutant IHCs. (E) Immunolocalization of ADF/CFL1 (red or grey) and β-actin (green) in adult (P26) organ of Corti with a selected cell magnified below to better show stereocilia labeling. Scale bars equal 5μm for the panels showing the sensory epithelium in (A) and (D), and 1μm for all other images.

Figure 5.. ADF/CFL1 localization and rhodamine-actin incorporation in Myo15^sh2/sh2 and Eps8^−/− mutants.

(A) ADF/CFL1 immunolocalization (grey, red) in inner hair cells (IHCs) from P6 Myo15^+/sh2 controls and Myo15^sh2/sh2 mutants imaged by Airyscan or (B) P6 Eps8^+/− control or Eps8^−/− mutant IHCs. In merged images β-actin is green. (C) Rhodamine-actin (red or grey) incorporation in permeabilized P6 Eps8^+/− control and Eps8^−/− mutant IHCs. In the merged image, phalloidin stained F-actin is green. (D) Quantification of rhodamine-actin intensity at stereocilia tips normalized to border values for Eps8^+/− controls (n = 42 cells from 3 mice) and Eps8^−/− mutants (n = 44 cells from 3 mice). Black bars on the graph indicate mean ± SD. **** denotes significance (p < 0.0001) and * denotes significance (p < 0.05). All scale bars equal 1 μm. See also Figure S2 and STAR Methods.

Figure 6.. Rhodamine-actin incorporation in Adf/Cfl1 mutants.

(A-C) Rhodamine-actin (red or grey) incorporation in permeabilized inner hair cells (IHCs) and outer hair cells (OHCs) of (A) P6 Adf^+/− CFL1^fl/+ control or (B) Adf^−/− CFL1^fl/+ Atoh1-cre mutant mice, which retain one intact allele of the Cfl1 gene. Scale bars equal 5 μm and 1 μm for IHC and OHC panels, respectively. (C) Magnified insets of IHCs. (D) On the left, rhodamine-actin fluorescence intensity at row 1 and row 2 IHC stereocilia tips, normalized to the cell border. Each data point represents a cell and the black bars indicate mean ± SD. On the right, the mean ± SD of the ratio of rhodamine-actin fluorescence intensities at row 2 stereocilia tips relative to row 1 from control and mutant hair cells. Control and mutant data points are colored grey and orange, respectively. Data represents 57 and 53 cells from 3 control and 4 mutant mice, respectively. **** denotes significance (p < 0.0001) and *** denotes significance (p < 0.001). See also Figure S3.

Figure 7.. Stereocilia morphology in Adf/Cfl1 induced double knockouts.

Adf^+/− Cfl1^fl/+ mice (control) and Adf^−/− Cfl1^fl/fl Cagg-creER mice (induced double knockout, iDKO) were used at P5, three days after tamoxifen injection. (A) SEM micrographs of inner hair cell (IHC) stereocilia, note the tapered tip morphology in row 3 stereocilia of iDKO cells. Boxed regions are magnified to the right and row 3 stereocilia tips are highlighted by yellow pseudocolor. (B) SEM of outer hair cell (OHC) stereocilia from control and iDKO mice, boxed regions magnified to the right. (C) Stereocilia lengths for control and iDKO IHCs were measured from digitally resliced image stacks to show the z dimension. On the left, representative resliced images from controls (top) and iDKO mutants (bottom). The graph shows length measurements for each condition with each data point representing a cell. For each condition, 35 to 40 cells were measured from four mice. (D) EPS8 immunostaining and quantification. Data points represent stereocilia and have been normalized to the average row 1 intensity. For each condition, 361 to 503 stereocilia per row were measured from an average of 7.75 cells from each of four mice. (E) Widths of IHC (left) and OHC (right) stereocilia were quantified from the widest, visible portion of each stereocilium from scanning electron micrographs. For IHCs, 15–21 cells from each condition were measured. For OHCs, 16–27 cells were measured. Two mice were used for each condition. Black bars in all graphs indicate mean ± SD. All scale bars equal 1 μm. See also Figures S4 and S5.