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. 2015 Apr 21:6:6873.
doi: 10.1038/ncomms7873.

Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear

Meghan C Drummond  1 , Melanie Barzik  1 , Jonathan E Bird  1 , Duan-Sun Zhang  2 , Claude P Lechene  3 , David P Corey  2 , Lisa L Cunningham  4 , Thomas B Friedman  1
Affiliations

Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear

Meghan C Drummond et al. Nat Commun. .

Abstract

The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24-48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of (15)N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips.

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Figures

Figure 1
Figure 1. Live-cell imaging reveals different classes of EGFP-β-actin dynamics in hair cell stereocilia.
(a) Still frames and (b) schematic representations of stereocilia bundles demonstrating steady-state tip localization of EGFP-β-actin (green) and asynchronous elongation (red arrow). The majority of stereocilia in the bundle retained stable tip localization of EGFP-β-actin throughout the live-imaging experiment. The lengths of stereocilia with asynchronous elongation (a) (yellow dashed line) were measured and are shown in yellow at each time point. (c) Stereociliogenesis of nascent stereocilia bundles on developing hair cells is shown in still frames and (d) illustrated in a schematic. Over 72 h of live imaging, stereocilia lengthen (yellow dashed lines). (e) Schematic representing the stereociliar F-actin treadmill hypothesis. All length changes were measured in 3D using Volocity. Kinocilia are illustrated in purple. All 112 movies are available in Supplementary Movies 1, 2, 3, 4, 5, 6. Scale bar, 5 μm.
Figure 2
Figure 2. Quantification of intensity profiles along the lengths of stereocilia.
(a) Still frames from a live-imaged hair cell with stable localization of EGFP-β-actin in the distal tip compartment. Dashed lines (orange) indicate stereocilia that were line-traced every 8 h beginning 24 h post transfection with pEGFP-β-actin. Images without line traces are available in Supplementary Fig. 1. (b) Line traces from stereocilia 1 and 2 demonstrate increasing levels of EGFP-β-actin in the cuticular plate and distal tip compartment, but not along the stereocilia shaft and confirm steady-state tip localization. Over the course of the experiment, additional stereocilia move in close proximity of stereocilium 2 (arrows) (a), resulting in extra peaks in the line traces that correspond to the distal tip of the second stereocilium. Scale bar, 5 μm.
Figure 3
Figure 3. Biolistic gene gun transfection may result in damage to hair cells.
(a) Penetration of the 1 μm gold DNA-coated particles through the cell membrane and cuticular plate of hair cells results in successful transfection of plasmid DNA; however, bullets can strike and damage stereocilia bundles in the process. (b–d) Representative images of stereocilia bundles classified as damaged and excluded from further analyses. Cells were biolistically transfected with EGFP-β-actin (green), fixed and counterstained with rhodamine phalloidin (red) to visualize all F-actin. Phase contrast shows positions of gold bullets. Dotted lines indicate gold particles on the surface of a hair cell. Scale bar, 5 μm.
Figure 4
Figure 4. Localization of EGFP-β-actin at stereocilia tips is polymerization dependent.
Localization of wild-type EGFP-β-actin, EGFP alone (negative control), and mutant EGFP-β-actinG63D or mutant EGFP-β-actinG13R (green) at 4 and 24 h post transfection. (a) Wild-type EGFP-β-actin was enriched at stereocilia tips with diffuse labelling along the shafts at 4 and 24 h post transfection. Consistent with our live-cell imaging observations, two stereocilia appear to have elongated from the distal end (arrowheads). In contrast, (b–d) EGFP-β-actinG63D, EGFP-β-actinG13R and the EFGP control were present diffusely throughout the hair cell body, stereocilia bundle and occasionally the kinocilium (arrows) at 4 and 24 h. F-actin is labelled with rhodamine phalloidin (red). Scale bar, 5 μm.
Figure 5
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.
Figure 6
Figure 6. Postnatal stereociliogenesis is detected with MIMS.
Representative images of mouse utricles at postnatal day 4 (P4) and P15. The 15N/14N ratio is illustrated in pseudocolour, with blue indicating low and red indicating high incorporation of 15N into newly synthesized proteins. (a) At P4, three of the six stereocilia bundles in the field are small and show high levels of protein incorporation (asterisks); they are consistent with being immature bundles that developed after 15N feeding began at P0. (b) At P15, bundles are approaching their mature sizes, but one has little incorporation of new protein (centre), whereas two show high incorporation of new protein (asterisks) consistent with development after P0. (c,d) Images of the same fields, indicating total protein. Scale bar, 5 μm.
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