Figure 1.
Fluo-4 imaging of cochlear hair cells.
(a) Fluo-4 AM dye preparation and loading protocol. Between each of the steps highlighted with pink stars, the dye solution was rigorously vortexed for 1 minute, bath sonicated for 1 minute, and centrifuged. (b–d) Confocal imaging of cochlear hair cells loaded with Fluo-4 AM Ca2+ indicator. (b) Low-magnification view of the cochlear epithelium shows that hair cells selectively load with Fluo-4 AM dye. The red box indicates the mid-apical region of the epithelium, which was used for all experiments. (c–d) Side view of a single hair bundle loaded with Fluo-4 (c), with corresponding DIC image (d). Arrow in (c) indicates a tip blush of Ca2+ entering through mechanotransduction channels. Scale bar in b is 100 µm and scale bar in c is 5 µm.
Figure 2.
Live-cell Ca2+ imaging correlates with SEM ultrastructure.
Organs imaged with Fluo-4 (a,d,g) were fixed and processed for SEM (b,c,e,f,h,i). (a, b, c) Cells with very bright bundle Fluo-4 represent dead or dying cells; in this example, a very bright cell became completely extruded from the ephithelium in the time between live cell imaging and fixation. (d) Hair bundles with average Fluo-4 signal have ordered stereocilia and tip links (e, f), while bundles with very low Fluo-4 signal (g) have damaged bundles (h, i). For all panels, colored boxes highlight corresponding cells in live-cell and SEM images. Orange arrowheads indicate tip links in (f). Scale bars in a, b, d, e, g and h are 5 µm, c, f, and i are 1 µm.
Figure 3.
High-resolution SEM analysis of tip links.
(a, b) Control hair bundles had moderate levels of bundle Fluo-4 fluorescence and many tip links at their stereocilia tips (c, d; orange arrowheads point to tip links). (e, f) Bundles treated with 5 mM EGTA for 5 min showed a marked decrease in bundle Fluo-4 fluorescence and a significant reduction in the number of tip links (g, h). Red stars in e and f highlight cell body Fluo-4 fluorescence. (i) Tip links were quantified by dividing the number of tip links in a bundle by the number of observable tip link positions (**** p<0.0001). Scale bars in a and e are 10 µm, c and g are 1 µm, and d and h are 0.5 µm.
Figure 4.
Mechanical stimulation of the hair bundle increases Fluo-4 Ca2+ signal.
(a) A stimulating pipette was positioned parallel to the epithelium, and oriented to maximally stimulate bundles in the mid-apical region. (b) CellTracker Red (CT-Red) was used as a cell-fill to normalize the Fluo-4 bundle fluorescence (c) A single bundle region of interest (ROI; yellow box) was continually imaged. (d) Fluid jet deflection of a bundle with a 0.2 psi pulse for 10 seconds caused an increase in the bundle Fluo-4 Ca2+ signal, which was timed with the stimulus (black bars). Normalized ΔG/R was calculated by dividing Fluo-4 by CT-Red fluorescence, then normalizing to the average response from the first 40 seconds of recording. Scale bars in a, b, and c are 5 µm.
Figure 5.
Blocking the transduction channel decreases hair-bundle and cell body Ca2+.
(a–d) Control hair cells have Fluo-4 signal in the bundle (b) and cell body (d). (e–h) Hair cells incubated with 100 µM tubocurarine to block the transduction channel have decreased Fluo-4 signal in the bundle (f) and cell body (h). (i–j) To quantify Fluo-4 fluorescence, individual bundle and cell body ROIs were selected and integrated density of Fluo-4 channel was calculated (see Figure 6), then normalized to the control mean for each group. (a, c, e, g) Corresponding DIC images. (au = arbitrary units; control bundles n = 63, tubocurarine bundles n = 73, control cell bodies n = 72, tubocurarine cell bodies n = 77; **** p<0.0001).
Figure 6.
Breaking the tip links decreases hair-bundle and cell body Ca2+.
(a–d) Control hair cells have Fluo-4 signal in the bundle (b) and cell body (d). (e–h) Hair cells pre-treated with 5 mM EGTA to break tip links have decreased Fluo-4 signal in the bundle (f) and cell body (h). (i, j) Fluo-4 fluorescence was quantified as in Figures 5 and 2. (a, c, e, g) Corresponding bundle and cell body DIC images. (AU = arbitrary units; control bundles n = 72, EGTA bundles n = 65, control cell bodies n = 79, EGTA cell bodies n = 76; **** p<0.0001). YZ-reslice images of control (k) and EGTA-treated (l) hair cells, with examples of bundle (orange square) and cell body (yellow rectangle) ROIs used for quantifying fluorescence. (m) Representative line scan profiles for the ROIs indicated by white rectangles in (k) and (l). Control hair cell (blue trace) has fluorescence in the bundle, top half of the cell body (CB top) and bottom half of the cell body (CB bottom). EGTA-treated hair cell (red trace) has fluorescence concentrated in the top half of the cell, where the dye is trapped in intracellular compartments, but lacks hair bundle and cytoplasmic CB bottom Fluo-4 signal.
Figure 7.
Expected physiological changes in the hair cell when tip links are broken.
In control conditions, Ca2+ enters the hair bundle through mechanoelectrical transduction (MET) channels that are open at rest. Inward transduction current depolarizes the cell to open voltage gated Ca2+ channels (VGCC), which allow Ca2+ influx into the cell body. After breaking tip links with EGTA, the transduction channel closes, which leads to hyperpolarization of the cell and closure of voltage-gated Ca2+ channels, resuting in a decrease in both bundle and cell body Ca2+.