FM 1-43 penetrates rapidly into IHCs.

<p>(<b>A</b>) Scheme of the organ of Corti, indicating the areas of the IHCs that were imaged. (<b>B</b>) FM 1-43 (10 µM) labeling of IHCs was imaged at room temperature at four cellular levels: stereocilia bundle, top, nuclear and basal. FM 1-43 reaches the cytoplasm, diffusing in an apex-to-base fashion (right panel). This observation is typical for 16 independent experiments.</p

3D reconstructions confirm the observations derived from single IHC sections.

<p>3D reconstructions of resting (<b>A</b>), stimulated (<b>B</b>) and recovered IHCs (<b>C</b> and <b>D</b>). Endocytotic organelles are shown in purple. Note the presence of tubular organelles both before and after stimulation. Most organelles, including the tubular ones, are replaced by small vesicles during the recovery periods. Insets show magnified regions from the four different cell region (cuticular plate, top, nuclear and basal regions). Note the increased number of endosome-like organelles at the base of the cell after stimulation and during recovery.</p

The vesicles forming during recovery in the top region of the IHCs are significantly larger than synaptic vesicles.

<p>(A) Analysis of the organelle numbers from the 3D reconstructions presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088353#pone-0088353-g005" target="_blank">Fig. 5</a>. Organelles containing the photo-oxidation product were classified in three categories: tubules (large elongated or flattened organelles), cisterns (round or elliptic organelles) and small vesicles. Tubules dominate at rest. Cisterns are induced to form during stimulation. Almost all organelles were processed to small vesicles during recovery. Images show representative organelles for each of the categories. Scale bar, 200 nm. (B) Quantification of the size of small vesicles (<120 nm) at the top and the base of the cell, after a 5-minute recovery. The latter are significantly smaller, and very similar to <i>bona fide</i> synaptic vesicles from the efferent boutons. The bars show means ± SEM, from 260, 690 and 219 vesicles (top, base and efferent, respectively). A one-way ANOVA test indicated that the vesicle populations were significantly different. A <i>post hoc</i> Bonferroni test revealed that the vesicles at the top of the IHCs were significantly larger than those in the basal region, and than those from the efferent boutons (<i>P</i><0.0001). The latter were not significantly different from each other (<i>P</i>>0.05). The analysis included all small vesicles found in the relevant IHC regions in the 3D reconstructions presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088353#pone-0088353-g005" target="_blank">Fig. 5</a>. The data points were sufficiently numerous to allow us to verify that the distributions were bell-shaped (using histograms of vesicle size). The variance was similar between the data groups: approximately 72, 190 and 143 units for top, base and efferent, respectively. The graphs show the entire data set we obtained in the 3D reconstructions; each 3D reconstruction is derived from one representative experiment.</p

FM 1-43 photo-oxidation suggests that synaptic vesicles recycle at the base of IHCs.

<p>(<b>A</b>)–(<b>D</b>) The electron micrographs show organelles containing the photo-oxidation product (DAB precipitate) at rest (<b>A</b>), during stimulation (<b>B</b>), or after a 5-minute (<b>C</b>) or 30-minute (<b>D</b>) recovery period. Some typical organelles are indicated by red arrowheads. Large tubular organelles are indicated by the dashed red traces. Mitochondria are indicated by white asterisks. Note that the basal region contains few labeled organelles at rest; their number increases after stimulation. Note also that the tubular organelles from the top and nuclear regions, found both at rest and during stimulation, disappear during the recovery periods. Scale bars, 200 nm. (<b>E</b>–<b>H</b>) Analysis of organelle labeling under the different experimental conditions. The percentage of the cell volume occupied by labeled organelles was measured in four different cellular regions: cuticular plate (<b>E</b>), top (<b>F</b>), nuclear (<b>G</b>) and basal (<b>H</b>). The graphs show averages performed for electron micrographs from 14 (resting), 12 (stimulation), 4 (5 minute recovery) and 4 (30 minute recovery) independent experiments. We used one-way ANOVA tests to check whether stimulation produced any changes in the different cellular regions. No significant differences were found for the cuticular plate region (<b>E</b>), for the top region (<b>F</b>), and for the nuclear region (<b>G</b>); all <i>P</i> values were higher than 0.05. In contrast, the ANOVA test revealed significant differences for the basal region (<b>H</b>); <i>P</i><0.05. A <i>post hoc</i> Bonferroni test indicated that stimulation increases the amount of label significantly in this region, compared to the resting control (<i>P</i><0.001).</p

Most commercial fluorescent markers fail in reporting endocytosis in IHCs.

<p>(<b>A</b>) FM 1-43 labels IHCs strongly in conditions where endocytosis is strongly reduced by low temperature (on ice, middle panel) or abolished by fixation (right panel). The labeling pattern is identical to that obtained at room temperature (RT, left panel). (<b>B</b>) Styryl dyes from the FM family (FM 1-84, FM 4-64, FM 4-64FX, AM 1-43), ranging in sizes between 450 and 470 Da, permeated IHCs in a similar fashion to FM 1-43 at room temperature. In contrast, the larger FM 3-25 (843 Da) did not penetrate into the tissue, producing a very faint labeling. The membrane-binding dye 5-dodecanoylaminofluorescein (DCF, 530 Da) had a similar effect. (<b>C</b>) An additional membrane dye, Di-2-ANEPEQ (549 Da), labeled IHCs in the same fashion as FM 1-43. Soluble compounds, such as calcein (623 Da) and 3000 Da Dextran coupled to fluorescein, labeled the intercellular spaces throughout the organ of Corti, but were not taken up efficiently by the cells. All dyes were used at a concentration of 10 µM, with the exception of Di-2-ANEPEQ, which was used at 100 µM, and DCF, used at 180 µM. Scale bars 10 µm. (<b>D</b>) Analysis of labeling intensity for all dyes that appeared to penetrate into the IHCs. The fluorescence intensity of the cells (excluding the nucleus) was normalized to the background intensity, and is expressed as fold over background. The black bars show results from cells incubated with the different dyes at room temperature. The gray bars show cells incubated on ice, at 2–4°C. The following numbers of IHCs were analyzed. FM 1-43: 26 at room temperature, 19 on ice; FM 1-84: 7, 27; FM 4-64: 13, 23; FM 4-64FX: 8, 20; AM 1-43: 20, 16; DCF: 8, 15; Di-2-ANEPEQ: 30, 35.</p

FM 1-43 can be used as an endocytosis tracer using photo-oxidation electron microscopy.

<p>(<b>A</b>) Experimental outline: IHCs incubated with FM 1-43 take up the dye (red symbols) in endocytotic organelles, but also in the cytosol, as a result of dye penetration through mechanotransducer channels. The dye in the cytosol attaches itself to the external (cytosolic) leaflets of the organelle membranes. After fixation and incubation with DAB (indicated as the blue background), the cells are illuminated with blue light; the FM molecules bleach (gray symbols) and emit reactive oxygen species that cause DAB precipitation (represented as black symbols). The DAB precipitate diffuses in the cytosol, unless it is trapped within the organelles. Therefore, endocytotic organelles are seen as black objects, while the FM dye in the cytosol only causes a general darkening of the cell volume. (<b>B</b>) In presence of the dye numerous dark organelles appear (some examples are indicated by red arrowheads). (<b>C</b>) In the absence of the dye only mitochondria are visibly labeled. Mitochondria are labeled by photo-oxidation by mechanisms independent of the addition of fluorescent markers, and are indicated by white asterisks. (<b>D</b>) Apart from mitochondria, no labeled organelles were found in cells incubated with FM 1-43 at low temperature (2–4°C), confirming that dye photo-oxidation selectively reports endocytotic events – despite the fact that FM 1-43 penetrates into the IHC cytosol at low temperature. The image is over-exposed, to increase the contrast as much as possible, which should allow the detection of any labeled organelles. Nevertheless, none are present, other than mitochondria. Scale bars, 500 nm.</p