Figure 1.
Uptake of styryl dye by vestibular hair cells.
(A,B,D,E) Styryl dyes AM1-43 and FM1-43FX distinctively label sensory hair cells of utricles and saccules (images for FX1-43FX are shown). (B,E) After incubation in thermolysin, stained sensory epithelia of utricles and saccules were peeled off the underlying stromal tissue. Shown are transverse projections of utricle whole mounts and peeled sensory epithelia of utricles and saccules. To better visualize styryl dye labeled tissue, we combined light microscopic images and fluorescence FM1-43FX visualization. (C,F) Shown are a cross sections of E18 chicken utricle and saccule with FM1-43FX labeled hair cells (green), co-labeled with antibodies to the hair cell marker myosin VIIa (red) and Sox2 (blue), which is detectable in hair cells and supporting cells.
Figure 2.
Dissociation of vestibular sensory epithelia into single hair cells and non-sensory cells.
AM1-43-stained sensory epithelia underwent different enzymatic and non-enzymatic treatments to test for optimal conditions to separate hair cells and non-sensory cells and to preserve hair cell morphology. Conditions were: 0.25% trypsin (A), 0.05% trypsin (B), accutase (C), accumax (D), enzyme-free (E) and 50% accumax + 0.025% trypsin (F). Shown are representative images of cells after mild trituration following 7 min incubations at 37°C.
Figure 3.
Flow cytometric separation of AM1-43 labeled and unlabeled cells.
Single cell suspensions generated from AM1-43-exposed vestibular sensory epithelia were subjected to one-color fluorescence-activated cell sorting. (A) to (C) depicts the gating strategy: (A) cell debris was excluded on based low side (SSC) and forward scatter (FSC) parameters. (B) From the debris-negative population, doublets were removed based on their divergence from a linear FSC-height and FSC-area gate. (C) From the debris-free and doublet-negative population, AM1-43-high cells (HC, presumptive hair cells) and AM1-43-low cells (NSC, non-sensory cells) were gated for collection. (D) Analysis of unstained cells revealed a single population and no AM1-43 fluorescence background. (E,F) Re-sort analyses of the two populations shown in (C), demonstrated >90% purity.
Figure 4.
Shotgun proteomic analysis of isolated hair cells and non-sensory cells.
(A) False discovery rates (FDR) at both protein and peptide levels were applied to filter the data to a representative number of PSMs, which result in the number of proteins identified. The majority of proteins were represented by more than one PSM. (B) Demonstration of a MSMS peptide spectral match (PSM) of the peptide ETLYGQEIDQASFLTILK from the protein otolin-1 (OTOL1). The peptide was identified with better than 2 parts-per-million (ppm) mass accuracy, where the experimental mass/charge (mz) was 1035.0452 Da and the theoretical 1035.0437 Da. The annotated b and y ions are indicative of peptide backbone bond cleavage between carbonyl carbon and nitrogen, whereas in the case of the amino-terminus, b-ions result, and for the carboxyl-terminus, y-ions. OTOL1 was only identified in the AM1-43-low cell fraction (see Fig. 3C), and suggests that this protein is present in non-sensory cells, presumably supporting cells, and not in hair cells. (C) Venn diagram displaying the number of proteins unique to hair cells (64), non-sensory cells (104) and the number of proteins shared between the two cell types (467) at the stringent filter setting of requiring at least 2 different peptides per protein. This corresponds to the FDR settings shown in the third line of the table shown in (A).
Table 1.
Proteins exclusively identified in hair cells.
Table 2.
Proteins exclusively identified in non-sensory cells.
Table 3.
Highly enriched hair cell proteins.
Table 4.
Highly enriched non-sensory cell proteins.
Figure 5.
Categorization of subcellular localization and predicted cellular function of identified hair cell and non-sensory cell proteins.
(A) All 634 identified hair cell and non-sensory cell proteins were classified according to gene ontology annotations and classified according to their subcellular localizations (upper bar graphs, before quantification). Taking into account the spectral counts of each identified protein resulted in slight changes of the distribution (lower bar graphs, after quantification). (B) Display of the gene ontology classifications of predicted cellular functions (upper bar graphs, before quantification) and after taking into account the spectral counts of each identified protein (lower bar graphs, after quantification).
Figure 6.
Categorization of subcellular localization and predicted cellular function for proteins unique to or highly enriched in hair cells and non-sensory cells.
(A) Shown is the subcellular distribution of 116 proteins specific to or enriched in hair cells (Table 1) compared to 170 proteins exclusively identified or enriched in non-sensory cells (Table 2) (upper bar graphs, before quantification). Taking into account the spectral counts of each identified protein resulted in changes of the distribution (lower bar graphs, after quantification). (B) Display of the gene ontology classifications of predicted cellular functions of the proteins unique to hair cells and supporting cells (upper bar graphs, before quantification) and after taking into account the spectral counts of each identified protein (lower bar graphs, after quantification).
Figure 7.
Qualitative analysis of identified hair cell markers by immunocytochemistry.
Shown are cross sections of E18 chicken utricles (A and C) and transverse projections of utricle whole mounts (B and D). (A, B) Co-immunolabeling of the identified hair cell markers otoferlin (green), myosin VIIA (red), and Sox2 (blue), which is detectable in hair cells and supporting cells. (C, D) Co-immunolabeling with antibodies to otoferlin (green), the identified hair cell marker AIFM1 (red), and Sox2 (blue).
Figure 8.
Qualitative analysis of identified non-sensory cell markers by immunocytochemistry.
Shown are cross sections of E18 chicken utricles. (A) Co-immunolabeling with antibodies to collagen XVII (green), the hair cell marker myosin VIIA (red), and Sox2 (blue). Collagen XVII immunoreactivity was detected in the basal lamina and below the basal lamina in the mesenchymal stromal cell layer. (B) Antibodies to talin detected the protein in supporting cells, which are Sox2 (blue) immunopositive and myosin VIIA (green)-negative. Talin was also detectable in mesenchymal stromal cells. (C) To better visualize the location of talin immunoreactivity in supporting cells, we used light microscopic images and the Sox2 immunostaining to outline the supporting cells’ nuclei (labeled 1–4), and found association of talin immunoreactivity (green) perinuclear and toward the basal end of supporting cells (arrows).