Limits...
A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

Herget M, Scheibinger M, Guo Z, Jan TA, Adams CM, Cheng AG, Heller S - PLoS ONE (2013)

Bottom Line: Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level.Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells.Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology - HNS, Stanford University, Stanford, California, USA.

ABSTRACT
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

Show MeSH

Related in: MedlinePlus

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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3672136&req=5

pone-0066026-g006: 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).

Mentions: Based on these considerations, we hypothesized that potential differences between the two populations would be even more obvious if we focus our analysis on proteins that are either exclusively detectable in each group (Tables 1 and 2) and proteins that are highly enriched or specific for each group (Tables 3 and 4). Comparisons with respect to subcellular localization revealed that of the specific hair cell and non-sensory cell proteomes 40% to 50% of all unique proteins were of cytoplasmic origin (Fig. 6A, before quantification). A higher percentage of unique non-sensory cell proteins over unique hair cell proteins was assigned to the ER (12% compared to 3% for hair cell specific proteins), or were not annotated. In contrast, slightly higher percentages of unique or upregulated hair cell proteins were found to be of mitochondrial, vesicular, Golgi and lysosomal origin. After quantification, a main difference arose for the vesicle proteins where an increase of 26% points was revealed for unique or upregulated hair cell proteins compared to 1% of unique or upregulated non-sensory cell proteins (Fig. 6A, after quantification). This increase mainly arose from the high number of spectral counts of the two proteins otoferlin and clathrin, both shown to be involved in hair cell vesicle trafficking [26], [34]. Accordingly, for the cellular function, a notably strong upregulation was observed for hair cell specific proteins involved in trafficking to 50% of all hair cell specific/enriched proteins versus 4% of all non-sensory cell specific/enriched proteins (Fig. 6B, after quantification). These quantitative assessments demonstrate that in comparison to non-sensory cells, protein trafficking is strongly reflected in the hair cell proteome. As discussed earlier, this might reflect the high turnover of synaptic vesicles due to sustained exocytosis at the ribbon synapses, with otoferlin as a key player in vesicle recycling and replenishment, as well trafficking of proteins to the stereociliary hair bundle. Conversely, based on the quantification, the non-sensory cells’ proteome appears to be enriched for proteins involved in synthesis, degradation, folding and particularly cytoskeletal proteins, which could be an indication for a higher protein turnover and cytoskeletal specializations in these cells, despite the well-known cytoskeletal structures of hair cells.


A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

Herget M, Scheibinger M, Guo Z, Jan TA, Adams CM, Cheng AG, Heller S - PLoS ONE (2013)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672136&req=5

pone-0066026-g006: 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).
Mentions: Based on these considerations, we hypothesized that potential differences between the two populations would be even more obvious if we focus our analysis on proteins that are either exclusively detectable in each group (Tables 1 and 2) and proteins that are highly enriched or specific for each group (Tables 3 and 4). Comparisons with respect to subcellular localization revealed that of the specific hair cell and non-sensory cell proteomes 40% to 50% of all unique proteins were of cytoplasmic origin (Fig. 6A, before quantification). A higher percentage of unique non-sensory cell proteins over unique hair cell proteins was assigned to the ER (12% compared to 3% for hair cell specific proteins), or were not annotated. In contrast, slightly higher percentages of unique or upregulated hair cell proteins were found to be of mitochondrial, vesicular, Golgi and lysosomal origin. After quantification, a main difference arose for the vesicle proteins where an increase of 26% points was revealed for unique or upregulated hair cell proteins compared to 1% of unique or upregulated non-sensory cell proteins (Fig. 6A, after quantification). This increase mainly arose from the high number of spectral counts of the two proteins otoferlin and clathrin, both shown to be involved in hair cell vesicle trafficking [26], [34]. Accordingly, for the cellular function, a notably strong upregulation was observed for hair cell specific proteins involved in trafficking to 50% of all hair cell specific/enriched proteins versus 4% of all non-sensory cell specific/enriched proteins (Fig. 6B, after quantification). These quantitative assessments demonstrate that in comparison to non-sensory cells, protein trafficking is strongly reflected in the hair cell proteome. As discussed earlier, this might reflect the high turnover of synaptic vesicles due to sustained exocytosis at the ribbon synapses, with otoferlin as a key player in vesicle recycling and replenishment, as well trafficking of proteins to the stereociliary hair bundle. Conversely, based on the quantification, the non-sensory cells’ proteome appears to be enriched for proteins involved in synthesis, degradation, folding and particularly cytoskeletal proteins, which could be an indication for a higher protein turnover and cytoskeletal specializations in these cells, despite the well-known cytoskeletal structures of hair cells.

Bottom Line: Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level.Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells.Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology - HNS, Stanford University, Stanford, California, USA.

ABSTRACT
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

Show MeSH
Related in: MedlinePlus