Dataset Information

Mass spectrometry identification of proteins of isolated auditory and vestibular hair cells using data-independent and data-dependent acquisition

ABSTRACT: Hair cells undergo postnatal development that leads to formation of their sensory organelles, synaptic machinery, and in the case of cochlear outer hair cells, their electromotile mechanism. To examine the proteome changes over development, we isolated pools of 5000 Pou4f3-Gfp positive or negative cells from the cochlea or utricles; these cell pools were analyzed by data-dependent and data-independent acquisition (DDA and DIA) mass spectrometry. DDA data were used to generate spectral libraries, which enabled identification and accurate quantitation of specific proteins using the DIA datasets. We also isolated and pooled individual inner and outer hair cells from adult cochlea and compared their proteomes to those of developing hair cells. The DDA and DIA datasets will be valuable for accurately quantifying proteins in hair cells and non-hair cells over this developmental window.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Supporting Cell Of Cochlea, Supporting Cell Of Vestibular Epithelium, Ear Hair Cell, Cochlea, Auditory Hair Cell, Utricle, Inner Ear, Vestibular Hair Cell

SUBMITTER: Peter Barr-Gillespie

LAB HEAD: Peter Barr-Gillespie

PROVIDER: PXD006240 | Pride | 2017-09-18

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	0_DDA_KREY_FACs_14_G-P0_Coch_95min_DDA_20160817_QE.raw	Raw
	10_DDA_KREY_FACs_35_GP0_V_95min_DDA_20160817_QE.raw	Raw
	10_DIA_KREY_FACs_sample_10_G-P4_V_95min_20160819_QE.raw	Raw
	11_DDA_KREY_FACs_34_GP0_V_95min_DDA_20170213_QE.raw	Raw
	11_DIA_KREY_FACs_sample_11_G-P4_V_95min_20160817_QE.raw	Raw

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Publications

Heterodimeric capping protein is required for stereocilia length and width regulation.

Avenarius Matthew R MR Krey Jocelyn F JF Dumont Rachel A RA Morgan Clive P CP Benson Connor B CB Vijayakumar Sarath S Cunningham Christopher L CL Scheffer Deborah I DI Corey David P DP Müller Ulrich U Jones Sherri M SM Barr-Gillespie Peter G PG

The Journal of cell biology 20170912 11

Control of the dimensions of actin-rich processes like filopodia, lamellipodia, microvilli, and stereocilia requires the coordinated activity of many proteins. Each of these actin structures relies on heterodimeric capping protein (CAPZ), which blocks actin polymerization at barbed ends. Because dimension control of the inner ear's stereocilia is particularly precise, we studied the CAPZB subunit in hair cells. CAPZB, present at ∼100 copies per stereocilium, concentrated at stereocilia tips as h ...[more]

PMID: 28899994

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Project description:Hearing loss is most commonly caused by the destruction of mechanosensory hair cells in the ear. This condition is usually permanent: Despite the presence of putative hair-cell progenitors in the cochlea, hair cells are not naturally replenished in adult mammals. Unlike those of the mammalian ear, the progenitor cells of nonmammalian vertebrates can regenerate hair cells through- out life. The basis of this difference remains largely unexplored but may lie in molecular dissimilarities that affect how progenitors respond to hair-cell death. We analyzed gene expression in hair-cell progenitors of the lateral-line system. We developed a transgenic line of zebrafish called alpl:mCherry that expresses a red fluorescent protein in the presumptive hair-cell progenitors known as mantle cells. Fluorescence-activated cell sorting from the skins of transgenic larvae, followed by microarray-based expression analysis, revealed a constellation of transcripts that are specifically enriched in these cells versus hair cells and non-fluorescent skin cells. Gene expression analysis after hair-cell ablation uncovered a cohort of genes that are differentially regulated early in regeneration, suggesting possible roles in the response of progen- itors to hair-cell death. These results provide a resource for studying hair-cell regeneration and the biology of sensory progenitor cells. Two sets of analyses were performed. The first compared baseline expression levels in four sorted cell types from two different transgenic lines of zebrafish, alpl:mCherry;pou4f3:GFP and alpl:mCherry:ET20. alpl:mCherry;pou4f3:GFP larvae express GFP in hair cells and mCherry in mantle cells. alpl:mCherry:ET20 larvae express GFP in most mantle cells and mChery in all mantle cells. The four cell types compared were GFP+ hair cells, mCherry+ mantle cells, mCherry+/GFP+ mantle cells from alpl:mCherry:ET20 larvae, and non-fluorescent (NF) cells. Two hair-cell, five mCherry+ mantle cell, four mCherry+/GFP+ mantle cell, and six NF cell samples were analyzed. This excludes a few samples that were discardced based on failure to cluster by principal component analysis. A second analysis, separately imported, compared gene expression in mCherry+ mantle cells and NF cells at four time points following chemical ablation of hair cells with copper sulfate. These were one, three, five, and eleven hours after treatment (hpCu). Untreated samples served as controls. For mCherry+ cells, five untreated and four each of 1 hpCu, 3 hpCu, 5 hpCu and 11 hpCu were analyzed. For NF cells, six untreated, seven 1 hpCu, and four each of 3 hpCu, 5 hpCu and 11 hpCu samples were analyzed. Untreated mCherry+ and NF cells were shared between both analyses.