Bundle isolation from E20 utricles of White Leghorn chicken (Gallus domesticus
) embryos was carried out as described previously (Gillespie and Hudspeth, 1991
) with modifications for chicken (Supplemental Data
). While the White Leghorn strain is different from the Red Jungle Fowl strain (Gallus gallus
) used for genome sequencing, DNA from the two strains differs in sequence by only ~1% (http://www.ensembl.org/Gallus_gallus/index.html
). Because mass spectrometry peptide identification tolerates small numbers of amino-acid differences (specified when setting the search parameters), this level of sequence difference should have had little effect on protein identification. Moreover, amino-acid sequences differences between proteins of the two strains will likely be much less than the 1% difference seen at the DNA level. Measurement of total protein and quantitative immunoblotting was performed as described in Supplemental Data
Mass spectrometry via LC-MS/MS and MuDPIT
Agarose plugs containing hair bundles were dried in a vacuum centrifuge. The agarose was rehydrated with 10 mM DTT, minced, and incubated at 35°C for 30 min. Iodoacetamide was added to 15 mM and the samples incubated in the dark for 30 min. The samples were dried, then trypsin (20 ng/ml; Sequencing Grade Modified Trypsin, Promega) was added to reswell the agarose; the sample was sonicated during rehydration. The sample was then adjusted to 10% acetonitrile and digested overnight at 37°C. Peptides were extracted via two additions of 80 μl of 1% formic acid, each followed by sonication. Acidified peptides were desalted on a StageTip (Proxeon), dried, and resuspended in 1% formic acid containing 2.5 mM ammonium acetate.
Tryptic peptides from digests were analyzed by nano-LC-MS/MS. Chromatography was achieved using an Eksigent nanoLC to generate a gradient using the following chromatographic conditions; mobile phase A; water, acetonitrile, formic acid, trifluoroacetic acid (95, 4.89, 0.1, 0.01, v/v/v/v) mobile phase B; acetonitrile, isopropanol, water, formic acid, trifluoroacetic acid (80, 10, 9.89, 0.1, 0.01, v/v/v/v/v). Mobile phase B was ramped from 2% to 45% over 40 minutes, increased to 80% in 5 minutes and held for five minutes before being returned to starting conditions. Flow was regulated at 200 nl/min and directed through a 75 μm x 15 cm column packed in-house with Astrosil (5 μm particle size, 100 Å pore size, C18 reverse phase chemistry; Stellar Phases) coupled to a 5 μm tapered emitter (New Objectives). Prior to analytical chromatography, 5 μl of tryptic digest was injected onto a 150 μm x 2 cm sample trap packed with Poros R10; the trap was washed with mobile phase A to remove salts and contaminants, then was switched in-line with the analytical column. Tandem mass spectrometry data was collected using a QSTAR XL hybrid time-of-flight mass spectrometer (Applied Biosystems) under the following conditions; spray voltage 1800–1900V; TOF-MS scan m/z 400–1600, 0.5 sec; TOF-MS/MS scan m/z 50–2000, 2.0 sec, 9 sec exclusion; data dependent product ion acquisition of the three most abundant +2 and +3 ions from the TOF-MS scan.
For MudPIT, the instrumentation described above was used with the following changes. A 5 cm, lab-packed SCX column was prepared for ion-exchange chromatography (Polysulfoethyl A, 5 μm particle size, 300 Å) as instructed by the manufacturer. Analytical columns of 75 and 50 μm diameter were used at flow rates of 200 or 100 nl/min, respectively. Tryptic peptides from a hair bundle digest were injected onto the SCX column and the breakthrough was analyzed as the first fraction. Additional pools of peptides were displaced from the ion exchange column via ammonium acetate injections (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, and 300 mM); each eluate was separately run on nano-LC-MS/MS as described above.
Mass spectrometry via GeLC
Hair-bundle proteins were resolved by 1D SDS-PAGE; individual lanes of Sypro Orange stained-gels were cut into ~45 individual slices using a 2DiDx sample preparation robot (Leap Technologies). Individual gel slices were placed in a ZipPlate (Millipore), which was used as a filter to facilitate tryptic digestion. Gel slices were destained in two changes of 100 mM ammonium bicarbonate in 30% methanol, dried with neat acetonitrile, reduced with 10 mM DTT, and alkylated with 50 mM iodoacetamide. After washing and drying, approximately 100 ng of sequencing grade trypsin in 20 mM ammonium bicarbonate was added to each sample along with sufficient buffer to completely immerse the gel slices in buffer. Digestion proceeded overnight at 37°C. After acidifying samples with 10 μl of 1% formic acid, the peptide extract was collected by centrifugation; the gel slices were further extracted with two additions each of 50% and 70% acetonitrile. Peptide extracts were brought to near dryness before being resuspended in 10 μl of 0.1% formic acid in preparation for nano-LC-MS/MS.
Monoisotopic masses for database searching were generated using Distiller (Matrix Science) and submitted to X! Tandem for protein identification. Masses were searched against the Ensembl database (Ensembl Gallus gallus WASHUC 1, v 37.1) with the following parameters: fixed modification, cysteine carbamidomethylation; variable modification, methionine oxidation; one missed cleavage allowed; digest agent, trypsin; refinement modifications, methionine oxidation and N/Q deamidation, as well as one point mutation allowed; no removal of redundant spectra; precursor and fragment ion mass tolerances of 100 ppm and 0.2 Da respectively.
We generated Table 2 as follows. Protein isoforms identified with the same set of peptides were inspected manually to determine the most abundant isoform; this step applied to actin, creatine kinase, tubulin, and enolase. Because of contamination by human skin keratin, we manually deleted all intermediate filament identifications. Likewise, hemoglobin identifications were removed, as they resulted from red blood cell contamination (data not shown). We wrote a Mathematica 5.0 program to automatically select proteins that fit the criteria we used for .
Auditory and vestibular organs from chicken embryos (E20) were dissected in chicken saline and processed for immunocytochemistry (Supplemental Data
). The antibodies for GAPDH, CLIC5, NHERF and B-CK required an unmasking procedure to expose the antigenic sites. Organs were fixed as usual in formaldehyde, then boiled in a citrate-based antigen unmasking solution (Vector Laboratories, Burlingame CA) for 5–10 min in a microwave. Organs were then washed in PBS and blocked in blocking solution as usual. The unmasking procedure inhibits phalloidin binding, presumably by disrupting F-actin structure; in experiments requiring antigen unmasking, actin counterstaining was achieved using a monoclonal anti-β-actin antibody (AC-15, Sigma; 1:400). AC-15 only detected stereocilia actin after unmasking.
Bullfrog hair cells were isolated in low-calcium saline (110 mM NaCl, 2 mM KCl, 2 mM MgCl2
, 0.1 mM CaCl2
, 3 mM D-glucose, 10 mM HEPES, pH 7.25) as described previously (Hirono et al., 2004
). Total ATP was quantified using the Enliten luciferin/luciferase kit (Promega) using methods described in Supplemental Data
Magnesium Green fluorescence
Bullfrog hair cells were isolated as described above; 3 μM Magnesium Green AM dye (Invitrogen Molecular Probes) was included in the dissociation solution. After hair cells had settled, the solution was replaced by standard saline (low-calcium saline with 4 mM CaCl2) containing 6.66 μM Magnesium Green AM. After 20 min of dye loading time, cells were washed with standard saline and allowed to de-esterify for an additional 20 min. Cells were viewed with a Plan Apochromat 60x (1.40 NA) oil lens on a Nikon TE 300 inverted microscope with a Bio-Rad MRC 1024 confocal imaging system. For inhibition of creatine kinase, a 10 mM stock solution of DNFB was diluted to a final concentration of 10 μM in the experimental chamber. Before adding DNFB, the basal Magnesium Green fluorescence was monitored for at least 6 min in order to ensure a relatively stable baseline. Image analysis was performed using ImageJ software; data were fitted to single-exponential functions. The error bars represent standard errors.
Analysis of creatine kinase knockout mice
To obtain heterozygous B-CK+/−
single knockout mice (Jost et al., 2002
) and UMi-CK−/−
single knockout mice (both 25% 129/Ola and 75% C57BL/6) were interbred. Breeding between heterozygous pairs generated heterozygous siblings, homozygous siblings with wild-type CK alleles (B-CK+/+
), and homozygous double-knockout siblings (B-CK−/−
). Homozygous siblings were interbred to generate two separate lines with the same genetic background: (1) wild-type mice, and (2) mice lacking both CK isoforms (CK=/=
double knockout mice; Streijger et al., 2005
). Histological analysis, ABR measurements, and vestibular tests were carried out as described in Supplemental Data
Mechanotransduction and adaptation