With their essential role in inner ear function, stereocilia of sensory hair cells demonstrate the importance of cellular actin protrusions. Actin packing in stereocilia is mediated by cross-linkers of the plastin, fascin, and espin families. Although mice lacking espin (ESPN) have no vestibular or auditory function, we found that mice that either lacked plastin 1 (PLS1) or had nonfunctional fascin 2 (FSCN2) had reduced inner ear function, with double-mutant mice most strongly affected. Targeted mass spectrometry indicated that PLS1 was the most abundant cross-linker in vestibular stereocilia and the second most abundant protein overall; ESPN only accounted for ∼15% of the total cross-linkers in bundles. Mouse utricle stereocilia lacking PLS1 were shorter and thinner than wild-type stereocilia. Surprisingly, although wild-type stereocilia had random liquid packing of their actin laments, stereocilia lacking PLS1 had orderly hexagonal packing. Although all three cross-linkers are required for stereocilia structure and function, PLS1 biases actin toward liquid packing, which allows stereocilia to grow to a greater diameter.

Peptide samples were analyzed with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) coupled to a Thermo/Dionex Ultimate 3000 Rapid Separation UPLC system and EasySpray nanosource. Samples were loaded onto an Acclaim PepMap C18, 5 μm particle, 100 μm x 2 cm trap using a 5 μl/min flow rate and then separated on a EasySpray PepMap RSLC, C18, 2 μm particle, 75 μm x 25 cm column at a 300 nl/min flow rate. Solvent A was water and solvent B was acetonitrile, each containing 0.1% (v/v) formic acid. After loading at 2% B for 5 min, peptides were separated using a 55-min gradient from 7.5-30% B, 10-min gradient from 30-90% B, 6-min at 90% B, followed by a 19 min re-equilibration at 2% B. Peptides were analyzed using the targeted MS2 mode of the Xcalibur software in which the doubly or triply charged precursor ion corresponding to each peptide was isolated in the quadrupole, fragmented by HCD, and full m/z 350-1600 scans of fragment ions at 30,000 resolution collected in the Orbitrap. Targeted MS2 parameters included an isolation width of 2 m/z for each precursor of interest, collision energy of 30%, AGC target of 5 x 104, maximum ion injection time of 100 ms, spray voltage of 2400 V, and ion transfer temperature of 275°C. No more than 75 precursors were targeted in each run and no scheduling was used. Three unique peptides for each protein of interest were chosen for isolation based on previous data-dependent discovery data or from online peptide databases (www.peptideatlas.org, www.thegpm.org). Precursor isolation lists for all peptides of interest were exported from the software package Skyline (http://proteome.gs.washington.edu/software/ skyline/) and imported into the Orbitrap control software. Skyline was used to analyze targeted MS/MS data. Chromatographic and spectral data from the RAW files were loaded into Skyline and analyzed to determine fragment ion peaks corresponding to each peptide. RAW files were also processed using Proteome Discoverer (Thermo Scientific) software in order to match MS/MS spectra to an Ensembl spectral database using Sequest HT. Fragment ion peaks that co-eluted with the fragment ion peaks for the corresponding heavy peptide were chosen for analysis. The type and proportion of daughter ions contributing to the peptide peak were required to match that of the heavy peptide peak. In addition, one or more spectra within the light or heavy peptide peak were matched to the correct peptide sequence within the spectral database. If spectra within a specific sample were not identified then a) the retention time of the chosen peak must be within 2 minutes of the retention time of an identified peak for that peptide from another sample and b) the type of daughter ions contributing to the peak must match the identified peptide peak from another sample. Chromatographic peak areas from all detected fragment ions for the light and heavy version of each peptide were integrated and summed, and then the peak area ratio between the light and heavy peptides was calculated. This ratio was multiplied by the amount of spiked heavy peptide to give the fmol amount of each light peptide in the sample. The peptide fmol amounts for each protein of interest were averaged for each sample (and normalized to the average fmol amount for actin within the same sample). The normalized peak areas were then averaged for the four biological replicates of each age/mouse strain to give an average protein intensity measurement for each protein of interest. For the calibration curve samples, a linear regression of the heavy peptide peak area in each of the 4 calibration samples was performed and tested for linearity around the measurement range. Peptides that did not perform linearly (R2 > 0.98) were excluded from analysis. For targeted MS/MS experiments comparing relative protein expression in WT versus Pls1-/- bundles, peptides were measured from three (wild type) or two (Pls1-/-) preparations of 10 ear-equivalents of hair bundles from either genotype. Peptides were generated using in-gel digestion methods, which have been previously described (Krey et al, 2015). Prior to digestion with trypsin, 150 fmol of heavy ACT peptides (see above) were added, and light peptide peak areas were measured for all other proteins of interest. Peptide peak areas for each protein were averaged for each sample and normalized to the average peptide peak area for actin within the same sample. The normalized peak areas were then averaged for the three biological replicates of each genotype to give an average protein intensity measurement for each protein of interest.

For targeted MS/MS, we measured ACT, PLS1, FSCN2, ESPN and ESPN-1 peptides from four preparations of 10 ear-equivalents of hair bundles isolated from P21-25 C57BL/6 mice and from four preparations from 2 different ages of CD-1 mice (P4-P6, P21-P23), each of 13-14 ear-equivalents of hair bundles. In-solution tryptic digests of the samples were prepared using an enhanced filter-aided sample preparation (eFASP) method (Erde et al, 2014). Proteins were digested in the filter unit in 100 µl digestion buffer with 200 ng sequencing-grade modified trypsin (Promega) at 37°C for 12-16 hours. Three quantified synthetic stable-isotope labeled peptides (SpikeTides-TQL) corresponding to each mouse protein sequence (ACT: EITALAPSTMK, GYSFTTTAER, AGFAGDDAPR, PLS1: IYALPDDLVEVKPK, MINLSEPDTIDER, VAFVNWINK, FSCN2: FFGGIEDR, FLVLPQPDGR, YLAPVGPAGTLK, ESPN: LAPWQR, LASLPAWR, TLGYDEAK, ESPN1: DNSGATVLHLAAR, YLVEEVALPAVSR, YLVQECSADPHLR) were obtained from JPT Peptide Technologies (Berlin, Germany) and used as internal standards; any cysteine residues were substituted by carbamoylmethylated cysteines during synthesis. The following amounts of each peptide were added along with the trypsin solution prior to digestion of each sample: ACT peptides, 500 fmol; PLS1 peptides, 50 fmol; FSCN2 peptides, 50 fmol; ESPN peptides 10 fmol; and ESPN1 peptides, 1 fmol. Calibration curves were run for all peptides by adding four dilutions of each peptide (centered around the amount spiked into the sample) to four mouse utricular lysate samples (0.5 ear equivalents) prepared in the same way as the bundle samples. Peptides were isolated by centrifugation and were extracted with ethyl acetate to remove remaining deoxycholic acid [31]. Heavy and endogenous forms of each peptide were monitored by targeted MS/MS. For targeted MS/MS experiments comparing relative protein expression in WT versus Pls1-/- bundles, peptides were measured from three (wild type) or two (Pls1-/-) preparations of 10 ear-equivalents of hair bundles from either genotype. Peptides were generated using in-gel digestion methods, which have been previously described (Krey et al, 2015). Prior to digestion with trypsin, 150 fmol of heavy ACT peptides (see above) were added, and light peptide peak areas were measured for all other proteins of interest.