Statistics and reproducibility
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. All experiments were performed at least twice unless otherwise noted. P < 0.05 was accepted as statistically significant. Data are displayed as mean ± SEM unless indicated otherwise. Data in Figs. 1c, 1d and 2i were replicated once with similar results. Data in Supplementary Fig. 1c were replicated >10× as part of standard genotyping procedures. Supplementary Figs. 1i–l and 2k–m were each replicated at least once with similar results.
All experiments involving animals were performed following the rules for Animal Welfare of the German Society for Laboratory Animal Science and received ethical approval by the Landesamt für Gesundheit und Soziales (LAGeSo, Berlin). Animals were housed in individually ventilated cages with free access to food and water, constant 22 ± 2 °C temperature and 55 ± 10% humidity, and a 12 h : 12 h light/dark cycle (light from 6:30 a.m. to 18:30 p.m.).
The animal model to probe the Z-disc and titin interactome was generated using homologous recombination in ES cells and blastocyst injection34 and backcrossed on a 129/S6 background (Taconic Biosciences). We inserted BirA downstream of Exons 28 at the transition of the Z-disc into the I-band—the identical position that we used for our integration of DsRed, as it did not interfere with sarcomere assembly, remodeling, or mechanics17. Animals received continuous biotin supplementation (3.7 µg/ml added to the drinking water) to improve levels of free Biotin and facilitate protein biotinylation. Animals were sacrificed by cervical dislocation. Heart weight, body weight, and tibia length was measured from wild-type, heterozygous, and homozygous knock-in animals at the indicated timepoints in male and female animals. All experimental animals were male adults (12–15-week-old) or neonatals (P1/2). For controls, we used wild-type animals to distinguish unspecific endogenous biotinylation. As this is the first BioID knock-in mouse, there are no suitable controls available with biotinylation mediated by a fusion protein outside the sarcomere. Unlike in cell culture experiments, we are not able to control for biotin in our mice, as biotin deficiency is not tolerated in vivo16.
Genomic DNA was prepared from ear tags following standard procedures. Proper integration of the BirA was verified by PCR using specific primers. The BirA construct into the titin locus was monitored by PCR using primers BirA-SeqFw (5′-CATCTCCAGAGGAATCGACAAG-3′) and rMA dsredKITV (5′-AAGCTTGATAAGGGATAGTCTTGGGCATAC-3′). Excision of the Neo selection and to distinguish between heterozygous and homozygous animals a PCR with the primers fDsRedrecF (5′-CAGCATCATGGTAAAGGCCATCAA -3′) and rDsRedrecF (5′-CATTCAAATGTTGCCATGGTGTCC-3′) was done. The Flp recombinase allele was detected using primers Flp-for (5′-GTCACTGCAGTTTAAATACAAGACG-3′) and Flp-rev (5′-GTTGCGCTAAAGAAGTATATGTGCC-3′).
SDS-agarose electrophoresis and western blotting
Tissues were collected, pulverized under liquid nitrogen using mortar and pestle, and extracted in titin sample buffer (8 M urea, 2 M thiourea, 3% SDS, 0.05 M Tris-HCl, 0.03% bromophenol blue, 75 mM dithiothreitol (DTT), pH 6.8) for 30 min on ice. Debris was removed by centrifugation. Proteins were separated on agarose gels and blotted on polyvinylidene difluoride membranes. Primary BirA antibody were purchased from BioFront Technologies (Chicken Polyclonal Ab to Escherichia coli Biotin Ligase/BirA; BID-CP-100, 1 : 5000) and the Streptavidin–horseradish peroxidase (HRP) conjugate from GE Healthcare (RPN1231V, 1 : 1000). The secondary HRP-conjugated antibody (ECL Rabbit IgG, HRP-linked whole Ab (from donkey) Amersham NA934V, 1 : 5000) was detected by chemiluminescence staining with ECL (Supersignal West Femto Chemiluminescent Substrate; Pierce Chemical, Co.).
Heart and quadriceps tissue of all genotypes was collected, snap frozen, and grinded. Total RNA was isolated from tissue powder with Trizol followed by a cleaning step with the Qiagen RNeasy isolation Kit. To convert RNA into complementary DNA (cDNA), a reversed transcription PCR with a viral RNA-dependent DNA polymerase was performed using the RNA to cDNA Kit from Applied Biosystem or ThermoScientificTM RevertAid RT Kit according to the manufacturer’s specifications. Taqman probe against the BirA was used to monitor the RNA expression in all genotypes (BirAfw 5′-ATCGGACTGAGTCTGGTGAT-3′; BirArev 5′-GTACAGGTCATTAGGCCACTTC-3′; BirAprobe FAM-5′-CTGAGAAAGCTGGGAGCCGACAAG-3′-TAMRA).
Hearts and quadriceps from BirA homozygous and wild-type animals were dissected and prepared for immunofluorescence staining35. Flexor digitorum brevis muscle was isolated and transferred to MEM medium containing Biotin. One muscle was stretched with Minutien Pins on Sylgard dishes the other muscle was pinned without stretch as control. Stretch experiments were performed for 15 min at 37 °C. Contraction was induced by skinning the muscles first with 2% Triton X-100 for 30 min followed by incubation with 4 mM CaCl2. The muscle was fixed with 4% paraformaldehyde (PFA) at room temperature, followed by fixation for 2 h at 4 °C. The muscles were transferred into 30% sucrose phosphate-buffered saline (PBS) solution overnight and embedded in TissueTek. Cryosections of 8 µm were fixed with 4% PFA at room temperature for 10 min, and permeabilized and blocked with 2% goat serum, 0.3% Triton X-100, and 2 % bovine serum albumin in PBS for 2 h. The incubation with the primary antibody (1 : 50 anti-α-actinin, Sigma-Aldrich EA-53; 1 : 50 anti-Myh8, Invitrogen PA5-72846; 1 : 50 anti-Myosin; Sigma-Aldrich M4276; 1 : 50 anti-Nebl, SCBT sc-393784; 1 : 50 anti-Pgam2, Abcam ab97800; 1 : 50 anti-titin BirA, BioFront Technologies) or Streptavidin Star conjugate (1 : 200, Abberior) was performed at 4 °C overnight followed by labeling with a fluorescent secondary antibody (1 : 200 goat anti-chicken, goat anti-mouse, or goat anti-rabbit STAR from Abberior) for 2 h at room temperature or overnight at 4 °C.
Images were acquired with a confocal laser-scanning microscope (LSM710, Carl Zeiss) with a Plan-Apochromat ×63/1.4 oil Ph3 objective. Super-resolution imaging was performed with a 3D-STED microscope (Abberior Instruments) and a ×100 oil objective (UPLANSAPO) or a TCS SP8 STED microscope (Leica) with a HC PL APO C2S ×100 oil objective (1.4 NA) used in 2D mode17. For imaging with the Leica STED system, Abberior STAR 635P was excited at a wavelength of 635 nm and fluorescence was detected between 650 nm and 700 nm. Abberior STAR 580 was excited at 580 nm and fluorescence was detected between 590 nm and 630 nm. For the Abberior system, the fluorescence excited at 635 nm was detected between 605 and 625 nm, and the fluorescence excited at 580 nm between 650 and 720 nm. For both dyes and systems, STED imaging was performed with a 775 nm depletion laser. Imaging with the Leica STED system was executed with a gating between 0.5 ns and 6 ns, and images were acquired with a pixel size of 23 nm × 23 nm and a scanning speed of 600 Hz (pixel dwell time 0.4 µs). With the Abberior system, the depletion laser was used with a laser power of 15% and images were made with a pixel size of 15 nm × 15 nm. All images not labeled as STED are confocal images. Abberior STAR 635 P signal is displayed in red, Abberior STAR 580 in green. Images were analyzed using ImageJ Fiji Software. Traces are displayed as mean (opaque) ± SEM (SEM, transparent lines).
For the identification of protein complexes, the protocol from Roux et al.16 was modified. Fifty milligrams of cryo-fractured tissue powder was resuspended with 0.5 ml BioID lysis buffer (50 mM Tris pH 7.5; 500 mM NaCl; 0.4% SDS; 5 mM EDTA; 1 mM DTT; 2% Triton X and 1× PICS I). The samples were incubated for 60 min on ice and snapped in between followed by sonication. To remove insoluble material the samples were centrifuged for 10 min at 12,000 r.p.m. at 8 °C. The supernatant was transferred into a new tube and incubated with the beads (MyoOne Streptavidin C1; Invitrogen) overnight16. Beads were collected with a magnetic rack and washed 2 times for 10 min with 0.1% deoxycholate; 1% NP-40; 500 mM NaCl; 1 mM EDTA and 50 mM HEPES pH 7.5 at 4 °C, 2 times washed with 250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, and 10 mM Tris pH 8.1 at 4 °C, and 2 times washed with 50 mM Tris pH 7.4 and 50 mM NaCl at 4 °C. Beads were resuspended in 20 μl urea buffer (6 M urea, 2 M thiourea, 10 mM HEPES pH 8.0), reduced in 10 mM DTT solution, followed by alkylation using 40 mM chloroacetamide. Samples were first digested with 1 µg endopeptidase LysC (Wako, Osaka, Japan) for 4 h and, after adding 80 µl 50 mM ammonium bicarbonate (pH 8.5), digested with 1 µg sequence-grade trypsin (Promega) overnight. The supernatant was collected and combined with the supernatant from an additional bead wash in 50 mM ammonium bicarbonate (pH 8.5). Samples were acidified with formic acid and peptides were desalted using StageTip purification36.
To capture biotinylated peptides we use a modified anti-Biotin AB protocol19. Complete heart and quadriceps cryo-fractured tissue powder was lysed for 1 hour at 4 °C (8 M urea, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, Protease inhibitor PICS I, 10 mM sodium azide, 10 mM sodium ascorbate). The samples were sonicated samples 3 and the protein concentration was determined using the bicinchoninic acid (BCA) assay. To reduce the proteins 5 mM DTT was added for 45 min at room temperature (RT) followed by carbamidomethylation with 10 mM iodoacetamide for 30 min at RT in the dark. To reduce the urea concentration to 2 M we used 50 mM Tris-HCl pH 8. The samples were digested overnight at 25 °C with sequencing-grade trypsin at an enzyme:substrate ratio of 1 : 50 or 1 : 100. To remove the insoluble material the samples were centrifuged at 4000 × g for 5 min at 4 °C until the supernatant was clear. The peptide concentration was measured with BCA assay and the samples were acidified with formic acid (1% final concentration) and desalted on a 500 mg C18 Sep-Pak SPE cartridge (Waters). Before the cartridges were washed 1× with 5 ml of 100% MeCN, 1× with 5 ml 50% MeCN:0.1% FA, 4× with 5 ml of 0.1% TFA. After sample loading, the cartridges were washed 3× with 5 ml of 0.1% TFA and then 1× with 5 ml of 1% FA. For elution of the peptides we used 6 ml of 50% MeCN:0.1% FA and dried by vacuum centrifugation (possible to store the peptides at −80 °C). For the enrichment of biotinylated peptides, a biotin antibody was used (anti-biotin ImmuneChem Pharmaceuticals, Inc.; ICP0615; 50 μg per sample). The antibody bound agarose beads were washed 3× in IAP buffer (50 mM MOPS pH 7.2, 10 mM sodium phosphate, and 50 mM NaCl). After washing, biotinylated peptides were eluted with 50 μl of 0.15% TFA, cleaned up, and stored on StageTips36.
Peptides were eluted using Buffer B (80% Acetonitrile and 0.1% formic acid) and organic solvent was evaporated using a speedvac (Eppendorf). Peptide samples were diluted in Buffer A (3% acetonitrile and 0.1% formic acid) and separated on a 20 cm reversed-phase column (ReproSil-Pur C18-AQ; Dr. Maisch GmbH) using a 98 min or 200 min gradient with a 250 nl/min flow rate of increasing Buffer B concentration (from 2% to 60%) on a high-performance liquid chromatography system (ThermoScientific). Peptides were measured on a Thermo Orbitrap Fusion or Q Exactive Plus instrument (Thermo). On the Orbitrap Fusion instrument, peptide precursor survey scans were performed at 120 K resolution with a 2 × 105 ion count target. Tandem MS was performed by isolation at 1.6 m/z with the quadrupole, higher energy collisional dissociation fragmentation with normalized collision energy of 32, and rapid scan MS analysis in the ion trap. The MS2 ion count target was set to 1 × 104 and the max injection time was 300 ms. The instrument was run in top speed mode with 3 s cycles. The Q Exactive Plus instrument (Thermo Fisher Scientific) was operated in the data-dependent mode with a full scan in the Orbitrap (70 K resolution; 3 × 106 ion count target; maximum injection time 50 ms) followed by top 10 MS2 scans using higher-energy collision dissociation (17.5 K resolution, 1 × 105 ion count target; 1.6 m/z isolation window; maximum injection time: 50 ms for streptavidin enriched samples and 250 ms for anti-biotin antibody enriched samples).
Raw data were processed using MaxQuant software package (v188.8.131.52)37. The internal Andromeda search engine was used to search MS2 spectra against a decoy mouse UniProt database (MOUSE.2017-01) containing forward and reverse sequences, including the sequence for the Titin-BirA construct. The search included variable modifications of methionine oxidation and N-terminal acetylation, deamidation (N and Q), biotin (K) and fixed modification of carbamidomethyl cysteine. Minimal peptide length was set to seven amino acids and a maximum of two missed cleavages was allowed. The false discovery rate (FDR) was set to 1% for peptide and protein, and site identifications. To filter for confidently identified peptides, the MaxQuant score was set to a minimum of 40. Unique and razor peptides were considered for quantification. Retention times were recalibrated based on the built-in nonlinear time-rescaling algorithm. MS2 identifications were transferred between runs with the “Match between runs” function of MaxQuant (only within replicates of the same batch and tissue). IBAQ intensities were calculated using the built-in algorithm. The resulting proteinGroups and biotinSites text files were filtered to exclude reverse database hits, potential contaminants, and proteins only identified by site. Statistical data analysis was performed using Perseus software (v184.108.40.206)38.
For the streptavidin protein pulldown data biological replicates for each genotype were defined as groups and proteinsGroups identifications with <3 valid values (>0) for at least one group were filtered out. After log2 transformation, missing values were imputed with random values taken from a normal distribution with 0.3× the SD of the measured, logarithmized values, down-shifted by 1.8 SDs. Differences in protein abundance between BirA-titin samples and wild-type control samples were calculated using two-sample Student’s t-test. Proteins enriched in the BirA-titin group and passing the significance cut-off (permutation based FDR < 5%, minimum three peptides identified) were considered titin-associated proteins. GO term enrichment analyses for titin-associated proteins were perfomed using Cytoscape and the ClueGO plugin39,40.
BirA-Titin-specific biotinylated peptides and their corresponding sites were extracted from the biotinSite text file, using a minimum of three valid values (Intensity > 0) in the BirA-Titin group and at least two more valid values than in the corresponding wild-type group. Annotated MS2 spectra for confident biotinylated peptides were extracted using MaxQuant Viewer application.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.