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Download were performed essentially as described previously (Witt et al
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S3 – FURTHER DETAILS ON METHODS USED Expression profiling (RT-PCR and Affymetrix gene chip analysis) were performed essentially as described previously (Witt et al., 2004). We used the Mouse Genome 430 2.0 array and compared two wt /KO pairs from two litters. Data was analyzed with the Affymetrix software suite. Typing of nebulin isoforms was performed essentially as previously described for titin (Lahmers et al., 2004), except that a new array was printed based upon on the murine nebulin gene sequence (obtained from www.ensembl.org). RT-PCR used the following primer sequences: Gene/construct sense F:mneb5'/NotI tttgcggccgcggaggctgggactagaggtcttgagtctgaggc R:mneb5'/XhoI F:mneb3'/BamHI antisense tttctcgagttgctcaaatttcagaagctaaatatactgg tttggatcccacctggaagtgaaagggggcatcactacttcc R:mneb3'/SalI tttgtcgacggtgtatctctgtcctggcaggcactgccc Genotyping Nebulin (Neb) AY189120 Neomycin resistance cassette (NEO) gaaacctgtggacctctgtgaagatcag ctctcccagagttgcttgactgtaaac cgaattcgccaatgacaagacgctgg RT PCR GAPDH ctcactcaagattgtcagcaatg NM008084 Nebulin (Neb) gatatccgtggaagccaatggttttccg AY189120 Desmoplakin (DSP) gctgcaataaaatcccagtagtaaaggctc AK077574 Sarcolipin (SLN) cttccctcagactacattaggccctgcc AK009005 AGRP cggaggtgctagatccacagaaccg BC079902 Ankrd2 ctgagcactcccctgcatgtggccgtccg AJ249346 Csrp3/MLP ggagcctgtgaaaagacggtctacc D88791 CARP cggacggcactccaccgagcatgc AF041847 TTN tcgccactgctcattcgcaagactcagacc NM011652 Used Oligos for RT-PCR studies gagggagatgctcagtgttgg tgtgttcttgacggcttggacaagttcagg gccaagattgctgctattgtggacatagtc gtattggtaggacctcacgaggagcc cagcaaggtacctgctgtcccaagc gccactcccaggcctctccagtccgttctg ccacttgctgtgtaagccctcc gtaggcattctccttggggctgtcg tcctccacatgcgtaggctctctgggttcc 1 Immunofluorescence and electron microscopy. NEB KO and wt mice were sacrificed at day 14, and quadriceps muscle were stretched, frozen and sectioned using routine methods. Immuno-labeling and confocal microscopy was performed essentially as described previously (Bang et al., 2001). All secondary antibodies did not show signals when used without first antibodies (data not shown). Images were produced using a BioRad MRC 1024 confocal laser scanning microscope using the LaserSHARP 2000 software package (Hercules, CA). protein species nomenclature origin CapZα Carp ankrd2 Alpha-actinin Desmoplakin MHC Myopalladin Nebulin N-term Nebulin C-term Nebulin SH3 Tropomodulin-1 mouse monoclonal rabbit polyclonal rabbit polyclonal mouse monoclonal mouse monoclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal mAB 5B12.3 #3119 #3434 A-7811 2.15 sc-20641 H300 #3118 x35-x36a 1843x #6963 #6142 #8626 DSHB, Iowa Labeit Labeit Sigma Dr. Herrmann Santa Cruz Labeit Dr. Gregorio Labeit Labeit Labeit Primary Antibodies Antibody species origin AlexaFluor 488 AlexaFluor 568 AlexaFluor 568 mouse IgG1 mouse IgG2A rabbit Molecular probes/Invitrogen Molecular probes/Invitrogen Molecular probes/Invitrogen Secondary Antibodies For electron microscopy, whole tibialis cranialis muscles were dissected from NEB KO, wt, and ht 10-day-old mice. Fibers were skinned, fixed in 3.7% 2 paraformaldehyde labelled overnight with phalloidin-biotin (Molecular Probes, B7474, biotin-XX phalloidin), followed by streptavidin-nanogold (Nanoprobes, 2016, nanogold streptavidin conjugate), silver enhancement as per instructions with Nanogold HQ silver kitfixing, and embedding. EM was performed as previously described (Trombitas & Granzier, 1997). Physiology. Tibialis cranialis muscle from 10-day-old KO and wt animals were used after skinning in relaxing solution (RS) containing 1% (w/v) Triton X-100 for 6 hrs at ~4 C. Preparations were washed thoroughly with RS and stored in 50% glycerol RS at -20C for less than 2 weeks. Small muscle bundles (diameter ~ 0.2 mm) were dissected from skinned muscles. For force-pCa measurements, fibers were attached to a strain gauge and a high-speed motor. Experiments were performed at room temperature (20-22 °C). Sarcomere length (SL) was measured with laser-diffraction using a He-Ne laser beam and adjusted to 2.0 m. The preparation was first activated at pCa 4.5 to obtain maximal Ca2+-activated tension. The pCa-tension relationship was obtained by switching the perfusate to activation solutions with different free calcium concentration. Active tension was normalized to the maximal tension (pCa = 4.5) at the end of the force-pCa protocol. The pCa-tension relationship was fitted to the Hill equation. The Hill coefficient, nH, was used as an index of cooperativity. Run-down of active tension was less than 15%. For additional details see (Cazorla et al., 2000). 3 Yeast two-hybrid screens – interaction of nebulin and titin A fragment encompassing exons 4 to 7 of human titin (corresponding to the Ig domain Z2 and the unique domain Z-is1; (Labeit & Kolmerer, 1995a)) was PCRamplified from total human skeletal muscle cDNA with Ex4-S and Ex-7R: (Ex-4S;tttccatgGCACCACCCAACTTCGTTCAACGACTG); (Ex-7R;tttggatcctaCACCTCTTTAGCACCAGTGGCAACAGC). The fragment was inserted into bait vector pGBKT7 (BD Bioscience). For Y2H screening, the bait TTN4-7-pGBKT7 and 40 µg of an adult skeletal cDNA library (inserted into pACT2 vector; BD Bioscience: 638818) were co-transformed into AH109 yeast cells (Yeastmaker Transformation System; BD Bioscience 630439). Co-transformed cells were incubated for five days at 30 °C on SD-Leu/-Trp/-His/-Ade plates. Plasmids from yeast clones were isolated, transferred into E.Coli and sequenced (for details see (Witt et al., 2005)). Screening of ~200,000 clones isolated 43 clones. Their sequences indicated that 16 prey clones (~38%) had nebulin inserts. All 16 clones extended to the C-terminus and contained the SH3 domain, whereas towards the N-terminus, variable numbers of nebulin repeats were included. The prey clone with the shortest insert had its 5’ end at bp 19,702 (in accession X83957, corresponding to incomplete M184). Expression of nebulin fragments in E. coli. Six nebulin fragments encompassing different parts from nebulin’s C-terminal region were amplified by PCR from human skeletal cDNA 1) repeats M155-M163; 2) M163-M170; 3) M171-M177; 4) M177-Cterm; 5) SH3 domain alone; 6) M185Cterm. For the expression of M185-Cterm in SPOTS blots and ITC experiments, the following primer pair was used to amplify nebulin: M185-sense:tttccatg-GATCCTATCACTGAACGAGTAAAGAAGA; NEB-SH3-reverse:tttggatc-CTAAATAGCTTCAACGTAGTTGGC. 4 (for the other respective oligonucleotide pairs #1-5, see supplement; for a sequence alignment and the actual peptide sequences of the nebulin repeats M1 to M185, see (Pfuhl et al., 1995)). Fragments were then cloned into the NcoI and BamHI sites of pET-M11 and expressed essentially as described in BL21 cells (Studier et al., 1990) either alone, or co-expressed with avian CapZ in pET3d (Soeno et al., 1998). The expressed peptides were purified from the soluble fractions as His-tagged fusion proteins on Ni-NTA agarose as described by the manufacturer Qiagen (the QIAexpressionist: www1.qiagen.com/literature/handbooks). Protein interaction studies Interaction of between nebulin and CapZ. BL21-codon plus cells (Stratagene) were transformed with a bi-cistronic construct, containing both the - and - subunits for avian CapZ (Soeno et al., 1998). In this pET3-derivate with Ampicillin-resistance, no histidine tags are included. Ampicillin-resistant BL21 cells were then co-transformed with Kanamycin-resistance pET-M11 constructs, harbouring the nebulin inserts 1) to 6). Triple-resistant colonies were then grown in LB supplemented with Chloramphenicol (10 µg/ml), Ampicillin (50 µg/ml), and Kanamycin (20 µg/ml) to an O.D. of 0.6, and T7-driven expression of insert proteins was induced by the addition of IPTG to a final concentration of 200 µM. Harvesting of cells, their lysis and purification of His-tagged protein complexes on Ni-NTA agarose was essentially as described by manufactures for induction of protein expression (see Novagen: www.emdbiosciences.com/html/NVG/literature; for His-tag fusion protein purification, see above Qiagen). After Ni-NTA purification, specifically bound proteins were eluted with 250 mM imidazole; aliquots of eluted proteins were separated by 10 % SDS-PAGE, transferred to a nitrocellulose membrane (Protran / Schleicher & Schuell). Blots were then reacted with anti-avian CapZ monoclonal antibody, using a dilution of 1:200 of the supernatant, provided by DSH Iowa and 5 secondary antibody alkaline-phosphatase-conjugated donkey anti-chicken (Jackson Immuno Research # 703-055-155). Detection substrate was NBT/BCIP as recommended (Roche). Interaction of between nebulin and titin. To survey for the residues in titin mediating binding to nebulin, we used a SPOTS blot membrane (JPT, Berlin) that displays exon 4 – exon 7 of titin (see also EMBL data library, accession AJ277892) as a series of 31 overlapping residues (peptides were acetylated at their amino terminus, to enhance stability). Initially, the membrane was washed for one minute with ethanol and thereafter three times for ten minutes with TBS, followed by an over night blocking step in TBS supplemented with 5 % (v/v) milk powder. For binding, nebulin fragment M177–Cterm was added to 50µg/ml final concentration. Unbound proteins were removed by three washes in TBS, supplemented with 0.05 % (v/v) Tween (Sigma). Bound fragments were reacted with specific antibodies to Nebulin M177–C-term (rabbit polyclonal; 0.7 µg/ml). Bound antibodies were detected by the Vectastain Elite Kit (Vectastain) by using biotinylated anti-rabbit IgG (PK6101), and the detection kit LumiLight Plus (Roche Diagnostics) essentially as recommended by the supplier. To further confirm the interaction of the titin residues contained in SPOTS 15 and 16 with nebulin, we used isocalorimetric titration (ITC). The nebulin fragment M185Cterm was expressed in E. coli, purified, dialyzed into PBS, and its concentration determined by Bradford assay. For ITC, the VP-ITC from MicroCal was used; nebulin M185-Cterm in the cell was used at 50 µM (corresponding to the highest concentration we could maintain this fragmenthighest concentration that was soluble). The syringe contained a synthetic 26mer peptide, corresponding to the positive SPOTS 15/16: N-QQLPHKTPPRIPPKPKSRSPTPPSIA-C. The peptide was from 6 PSL (Heidelberg, Germany; HPLC-grade >99 %; acetylated aminoterminally) and was used at 400 µM (also dissolved in PBS) 7