Download were performed essentially as described previously (Witt et al

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 -20C 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