Two alternatively-spliced human nebulin isoforms with either exon 143 or exon 144 and their developmental regulation

Figures

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  Figure 1. Phage display epitope mapping of three nebulin mAbs. Biopanning with a mixture of the 11 mAbs succeeded in selecting from the library, 2 phage-displayed peptides recognized by 3 of the mAbs. The peptide sequences obtained by sequencing the phage DNA inserts are aligned with the protein sequence encoded by nebulin exons 138-139-140-141-142-144 and they show an excellent match of 6 or 7 amino-acids within each of the two overlapping 15-mer peptides. Peptide PEP3 was recognized by both NEB-6B5 and NEB-9C4, whereas PEP6 was recognized by both NEB-9C4 and NEB-8D2. Both peptides are encoded by exon 140 of the NEB gene.

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  Figure 2. Monoclonal antibodies against different nebulin epitopes recognize a protein of ca 800 kD on western blots. (A) Different mAb supernatants were applied to a strip blot of human muscle extract using a miniblotter. Lanes 1–7 and 9 are the eight mAbs in Table 1A that recognize exons 138–142. Lane 8 is a negative control and lane 10 is MANDYS1 mAb which recognizes 427 kD dystrophin. (B) In this case, the blot was cut into strips which were exposed to different mAb supernatants separately. The first lane is NEB-6B5 from lane 9 in (A) and lane 2 is MANDYS1 anti-dystrophin marker. NEB-9H1 is the exon 144 mAb and the two flanking lanes labelled “X” are two supernatants from the hybridoma fusion that were not specific for nebulin and not cloned; these are followed by two sub-clones each of the exon 143 mAbs, NEB-3F4 and NEB-6E6. (C) A freshly-made extract of human muscle was loaded into 3 lanes separated by pre-stained Mr markers. The blot was cut vertically into 3 sections through the Mr lanes and each section was developed with MANNES2A anti-nesprin-2 (792 kD), NEB-6B5 or MANDYS1 anti-dystrophin (427 kD), as described in Methods. The largest prestained Mr marker (250 kD) is also shown.…

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  Figure 3. Nebulin with S21b (exon 144) is found in cross-striations of all muscle fibres, but nebulin with S21a (exon 143) is found in some fibres only. Muscle sections (quadriceps) were labelled with either an exon 143 mAb (NEB-3F4) or the exon 144 mAb (NEB-9H1) and confocal images were acquired with either high magnification (63x objective: upper two frames) or at lower magnification (20x objective: lower two frames). For NEB-3F4, fibres are variably labelled strongly, weakly or not at all.

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  Figure 4. Multinucleate human muscle myotubes in cell culture only produce the S21b (exon 144) isoform of nebulin and not the alternatively-spliced S21a isoform (exon 143). Two different immortalised cell lines were used, one derived from a 5-day old infant (left column) and one from a 53-year-old donor (right column). The exon 143 mAb shown is NEB-6E6 but identical results were obtained with NEB-3F4. The size bars are 25 microns in each frame.

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  Figure 5. Expression of nebulin protein S21a (with exon 143) increases progressively during human fetal muscle development, becoming as strong as S21b (exon 144) in some fibres by 17-weeks of gestation. Frozen sections of (A) 12-week or (B) 17-week gestation fetal muscle were stained with mAbs against S21a (NEB-3F4 or “NEB143”), S21b (NEB-9H1 or “NEB144”) or fast, fetal and developmental myosin isoforms as indicated. Myosin stainings were counter-stained for nuclei with haematoxylin. Note the central nuclei in fetal myotubes. Surrounding connective tissue, revealed by haematoxylin-stained nuclei, is not stained by any of the mAbs. The 50 micron size bar in the first frame applies to all 8 frames.

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  Figure 6. Fibres in human quadriceps muscle expressing the fast myosin isoform are usually also stained by the mAbs specific for the S21a isoform (NEB-3F4). Serial sections from a quadriceps sample were immunostained by the peroxidase method with mAbs against nebulin encoded by exon 143, fast myosin or slow myosin (see Methods). Some of the fibres are numbered to enable easier comparison of the three serial sections. Most fibres are either “fast” or “slow” but some “hybrid” fibres also occur (e.g. 17 and 40). Most fibres with fast myosin are also labelled with NEB-3F4. The 200 micron size bar in the bottom frame applies to all 3 frames.