The obscurin gene, domains in the protein and isoforms in the muscles. (A) Exons in the gene are numbered 1–36. Epgy2 marks the position of the P-element insertion. Lines above the gene show exons encoding the RhoGEF (DH-PH) and the two kinase domains (Kin1 and Kin2). (B) Domains predicted in the protein. Lines above domains show regions that were used to raise antibodies. (C) Western bots of isoforms in larva (L), adult thorax (T) and IFM (F). Blots were incubated with anti-Ig14-16 or anti-kinase1 antibodies. There are at least four isoforms in the thorax. Isoforms A and C are in IFM; the larva has isoform E only. Both antibodies reacted with all five obscurin isoforms.
Obscurin in the embryo, larva and pupa of wild-type flies. Muscles were labelled with anti-obscurin. (A) Wild-type embryo (late stage 17) showing striations across the muscles, with a gap at the epidermal attachment site (arrows). (B) Embryo labelled with anti-obscurin (green) and anti-kettin (an isoform of Sls) (red). Obscurin is either side of the epidermal attachment site. (C) Embryo of P-element mutant. Labelling is similar to that of the wild-type embryo. (D) Larval body wall muscle, from a fly line with a GFP-sls gene. Anti-obscurin (red); GFP-kettin (green) marks the Z-discs and is in a doublet closer to the epidermal attachment site than obscurin in the M-line. (E) IFM in pupa at 30 hours, 48 hours and 72 hours APF and after eclosion (at 25°C); anti-obscurin (green) and anti-kettin (magenta). At 30 hours, obscurin has a punctate distribution and kettin is in amorphous strands. At 48 hours, myofibrils are closely packed and obscurin labelling appears as a broad band in the M-line and kettin at the Z-disc; at 72 hours, myofibrils are further apart and labelling of the narrow myofibrils appears as dots at the M-line and Z-disc. Myofibrils become wider in the adult, and obscurin and kettin are labelled in striations. Scale bars: 5 µm.
Obscurin and myosin in the IFM of the pupa. IFM was labelled with anti-obscurin (green), anti-myosin (magenta) and phalloidin (red). Pupae were grown at 29°C. (A) Wild-type pupa. At 30 hours APF, obscurin is labelled in broad striations and myosin is in amorphous strands; at 48 hours, there are H-zones and obscurin and myosin are labelled in dots at the M-line; at 72 hours, obscurin and myosin striations are sharper and there is a doublet in the myosin label near the Z-disc; in the adult, myosin is labelled at the Z-disc in a single band. From 48 hours APF to the adult, myofibrils become wider, the H-zone better defined and sarcomeres longer. (B) Mef2-GAL4; UAS-unc89-IR pupa. There is no obscurin in the IFM at any stage. At 30 hours APF, myosin is in amorphous strands; at 48 hours, there are no H-zones and myosin appears in broad striations (the fibres were too fragile to isolate single myofibrils); at 72 hours, myosin striations are in the M-line region; in the adult, myosin labelling is in a dot at the M-line and in a single band at the Z-disc. Myofibrils are narrower than in the wild-type. Inserts are single myofibrils at 1.5× the magnification of the main panels. Scale bars: 5 µm.
Effect of reduced obscurin expression. (A) Flight tests of wild-type, P-element, and RNAi knockdown flies; the proportion of flies able to fly is shown, n = 20 for each genotype. The unc89[EY15484] mutant and RNAi lines were flightless; flight was restored by excising the P-element. Control flies with UAS-RNAi (dsRNAi) or GAL4 drivers flew. (B) Obscurin isoforms in P-element and obscurin RNAi knockdown flies. Western blots of thorax (T), IFM (F) and larva were incubated with anti-obscurin (top) or anti-Sls as a loading control (bottom). The P-element mutant had the smallest thoracic isoform, reduced IFM isoforms and a wild-type larval isoform. UH3-GAL4; UAS-unc89-IR flies had the two non-IFM isoforms; there was no obscurin in the Mef2-GAL4; UAS-unc89-IR flies. Thoraces had three isoforms of Sls (kettin is the 540 kDa isoform): the lack of the largest Sls isoform in IFM confirms the purity of the sample. (C) Obscurin in the IFM. Myofibrils were labelled with anti-obscurin (green), anti-α-actinin (magenta) antibodies, and phalloidin (red). Obscurin in the wild-type M-line is reduced in the unc89[EY15484] mutant and missing in the RNAi lines. H-zones (arrowheads) are missing in the RNAi lines. Z-stacks (top) show obscurin across the diameter of the myofibril. Scale bar: 5 µm.
Structure of the IFM in flies with reduced obscurin. (A–F) EM images of (A) wild-type, (B,C) unc89[EY15484], (D) unc89[EY15484] rescued, (E) UH3-GAL4; UAS-unc89-IR and (F) Mef2-GAL4; UAS-unc89-IR flies. The H-zone of the P-element mutant is wavy compared with the wild-type and there are aggregates in the H-zone (white arrow). In a more severe case, bare zones of thick filaments (black arrows) are shifted from the middle of the sarcomere. Excision of the P-element rescues the phenotype. IFMs in the RNAi lines have more irregular H-zones and shifted bare zones. Some filaments bypass the Z-disc at the myofibril periphery (asterisks). Scale bar: 0.6 µm.
Symmetry of thick and thin filaments in the IFM. (A) In the wild-type, the M-line appears as a broadening of the thick filament. In unc89[EY15484] flies, the M-line is replaced by dense aggregates between thick filaments (white arrow). Bare zones (black arrows) are more shifted in Mef2-GAL4; UAS-unc89-IR flies. Myofibrils are in rigor and chevrons (crossbridges) on thin filaments show that the polarity of filaments changes either side of a shifted thick filament bare zone. M, M-line or mid-point of the sarcomere. Rows of chevrons (indicated by asterisks and arrowheads) are best viewed obliquely. Scale bar, 200 nm. (B) Model for the structure of the IFM sarcomere. Top, wild-type: the bare zone on thick filaments is central and the polarity of crossbridges on thin filaments is symmetrical. Variation in the length of thin filaments in the bare zone by a tropomyosin unit is shown (Haselgrove and Reedy, 1984). Bottom, flies lacking obscurin: thin (actin) filaments are longer on one side of a shifted bare zone than the other. The polarity of crossbridges is determined by the thin filament, and stays the same up to the edge of a shifted bare zone. My, myosin; Ac, actin.
Obscurin in the IFM. Cryosections were labelled with anti-obscurin and Protein-A–gold. Histograms below the EM images show the distribution of gold particles. (A) In wild-type IFM, obscurin is in the M-line. (B) In the unc89[EY15484] mutant, remaining obscurin is in the M-line region. (C) There are obscurin remnants in the distorted H-zone of the UH3-GAL4; UAS-unc89-IR sarcomere. (D) The Mef2-GAL4; UAS-unc89-IR sarcomere has negligible obscurin. Knockdown of obscurin in IFM is partial in the P-element mutant and effectively complete in the RNAi lines. Scale bars: 500 nm.
Structure of thick filaments in the IFM. (A) Isolated thick filaments negatively or positively-stained: the bare zone of the wild-type filament is central and those of RNAi lines driven by UH3-GAL4 or Mef2-GAL4 are offset. The filament in the bottom panel is 7.8 µm long, over twice the wild-type length. Scale bars, 500 nm. (B, top) The shortest distance from the end of the filament to the centre of the bare zone, relative to the length of the filament was measured. All samples had some filaments with central bare zones (shown as 0.5 in the figure). The position of the bare zone varied most in the RNAi lines. (B, bottom) The length of the majority of filaments in IFM of knockdown flies was close to that of wild-type flies (3.2 µm).
Association of obscurin with myosin. (A) SDS-PAGE of proteins bound to anti-obscurin. Protein-A beads with (lanes 1 and 3) and without (lane 2) anti-obscurin, were incubated with an extract of proteins from the fly thorax. Proteins pelleted with anti-obscurin were obscurin (Obs) and myosin (Myo). (B) EM image of a cryosection of thick filaments in IFM labelled with anti-obscurin antibody and Protein–A-gold. Obscurin is confined to a small area of the thick filaments. Scale bar: 200 nm.