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First published online 16 March 2004
doi: 10.1242/jcs.01024

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Troponin I is required for myofibrillogenesis and sarcomere formation in Drosophila flight muscle

Department of Biology, University of York, York, YO10 5DD, UK

View larger version (26K): [in a new window]   Fig. 1. Fibre formation and the cellular processes involved during DLM development. (A) The DLM develop by fusion of myoblasts to the remnants of larval oblique muscles (LOM). (B) The LOM escape complete histolysis to serve as templates (TEM). (C) The IFM elongate by myoblast fusion, attach to the tendon cells (TC). (D) Following a fibre shortening, myofibrillogenesis begins. (E) The interdigitating region of tendon and muscle processes (TCM) retract as the muscles increase in length and size until (F) functional myofibres are formed. Anterior is to the right and dorsal to the top. Not to scale.

View larger version (51K): [in a new window]   Fig. 2. IFM fibres formed in actin and myosin null mutants. (A) DLM of the myosin null mutant Mhc7. All six DLM fibres are visible (the star indicates the middle fibre). (B) DLM of the Actin88FKM88 null mutant fibres. They are `wiggly' and slightly elongated compared with the myosin null fibres. The fibres bulge (arrowheads) in many places. (C) DLM fibres of the double homozygote null, Mhc7; Actin88FKM88. Externally the fibres appear no different from those of Mhc7 or wildtype (not shown). Anterior towards left; dorsal to the top. Bar, 0.159 mm.

View larger version (76K): [in a new window]   Fig. 3. Development of IFM fibres in hdp3 flies. (A) Fusion of myoblasts (red, Dmef2 immuno-localised expression) with the DLM templates (green, Act88F-GFP) in the hdp3 mutant. The fusion process is not affected (yellow colour is as a result of overlaying of the red and green). Anterior to the lower left-hand corner, posterior to upper right-hand corner. Bar, 0.076 mm. (B) Wild-type DLM myotubes at around 40 hours APF (immunolocalised with Dmef2 expression). Fibres are elongated with syncytial nuclei arrayed in lines. Bar, 0.09 mm. (C) hdp3 myotubes at the same stage with the nuclei in arrays, indicating that myoblast fusion is not affected. hdp3 fibres are shorter than wildtype and never extend completely to reach the attachment sites. Bar, 0.115 mm. For both B and C, anterior is to the top; dorsal towards the left. (D) Wild-type IFM development at 42 hours APF monitored with Act88F-GFP expression. The fibres completely extend to the cuticle. The star indicates a DLM and `I' overlays one of the DVM. (E) hdp3 mutant DLM and DVM (I) fibres at 42 hours APF are shorter than wildtype and start degrading (arrow) from the fibre ends. (F) hdp3 DLM fibres at 46-48 hours APF. Fibres are clumped together and pulled to sides (arrowheads) and completely degrade with time. No fibres are seen in newly eclosed adults (see Fig. 7A). Anterior to the right-hand side for panels D-F. Only one hemi-segment shown in all figures. Bars, 0.1 mm (D-F).

View larger version (151K): [in a new window]   Fig. 4. Myofibril assembly in wild-type and hdp3 flies. Immunolocalisation of myosin (green: FITC-labelled antibody) and F-actin filaments (red, rhodamine-phalloidin labelled). (A) Wild-type myofibrils: single myofibrils are visible with thick (arrowheads showing two alternate A-band regions) and thin (arrows indicating two alternate I-band regions) filaments assembled as clearly demarcated repetitive I-A-I bands. (B) hdp3 mutant myofibrils are clumped as a bundle of barely distinguishable myofibrils with no clear sarcomere-like structures. Thick and thin filaments are seen, but scattered without a definite structure. The asterisk indicates the degenerating region of the developing fibre. Bars, 1.6 µm.

View larger version (124K): [in a new window]   Fig. 5. Electron micrographs of developing myofibrils. (A) Longitudinal section (LS) of 42 hours APF wild-type myofibrils, seen singly with uniform length sarcomeres demarcated by straight electron dense Z-discs (arrowheads). (B) LS of 46-48 hours APF wild type myofibrils, well-defined sarcomeres are seen (arrowheads to show well-formed, Z-disc demarcated sarcomeres). (C) LS of hdp3 mutant myofibril at 42 hours APF. No myofibrillar or sarcomeric structures are present but larger, stretched and rather bulbous Z-discs (arrow) are seen. (D) LS of hdp3 myofibril at 46-48 hours APF. Myofilaments (now in bundled fibres) show no definite sarcomeric structure or Z-discs (asterisk). Bars, 0.58 µm

View larger version (96K): [in a new window]   Fig. 6. The hypercontraction phenotype of hdp3/+ heterozygotes. Polarized light micrographs of (A) wild-type IFM (the star indicates a single DLM and the diamond indicates the TDT) and (B) hypercontracted (arrowheads) IFM from hdp3/+ flies; note the hypercontracted TDT (arrow). Electron micrograph of transverse section (TS) of (C) normal, wild-type and (D) hdp3/+ heterozygote myofibrils showing the precise myofibrillar lattice of wildtype and the irregular and loose packing of the mutant myofibril, but with normal thick-thin filament lattice at its centre. Electron micrograph of LS of (E) wild-type myofibril showing the regular sarcomeres with their Z discs and M lines, and (F) of hdp3/+ myofibrils in which Z-discs (arrowheads) and M-lines (arrow) appear streamed. Bars, 0.12 mm (A,B); 0.63 µm (C-F).

View larger version (83K): [in a new window]   Fig. 7. Reducing acto-myosin force suppresses hdp3 fibre hypercontraction. Polarised light micrograph hemithoraces of (A) hdp3, with no IFM and TDT is visible [asterisk shows where IFM and TDT are normally present (compare to Fig. 6A)], (B) hdp3/Y; Mhc10/+, Y97, with only a small bunch of muscle mass visible (arrow), (C) hdp3/Y; Mhc10/+, Y97/Y97, with increased muscle mass (arrow) and (D) hdp3/Y; Mhc10/Mhc10, Y97/Y97, where muscle fibres are present comparable with the Mhc10/Mhc10, Y97/Y97 genotype (not shown), in which fibres extend the length of the thorax. The star indicates a DLM; thinned fibre ends (arrowheads) are still visible. Anterior to left; dorsal to top in all the figures. Bars, 0.138 mm (A-D). Electron micrographs of LS of (E) hdp3/Y; Mhc7/MhcP401S IFM to show presence of Z-discs (absent in hdp3 homozygotes) showing the presence of `tiger-tail' assemblies (arrowheads) and thick filaments (arrows), and (F) hdp3; Mhc10; Y97 IFM to illustrate the increased number of thick filaments (arrows), scant thin filaments and Z-disc like bodies (arrowheads), although these are associated with only very small amounts of short thin filaments. Bars, 0.5 µm (E-F).

View larger version (24K): [in a new window]   Fig. 8. Expression of thin filament muscle protein and mRNA are reduced in the absence of TnI. (A) RT-PCR amplification shows decreased thin filament mRNA levels but no reduction in the myosin heavy chain control. (B) Immuno-western blots of IFM proteins from different genotypes show significant reductions in the amount of all the thin filament proteins. Note that arthrin, the IFM-specific, mono-ubiquitinated actin isoform, is missing and that the muscle samples used for the TnI immunoblotting are of whole thoracic proteins to show that the smaller, non-IFM isoform (TnI-E*) is expressed at normal levels in hdp3 flies, while the IFM-specific isoform (TnI-A*), present in the Canton-S and Mhc12 samples, is missing. (C) Immunoblotting of dissected `skinned' IFM samples from wild-type and hdp3; Mhc12 flies at 40-48, 60-75 hours APF and from adult flies. Ponceau-staining of the glutathione suphuryl transferase 2 (GST-2) band (Clayton et al., 1998) was used to indicate relative protein loadings.

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