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John C Sparrow

Publications and source records attributed to John C Sparrow.

11 recordsLinked to original sources
PubMed2004

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

Myofibrillar proteins assemble to form the highly ordered repetitive contractile structural unit known as a sarcomere. Studies of myogenesis in vertebrate cell culture and embryonic developmental systems have identified some of the processes involved during sarcomere formation. However, isoform changes during vertebrate muscle development and a lack of mutants have made it difficult to determine how these proteins assemble to form sarcomeres. The indirect flight muscles (IFMs) of Drosophila provide a unique genetic system with which to study myofibrillogenesis in vivo. We show in this paper that neither sarcomeric myosin nor actin are required for myoblast fusion or the subsequent morphogenesis of muscle fibres, i.e. fibre morphogenesis does not depend on myofibrillogenesis. However, fibre formation and myofibrillogenesis are very sensitive to the interactions between the sarcomeric proteins. A troponin I (TnI) mutation, hdp(3), leads to an absence of TnI in the IFMs and tergal depressor of trochanter (TDT) muscles due to a transcript-splicing defect. Sarcomeres do not form and the muscles degenerate. TnI is part of the thin filament troponin complex which regulates muscle contraction. The effects of the hdp(3) mutation are probably caused by unregulated acto-myosin interactions between the thin and thick filaments as they assemble. We have tested this proposal by using a transgenic myosin construct to remove the force-producing myosin heads. The defects in sarcomeric organisation and fibre degeneration in hdp(3) IFMs are suppressed, although not completely, indicating the need for inhibition of muscle contraction during muscle development. We show that mRNA and translated protein products of all the major thin filament proteins are reduced in hdp(3) muscles and discuss how this and previous studies of thin filament protein mutants indicate a common co-ordinated control mechanism that may be the primary cause of the muscle defects.

Actins↗
PubMed2004

Actin mutations are one cause of congenital fibre type disproportion.

We report three heterozygous missense mutations of the skeletal muscle alpha actin gene (ACTA1) in three unrelated cases of congenital fiber type disproportion (CFTD) from Japan and Australia. This represents the first genetic cause of CFTD to be identified and confirms that CFTD is genetically heterogeneous. The three mutations we have identified Leucine221Proline, Aspartate292Valine, and Proline332Serine are novel. They have not been found previously in any cases of nemaline, actin, intranuclear rod, or rod-core myopathy caused by mutations in ACTA1. It remains unclear why these mutations cause type 1 fiber hypotrophy but no nemaline bodies. The three mutations all lie on one face of the actin monomer on the surface swept by tropomyosin during muscle activity, which may suggest a common pathological mechanism. All three CFTD cases with ACTA1 mutations had severe congenital weakness and respiratory failure without ophthalmoplegia. There were no clinical features specific to CFTD cases with ACTA1 mutations, but the presence of normal eye movements in a severe CFTD patient may be an important clue for the presence of a mutation in ACTA1.

Actins↗
PubMed2004

Drosophila muscle regulation characterized by electron microscopy and three-dimensional reconstruction of thin filament mutants.

Wild-type and mutant thin filaments were isolated directly from "myosinless" Drosophila indirect flight muscles to study the structural basis of muscle regulation genetically. Negatively stained filaments showed tropomyosin with periodically arranged troponin complexes in electron micrographs. Three-dimensional helical reconstruction of wild-type filaments indicated that the positions of tropomyosin on actin in the presence and absence of Ca(2+) were indistinguishable from those in vertebrate striated muscle and consistent with a steric mechanism of regulation by troponin-tropomyosin in Drosophila muscles. Thus, the Drosophila model can be used to study steric regulation. Thin filaments from the Drosophila mutant heldup(2), which possesses a single amino acid conversion in troponin I, were similarly analyzed to assess the Drosophila model genetically. The positions of tropomyosin in the mutant filaments, in both the Ca(2+)-free and the Ca(2+)-induced states, were the same, and identical to that of wild-type filaments in the presence of Ca(2+). Thus, cross-bridge cycling would be expected to proceed uninhibited in these fibers, even in relaxing conditions, and this would account for the dramatic hypercontraction characteristic of these mutant muscles. The interaction of mutant troponin I with Drosophila troponin C is discussed, along with functional differences between troponin C from Drosophila and vertebrates.

Actin Cytoskeleton↗
PubMed2004

Distance and force production during jumping in wild-type and mutant Drosophila melanogaster.

In many insects renowned for their jumping ability, elastic storage is used so that high forces can be developed prior to jumping. We have combined physiological, behavioural and genetic approaches to test whether elastic energy storage makes a major contribution to jumping in Drosophila. We describe a sensitive strain gauge setup, which measures the forces produced by tethered flies through their mesothoracic legs. The peak force produced by the main jumping muscle of female flies from a wild-type (Canton-S) strain is 101+/-4.4 microN [and this is indistinguishable from a second wild-type (Texas) strain]. The force takes 8.2 ms to reach its peak. The peak force is not affected significantly by altering the leg angle (femur-tibia joint angle) in the range of 75-120 degrees, but the peak force declines as the leg is extended further. Measurements of jumping ability (distance jumped) showed that female Drosophila (with their wings removed) of two wild-type strains, Canton-S and Texas, produced jumps of 28.6+/-0.7 and 30.2+/-1.0 mm (mean +/- s.e.m.). For a female wild-type Drosophila, a jump of 30 mm corresponds to a kinetic energy of 200 nJ on take-off (allowing 20% of the energy to overcome air resistance). We develop equations of motion for a linear force-time model of take-off and calculate that the time to take-off is 5.0 ms and the peak force should be 274 microN (137 microN leg(-1)). We predicted, from the role of octopamine in enhancing muscle tension in several locust muscles, that if stored elastic energy plays no part in force development, then genetic manipulation of the octopaminergic system would directly affect force production and jumping in Drosophila. Using two mutants deficient in the octopaminergic system, TbhnM18 (M18) and TyrRhono (hono), we found significantly reduced jumping distances (20.7+/-0.7 and 20.7+/-0.4 mm, respectively) and force production (52% and 55%, respectively) compared with wild type. From the reduced distance and force production in M18, a mutant deficient in octopamine synthesis, and in hono, a tyramine/octopamine receptor mutant, we conclude that in Drosophila, as in locusts, octopamine modulates escape jumping. We conclude that the fly does not need to store large quantities of elastic energy in order to make its jump because (1) the measured and calculated forces agree to within 40% and (2) the reduction in distances jumped by the mutants correlates well with their reduction in measured peak force.

Animals↗
PubMed2003

Molecular evolutionary convergence of the flight muscle protein arthrin in Diptera and hemiptera.

Uniquely, the asynchronous flight muscle myofibrils of many insects contain arthrin, a stable 1:1 conjugate between actin and ubiquitin. The function of arthrin is still unknown. Here we survey for the presence of arthrin in 63 species of insect across nine orders using Western blotting. Analysis of the evolutionary distribution shows that arthrin has evolved a limited number of times but at least once in the Diptera and once in the Hemiptera. However, the presence of arthrin does not correlate with any observed common features of flight mechanism, natural history, or morphology. We also identify the site of the isopeptide bond in arthrin from Drosophila melanogaster (Diptera) and Lethocerus griseus (Hemiptera) using mass spectrometry. In both species, the isopeptide bond is formed between lysine 118 of the actin and the C-terminal glycine 76 of ubiquitin. Thus, not only the ubiquitination of actin but also the site of the isopeptide bond has evolved convergently in Diptera and Hemiptera. In terms of the actin monomer, lysine 118 is near neither the binding sites of the major actin-binding proteins, myosin, tropomyosin, or the troponins, nor the actin polymerization sites. However, molecular modeling supports the idea that ubiquitin bound to an actin in one F-actin strand might be able to interact with tropomyosin bound to the actin monomers of the other strand and thereby interfere with thin filament regulation.

Actins↗
PubMed2003

Muscle disease caused by mutations in the skeletal muscle alpha-actin gene (ACTA1).

Mutations in the skeletal muscle alpha-actin gene (ACTA1) associated with congenital myopathy with excess of thin myofilaments, nemaline myopathy and intranuclear rod myopathy were first described in 1999. At that time, only 15 different missense mutations were known in ACTA1. More than 60 mutations have now been identified. This review analyses this larger spectrum of mutations in ACTA1. It investigates the molecular consequences of the mutations found to date, provides a framework for genotype-phenotype correlation and suggests future studies in light of results of investigation of normal and mutant actin in other systems, notably the actin specific to the indirect flight muscles of Drosophila. The larger series confirms that the majority of ACTA1 mutations are dominant, a small number are recessive and most isolated cases with no previous family history have de novo dominant mutations. The severity of the disease caused ranges from lack of spontaneous movements at birth requiring immediate mechanical ventilation, to mild disease compatible with life to adulthood. Overall, the mutations within ACTA1 are randomly distributed throughout the protein. However, the larger series of mutations now available indicates that there may be clustering of mutations associated with some phenotypes, e.g. actin myopathy. This would suggest that interference with certain actin functions may be more associated with certain phenotypes, though the exact pathophysiology of the actin mutations remains unknown.

Actins↗
PubMed2003

Isolation and kinetic characterisation of myosin and myosin S1 from the Drosophila indirect flight muscles.

The potential to explore myosin function through the alternative exons and mutations of the single muscle myosin heavy chain gene, Mhc of Drosophila requires detailed kinetic analysis of the myosins. We have obtained microgram quantities of enzymatically active Drosophila myosin and subfragment 1 (S1) from dissected indirect flight muscles. Using recent developments in stopped-flow and flash-photolysis methods combined with fluorescent/light scattering technologies we have determined some of the key kinetic parameters of actin-myosin and myosin-nucleotide interactions. The rate of ATP-induced dissociation of actin from Drosophila myosin (0.23 microM(-1) s(-1)) and subfragment 1 (S1, 0.82 microM(-1) s(-1)) are both fast and similar to values measured for mammalian skeletal muscle myosins and S1 fragments respectively. The ATP-induced cross bridge dissociation of Drosophila acto.S1 is expected to be fast since, for a rapidly contracting muscle like the Drosophila flight muscle, the post power stroke cross bridge must detach rapidly from actin or become a drag on the contracting filament. ATP-induced detachment is preceded by ADP release and this is proposed as the rate-limiting step that defines muscle shortening velocity. We show that the affinity of ADP for acto.S1 at 400 microM is 2-3 fold weaker than fast vertebrate myosins. This leads to an estimate of the ADP release rate constant of 4000 s(-1). We show that this predicts a maximum shortening velocity very similar to that obtained from in vivo estimates of indirect flight muscle shortening. The data is therefore compatible with ADP dissociation limiting the in vivo shortening velocity.

Actins↗
PubMed2003

Suppression of muscle hypercontraction by mutations in the myosin heavy chain gene of Drosophila melanogaster.

The indirect flight muscles (IFM) of Drosophila melanogaster provide a good genetic system with which to investigate muscle function. Flight muscle contraction is regulated by both stretch and Ca(2+)-induced thin filament (actin + tropomyosin + troponin complex) activation. Some mutants in troponin-I (TnI) and troponin-T (TnT) genes cause a "hypercontraction" muscle phenotype, suggesting that this condition arises from defects in Ca(2+) regulation and actomyosin-generated tension. We have tested the hypothesis that missense mutations of the myosin heavy chain gene, Mhc, which suppress the hypercontraction of the TnI mutant held-up(2) (hdp(2)), do so by reducing actomyosin force production. Here we show that a "headless" Mhc transgenic fly construct that reduces the myosin head concentration in the muscle thick filaments acts as a dose-dependent suppressor of hypercontracting alleles of TnI, TnT, Mhc, and flightin genes. The data suggest that most, if not all, mutants causing hypercontraction require actomyosin-produced forces to do so. Whether all Mhc suppressors act simply by reducing the force production of the thick filament is discussed with respect to current models of myosin function and thin filament activation by the binding of calcium to the troponin complex.

Animals↗
PubMed2002

Contractility of single human dermal myofibroblasts and fibroblasts.

Human dermal myofibroblasts, characterised by the expression of alpha-smooth muscle actin, are part of the granulation tissue and implicated in the generation of contractile forces during normal wound healing and pathological contractures. We have compared the contractile properties of single human dermal fibroblasts and human dermal myofibroblasts by culturing them on flexible silicone elastomers. The flexibility of the silicone substratum permits the contractile forces exerted by the cells to be measured [Fray et al., 1998: Tissue Eng. 4:273-283], without changing their expression of alpha-smooth muscle actin. The mean contractile force produced by myofibroblasts (2.2 microN per cell) was not significantly different from that generated by fibroblasts (2.0 microN per cell) when cultured on a substrata with a low elastomer stiffness. Forces produced by fibroblasts were unaffected by increases in elastomer stiffness, but forces measured for myofibroblasts increased to a mean value of 4.1 microN/cell. This was associated with a higher proportion of myofibroblasts being able to produce wrinkles on elastomers of high stiffness compared to fibroblasts. We discuss the force measurements at the single cell level, for both fibroblast and myofibroblasts, in relation to the proposed role of myofibroblasts in wound healing and pathological contractures.

Adult↗
PubMed2002

Molecular motors: force and movement generated by single myosin II molecules.

Muscle myosin II is an ATP-driven, actin-based molecular motor. Recent developments in optical tweezers technology have made it possible to study movement and force production on the single-molecule level and to find out how different myosin isoforms may have adapted to their specific physiological roles.

Adenosine Triphosphate↗