Transformation of Drosophila melanogaster with the wild-type myosin heavy-chain gene: rescue of mutant phenotypes and analysis of defects caused by overexpression.

Cripps, Richard M.; Becker, Kurt; Mardahl, Maibritt; Kronert, William A.; Hodges, Dianne; Bernstein, Sanford I.

doi:10.1083/jcb.126.3.689

Cited by 48 publications

(50 citation statements)

References 45 publications

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“…Consistent with an imbalance between thin and thick filaments Cripps et al, 1994), we have observed fraying of their hexagonal packing with unintegrated thick filaments only residing at the periphery of myofibrils.…”

Section: Tags At the N Terminus Of Actin May Influence Actin-myosin Isupporting

confidence: 62%

Differential Epitope Tagging of Actin in TransformedDrosophilaProduces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

Brault¹,

Sauder

Reedy

et al. 1999

MBoC

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We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments. INTRODUCTIONActins, a highly conserved family of cytoplasmic proteins, are among the most abundant proteins in eukaryotic cells. As a major component of the cytoskeleton, they control shape and motility in nonmuscle cells. In muscle, actin assembles into thin filaments, which together with interdigitating myosin thick filaments provide the framework for muscle contraction.Many organisms synthesize multiple isoforms of actin that are very similar in amino acid sequence even within the same cell. The differential expression of distinct actins as well as the high conservation of specific isoforms across species emphasize the functional importance of isoforms. In the case of actin, the question of how structure determines function appears to be particularly challenging. Considerable efforts have been made to understand how the different isoforms fulfill their various functions despite their extremely high sequence identity, and yet the basis of their functional diversity remains elusive. § Corresponding author. E-mail address: Schoenenberg@ ubaclu.unibas.ch. © 1999 by The American Society for Cell Biology 135Studying the specific role of a particular actin isoform has always been hampered by the difficulty of discriminating between the introduced a...

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Section: Tags At the N Terminus Of Actin May Influence Actin-myosin Isupporting

confidence: 62%

Differential Epitope Tagging of Actin in TransformedDrosophilaProduces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

Brault¹,

Sauder

Reedy

et al. 1999

MBoC

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“…Our model can also explain the sequential activation of different genes, because those activated at low thresholds of mef2 activity would be expressed before genes that are activated at higher thresholds. Last, this temporal regulation by Mef2 may also contribute to the absolute level and relative stoichiometry of expression of sarcomeric proteins, which is known to be important in myogenesis (14)(15)(16)(17)(18).…”

Section: Discussionmentioning

confidence: 99%

“…This highlighted that the analysis of how mef2 functions is central to understanding how muscle is made. Important characteristics of the underlying genetic program include the temporal coordination of muscle gene expression (11)(12)(13) and the regulation of levels and relative stoichiometries of gene expression during myogenesis (14)(15)(16)(17)(18)(19). Although these basic features have been known for many years, much remains to be understood about them.…”

mentioning

confidence: 99%

mef2 activity levels differentially affect gene expression during Drosophila muscle development

Elgar

Han

Taylor

2008

Proc. Natl. Acad. Sci. U.S.A.

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Cell differentiation is controlled by key transcription factors, and a major question is how they orchestrate cell-type-specific genetic programs. Muscle differentiation is a well studied paradigm in which the conserved Mef2 transcription factor plays a pivotal role. Recent genomic studies have identified a large number of mef2-regulated target genes with distinct temporal expression profiles during Drosophila myogenesis. However, the question remains as to how a single transcription factor can control such diverse patterns of gene expression. In this study we used a strategy combining genomics and developmental genetics to address this issue in vivo during Drosophila muscle development. We found that groups of mef2-regulated genes respond differently to changes in mef2 activity levels: some require higher levels for their expression than others. Furthermore, this differential requirement correlates with when the gene is first expressed during the muscle differentiation program. Genes that require higher levels are activated later. These results implicate mef2 in the temporal regulation of muscle gene expression, and, consistent with this, we show that changes in mef2 activity levels can alter the start of gene expression in a predictable manner. Together these results indicate that Mef2 is not an all-or-none regulator; rather, its action is more subtle, and levels of its activity are important in the differential expression of muscle genes. This suggests a route by which mef2 can orchestrate the muscle differentiation program and contribute to the stringent regulation of gene expression during myogenesis. muscle differentiation program ͉ transcription factor levels F or several decades it has been appreciated that the controlled regulation of gene expression, including the coordinated activation of batteries of genes, lies behind cell differentiation programs (1-3). It is now clear that a principle tier of control of cell differentiation is through key transcription factors, and an important general question is how these factors coordinate the genetic program of such complex processes. A classic paradigm is muscle, in which the conserved Mef2 transcription factor is a major regulator of gene expression and differentiation (4). Mef2 was first identified in mammalian cell culture (5-7), but because mammals possess four closely related mef2 genes functional analyses during development are complicated. In contrast, Drosophila has a single mef2 gene and was the first organism to be used to show that mef2 is required for muscle development in vivo (8)(9)(10). This highlighted that the analysis of how mef2 functions is central to understanding how muscle is made. Important characteristics of the underlying genetic program include the temporal coordination of muscle gene expression (11-13) and the regulation of levels and relative stoichiometries of gene expression during myogenesis (14-19). Although these basic features have been known for many years, much remains to be understood about them. However, the identificati...

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“…Thoraces from late pupa and 2-d-old female flies were isolated and prepared for transmission electron microscopy according to Cripps et al (1994). Fixatives and Embed812 resin were purchased from Electron Microscopy Sciences (Fort Washington, PA); other reagents were purchased from Sigma-Aldrich (St. Louis, MO).…”

Section: Electron Microscopy Of Drosophila Ifmmentioning

confidence: 99%

Myosin Transducer Mutations Differentially Affect Motor Function, Myofibril Structure, and the Performance of Skeletal and Cardiac Muscles

Cammarato

Dambacher

Knowles

et al. 2008

MBoC

116

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Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc 5 affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc 5 (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc 5 myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc 5 mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders. INTRODUCTIONThe myosin molecular motor of striated muscle is a hexameric molecule composed of two myosin heavy chains (MHCs) and four light chains. The N-terminal globular motor domain is a product inhibited ATPase comprised of several communicating domains and functional units (reviewed by Holmes, 1999, 2005;. Alterations to the various domains dramatically affect the biochemical and mechanical properties of the motor in vitro, and mutations that diminish or enhance the molecular performance of myosin in vivo are associated with the pathogenesis of both skeletal and cardiac myopathies (reviewed by Sellers, 1999;Redowicz, 2002;Oldfors et al., 2004;Chang and Potter, 2005;Laing and Nowak, 2005;Tardiff, 2005;Oldfors, 2007). Central to an understanding of myosinbased myopathies is a fundamental appreciation for how depressed or enhanced molecular motor function differentially affects diverse striated muscles.Among the communicating functional units of myosin is the recently described transducer region (Coureux et al., 2004). Myosin V crystal structures reveal the transducer's central location within the motor domain near the nucleotide binding site. The transducer includes the last three strands of a seven-stranded ␤-sheet, which undergoes distortion essential for rearrangements within the nucleotide pocket during the ATPase cycle, and the loops an...

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Transformation of Drosophila melanogaster with the wild-type myosin heavy-chain gene: rescue of mutant phenotypes and analysis of defects caused by overexpression.

Cited by 48 publications

References 45 publications

Differential Epitope Tagging of Actin in TransformedDrosophilaProduces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

Differential Epitope Tagging of Actin in TransformedDrosophilaProduces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

mef2 activity levels differentially affect gene expression during Drosophila muscle development

Myosin Transducer Mutations Differentially Affect Motor Function, Myofibril Structure, and the Performance of Skeletal and Cardiac Muscles

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