UVM Theses and Dissertations
Format:
Print
Author:
Barton, Byron E.
Dept./Program:
Biology
Year:
2007
Degree:
PhD
Abstract:
Drosophila indirect flight muscle (IFM) is a highly ordered tissue composed of many myofibrils that generate the high power required for insect flight. Because of its precise structure, a mutation or the improper expression of a myofibrillar protein can have profound consequences on the function of the IFM. Flightin is a multiphosphorylated, myosin binding protein that in Drosophila is expressed specifically in IFM. Mutant flies that contain a genetic deficiency which deletes several genes, including flightin, are homozygous lethal and in heterozygotes have reduced flightin expression that results in decreased flight ability, alterations in myofibrillar structure and impaired fiber mechanics. A null mutation in flightin (fln⁰) results in flies with abnormally developed IFM sarcomeres, muscle fibers with altered viscoelastic properties in newly eclosed flies, hypercontracted fibers and disrupted myofibrils in adult flies, and flightlessness.
The first goal of this study was to create transgenic flies containing a wild type flightin transgene to determine if expression of flightin is sufficient to rescue the phenotype of fln⁰ flies. Two wild type transgene copies infln⁰ flies were sufficient to "rescue" the flight impairment, ultrastructural, and fiber mechanical defects observed in fln⁰ IFM. Tetraploid flies, containing two endogenous wild type flightin gene copies and two transgenic wild type genes, did not have an apparent affect on IFM function. These results suggest that flightin stoichiometry is determined post-transcriptionally and is likely to be dictated by the number of available myosin binding sites for flightin.
The second goal of this study was to determine the importance of flightin phosphorylation and the affects that individual phosphorylation sites may have on overall IFM function. Five phosphorylation sites (S139, S141, S145, T158, S162) were identified by MALDI-TOF analysis. Transgenic constructs were created in which two (fln²[superscript TSA]) three, (fln³[superscript SA]) and five (fln⁵[superscript STA])p hospho lated sites were mutated to alanine. In two to five day old adult flies, expression of fln⁵[superscript STA] in a fln⁰ background resulted in severe IFM hypercontraction and myofibrillar degeneration that closely resembled fln⁰. In a fln⁰ background, both flies with the fln²[superscript TSA] and fln³[superscript SA] were flightless, but fln²[superscript TSA] had a more severe hypercontracted phenotype, more myofibrillar degradation and fewer flightin isovariants detected on two-dimensional gels.
Mechanical analysis of newly eclosed mutant fibers indicated that while fibers of fln⁵[superscript STA] and fln²[superscript TSA] in a fln⁰ background were abnormal, fibers from flies with the fln³[superscript SA] transgene in afln⁰ background were normal. Expression of fln⁵[superscript STA] and fln²[superscript TSA] in a wild type background (fln/fln⁺) resulted in a dominant negative effect manifested as flight impairments, myofibrillar abnormalities and hypercontraction while expression of fln³[superscript SA] in a wild type background was normal. These results suggest that different phosphorylated residues in flightin, particularly T158 and S162, are important in the function of Drosophila IFM.
The first goal of this study was to create transgenic flies containing a wild type flightin transgene to determine if expression of flightin is sufficient to rescue the phenotype of fln⁰ flies. Two wild type transgene copies infln⁰ flies were sufficient to "rescue" the flight impairment, ultrastructural, and fiber mechanical defects observed in fln⁰ IFM. Tetraploid flies, containing two endogenous wild type flightin gene copies and two transgenic wild type genes, did not have an apparent affect on IFM function. These results suggest that flightin stoichiometry is determined post-transcriptionally and is likely to be dictated by the number of available myosin binding sites for flightin.
The second goal of this study was to determine the importance of flightin phosphorylation and the affects that individual phosphorylation sites may have on overall IFM function. Five phosphorylation sites (S139, S141, S145, T158, S162) were identified by MALDI-TOF analysis. Transgenic constructs were created in which two (fln²[superscript TSA]) three, (fln³[superscript SA]) and five (fln⁵[superscript STA])p hospho lated sites were mutated to alanine. In two to five day old adult flies, expression of fln⁵[superscript STA] in a fln⁰ background resulted in severe IFM hypercontraction and myofibrillar degeneration that closely resembled fln⁰. In a fln⁰ background, both flies with the fln²[superscript TSA] and fln³[superscript SA] were flightless, but fln²[superscript TSA] had a more severe hypercontracted phenotype, more myofibrillar degradation and fewer flightin isovariants detected on two-dimensional gels.
Mechanical analysis of newly eclosed mutant fibers indicated that while fibers of fln⁵[superscript STA] and fln²[superscript TSA] in a fln⁰ background were abnormal, fibers from flies with the fln³[superscript SA] transgene in afln⁰ background were normal. Expression of fln⁵[superscript STA] and fln²[superscript TSA] in a wild type background (fln/fln⁺) resulted in a dominant negative effect manifested as flight impairments, myofibrillar abnormalities and hypercontraction while expression of fln³[superscript SA] in a wild type background was normal. These results suggest that different phosphorylated residues in flightin, particularly T158 and S162, are important in the function of Drosophila IFM.