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Flightin is essential for thick filament assembly and sarcomere stability in Drosophila flight muscles.

Reedy MC, Bullard B, Vigoreaux JO - J. Cell Biol. (2000)

Bottom Line: We have created a mutation for flightin, fln(0), that results in loss of flight ability but has no effect on fecundity or viability.That myosin is cleaved in the absence of flightin is consistent with the immunolocalization of flightin on the thick filament and biochemical and genetic evidence suggesting it is associated with the myosin rod.Our results indicate that flightin is required for the establishment of normal thick filament length during late pupal development and thick filament stability in adult after initiation of contractile activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Flightin is a multiply phosphorylated, 20-kD myofibrillar protein found in Drosophila indirect flight muscles (IFM). Previous work suggests that flightin plays an essential, as yet undefined, role in normal sarcomere structure and contractile activity. Here we show that flightin is associated with thick filaments where it is likely to interact with the myosin rod. We have created a mutation for flightin, fln(0), that results in loss of flight ability but has no effect on fecundity or viability. Electron microscopy comparing pupa and adult fln(0) IFM shows that sarcomeres, and thick and thin filaments in pupal IFM, are 25-30% longer than in wild type. fln(0) fibers are abnormally wavy, but sarcomere and myotendon structure in pupa are otherwise normal. Within the first 5 h of adult life and beginning of contractile activity, IFM fibers become disrupted as thick filaments and sarcomeres are variably shortened, and myofibrils are ruptured at the myotendon junction. Unusual empty pockets and granular material interrupt the filament lattice of adult fln(0) sarcomeres. Site-specific cleavage of myosin heavy chain occurs during this period. That myosin is cleaved in the absence of flightin is consistent with the immunolocalization of flightin on the thick filament and biochemical and genetic evidence suggesting it is associated with the myosin rod. Our results indicate that flightin is required for the establishment of normal thick filament length during late pupal development and thick filament stability in adult after initiation of contractile activity.

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Generation and characterization of a  allele of flightin. (A) Summary of genetic scheme to isolate  mutation in the flightin gene. Male flies fed the mutagen EMS were mated to females heterozygous for Df(3L)fln and the hemizygous progeny were scored for the absence of flightin by immunodot blots. (B) A representative dot blot. The sample encircled, 2987, did not react with an antiflightin mAb. (C) Strain 2987 does not express flightin. Western blot showing the accumulation of α-actinin (top) and flightin (bottom) in IFM of the following strains: (1) Oregon R (wild-type); (2) Df(3L)kto1/+, a deficiency of the 76BD region that does not delete the flightin gene; (3) Df(3L)fln/+; (4) 2987/+; (5) 2987/Df(3L)fln; (6) 2987/2987; (7) +/+ pupa. Note that flightin is not detected in 2987 homozygotes nor 2987/Df(3L)fln transheterozygotes. (D) DNA sequence analysis of the flightin gene from wild-type flies (top) and 2987 (bottom). Note the G to A transition converts a Trp codon at the eighth amino acid position into a stop codon. Herein we will refer to line 2987 as fln0.
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Figure 3: Generation and characterization of a allele of flightin. (A) Summary of genetic scheme to isolate mutation in the flightin gene. Male flies fed the mutagen EMS were mated to females heterozygous for Df(3L)fln and the hemizygous progeny were scored for the absence of flightin by immunodot blots. (B) A representative dot blot. The sample encircled, 2987, did not react with an antiflightin mAb. (C) Strain 2987 does not express flightin. Western blot showing the accumulation of α-actinin (top) and flightin (bottom) in IFM of the following strains: (1) Oregon R (wild-type); (2) Df(3L)kto1/+, a deficiency of the 76BD region that does not delete the flightin gene; (3) Df(3L)fln/+; (4) 2987/+; (5) 2987/Df(3L)fln; (6) 2987/2987; (7) +/+ pupa. Note that flightin is not detected in 2987 homozygotes nor 2987/Df(3L)fln transheterozygotes. (D) DNA sequence analysis of the flightin gene from wild-type flies (top) and 2987 (bottom). Note the G to A transition converts a Trp codon at the eighth amino acid position into a stop codon. Herein we will refer to line 2987 as fln0.

Mentions: A genetic screen was conducted to identify mutations that prevent flightin expression in the IFM (Fig. 3 A). Male flies were fed EMS (a depurinating agent), mated to female Df(3L)fln flies, and the hemizygous progeny was tested for the presence of flightin via dot blots. Of 1,280 flies tested, one (2987) did not express flightin (Fig. 3 B). A homozygous line established from a sibling did not express flightin (Fig. 3 C).


Flightin is essential for thick filament assembly and sarcomere stability in Drosophila flight muscles.

Reedy MC, Bullard B, Vigoreaux JO - J. Cell Biol. (2000)

Generation and characterization of a  allele of flightin. (A) Summary of genetic scheme to isolate  mutation in the flightin gene. Male flies fed the mutagen EMS were mated to females heterozygous for Df(3L)fln and the hemizygous progeny were scored for the absence of flightin by immunodot blots. (B) A representative dot blot. The sample encircled, 2987, did not react with an antiflightin mAb. (C) Strain 2987 does not express flightin. Western blot showing the accumulation of α-actinin (top) and flightin (bottom) in IFM of the following strains: (1) Oregon R (wild-type); (2) Df(3L)kto1/+, a deficiency of the 76BD region that does not delete the flightin gene; (3) Df(3L)fln/+; (4) 2987/+; (5) 2987/Df(3L)fln; (6) 2987/2987; (7) +/+ pupa. Note that flightin is not detected in 2987 homozygotes nor 2987/Df(3L)fln transheterozygotes. (D) DNA sequence analysis of the flightin gene from wild-type flies (top) and 2987 (bottom). Note the G to A transition converts a Trp codon at the eighth amino acid position into a stop codon. Herein we will refer to line 2987 as fln0.
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Figure 3: Generation and characterization of a allele of flightin. (A) Summary of genetic scheme to isolate mutation in the flightin gene. Male flies fed the mutagen EMS were mated to females heterozygous for Df(3L)fln and the hemizygous progeny were scored for the absence of flightin by immunodot blots. (B) A representative dot blot. The sample encircled, 2987, did not react with an antiflightin mAb. (C) Strain 2987 does not express flightin. Western blot showing the accumulation of α-actinin (top) and flightin (bottom) in IFM of the following strains: (1) Oregon R (wild-type); (2) Df(3L)kto1/+, a deficiency of the 76BD region that does not delete the flightin gene; (3) Df(3L)fln/+; (4) 2987/+; (5) 2987/Df(3L)fln; (6) 2987/2987; (7) +/+ pupa. Note that flightin is not detected in 2987 homozygotes nor 2987/Df(3L)fln transheterozygotes. (D) DNA sequence analysis of the flightin gene from wild-type flies (top) and 2987 (bottom). Note the G to A transition converts a Trp codon at the eighth amino acid position into a stop codon. Herein we will refer to line 2987 as fln0.
Mentions: A genetic screen was conducted to identify mutations that prevent flightin expression in the IFM (Fig. 3 A). Male flies were fed EMS (a depurinating agent), mated to female Df(3L)fln flies, and the hemizygous progeny was tested for the presence of flightin via dot blots. Of 1,280 flies tested, one (2987) did not express flightin (Fig. 3 B). A homozygous line established from a sibling did not express flightin (Fig. 3 C).

Bottom Line: We have created a mutation for flightin, fln(0), that results in loss of flight ability but has no effect on fecundity or viability.That myosin is cleaved in the absence of flightin is consistent with the immunolocalization of flightin on the thick filament and biochemical and genetic evidence suggesting it is associated with the myosin rod.Our results indicate that flightin is required for the establishment of normal thick filament length during late pupal development and thick filament stability in adult after initiation of contractile activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Flightin is a multiply phosphorylated, 20-kD myofibrillar protein found in Drosophila indirect flight muscles (IFM). Previous work suggests that flightin plays an essential, as yet undefined, role in normal sarcomere structure and contractile activity. Here we show that flightin is associated with thick filaments where it is likely to interact with the myosin rod. We have created a mutation for flightin, fln(0), that results in loss of flight ability but has no effect on fecundity or viability. Electron microscopy comparing pupa and adult fln(0) IFM shows that sarcomeres, and thick and thin filaments in pupal IFM, are 25-30% longer than in wild type. fln(0) fibers are abnormally wavy, but sarcomere and myotendon structure in pupa are otherwise normal. Within the first 5 h of adult life and beginning of contractile activity, IFM fibers become disrupted as thick filaments and sarcomeres are variably shortened, and myofibrils are ruptured at the myotendon junction. Unusual empty pockets and granular material interrupt the filament lattice of adult fln(0) sarcomeres. Site-specific cleavage of myosin heavy chain occurs during this period. That myosin is cleaved in the absence of flightin is consistent with the immunolocalization of flightin on the thick filament and biochemical and genetic evidence suggesting it is associated with the myosin rod. Our results indicate that flightin is required for the establishment of normal thick filament length during late pupal development and thick filament stability in adult after initiation of contractile activity.

Show MeSH
Related in: MedlinePlus