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Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle.

Kulke M, Neagoe C, Kolmerer B, Minajeva A, Hinssen H, Bullard B, Linke WA - J. Cell Biol. (2001)

Bottom Line: After extraction of the kettin-associated actin, the A-band edges were also stained.Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin.We conclude that kettin is attached not only to actin but also to the end of the thick filament.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology and Pathophysiology, University of Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and alpha-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM-I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with mu-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin- mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.

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Isoforms of large insect muscle proteins studied by SDS-PAGE and immunoblotting and dotblot to probe kettin-myosin binding. (A) Coomassie-stained 2% SDS-gels to separate projectin and kettin of Lethocerus IFM/leg muscle or Drosophila IFM myofibrils. Drosophila thoraces frozen in liquid nitrogen immediately after dissection were also included in the analysis (right two lanes; right lane is silver stain). For size comparison and to construct a calibration curve for high molecular weight proteins, large rat cardiac and rabbit psoas proteins were used. P, projectin; K, kettin; *unidentified band. (B) Immunoblots. Proteins in Drosophila thoraces (lane 1) or washed IFM myofibrils (lanes 2–6) were separated on 2.5–7.5 SDS-gradient gels. Lane 4 was run with IFM myofibrils from the Drosophila actin- mutant, KM88. Immunoblots in lanes 1–4 were incubated with α-kettin Ig16; lane 3 is a longer exposure of lane 2, whereas lanes 3 and 4 were exposed for the same time to compare relative amounts of 500-kD kettin in wild-type and actin-. Lane 5 was incubated with α-kettin Ig34/35 and lane 6 with 9D10 antibody to PEVK. Molecular masses are estimated relative to kettin (500 kD) and projectin (900 kD). (C) Dotblot to show binding of expressed kettin Ig34/35 domains to myosin. Upper strip, dots of 2.0, 1.0, and 0.5 μg of Lethocerus myosin incubated in Ig34/35 followed by anti-His and second antibody; lower strip, myosin dots incubated with anti-His and second antibody only.
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fig7: Isoforms of large insect muscle proteins studied by SDS-PAGE and immunoblotting and dotblot to probe kettin-myosin binding. (A) Coomassie-stained 2% SDS-gels to separate projectin and kettin of Lethocerus IFM/leg muscle or Drosophila IFM myofibrils. Drosophila thoraces frozen in liquid nitrogen immediately after dissection were also included in the analysis (right two lanes; right lane is silver stain). For size comparison and to construct a calibration curve for high molecular weight proteins, large rat cardiac and rabbit psoas proteins were used. P, projectin; K, kettin; *unidentified band. (B) Immunoblots. Proteins in Drosophila thoraces (lane 1) or washed IFM myofibrils (lanes 2–6) were separated on 2.5–7.5 SDS-gradient gels. Lane 4 was run with IFM myofibrils from the Drosophila actin- mutant, KM88. Immunoblots in lanes 1–4 were incubated with α-kettin Ig16; lane 3 is a longer exposure of lane 2, whereas lanes 3 and 4 were exposed for the same time to compare relative amounts of 500-kD kettin in wild-type and actin-. Lane 5 was incubated with α-kettin Ig34/35 and lane 6 with 9D10 antibody to PEVK. Molecular masses are estimated relative to kettin (500 kD) and projectin (900 kD). (C) Dotblot to show binding of expressed kettin Ig34/35 domains to myosin. Upper strip, dots of 2.0, 1.0, and 0.5 μg of Lethocerus myosin incubated in Ig34/35 followed by anti-His and second antibody; lower strip, myosin dots incubated with anti-His and second antibody only.

Mentions: In a more systematic effort to detect high molecular mass proteins, 2% SDS-PAGE was carried out on myofibrils from both Drosophila and Lethocerus IFM and Lethocerus leg muscle (Fig. 7 A). Vertebrate titin and nebulin served as size markers. The analysis confirmed that all insect muscle types contain various large proteins (Fig. 7 A, left). In Drosophila IFM, stronger protein bands likely correspond to 400-kD M-line protein, 500-kD kettin, and ∼900-kD projectin (Fig. 7 A, lane 3). A faint band appearing at ∼700 kD may correspond to another kettin isoform (see next paragraph). The possibility exists that larger titin-like proteins were not visualized because of degradation during myofibril preparation. (The ORFs from the genome sequence of D-titin predict that the maximum size of the protein is 1.8 MD [Machado and Andrew, 2000; Zhang et al., 2000].) Therefore, Drosophila thoraces were frozen in liquid nitrogen immediately after dissection and were also tested for their protein composition (Fig. 7 A, right). No protein larger than ∼1,100–1,200 kD was detectable, even on SDS-gels stained with silver to improve sensitivity. Thus, Drosophila flight muscles, which make up a majority of the thoracic muscles, may not express a protein of 1.8-MD size corresponding to a full-length D-titin. Lethocerus leg muscle has a protein of high molecular mass not present in the IFM.


Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle.

Kulke M, Neagoe C, Kolmerer B, Minajeva A, Hinssen H, Bullard B, Linke WA - J. Cell Biol. (2001)

Isoforms of large insect muscle proteins studied by SDS-PAGE and immunoblotting and dotblot to probe kettin-myosin binding. (A) Coomassie-stained 2% SDS-gels to separate projectin and kettin of Lethocerus IFM/leg muscle or Drosophila IFM myofibrils. Drosophila thoraces frozen in liquid nitrogen immediately after dissection were also included in the analysis (right two lanes; right lane is silver stain). For size comparison and to construct a calibration curve for high molecular weight proteins, large rat cardiac and rabbit psoas proteins were used. P, projectin; K, kettin; *unidentified band. (B) Immunoblots. Proteins in Drosophila thoraces (lane 1) or washed IFM myofibrils (lanes 2–6) were separated on 2.5–7.5 SDS-gradient gels. Lane 4 was run with IFM myofibrils from the Drosophila actin- mutant, KM88. Immunoblots in lanes 1–4 were incubated with α-kettin Ig16; lane 3 is a longer exposure of lane 2, whereas lanes 3 and 4 were exposed for the same time to compare relative amounts of 500-kD kettin in wild-type and actin-. Lane 5 was incubated with α-kettin Ig34/35 and lane 6 with 9D10 antibody to PEVK. Molecular masses are estimated relative to kettin (500 kD) and projectin (900 kD). (C) Dotblot to show binding of expressed kettin Ig34/35 domains to myosin. Upper strip, dots of 2.0, 1.0, and 0.5 μg of Lethocerus myosin incubated in Ig34/35 followed by anti-His and second antibody; lower strip, myosin dots incubated with anti-His and second antibody only.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196178&req=5

fig7: Isoforms of large insect muscle proteins studied by SDS-PAGE and immunoblotting and dotblot to probe kettin-myosin binding. (A) Coomassie-stained 2% SDS-gels to separate projectin and kettin of Lethocerus IFM/leg muscle or Drosophila IFM myofibrils. Drosophila thoraces frozen in liquid nitrogen immediately after dissection were also included in the analysis (right two lanes; right lane is silver stain). For size comparison and to construct a calibration curve for high molecular weight proteins, large rat cardiac and rabbit psoas proteins were used. P, projectin; K, kettin; *unidentified band. (B) Immunoblots. Proteins in Drosophila thoraces (lane 1) or washed IFM myofibrils (lanes 2–6) were separated on 2.5–7.5 SDS-gradient gels. Lane 4 was run with IFM myofibrils from the Drosophila actin- mutant, KM88. Immunoblots in lanes 1–4 were incubated with α-kettin Ig16; lane 3 is a longer exposure of lane 2, whereas lanes 3 and 4 were exposed for the same time to compare relative amounts of 500-kD kettin in wild-type and actin-. Lane 5 was incubated with α-kettin Ig34/35 and lane 6 with 9D10 antibody to PEVK. Molecular masses are estimated relative to kettin (500 kD) and projectin (900 kD). (C) Dotblot to show binding of expressed kettin Ig34/35 domains to myosin. Upper strip, dots of 2.0, 1.0, and 0.5 μg of Lethocerus myosin incubated in Ig34/35 followed by anti-His and second antibody; lower strip, myosin dots incubated with anti-His and second antibody only.
Mentions: In a more systematic effort to detect high molecular mass proteins, 2% SDS-PAGE was carried out on myofibrils from both Drosophila and Lethocerus IFM and Lethocerus leg muscle (Fig. 7 A). Vertebrate titin and nebulin served as size markers. The analysis confirmed that all insect muscle types contain various large proteins (Fig. 7 A, left). In Drosophila IFM, stronger protein bands likely correspond to 400-kD M-line protein, 500-kD kettin, and ∼900-kD projectin (Fig. 7 A, lane 3). A faint band appearing at ∼700 kD may correspond to another kettin isoform (see next paragraph). The possibility exists that larger titin-like proteins were not visualized because of degradation during myofibril preparation. (The ORFs from the genome sequence of D-titin predict that the maximum size of the protein is 1.8 MD [Machado and Andrew, 2000; Zhang et al., 2000].) Therefore, Drosophila thoraces were frozen in liquid nitrogen immediately after dissection and were also tested for their protein composition (Fig. 7 A, right). No protein larger than ∼1,100–1,200 kD was detectable, even on SDS-gels stained with silver to improve sensitivity. Thus, Drosophila flight muscles, which make up a majority of the thoracic muscles, may not express a protein of 1.8-MD size corresponding to a full-length D-titin. Lethocerus leg muscle has a protein of high molecular mass not present in the IFM.

Bottom Line: After extraction of the kettin-associated actin, the A-band edges were also stained.Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin.We conclude that kettin is attached not only to actin but also to the end of the thick filament.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology and Pathophysiology, University of Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and alpha-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM-I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with mu-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin- mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.

Show MeSH
Related in: MedlinePlus