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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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Actin filaments in stretched Drosophila IFM sarcomeres. (A) Single myofibrils stained with rhodamine-phalloidin. Note actin filament breakage at Z-discs (arrows). (B) Electron micrographs of IFM sarcomeres. Myofilament breakage at the Z-disc (Z) was observed at modest stretch (arrow). M, M-line; (c) Actin staining of stretched single myofibrils after digestion with μ-calpain. Actin filaments of opposing half sarcomeres usually remained connected at the Z-disc (arrowheads); actin was seen rarely to be broken (arrow). (D) Actin staining of sarcomeres after extraction with 0.2–0.3 mg/ml (top) and >0.3 mg/ml (bottom) gelsolin fragment. Bars: (B) 0.5 μm; (all IF images) 5 μm.
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fig3: Actin filaments in stretched Drosophila IFM sarcomeres. (A) Single myofibrils stained with rhodamine-phalloidin. Note actin filament breakage at Z-discs (arrows). (B) Electron micrographs of IFM sarcomeres. Myofilament breakage at the Z-disc (Z) was observed at modest stretch (arrow). M, M-line; (c) Actin staining of stretched single myofibrils after digestion with μ-calpain. Actin filaments of opposing half sarcomeres usually remained connected at the Z-disc (arrowheads); actin was seen rarely to be broken (arrow). (D) Actin staining of sarcomeres after extraction with 0.2–0.3 mg/ml (top) and >0.3 mg/ml (bottom) gelsolin fragment. Bars: (B) 0.5 μm; (all IF images) 5 μm.

Mentions: Low extensibility and stretch-induced high SL inhomogeneity were consistently observed in relaxed Drosophila IFM myofibrils. Somewhat surprisingly, when nonactivated myofibrils were stretched by ∼10% from slack SL and stained with rhodamine-phalloidin, actin always appeared to be broken at the Z-disc (Fig. 3 A); it seemed impossible to pull the actin filaments out of the A-band. Myofilament breakage at the Z-disc could be observed also on electron micrographs of stretched IFM sarcomeres (Fig. 3 B). In addition, actin-filament breakage was seen in stretched myofibrils stained with α-actin antibodies (unpublished data). A possible explanation of this result is that some actin-myosin connections remained in the A-band, despite the presence of 20 mM BDM (an active force-suppressing agent) in the relaxing buffer. Alternatively, some additional structure(s) could link actin and myosin filaments thereby stiffening the sarcomere.


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)

Actin filaments in stretched Drosophila IFM sarcomeres. (A) Single myofibrils stained with rhodamine-phalloidin. Note actin filament breakage at Z-discs (arrows). (B) Electron micrographs of IFM sarcomeres. Myofilament breakage at the Z-disc (Z) was observed at modest stretch (arrow). M, M-line; (c) Actin staining of stretched single myofibrils after digestion with μ-calpain. Actin filaments of opposing half sarcomeres usually remained connected at the Z-disc (arrowheads); actin was seen rarely to be broken (arrow). (D) Actin staining of sarcomeres after extraction with 0.2–0.3 mg/ml (top) and >0.3 mg/ml (bottom) gelsolin fragment. Bars: (B) 0.5 μm; (all IF images) 5 μm.
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fig3: Actin filaments in stretched Drosophila IFM sarcomeres. (A) Single myofibrils stained with rhodamine-phalloidin. Note actin filament breakage at Z-discs (arrows). (B) Electron micrographs of IFM sarcomeres. Myofilament breakage at the Z-disc (Z) was observed at modest stretch (arrow). M, M-line; (c) Actin staining of stretched single myofibrils after digestion with μ-calpain. Actin filaments of opposing half sarcomeres usually remained connected at the Z-disc (arrowheads); actin was seen rarely to be broken (arrow). (D) Actin staining of sarcomeres after extraction with 0.2–0.3 mg/ml (top) and >0.3 mg/ml (bottom) gelsolin fragment. Bars: (B) 0.5 μm; (all IF images) 5 μm.
Mentions: Low extensibility and stretch-induced high SL inhomogeneity were consistently observed in relaxed Drosophila IFM myofibrils. Somewhat surprisingly, when nonactivated myofibrils were stretched by ∼10% from slack SL and stained with rhodamine-phalloidin, actin always appeared to be broken at the Z-disc (Fig. 3 A); it seemed impossible to pull the actin filaments out of the A-band. Myofilament breakage at the Z-disc could be observed also on electron micrographs of stretched IFM sarcomeres (Fig. 3 B). In addition, actin-filament breakage was seen in stretched myofibrils stained with α-actin antibodies (unpublished data). A possible explanation of this result is that some actin-myosin connections remained in the A-band, despite the presence of 20 mM BDM (an active force-suppressing agent) in the relaxing buffer. Alternatively, some additional structure(s) could link actin and myosin filaments thereby stiffening the sarcomere.

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