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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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Immunolabeling of relaxed Drosophila IFM sarcomeres with various antibodies. (A) Domain structure of kettin (Kolmerer et al., 2000) and position of kettin antibodies used. (B and C) IF microscopy of stretched single myofibrils stained with kettin antibodies and Cy-3–conjugated IgG. (D) IEM of sarcomeres at rest length and after small stretch, stained with α-kettin Ig34/35. (E and F) IF of stretched single myofibrils stained with α-projectin (E) and α-PEVK antibodies (F). pc, phase–contrast image; fl, fluorescence image. Bars: (D) 0.5 μm; (F, IF images) 5 μm.
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fig2: Immunolabeling of relaxed Drosophila IFM sarcomeres with various antibodies. (A) Domain structure of kettin (Kolmerer et al., 2000) and position of kettin antibodies used. (B and C) IF microscopy of stretched single myofibrils stained with kettin antibodies and Cy-3–conjugated IgG. (D) IEM of sarcomeres at rest length and after small stretch, stained with α-kettin Ig34/35. (E and F) IF of stretched single myofibrils stained with α-projectin (E) and α-PEVK antibodies (F). pc, phase–contrast image; fl, fluorescence image. Bars: (D) 0.5 μm; (F, IF images) 5 μm.

Mentions: The position of kettin in relaxed Drosophila IFM sarcomeres was analyzed by immunofluorescence (IF) and immunoelectron microscopy (IEM). Two different antibodies were used, one against the central part (Ig16) and the other against the COOH terminus (Ig34/35) of the published kettin sequence (Fig. 2 A). By IF, kettin epitopes appeared as a single stripe in the Z-disc region of sarcomeres independent of the stretch state (Fig. 2, B and C). IEM showed that α-Ig34/35 labels both nonstretched and modestly stretched sarcomeres at the periphery of the Z-disc (Fig. 2 D). In extremely stretched myofibrils, some rearrangement of kettin could be seen: IF with α-Ig16 or α-Ig34/35 revealed a “smear” near some Z-discs (Fig. 2, B and C, arrows).


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)

Immunolabeling of relaxed Drosophila IFM sarcomeres with various antibodies. (A) Domain structure of kettin (Kolmerer et al., 2000) and position of kettin antibodies used. (B and C) IF microscopy of stretched single myofibrils stained with kettin antibodies and Cy-3–conjugated IgG. (D) IEM of sarcomeres at rest length and after small stretch, stained with α-kettin Ig34/35. (E and F) IF of stretched single myofibrils stained with α-projectin (E) and α-PEVK antibodies (F). pc, phase–contrast image; fl, fluorescence image. Bars: (D) 0.5 μm; (F, IF images) 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196178&req=5

fig2: Immunolabeling of relaxed Drosophila IFM sarcomeres with various antibodies. (A) Domain structure of kettin (Kolmerer et al., 2000) and position of kettin antibodies used. (B and C) IF microscopy of stretched single myofibrils stained with kettin antibodies and Cy-3–conjugated IgG. (D) IEM of sarcomeres at rest length and after small stretch, stained with α-kettin Ig34/35. (E and F) IF of stretched single myofibrils stained with α-projectin (E) and α-PEVK antibodies (F). pc, phase–contrast image; fl, fluorescence image. Bars: (D) 0.5 μm; (F, IF images) 5 μm.
Mentions: The position of kettin in relaxed Drosophila IFM sarcomeres was analyzed by immunofluorescence (IF) and immunoelectron microscopy (IEM). Two different antibodies were used, one against the central part (Ig16) and the other against the COOH terminus (Ig34/35) of the published kettin sequence (Fig. 2 A). By IF, kettin epitopes appeared as a single stripe in the Z-disc region of sarcomeres independent of the stretch state (Fig. 2, B and C). IEM showed that α-Ig34/35 labels both nonstretched and modestly stretched sarcomeres at the periphery of the Z-disc (Fig. 2 D). In extremely stretched myofibrils, some rearrangement of kettin could be seen: IF with α-Ig16 or α-Ig34/35 revealed a “smear” near some Z-discs (Fig. 2, B and C, arrows).

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