Limits...
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.

Show MeSH

Related in: MedlinePlus

Actin-extracted, stretched, Drosophila IFM myofibrils. (A) Images of single myofibrils at different SLs stained with α-kettin Ig16. Both Z-discs (arrow) and A-band edges (arrowheads) are labeled. (B) Examples of intensity profiles and graph showing the SL-dependent spacing (mean ± SD; n = 22) of kettin-Ig16 epitopes at the A-band edge measured across the M-line (•) or Z-disc (▴). For comparison, the distance between A-band edges is also shown (○ and ▵). (C) Sequence of fluorescence images of a single myofibril at ∼4.0 μm SL stained with α-Ig34/35. Upon exposure of the myofibril to 20 Hz sinusoidal length oscillations, the intensity of Z-disc epitopes gradually decreased (arrows), whereas that of epitopes at the A-band edge increased (arrowheads). (D) IEM of actin-extracted fibers stained with α-Ig34/35. A larger area is shown in the top left image. Images at bottom depict sarcomeres at two different degrees of stretch: ∼3.8 and ∼4.2 μm SL. Nanogold particles indicate the position of kettin epitopes at the Z-disc periphery (arrows) and A-band edge (arrowheads). The histogram shows the nanogold particle distance from the center of the Z-disc measured in ∼3.8-μm-long sarcomeres. A major peak is at ∼60 nm, a minor peak at 360–400 nm, out from the Z-disc center. (E) IF of single myofibril stained with α-projectin antibody. The A-band edge is stained strongly (arrowheads) and the Z-disc faintly (arrow). (F) α-PEVK antibody did not stain actin-extracted myofibrils. Bars: (D) 0.5 μm; (all IF images) 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196178&req=5

fig4: Actin-extracted, stretched, Drosophila IFM myofibrils. (A) Images of single myofibrils at different SLs stained with α-kettin Ig16. Both Z-discs (arrow) and A-band edges (arrowheads) are labeled. (B) Examples of intensity profiles and graph showing the SL-dependent spacing (mean ± SD; n = 22) of kettin-Ig16 epitopes at the A-band edge measured across the M-line (•) or Z-disc (▴). For comparison, the distance between A-band edges is also shown (○ and ▵). (C) Sequence of fluorescence images of a single myofibril at ∼4.0 μm SL stained with α-Ig34/35. Upon exposure of the myofibril to 20 Hz sinusoidal length oscillations, the intensity of Z-disc epitopes gradually decreased (arrows), whereas that of epitopes at the A-band edge increased (arrowheads). (D) IEM of actin-extracted fibers stained with α-Ig34/35. A larger area is shown in the top left image. Images at bottom depict sarcomeres at two different degrees of stretch: ∼3.8 and ∼4.2 μm SL. Nanogold particles indicate the position of kettin epitopes at the Z-disc periphery (arrows) and A-band edge (arrowheads). The histogram shows the nanogold particle distance from the center of the Z-disc measured in ∼3.8-μm-long sarcomeres. A major peak is at ∼60 nm, a minor peak at 360–400 nm, out from the Z-disc center. (E) IF of single myofibril stained with α-projectin antibody. The A-band edge is stained strongly (arrowheads) and the Z-disc faintly (arrow). (F) α-PEVK antibody did not stain actin-extracted myofibrils. Bars: (D) 0.5 μm; (all IF images) 5 μm.

Mentions: Proteins potentially involved in the passive stiffening were extracted from IFM myofibrils by various procedures. Actin was removed with a Ca2+-independent gelsolin fragment, which extracted all but the Z-disc actin when used at concentrations of 0.2–0.3 mg/ml (Fig. 3 D, top). At concentrations >0.3 mg/ml, Z-disc actin was removed also (Fig. 3 D, bottom). Even after complete actin extraction, myofibrils did not disintegrate; rather, they could be stretched highly while showing reasonable SL homogeneity (Fig. 3 D and Fig. 4) . Removal of actin allowed the myofibrils to be extended reversibly over a wider SL range.


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-extracted, stretched, Drosophila IFM myofibrils. (A) Images of single myofibrils at different SLs stained with α-kettin Ig16. Both Z-discs (arrow) and A-band edges (arrowheads) are labeled. (B) Examples of intensity profiles and graph showing the SL-dependent spacing (mean ± SD; n = 22) of kettin-Ig16 epitopes at the A-band edge measured across the M-line (•) or Z-disc (▴). For comparison, the distance between A-band edges is also shown (○ and ▵). (C) Sequence of fluorescence images of a single myofibril at ∼4.0 μm SL stained with α-Ig34/35. Upon exposure of the myofibril to 20 Hz sinusoidal length oscillations, the intensity of Z-disc epitopes gradually decreased (arrows), whereas that of epitopes at the A-band edge increased (arrowheads). (D) IEM of actin-extracted fibers stained with α-Ig34/35. A larger area is shown in the top left image. Images at bottom depict sarcomeres at two different degrees of stretch: ∼3.8 and ∼4.2 μm SL. Nanogold particles indicate the position of kettin epitopes at the Z-disc periphery (arrows) and A-band edge (arrowheads). The histogram shows the nanogold particle distance from the center of the Z-disc measured in ∼3.8-μm-long sarcomeres. A major peak is at ∼60 nm, a minor peak at 360–400 nm, out from the Z-disc center. (E) IF of single myofibril stained with α-projectin antibody. The A-band edge is stained strongly (arrowheads) and the Z-disc faintly (arrow). (F) α-PEVK antibody did not stain actin-extracted myofibrils. Bars: (D) 0.5 μm; (all IF images) 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Actin-extracted, stretched, Drosophila IFM myofibrils. (A) Images of single myofibrils at different SLs stained with α-kettin Ig16. Both Z-discs (arrow) and A-band edges (arrowheads) are labeled. (B) Examples of intensity profiles and graph showing the SL-dependent spacing (mean ± SD; n = 22) of kettin-Ig16 epitopes at the A-band edge measured across the M-line (•) or Z-disc (▴). For comparison, the distance between A-band edges is also shown (○ and ▵). (C) Sequence of fluorescence images of a single myofibril at ∼4.0 μm SL stained with α-Ig34/35. Upon exposure of the myofibril to 20 Hz sinusoidal length oscillations, the intensity of Z-disc epitopes gradually decreased (arrows), whereas that of epitopes at the A-band edge increased (arrowheads). (D) IEM of actin-extracted fibers stained with α-Ig34/35. A larger area is shown in the top left image. Images at bottom depict sarcomeres at two different degrees of stretch: ∼3.8 and ∼4.2 μm SL. Nanogold particles indicate the position of kettin epitopes at the Z-disc periphery (arrows) and A-band edge (arrowheads). The histogram shows the nanogold particle distance from the center of the Z-disc measured in ∼3.8-μm-long sarcomeres. A major peak is at ∼60 nm, a minor peak at 360–400 nm, out from the Z-disc center. (E) IF of single myofibril stained with α-projectin antibody. The A-band edge is stained strongly (arrowheads) and the Z-disc faintly (arrow). (F) α-PEVK antibody did not stain actin-extracted myofibrils. Bars: (D) 0.5 μm; (all IF images) 5 μm.
Mentions: Proteins potentially involved in the passive stiffening were extracted from IFM myofibrils by various procedures. Actin was removed with a Ca2+-independent gelsolin fragment, which extracted all but the Z-disc actin when used at concentrations of 0.2–0.3 mg/ml (Fig. 3 D, top). At concentrations >0.3 mg/ml, Z-disc actin was removed also (Fig. 3 D, bottom). Even after complete actin extraction, myofibrils did not disintegrate; rather, they could be stretched highly while showing reasonable SL homogeneity (Fig. 3 D and Fig. 4) . Removal of actin allowed the myofibrils to be extended reversibly over a wider SL range.

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