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

Stiffness measurements on single Drosophila IFM myofibrils. (A) Protocol: 20 Hz sinusoidal oscillations were applied for 1–2 s; the rest interval (Δt) was 1–20 min. (B) Examples of oscillatory force response of control specimens and calpain-treated myofibrils. (C) Stiffness (mean ± SD; n = 3) versus time after calpain treatment (solid line) in comparison with control stiffness (dotted line). SL was 3.7 μm except after 30 min when myofibrils were stretched to 3.8 μm. (D) Examples of force response of control specimens, igase-digested, and gelsolin-treated single myofibrils. (E) Stiffness (mean ± SD; n = 3) versus time in control myofibrils (dotted curve), during igase digestion (gray solid curve), and during actin extraction (black solid curve). SL before the stretch at the end of the experimental protocol was 3.7 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196178&req=5

fig6: Stiffness measurements on single Drosophila IFM myofibrils. (A) Protocol: 20 Hz sinusoidal oscillations were applied for 1–2 s; the rest interval (Δt) was 1–20 min. (B) Examples of oscillatory force response of control specimens and calpain-treated myofibrils. (C) Stiffness (mean ± SD; n = 3) versus time after calpain treatment (solid line) in comparison with control stiffness (dotted line). SL was 3.7 μm except after 30 min when myofibrils were stretched to 3.8 μm. (D) Examples of force response of control specimens, igase-digested, and gelsolin-treated single myofibrils. (E) Stiffness (mean ± SD; n = 3) versus time in control myofibrils (dotted curve), during igase digestion (gray solid curve), and during actin extraction (black solid curve). SL before the stretch at the end of the experimental protocol was 3.7 μm.

Mentions: To investigate how μ-calpain and gelsolin may affect passive stiffness, we measured the force response of single nonactivated Drosophila IFM myofibrils to sinusoidal motor movement (Fig. 6 A). Calpain-treated myofibrils were stretched in relaxing buffer to 3.7 μm SL, and stiffness was recorded immediately after the stretch and after a waiting period of 20 and 30 min, respectively (Fig. 6 B). Fig. 6 C shows that stiffness of μ-calpain–treated myofibrils dropped by 50–60% within 20–30 min, whereas that of control specimens decreased only by ∼20% (normal stress relaxation). In contrast to controls, digested myofibrils exhibited no stiffness increase upon further stretch. Thus, cleavage of kettin significantly depresses myofibril stiffness. Whereas passive stiffness was recorded after μ-calpain digestion (because digestion requires Ca2+), it was measured during treatment with gelsolin fragment in relaxing buffer (Fig. 6 D, bottom panels). As shown in Fig. 6 E (black solid curve), stiffness decreased greatly within the first minute of gelsolin application. This initial stiffness drop could be as large as 50%. Then, stiffness decreased only a little and after 20–30 min reached values comparable to those measured in calpain-treated myofibrils (Fig. 6, C and E). Further stretch of actin-extracted specimens failed to increase stiffness appreciably.


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)

Stiffness measurements on single Drosophila IFM myofibrils. (A) Protocol: 20 Hz sinusoidal oscillations were applied for 1–2 s; the rest interval (Δt) was 1–20 min. (B) Examples of oscillatory force response of control specimens and calpain-treated myofibrils. (C) Stiffness (mean ± SD; n = 3) versus time after calpain treatment (solid line) in comparison with control stiffness (dotted line). SL was 3.7 μm except after 30 min when myofibrils were stretched to 3.8 μm. (D) Examples of force response of control specimens, igase-digested, and gelsolin-treated single myofibrils. (E) Stiffness (mean ± SD; n = 3) versus time in control myofibrils (dotted curve), during igase digestion (gray solid curve), and during actin extraction (black solid curve). SL before the stretch at the end of the experimental protocol was 3.7 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Stiffness measurements on single Drosophila IFM myofibrils. (A) Protocol: 20 Hz sinusoidal oscillations were applied for 1–2 s; the rest interval (Δt) was 1–20 min. (B) Examples of oscillatory force response of control specimens and calpain-treated myofibrils. (C) Stiffness (mean ± SD; n = 3) versus time after calpain treatment (solid line) in comparison with control stiffness (dotted line). SL was 3.7 μm except after 30 min when myofibrils were stretched to 3.8 μm. (D) Examples of force response of control specimens, igase-digested, and gelsolin-treated single myofibrils. (E) Stiffness (mean ± SD; n = 3) versus time in control myofibrils (dotted curve), during igase digestion (gray solid curve), and during actin extraction (black solid curve). SL before the stretch at the end of the experimental protocol was 3.7 μm.
Mentions: To investigate how μ-calpain and gelsolin may affect passive stiffness, we measured the force response of single nonactivated Drosophila IFM myofibrils to sinusoidal motor movement (Fig. 6 A). Calpain-treated myofibrils were stretched in relaxing buffer to 3.7 μm SL, and stiffness was recorded immediately after the stretch and after a waiting period of 20 and 30 min, respectively (Fig. 6 B). Fig. 6 C shows that stiffness of μ-calpain–treated myofibrils dropped by 50–60% within 20–30 min, whereas that of control specimens decreased only by ∼20% (normal stress relaxation). In contrast to controls, digested myofibrils exhibited no stiffness increase upon further stretch. Thus, cleavage of kettin significantly depresses myofibril stiffness. Whereas passive stiffness was recorded after μ-calpain digestion (because digestion requires Ca2+), it was measured during treatment with gelsolin fragment in relaxing buffer (Fig. 6 D, bottom panels). As shown in Fig. 6 E (black solid curve), stiffness decreased greatly within the first minute of gelsolin application. This initial stiffness drop could be as large as 50%. Then, stiffness decreased only a little and after 20–30 min reached values comparable to those measured in calpain-treated myofibrils (Fig. 6, C and E). Further stretch of actin-extracted specimens failed to increase stiffness appreciably.

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