Actin removal from cardiac myocytes shows that near Z line titin attaches to actin while under tension

Károly Trombitás, Hendrikus "Henk" Granzier

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74 Citations (Scopus)

Abstract

The I band of cardiac sarcomeres contains both actin and titin/connectin filaments. Earlier work has suggested that titin binds to actin in situ. This interaction must be weak in the region of the I band where titin behaves elastically. On the other hand, titin may bind strongly to actin in the ~100-nm-wide region adjoining the Z line, where titin has been found to be inelastic. To study the putative interaction between titin and actin, techniques for selective removal of actin from different regions of the I band are needed. Here we report studies with a gelsolin fragment (FX-45) and extract actin from rat cardiac myocytes. Actin extraction was biphasic: the majority of actin was extracted in ~10 min, whereas actin near the Z line (where titin is inelastic) required a ~10-fold longer extraction time. Thus, by controlling the extraction time, we could remove either the full actin filament outside the Z line or just the segment of the actin filament that extends beyond the inelastic region of titin that adjoins the Z line. The actin filament-free I band contained titin filaments, typically with one filament extending from each thick filament. In addition, we observed a dark transverse line (junction line), the location of which in the sarcomere varied linearly with sarcomere length. The position in the sarcomere of the junction line coincided with the binding site of the anti-titin antibody 9D10. Actin removal significantly affected the slack sarcomere length. Slack sarcomere length was 1.85 ± 0.04/μm in control cells and decreased to 1.71 ± 0.05 μm after actin near the Z line was extracted. This length reduction may be caused by contraction of the titin segment that becomes exposed after actin removal near the Z line, indicating that titin is not only attached to the actin filament but is also under tension.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume273
Issue number2 42-2
StatePublished - 1997
Externally publishedYes

Fingerprint

Connectin
Cardiac Myocytes
Actins
Sarcomeres
Actin Cytoskeleton
Gelsolin

Keywords

  • Connectin
  • Entropic elasticity
  • Gelsolin
  • Nebulette
  • Passive force
  • Slack sarcomere length

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Physiology (medical)

Cite this

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title = "Actin removal from cardiac myocytes shows that near Z line titin attaches to actin while under tension",
abstract = "The I band of cardiac sarcomeres contains both actin and titin/connectin filaments. Earlier work has suggested that titin binds to actin in situ. This interaction must be weak in the region of the I band where titin behaves elastically. On the other hand, titin may bind strongly to actin in the ~100-nm-wide region adjoining the Z line, where titin has been found to be inelastic. To study the putative interaction between titin and actin, techniques for selective removal of actin from different regions of the I band are needed. Here we report studies with a gelsolin fragment (FX-45) and extract actin from rat cardiac myocytes. Actin extraction was biphasic: the majority of actin was extracted in ~10 min, whereas actin near the Z line (where titin is inelastic) required a ~10-fold longer extraction time. Thus, by controlling the extraction time, we could remove either the full actin filament outside the Z line or just the segment of the actin filament that extends beyond the inelastic region of titin that adjoins the Z line. The actin filament-free I band contained titin filaments, typically with one filament extending from each thick filament. In addition, we observed a dark transverse line (junction line), the location of which in the sarcomere varied linearly with sarcomere length. The position in the sarcomere of the junction line coincided with the binding site of the anti-titin antibody 9D10. Actin removal significantly affected the slack sarcomere length. Slack sarcomere length was 1.85 ± 0.04/μm in control cells and decreased to 1.71 ± 0.05 μm after actin near the Z line was extracted. This length reduction may be caused by contraction of the titin segment that becomes exposed after actin removal near the Z line, indicating that titin is not only attached to the actin filament but is also under tension.",
keywords = "Connectin, Entropic elasticity, Gelsolin, Nebulette, Passive force, Slack sarcomere length",
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T1 - Actin removal from cardiac myocytes shows that near Z line titin attaches to actin while under tension

AU - Trombitás, Károly

AU - Granzier, Hendrikus "Henk"

PY - 1997

Y1 - 1997

N2 - The I band of cardiac sarcomeres contains both actin and titin/connectin filaments. Earlier work has suggested that titin binds to actin in situ. This interaction must be weak in the region of the I band where titin behaves elastically. On the other hand, titin may bind strongly to actin in the ~100-nm-wide region adjoining the Z line, where titin has been found to be inelastic. To study the putative interaction between titin and actin, techniques for selective removal of actin from different regions of the I band are needed. Here we report studies with a gelsolin fragment (FX-45) and extract actin from rat cardiac myocytes. Actin extraction was biphasic: the majority of actin was extracted in ~10 min, whereas actin near the Z line (where titin is inelastic) required a ~10-fold longer extraction time. Thus, by controlling the extraction time, we could remove either the full actin filament outside the Z line or just the segment of the actin filament that extends beyond the inelastic region of titin that adjoins the Z line. The actin filament-free I band contained titin filaments, typically with one filament extending from each thick filament. In addition, we observed a dark transverse line (junction line), the location of which in the sarcomere varied linearly with sarcomere length. The position in the sarcomere of the junction line coincided with the binding site of the anti-titin antibody 9D10. Actin removal significantly affected the slack sarcomere length. Slack sarcomere length was 1.85 ± 0.04/μm in control cells and decreased to 1.71 ± 0.05 μm after actin near the Z line was extracted. This length reduction may be caused by contraction of the titin segment that becomes exposed after actin removal near the Z line, indicating that titin is not only attached to the actin filament but is also under tension.

AB - The I band of cardiac sarcomeres contains both actin and titin/connectin filaments. Earlier work has suggested that titin binds to actin in situ. This interaction must be weak in the region of the I band where titin behaves elastically. On the other hand, titin may bind strongly to actin in the ~100-nm-wide region adjoining the Z line, where titin has been found to be inelastic. To study the putative interaction between titin and actin, techniques for selective removal of actin from different regions of the I band are needed. Here we report studies with a gelsolin fragment (FX-45) and extract actin from rat cardiac myocytes. Actin extraction was biphasic: the majority of actin was extracted in ~10 min, whereas actin near the Z line (where titin is inelastic) required a ~10-fold longer extraction time. Thus, by controlling the extraction time, we could remove either the full actin filament outside the Z line or just the segment of the actin filament that extends beyond the inelastic region of titin that adjoins the Z line. The actin filament-free I band contained titin filaments, typically with one filament extending from each thick filament. In addition, we observed a dark transverse line (junction line), the location of which in the sarcomere varied linearly with sarcomere length. The position in the sarcomere of the junction line coincided with the binding site of the anti-titin antibody 9D10. Actin removal significantly affected the slack sarcomere length. Slack sarcomere length was 1.85 ± 0.04/μm in control cells and decreased to 1.71 ± 0.05 μm after actin near the Z line was extracted. This length reduction may be caused by contraction of the titin segment that becomes exposed after actin removal near the Z line, indicating that titin is not only attached to the actin filament but is also under tension.

KW - Connectin

KW - Entropic elasticity

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KW - Nebulette

KW - Passive force

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