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Vertebrate isoforms of actin capping protein beta have distinct functions In vivo.

Hart MC, Cooper JA - J. Cell Biol. (1999)

Bottom Line: The beta2 did not localize to the Z-line.Therefore, CPbeta1 and CPbeta2 each have a distinct function that cannot be provided by the other isoform.CPbeta1 attaches actin filaments to the Z-line, and CPbeta2 organizes the actin at the intercalated discs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Actin capping protein (CP) binds barbed ends of actin filaments to regulate actin assembly. CP is an alpha/beta heterodimer. Vertebrates have conserved isoforms of each subunit. Muscle cells contain two beta isoforms. beta1 is at the Z-line; beta2 is at the intercalated disc and cell periphery in general. To investigate the functions of the isoforms, we replaced one isoform with another using expression in hearts of transgenic mice. Mice expressing beta2 had a severe phenotype with juvenile lethality. Myofibril architecture was severely disrupted. The beta2 did not localize to the Z-line. Therefore, beta1 has a distinct function that includes interactions at the Z-line. Mice expressing beta1 showed altered morphology of the intercalated disc, without the lethality or myofibril disruption of the beta2-expressing mice. The in vivo function of CP is presumed to involve binding barbed ends of actin filaments. To test this hypothesis, we expressed a beta1 mutant that poorly binds actin. These mice showed both myofibril disruption and intercalated disc remodeling, as predicted. Therefore, CPbeta1 and CPbeta2 each have a distinct function that cannot be provided by the other isoform. CPbeta1 attaches actin filaments to the Z-line, and CPbeta2 organizes the actin at the intercalated discs.

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Immunofluorescence localization of vinculin in wild-type, TG-wtβ1 (line 3), TG-wtβ2 (lines 2 and 3), and TG-β1L262R (line 3) hearts. In wild-type myocardium, vinculin localized to a line at the intercalated discs (arrow) and weakly at the border of the myocytes. In TG-wtβ1 and TG-β1L262R hearts, the localization of vinculin to the intercalated disc was a curved line (arrow) that was not transverse relative to the orientation of the myofibrils. In TG-wtβ2 hearts, vinculin localized to intercalated discs in small truncated lines (arrows). Bar, 5 μm.
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Figure 11: Immunofluorescence localization of vinculin in wild-type, TG-wtβ1 (line 3), TG-wtβ2 (lines 2 and 3), and TG-β1L262R (line 3) hearts. In wild-type myocardium, vinculin localized to a line at the intercalated discs (arrow) and weakly at the border of the myocytes. In TG-wtβ1 and TG-β1L262R hearts, the localization of vinculin to the intercalated disc was a curved line (arrow) that was not transverse relative to the orientation of the myofibrils. In TG-wtβ2 hearts, vinculin localized to intercalated discs in small truncated lines (arrows). Bar, 5 μm.

Mentions: The intercalated discs of the TG-wtβ2 myocardium had a relatively normal ultrastructure, but were increased in number and decreased in length. To confirm this result, intercalated discs were examined by light microscopy using antivinculin to show the intercalated discs. In normal myocardium, antivinculin staining was found as a fine line at the intercalated discs and weakly at the border of the myocytes (see Fig. 11). In TG-wtβ2 myocardium, vinculin labeling appeared as small irregular lines consistent with the multiplicity of intercalated discs in ultrastructure analysis. This could be due to the addition of new intercalated discs or fragmentation of existing discs caused by the underlying myofibril dysgenesis (see Discussion).


Vertebrate isoforms of actin capping protein beta have distinct functions In vivo.

Hart MC, Cooper JA - J. Cell Biol. (1999)

Immunofluorescence localization of vinculin in wild-type, TG-wtβ1 (line 3), TG-wtβ2 (lines 2 and 3), and TG-β1L262R (line 3) hearts. In wild-type myocardium, vinculin localized to a line at the intercalated discs (arrow) and weakly at the border of the myocytes. In TG-wtβ1 and TG-β1L262R hearts, the localization of vinculin to the intercalated disc was a curved line (arrow) that was not transverse relative to the orientation of the myofibrils. In TG-wtβ2 hearts, vinculin localized to intercalated discs in small truncated lines (arrows). Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2168092&req=5

Figure 11: Immunofluorescence localization of vinculin in wild-type, TG-wtβ1 (line 3), TG-wtβ2 (lines 2 and 3), and TG-β1L262R (line 3) hearts. In wild-type myocardium, vinculin localized to a line at the intercalated discs (arrow) and weakly at the border of the myocytes. In TG-wtβ1 and TG-β1L262R hearts, the localization of vinculin to the intercalated disc was a curved line (arrow) that was not transverse relative to the orientation of the myofibrils. In TG-wtβ2 hearts, vinculin localized to intercalated discs in small truncated lines (arrows). Bar, 5 μm.
Mentions: The intercalated discs of the TG-wtβ2 myocardium had a relatively normal ultrastructure, but were increased in number and decreased in length. To confirm this result, intercalated discs were examined by light microscopy using antivinculin to show the intercalated discs. In normal myocardium, antivinculin staining was found as a fine line at the intercalated discs and weakly at the border of the myocytes (see Fig. 11). In TG-wtβ2 myocardium, vinculin labeling appeared as small irregular lines consistent with the multiplicity of intercalated discs in ultrastructure analysis. This could be due to the addition of new intercalated discs or fragmentation of existing discs caused by the underlying myofibril dysgenesis (see Discussion).

Bottom Line: The beta2 did not localize to the Z-line.Therefore, CPbeta1 and CPbeta2 each have a distinct function that cannot be provided by the other isoform.CPbeta1 attaches actin filaments to the Z-line, and CPbeta2 organizes the actin at the intercalated discs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Actin capping protein (CP) binds barbed ends of actin filaments to regulate actin assembly. CP is an alpha/beta heterodimer. Vertebrates have conserved isoforms of each subunit. Muscle cells contain two beta isoforms. beta1 is at the Z-line; beta2 is at the intercalated disc and cell periphery in general. To investigate the functions of the isoforms, we replaced one isoform with another using expression in hearts of transgenic mice. Mice expressing beta2 had a severe phenotype with juvenile lethality. Myofibril architecture was severely disrupted. The beta2 did not localize to the Z-line. Therefore, beta1 has a distinct function that includes interactions at the Z-line. Mice expressing beta1 showed altered morphology of the intercalated disc, without the lethality or myofibril disruption of the beta2-expressing mice. The in vivo function of CP is presumed to involve binding barbed ends of actin filaments. To test this hypothesis, we expressed a beta1 mutant that poorly binds actin. These mice showed both myofibril disruption and intercalated disc remodeling, as predicted. Therefore, CPbeta1 and CPbeta2 each have a distinct function that cannot be provided by the other isoform. CPbeta1 attaches actin filaments to the Z-line, and CPbeta2 organizes the actin at the intercalated discs.

Show MeSH