Structural and functional reconstitution of thin filaments in skeletal muscle

Takashi Funatsu, Takashi Anazawa, Shin'ichi Ishiwata

    Research output: Contribution to journalArticle

    22 Citations (Scopus)

    Abstract

    Thin filaments were reconstituted by incorporating exogenous actin, tropomyosin and troponin into glycerinated skeletal muscle fibres or myofibrils. Firstly, thin filaments except short fragments at the Z line were selectively removed by treatment with plasma gelsolin, an actin severing protein. As a result, the fibres (or fibrils) lost the ability to generate active tension. Next, actin filaments were reconstituted by adding purified G-actin which polymerizes onto the actin fragments which remained at the Z line. Rhodamine phalloidin staining of myofibrils showed that exogenous actin was incorporated into the position where the intrinsic thin filaments located. Thin section electron micrographs of fibres showed that reconstituted actin filaments ran from the Z line to the inside of the A band, with some reaching the H zone. The number density of reconstituted actin filaments in the A band was about 20% of that found in intact fibres. The actin filament-reconstituted fibres (or fibrils) generated active tension in a Ca2+-insensitive manner and the tension was reversibly suppressed by 2,3-butanedione 2-monoxime. The recovered active tension was about 20% of tension developed by intact fibres. These results indicate that reconstituted actin filaments bear active tension similar to that borne by intact thin filaments. Thin filament-reconstituted fibres, which were prepared by adding purified tropomyosin-troponin complexes into actin filament-reconstituted fibres, showed Ca2+-sensitive tension generation. The maximum tension generated was not affected by the presence of tropomyosin and troponin. SDS-PAGE analysis showed that more than 25% of actin and 20% of tropomyosin and troponin was incorporated into the reconstituted fibres. These results indicate that the structure and function of thin filaments are substantially reconstituted by self-assembly of actin, tropomyosin and troponin. The reconstituted fibres and fibrils will be useful for studying the molecular mechanism of muscle contraction and its regulation.

    Original languageEnglish
    Pages (from-to)158-171
    Number of pages14
    JournalJournal of Muscle Research and Cell Motility
    Volume15
    Issue number2
    DOIs
    Publication statusPublished - 1994 Apr

    Fingerprint

    Muscle
    Actins
    Actin Cytoskeleton
    Skeletal Muscle
    Tropomyosin
    Troponin
    Fibers
    Myofibrils
    Gelsolin
    Skeletal Muscle Fibers
    Muscle Contraction
    Polyacrylamide Gel Electrophoresis
    Electrons
    Diacetyl
    Staining and Labeling
    Self assembly
    Proteins
    Plasmas

    ASJC Scopus subject areas

    • Physiology
    • Endocrinology
    • Clinical Biochemistry
    • Cell Biology

    Cite this

    Structural and functional reconstitution of thin filaments in skeletal muscle. / Funatsu, Takashi; Anazawa, Takashi; Ishiwata, Shin'ichi.

    In: Journal of Muscle Research and Cell Motility, Vol. 15, No. 2, 04.1994, p. 158-171.

    Research output: Contribution to journalArticle

    Funatsu, Takashi ; Anazawa, Takashi ; Ishiwata, Shin'ichi. / Structural and functional reconstitution of thin filaments in skeletal muscle. In: Journal of Muscle Research and Cell Motility. 1994 ; Vol. 15, No. 2. pp. 158-171.
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    abstract = "Thin filaments were reconstituted by incorporating exogenous actin, tropomyosin and troponin into glycerinated skeletal muscle fibres or myofibrils. Firstly, thin filaments except short fragments at the Z line were selectively removed by treatment with plasma gelsolin, an actin severing protein. As a result, the fibres (or fibrils) lost the ability to generate active tension. Next, actin filaments were reconstituted by adding purified G-actin which polymerizes onto the actin fragments which remained at the Z line. Rhodamine phalloidin staining of myofibrils showed that exogenous actin was incorporated into the position where the intrinsic thin filaments located. Thin section electron micrographs of fibres showed that reconstituted actin filaments ran from the Z line to the inside of the A band, with some reaching the H zone. The number density of reconstituted actin filaments in the A band was about 20{\%} of that found in intact fibres. The actin filament-reconstituted fibres (or fibrils) generated active tension in a Ca2+-insensitive manner and the tension was reversibly suppressed by 2,3-butanedione 2-monoxime. The recovered active tension was about 20{\%} of tension developed by intact fibres. These results indicate that reconstituted actin filaments bear active tension similar to that borne by intact thin filaments. Thin filament-reconstituted fibres, which were prepared by adding purified tropomyosin-troponin complexes into actin filament-reconstituted fibres, showed Ca2+-sensitive tension generation. The maximum tension generated was not affected by the presence of tropomyosin and troponin. SDS-PAGE analysis showed that more than 25{\%} of actin and 20{\%} of tropomyosin and troponin was incorporated into the reconstituted fibres. These results indicate that the structure and function of thin filaments are substantially reconstituted by self-assembly of actin, tropomyosin and troponin. The reconstituted fibres and fibrils will be useful for studying the molecular mechanism of muscle contraction and its regulation.",
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