Changes in myosin and myosin light chain kinase during myogenesis

Sp, Scordilis; Bw, Uhlendorf; Scarpa, Stefano; Gl, Cantoni; Miles, JM; Rs, Adelstein

doi:10.1021/bi00515a032

Cited by 8 publications

(6 citation statements)

References 27 publications

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“…In contrast, levels of skeletal muscle myosin heavy chains (fast and slow isoforms) and skeletal muscle α-actin increase (Fig. 1c), as expected (Scordilis et al 1981; Lin and Lin 1986; Hayward et al 1988). Consistently with previous observations, we also observed a decrease in β-actin expression (Lin and Lin 1986; Hayward et al 1988), but we did not notice any substantial difference in γ-actin expression (Fig.…”

Section: Resultssupporting

confidence: 84%

Involvement of unconventional myosin VI in myoblast function and myotube formation

Karolczak

Pavlyk

Majewski

et al. 2015

Histochem Cell Biol

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The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873–885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation.

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Section: Resultssupporting

confidence: 84%

Involvement of unconventional myosin VI in myoblast function and myotube formation

Karolczak

Pavlyk

Majewski

et al. 2015

Histochem Cell Biol

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“…Immunochemistry: Myosin was metabolically labeled by incubating cultures with [35S]methionine (0.5 mCi in 3 ml medium/100-mm diam culture dish; New England Nuclear, Boston, MA; specific activity 1,000 Ci/mmol) in methionine-free medium (RPMI-1640) with 10% dialyzed (against Dulbecco's PBS) fetal calf serum for 12-18 h at 37"C. Cultures were washed in Hank's balanced salt solution (Ca *+-and Mg++-free) at 4"C to remove extracellular label. Cells were scrape-harvested in 0.5 ml of extraction buffer (0.5 M NaCI; 0.5 mM EDTA; 0.5 mM dithiothreitol (DTT); 30 mM Tris, pH 7.5; 0.1 mg/ ml BSA (Pentex ~, Miles Laboratories, Naperville, IL); and 0.1 t,g/ml phenylmethylsulfonyl fluoride (PMSF); as modified from Scordilis et al [26]), disrupted in a Dounce homogenizer on ice, and cleared of particulates by centrifugation at 100,000 g for 1 h at 4"C. In preliminary experiments, this extraction protocol yielded a mean recovery (supernatant) of 70 ± 17% (SD) of the total cellular myosin (as judged by comparison of supernatant and pellet fractions on quantitative SDS PAGE, as below). Recovery was not significantly greater when 10 mM ATP was added to the extraction buffer.…”

Section: Antiseramentioning

confidence: 99%

Myosin in cultured vascular smooth muscle cells: immunofluorescence and immunochemical studies of alterations in antigenic expression.

Larson¹,

Fujiwara²,

Alexander³

et al. 1984

The Journal of Cell Biology

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Vascular smooth muscle cells (VSMC) in the rat mesenteric artery show specific immunofluorescent staining with antisera against purified human uterine myosin (ASMM) but not human platelet myosin (APM). However, in primary cultures produced by enzymatic dissociation of this vessel, VSMC stain specifically with both ASMM and APM within 5 h after plating and throughout growth to confluence (4-10 d). In confluent cultures, APM staining remains bright while ASMM staining is reduced in intensity in most cells. In contrast, cellular myosin content, determined by quantitative SDS PAGE, is comparable in confluent and growing cultures. Immunoprecipitation of high salt extracts of cultured VSMC with ASMM and APM yields myosins with the same mobilities on SDS PAGE. When serial, exhaustive precipitations are performed with one antiserum, followed by reprecipitation with the other, myosin in subconfluent and confluent VSMC cultures is exhaustively precipitated by either antiserum, thus indicating complete immunological cross-reactivity. These results might be explained by synthesis of a new myosin isoform reactive with both ASMM and APM. However, the development of APM staining in cultured VSMC did not require protein synthesis. Therefore, it is more likely that the changes in immunofluorescent staining observed in vitro reflect conformational alterations, perhaps related to cytoskeletal rearrangements. These changes in myosin antigenic expression may be relevant to the problem of VSMC phenotypic modulation both in vitro and in vivo.

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“…In general two LC of fast muscle type, LC1f and LC2f (114,138) and one embryonic LC (LC1 emb) are characteristic of the early stages of rat muscle development (135 …”

Section: Fusionmentioning

confidence: 99%

Differentiation of skeletal muscle cells in culture.

Inestrosa

1982

Cell Struct. Funct.

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Changes in myosin and myosin light chain kinase during myogenesis

Cited by 8 publications

References 27 publications

Involvement of unconventional myosin VI in myoblast function and myotube formation

Involvement of unconventional myosin VI in myoblast function and myotube formation

Myosin in cultured vascular smooth muscle cells: immunofluorescence and immunochemical studies of alterations in antigenic expression.

Differentiation of skeletal muscle cells in culture.

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