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Related Experiment Videos

Isolation and characterization of a regulated form of actin depolymerizing factor

T E Morgan1, R O Lockerbie, L S Minamide

  • 1Department of Biochemistry, Colorado State University, Fort Collins 80523.

The Journal of Cell Biology
|August 1, 1993
PubMed
Summary

Muscle cells regulate actin depolymerizing factor (ADF) activity through pre- and post-translational mechanisms. A novel phosphorylated ADF (pADF) isoform is identified, impacting actin dynamics in muscle development.

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Muscle Development

Background:

  • Actin depolymerizing factor (ADF) is crucial for actin dynamics.
  • Developing muscle down-regulates ADF.
  • ADF activity is pH-dependent and involves F-actin binding and severing.

Purpose of the Study:

  • Investigate ADF expression and regulation in chick myocytes.
  • Identify and characterize novel ADF isoforms in muscle cells.
  • Determine the functional significance of ADF post-translational modifications.

Main Methods:

  • In vitro cell culture of chick myocytes.
  • Immunoreactivity and Western blot analysis for ADF.
  • Protein purification, peptide mapping, and partial sequencing.

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  • Radiolabeling with [32P]orthophosphate and immunoprecipitation.
  • Enzymatic treatment with alkaline phosphatase.
  • Quantification of ADF isoforms in various tissues.

    • Main Results:

    • ADF immunoreactivity increased in cultured myocytes due to a higher molecular weight isoform, pADF.
    • pADF is a phosphorylated serine residue of ADF, structurally related to ADF.
    • pADF does not bind G-actin or affect actin assembly, unlike ADF.
    • pADF levels range from 18% to 150% of unphosphorylated ADF in various cells/tissues.
    • Ubiquitous kinases did not phosphorylate ADF in vitro, suggesting a specific kinase.

    Conclusions:

    • Myocytes employ both pre- and post-translational mechanisms to regulate ADF activity.
    • Post-translational modification, specifically phosphorylation to form pADF, is a key regulatory mechanism for ADF in muscle.
    • This regulation is vital for muscle development and represents a general mechanism for cellular control of ADF activity.