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Related Experiment Videos

Cloning and characterization of actin depolymerizing factor from Toxoplasma gondii

M L Allen1, J M Dobrowolski, H Muller

  • 1Department of Molecular Pharmacology, Stanford University School of Medicine, CA 94305-5332, USA.

Molecular and Biochemical Parasitology
|September 1, 1997
PubMed
Summary

Toxoplasma gondii actin depolymerizing factor (ADF) was sequenced and found to be a small, single-copy gene. Recombinant ADF binds actin monomers and depolymerizes F-actin, localizing to the cytoplasm and plasma membrane.

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Area of Science:

  • Molecular Biology
  • Parasitology
  • Cell Biology

Background:

  • Actin dynamics are crucial for eukaryotic cell function.
  • Actin depolymerizing factor (ADF) proteins regulate actin polymerization.
  • Understanding ADF in parasites like Toxoplasma gondii is important for cell biology.

Purpose of the Study:

  • To determine the predicted amino acid sequence of ADF from Toxoplasma gondii.
  • To characterize the biochemical and cellular properties of T. gondii ADF.

Main Methods:

  • Full-length cDNA sequencing to determine the amino acid sequence.
  • Southern blot analysis to assess gene copy number.
  • Purification of homogeneous recombinant T. gondii ADF from E. coli.
  • In vitro assays for actin monomer binding and F-actin depolymerization.

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  • Immunofluorescence and immunoelectron microscopy for ADF localization.

    • Main Results:

    • The predicted amino acid sequence of T. gondii ADF comprises 118 amino acids (13.4 kDa).
    • T. gondii ADF shows high sequence similarity to other ADF/cofilin proteins but lacks a nuclear localization sequence.
    • Southern blot confirmed T. gondii ADF is a single-copy gene.
    • Recombinant T. gondii ADF actively binds actin monomers and depolymerizes F-actin.
    • ADF localizes to the cytoplasm and beneath the plasma membrane in T. gondii.

    Conclusions:

    • T. gondii ADF is a functional actin monomer-binding and F-actin-depolymerizing protein.
    • Its distinct features, such as smaller size and lack of nuclear localization, may reflect adaptations to the parasite's biology.
    • ADF's localization suggests roles in parasite motility and host cell invasion.