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Heterokaryon Technique for Analysis of Cell Type-specific Localization
09:31

Heterokaryon Technique for Analysis of Cell Type-specific Localization

Published on: March 11, 2011

p21ras. Heterogeneous localization in transformed cells

S E Myrdal, N Auersperg

    Experimental Cell Research
    |August 1, 1985
    PubMed

    Insights

    The cellular distribution of p21ras, a protein linked to Kirsten murine sarcoma virus (KiMSV) transformation, is heterogeneous in transformed cells. This specific p21ras localization correlates with cell morphology changes and is reversible.

    Area of Science:

    • Molecular Biology
    • Cell Biology
    • Virology

    Background:

    • The cellular targets and precise subcellular localization of the Kirsten murine sarcoma virus (KiMSV)-transforming protein, p21ras, remain largely unknown.
    • Previous studies suggested a diffuse distribution of mature p21 on the cytoplasmic face of the plasma membrane.

    Purpose of the Study:

    • To investigate the subcellular distribution of p21ras in normal rat kidney (NRK) cells transformed by KiMSV.
    • To determine if p21ras localization is associated with cellular transformation and morphology.
    • To explore factors influencing p21ras distribution, such as gene overexpression, phosphorylation, and the viral oncogene.

    Main Methods:

    • Indirect immunofluorescent staining of sparse cell cultures after fixation without buffer washes.
    • Analysis of various NRK cell lines: KiMSV-transformed, newly transformed, temperature-sensitive (ts) KiMSV-transformed, and spontaneously transformed.
    • Phase contrast microscopy and scanning electron microscopy (SEM) to observe cellular morphology and ruffling.

    Main Results:

    • A strikingly heterogeneous subcellular distribution of p21ras was observed in transformed NRK cells, contrasting with untransformed cells.
    • This transformation-associated p21ras distribution was independent of gene overexpression, protein phosphorylation, or the viral oncogene.
    • In temperature-sensitive cells, the heterogeneous p21ras distribution was reversible and correlated with acquired transformed morphology, particularly with cellular ruffles and extensions.

    Conclusions:

    • The subcellular localization of p21ras is dynamically regulated and closely linked to the acquisition of a transformed cellular phenotype.
    • The preferential accumulation of p21ras in areas of cellular ruffling suggests a functional role in mediating transformation-induced morphological changes.
    • p21ras's specific localization, rather than mere presence or overexpression, appears critical for its function as a transforming molecule.

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