Regulation of the microfilament system in normal and polyoma virus transformed cultured (BHK) cells.
The content and state of actin in baby hamster kidney (BHK) cells before and after transformation with polyoma virus were examined by deoxyribonuclease assay and gel electrophoresis followed by dye elution. The actin content of the transformed cells, relative to total cell protein, was lower than that of the normal cells by 30-50%. In both the normal and transformed cells the greater part of the total actin was found on lysis to be in the monomeric state. Cytoplasmic and membrane fractions of the two cell lines were, in qualitative terms, very similar in their protein compositions. The plasma membrane isolated from the transformed cells was richer in actin than that from the untransformed, and both membrane fractions contained proteins corresponding to myosin, filamin and alpha-actinin on SDS-polyacrylamide gels. The cell extract from both the normal and transformed lines formed an actin-based gel on incubation at 30 degrees C, although the amount of the cross-linked actin was much smaller in the latter. This was a consequence not only of the lower concentration of total actin in the cell, but also, presumably, of a gross relative deficiency in the concentration or activity of filament cross-linking protein(s) in the cytoplasm. Thus, small aliquots of cytoplasmic fractions from transformed cells, when added to an excess of exogenous F-actin, were able to cross-link the filaments to a much smaller extent than those from the normal cells. A similar range of proteins was found to be associated with the actin gels formed from both cell extracts. One conspicuous difference was that a species migrating in SDS-gel electrophoresis as a doublet with a subunit molecular weight of about 58,000, and tentatively identified as intermediate filament protein, was replaced in the transformed cells by a single band. Filament cross-linking activity of the cytoplasmic fractions was enhanced by addition of Triton extracts of crude membranes, although the latter were not capable of cross-linking exogenous F-actin on their own. The effect of Triton extracts was much greater in the case of membranes from the transformed cells. The cytoplasmic fractions of BHK cells contain capping protein(s) and/or complexes of such proteins with actin; these reveal themselves by the propensity of the extracts to nucleate polymerization of exogenous G-actin. This activity was more abundant in transformed cells, despite their lower actin content. Their membranes were also more effective in nucleating G-actin polymerization, indicating the presence of a greater number of filament ends.(ABSTRACT TRUNCATED AT 400 WORDS)
Koffer
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A
Daridan
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Clarke
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GD
D000199:Actins / Q000032:analysis*; D000818:Animals; D002352:Carrier Proteins; D002460:Cell Line; D002472:Cell Transformation, Viral; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D003432:Cross-Linking Reagents; D003599:Cytoskeleton / Q000032:analysis* / Q000378:metabolism; D004589:Electrophoresis, Disc; D007668:Kidney / Q000166:cytology*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000032:analysis; D008840:Microfilament Proteins / Q000032:analysis*; D011120:Polyomavirus
D000199:Actins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; C034037:F-actin-binding proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008840:Microfilament Proteins