Relative importance of beta(cyto)- and gamma(cyto)-actin in primary mouse embryonic fibroblasts

摘要

The highly homologous beta (beta(cyto)) and gamma (gamma(cyto)) cytoplasmic actins are hypothesized to carry out both redundant and unique essential functions, but studies using targeted gene knockout and siRNA-mediated transcript knockdown to examine beta(cyto)- and gamma(cyto)-isoform-specific functions in various cell types have yielded conflicting data. Here we quantitatively characterized actin transcript and protein levels, as well as cellular phenotypes, in both gene- and transcript-targeted primary mouse embryonic fibroblasts. We found that the smooth muscle alpha(sm)-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary beta(cyto)- or beta/gamma(cyto)-actin double-knockout fibroblasts. Gene targeting of beta(cyto)-actin, but not gamma(cyto)-actin, led to greatly decreased cell proliferation, decreased levels of cellular ATP, and increased serum response factor signaling in primary fibroblasts, whereas immortalization induced by SV40 large T antigen supported fibroblast proliferation in the absence of beta(cyto)-actin. Consistent with in vivo gene-targeting studies in mice, both gene-and transcript-targeting approaches demonstrate that the loss of beta(cyto)-actin protein is more disruptive to primary fibroblast function than is the loss of gamma(cyto)-actin.