Relative importance of βcyto- and γcyto-actin in primary mouse embryonic fibroblasts

摘要

The highly homologous β (β_cyto) and γ (γ_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 β_cyto- and γ_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 α_sm-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary β_cyto- or β/γ_cyto-actin double-knockout fibroblasts. Gene targeting of β_cyto-actin, but not γ_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 β_cyto-actin. Consistent with in vivo gene-targeting studies in mice, both gene- and transcript-targeting approaches demonstrate that the loss of β_cyto-actin protein is more disruptive to primary fibroblast function than is the loss of γ_cyto-actin.