Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

Abstract

Abstract. We have examined the role of feedbackregulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human ~-and ~-actin genes. In ~-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human T-actin expression resulted in a progressive down-regulation of mouse/~-and ~/-actin mRNAs. Transfection of the ~-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse/3-actin mRNA. In contrast, transfection of a/~-actin gene carrying a single-point mutation ~sm) produced a feedback-regulatory response similar to that of the 7-actin gene. Expression of a /~-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the downregulation of mouse ~,-actin in both the ~-and/3sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse/3-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse 3,-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of B-actin may be distinct from that governing 7-actin. We conclude that actin feedbackregulation provides a biochemical assay for differences between the two nonmuscle actin genes.

Autoregulation of Actin Synthesis in Hepatocytes by Transcriptional and Posttranscriptional Mechanisms

“…In line with the present study, down regulation of the endogenous actin synthesis was observed in human fibroblasts after transfection and expression of a mutant p-actin gene [36]. In contrast, transfection of mouse myoblasts with human P-actin and y-actin genes and consecutive expression resulted in a transcriptional down regulation of the endogenous actin synthesis in y-actin transfectants but not in p-actin transfectants [37]. These results indicate that a highly specific mechanism of autoregulation may exist.…”

Autoregulation of actin synthesis responds to monomeric actin levels

“…This notion is also supported by studies using other compounds that either decrease the level of polymerized actin, such as Clostridium botulinum C2 toxin, or increase its level, like phalloidin [Serpinskaya et al, 1990[Serpinskaya et al, , 1991Reuner et al, 1991;Bershadsky et al, 1995], or manipulate G-actin levels with hypotonic treatment [Reuner et al, 1995b], or overexpress ectopically introduced actin [Lloyd et al, 1992]. In contrast, cytochalasins that are known to disrupt actin filaments, but do not change the level of F-actin, or even elevate it in certain cells [Morris and Tannenbaum, 1980;Rao et al, 1982], do not affect, or may even enhance, actin synthesis [Tannenbaum and Goodman, 1983].…”

Cytoplasmic γ Actin as a Z-Disc Protein

“…BSA-MBS (lane 1), BSA-MBS conjugated with N-terminal peptide of cytoplasmic ␥ (lane 2), ␣-smooth muscle (lane 3) and ␥ smooth muscle actin (lane 4) were subjected to Western blot analysis. The membrane was stained with polyclonal antibody against the N-terminal peptide of cytoplasmic ␥ actin and subsequently exposed to X-ray film (lanes 1-4) at a higher exposure (lanes [5][6][7][8]. To visualize the samples loaded, the membrane was stained with CBB (lanes 9 -12).…”