Dominant negative effect of cytoplasmic actin isoproteins on cardiomyocyte cytoarchitecture and function.

Abstract

Abstract. The intracompartmental sorting and functional consequences of ectopic expression of the six vertebrate actin isoforms was investigated in different types of cultured cells. In transfected fibroblasts all isoactin species associated with the endogenous microfilament cytoskeleton, even though cytoplasmic actins also showed partial localization to peripheral submembranous sites. Functional and structural studies were performed in neonatal and adult rat cardiomyocytes. All the muscle isoactin constructs sorted preferentially to sarcomeric sites and, to a lesser extent, also to stressfiber-like structures. The expression of muscle actins did not interfere with cell contractility, and did not disturb the localization of endogenous sarcomeric proteins. In sharp contrast, ectopic expression of the two cytoplasmic actin isoforms resulted in rapid cessation of cellular contractions and induced severe morphological alterations characterized by an exceptional outgrowth of filopodia and cell flattening. Quantitative analysis in neonatal cardiomyocytes indicated that the levels of accumulation of the different isoactins are very similar and cannot be responsible for the observed isoproteinsspecific effects. Structural analysis revealed a remodeling of the cytoarchitecture including a specific alteration of sarcomeric organization; proteins constituting the sarcomeric thin filaments relocated to nonmyofibrillar sites while thick filaments and titin remained unaffected. Experiments with chimeric proteins strongly suggest that isoform specific residues in the carboxy-terminal portion of the cytoplasmic actins are responsible for the dominant negative effects on function and morphology.

Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

“…Transfected ␣-SMA was predominantly incorporated into stress fibers as evaluated by double-labeling with antibodies against ␣-SMA and against the VSV-G-tag at the actin C terminus ( Figure 7B). Stably transfected ␣-cardiac ( Figure 7D) and transiently transfected ␤-cytoplasmic actin were also incorporated into stress fibers, but the latter occasionally accumulated in cytoplasmic aggregates if strongly overexpressed, a phenomenon reported previously for transfected primary cells (von Arx et al, 1995;Mounier et al, 1997). Transfected ␥-cytoplasmic actin predominantly localized in lamellipodia and membrane ruffles ( Figure 7C) and sporadically appeared in stress fibers of high expressing clones.…”

“…The actin constructs used in our experiments were described by von Arx et al (1995) and their sorting in several cell types was reported previously (Mounier et al, 1997). For transient and stable transfections, we used a pCMV␤ vector (Clontech Laboratories AG, Basel, CH), in which the neomycin gene was cloned in EcoRI and in which the ␤-galactosidase gene was replaced by full-length cDNA encoding rat ␣-SMA, human ␥-and ␤-cytoplasmic actins, and chicken ␣-cardiac muscle actin.…”

The physiological significance of β-actin mRNA localization in determining cell polarity and directional motility

“…␤-actin does not substitute for muscle actin in either the formation of stress fibers (17) or myofibrils in cardiomyocytes (18). In addition, it seems to interact more tightly with certain actin binding proteins that may function at the leading edge of crawling cells.…”

Rescue of cardiac α-actin-deficient mice by enteric smooth muscle γ-actin

“…All four muscle actins can incorporate into the thin filaments of cultured adult rat cardiomyocytes (41), and vascular smooth muscle ␣-actin is normally present in the sarcomeres of the developing heart (15,17). Similarly, both vascular smooth muscle and skeletal muscle ␣-actins can be detected in myofibrils (Fig.…”