Overexpression of human fibroblast caldesmon fragment containing actin-, Ca++/calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments.

Abstract

Abstract. Fibroblast caldesmon is a protein postulated to participate in the modulation of the actin cytoskeleton and the regulation of actin-based motility. The cDNAs encoding the NH2-terminal (aa.1-243, CAD40) and COOH-terminal (aa.244-538, CAD39) fragments of human caldesmon were subcloned into expression vectors and we previously reported that bacterially produced CAD39 protein retains its actin-binding properties as well as its ability to enhance low Mr tropomyosin (TM) binding to actin and to inhibit TMactin-activated HMM ATPase activity in vitro (Novy, R. E., J. R. Sellers, L.-E Liu, and J. J.-C. Lin. 1993. Cell Motil. Cytoskeleton. 26:248-261). Bacterially produced CAD40 does not bind actin. To study the in vivo effects of CAD39 expression on the stability of actin filaments in CHO ceils, we isolated and characterized stable CHO transfectants which express varyhag amounts of CAD39. We found that expression of CAD39 in CHO cells stabilized microfilament bundles as well as endogenous TM. CaD39-expressing clones displayed an increased resistance to cytochalasin B and Triton X-100 treatments and yielded increased amounts of TM-containing actin filaments in microfilamerit isolation procedures. In addition, analysis of these clones with immunoblotting and indirect immunofluorescence microscopy with anti-TM antibody revealed that stabilized endogenous TM and enhanced TM-containing microfilament bundles parallel increased amounts of CAD39 expression. The increased TM observed corresponded to a decrease in TM turnover rate and did not appear to be due to increased synthesis of endogenous TM. Additionally, the phenomenon of stabilized TM did not occur in stable CHO clones expressing CAD40. Therefore, it is likely that CAD39 can enhance TM's binding to F-actin in vivo, thus reducing TM's rate of turnover and stabilizing actin microfilament bundles.

Caldesmon mutant defective in Ca2+-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility

“…Two overexpressing clones, m5-8 and m4-65, were used for further characterization of the cells expressing CaD39-AB, and the nonexpressing drug-resistant line C3 served as a negative control. To verify that mutant phenotypes observed in the overexpressing cells resulted from expression of CaD39-AB rather than that of wild-type CaD39, we also included a CaD39-overexpressing cell line, 39C15 (Warren et al, 1994) for comparison. The stable clones used in this study are summarized in Fig.…”

Sox4-mediated caldesmon expression facilitates skeletal myoblast differentiation

“…CaD can interact with F-actin, myosin, calmodulin (CaM) and tropomyosin (TM) (Graceffa, 1987;Smith et al, 1987;Sobue et al, 1981;Sobue and Sellers, 1991;Wang et al, 1997). Moreover, CaD enhances the binding of F-actin to TM and competes with gelsolin, an actin-severing protein that binds to F-actin (Ishikawa et al, 1989;Warren et al, 1994). Consequently, because it promotes stable actin-myosin structures in smooth muscle, CaD is an important regulatory protein for thin filament organization (Sobue and Sellers, 1991).…”

Mutant Caldesmon Lacking cdc2 Phosphorylation Sites Delays M-Phase Entry and Inhibits Cytokinesis

“…For example, caldesmon together with tropomyosin protects actin filaments against the actin-severing and -capping activities of gelsolin (Ishikawa et al, 1989a,b). In vivo, microfilaments became more resistant to cytochalasin when the COOH-terminal actinand calmodulin-binding domain of caldesmon was overexpressed in Chinese hamster ovary (CHO) cells (Warren et al, 1994). Caldesmon also is responsible for glucocorticoid-induced stabilization of the actin cytoskeleton in A549 cells (Castellino et al, 1995).…”