Characterization of cDNA clones encoding a human fibroblast caldesmon isoform and analysis of caldesmon expression in normal and transformed cells.

The Journal of Cell Biology

Lin

McDermott

et al. 1995

Abstract. Human fibroblasts generate at least eight tropomyosin (TM) isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsmo0 from four distinct genes, and we have previously demonstrated that bacterially produced chimera hTM5/3 exhibits an unusually high affinity for actin filaments and a loss of the salt dependence typical for TM-actin binding (Novy, R.E., J. R. Sellers, L.-E Liu, and J. J.-C. Lin, 1993. Cell Motil. & Cytoskeleton. 26: 248-261). TO examine the functional consequences of expressing this mutant TM isoform in vivo, we have transfected CHO cells with the full-length eDNA for hTM5/3 and compared them to cells transfected with hTM3 and hTM5. Immunofiuorescence microscopy reveals that stably transfected CHO cells incorporate force-expressed hTM3 and hTM5 into stress fibers with no significant effect on general cell morphology, microfilament organization or cytokinesis. In stable lines expressing hTM5/3, however, cell division is slow and sometimes incomplete. The doubling time and the incidence of multinucleate cells in the stable hTM5/3 lines roughly parallel expression levels. A closely related chimeric isoform hTM5/2, which differs only in the internal, alternatively spliced exon also produces defects in cytokinesis, suggesting that normal TM function may involve coordination between the amino and carboxy terminal regions. This coordination may be prevented in the chimeric mutants. As bacterially produced hTM5/3 and hTM5/2 can displace hTM3 and hTM5 from actin filaments in vitro, it is likely that CHO-expressed hTM5/3 and hTM5/2 can displace endogenous TMs to act dominantly in vivo. These results support a role for nonmuscle TM isoforms in the fine tuning of microfilament organization during cytokinesis.Additionally, we find that overexpression of TM does not stabilize endogenous microfilaments, rather, the hTM-expressing cells are actually more sensitive to cytochalasin B. This suggests that regulation of microfilament integrity in vivo requires stabilizing factors other than, or in addition to, TM.

Section: Discussionmentioning

confidence: 99%

Forced expression of chimeric human fibroblast tropomyosin mutants affects cytokinesis.

The Journal of Cell Biology

Lin

McDermott

et al. 1995

“…For example, caldesmon is phosphorylated and subsequently released from microfilaments in mitotic cells actively dissolving their actin filament bundies (Yamashiro et al, 1990;Hosoya et al, 1993). Also, in transformed cells which exhibit a diminished and diffuse actin cytoskeleton, caldesmon expression is downregulated (Novy et al, 1991;Owada et al, 1984). We are seeking to further understand the stabilizing role of nonmuscle caldes-mon and, in this paper, provide direct evidence for the first time, that the actin-, TM-, and Ca++/Calmodulin-binding COOH-terminal fragment of human fibroblast caldesmon can stabilize TMs and microfilament bundles in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…Monoclonal antibody C21 was used in this analysis because it recognizes an epitope, aa. 480-529 of human fibroblast caldesmon, that is conserved with 96% identity between human, rat and chicken caldesmon (Novy et al, 1991), and C21 does recognize CHO caldesmon to the same extent that it recognizes CAD39. To confirm that the differences seen between endogenous caldesmon levels and expressed CAD39 were not due to properties peculiar to C21, we used polyclonal anticaldesmon antibodies (Bretscher and Lynch, 1985) to stain control and CaD39-expressing cells (data not shown).…”

Section: High Amounts Of Cad39 and Cad40 Are Expressed In Stable Cho Cell Linesmentioning

confidence: 99%

“…CAD40, which does not possess actin or TM-binding sites, distributed throughout the cytoplasm and appeared to concentrate in the nucleus. The nuclear staining may be a nonspecific consequence of CaD40's small size, but, interestingly, CAD40 sequence does contain several stretches of positively charged amino acid residues, which may serve as a nuclear localization signal (for sequence detail, see Novy et al, 1991). Western blot analysis of total cell extracts, harvested after transient transfection, revealed C21-positive (CAD39) and C57-positive (CAD40) bands at the expected positions after gel electrophoresis (data not shown), indicating that CAD39 and CAD40 are being expressed in their entirety in these transfection experiments.…”

Section: Force-expressed Cad39 Localizes To Stress L~bers and Membrane Ruffle Regions In Cho Cellsmentioning

confidence: 99%

“…More evidence to suggest that nonmuscle caldesmon affects microfilament stability and organization comes from reports that document mitosis-specific phosphorylation of caldesmon, and caldesmon's subsequent release from microfilaments during mitotic cytoskeletal remodeling (Yamashiro et al, 1990;Hosoya et al, 1993). Many transformed lines also show a reorganized, diminished actin cytoskeleton, and such transformed lines also demonstrate a reduction in caldesmon expression (Novy et al, 1991;Owada et al, 1984).…”

mentioning

confidence: 99%

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

The Journal of Cell Biology

Lin

Wamboldt

1994

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.

Overexpression of microfilament-stabilizing human caldesmon fragment, CaD39, affects cell attachment, spreading, and cytokinesis

Cell Motil. Cytoskeleton

Shutt

McDermott

et al. 1996

Previous studies have demonstrated that overexpression of the carboxyl‐terminal fragment, CaD39, of human fibroblast caldesmon in Chinese hamster ovary cells protected endogenous tropomyosin from turnover and stabilized actin microfilament bundles [Warren et al., 1994: J. Cell Biol. 125:359–368]. To assess the consequences of having CaD39‐stabilized microfilaments in living cell, we characterized the motile behaviors of stable CaD39‐expressing lines. We here found that CaD39‐expressing cells adhered faster to plastic, glass, fibronectin‐coated glass, and collagen‐coated glass than control cells. Moreover, the CaD39‐expressing cells also exhibited enhanced spreading immediately after attachment. Despite these differences, overexpression of CaD39 had little effect on the velocity of intracellular granule movement, or the velocity and persistence of cellular translocation. However, CaD39‐expressing cells were more elongate and encompassed less area than non‐expressing cells during migration in a wound‐healing assay. In interphase cells, the expressed CaD39 fragments were found associated with tropomyosin‐enriched microfilaments. Like endogenous caldesmon, the CaD39 fragment was also modified at mitosis. Although a significant portion of CaD39 underwent only partial modification, the majority of the CaD39 was released from the microfilaments during mitosis. This is consistent with the finding that the CaD39‐induced advantage for attachment and spreading was lost during mitosis. In CaD39‐expressing cells, an incomplete release of the CaD39 from microfilaments at mitosis was found which may be responsible for the increase in the frequency of multinuclear cells in CaD39‐expressing lines. © 1996 Wiley‐Liss, Inc.