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

Abstract

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.

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

“…Similar defects were observed in another CaD39-AB-expressing cell line, m5-8 (Table 2). In contrast to CaD39-AB-expressing cells, no motility defects were detected in 39C15 cells overexpressing the wild-type CaD39 fragment (Warren et al, 1996). Taken together, these results demonstrated that expression of a CaD39 defective in Ca 2+ -CaM binding impairs speed and directionality during wound healing.…”

“…Compared with the control cells, phalloidin staining is more prominent in 39C15 cells (Fig. 7F), suggesting that CaD39 stabilizes the structure of stress fibers (Warren et al, 1996). However, the stress fiber structure in CaD39-AB-expressing cells appears to be disintegrated (Fig.…”

Cytoskeletal organization in tropomyosin-mediated reversion of ras-transformation: Evidence for Rho kinase pathway

“…This results in weakening of TM-actin association, exposing actin ®laments to the action of gel-severing proteins (Pittenger et al, 1995). Furthermore, increased expression of caldesmon stabilizes micro®laments (Warren et al, 1995(Warren et al, , 1996, and caldesmon levels are decreased in many transformed cells, as shown in Figure 3, and by others (Button et al, 1995;Novy et al, 1991). More recent studies implicate caldesmon in the regulation of actomyosin contractility and adhesion-dependent signaling in ®broblasts (Helfman et al, 1999).…”

Caldesmon is an integral component of podosomes in smooth muscle cells

“…In non-muscle cells and smooth muscle cells in culture, l-caldesmon plays a dual role in the regulation of cytoskeleton organisation by (1) modulating contractility (Helfman et al, 1999), and (2) stabilisation of the stress fibres and focal adhesions (Li et al, 2004;Ishikawa et al, 1998). By virtue of its involvement in cytoskeletal organisation, l-caldesmon plays a key role in cell movement (Helfman et al, 1999;Li et al, 2004), intracellular granule movement (Hegmann et al, 1991) and cytokinesis (Warren et al, 1996;Yamashiro et al, 2001).…”