Caldesmon mutant defective in Ca(2+)-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility

• Published in: Journal of cell science Vol. 117; no. Pt 16; pp. 3593 - 3604
• Main Authors: Li, Yan, Lin, Jenny L C, Reiter, Rebecca S, Daniels, Karla, Soll, David R, Lin, Jim J C
• Format: Journal Article
• Language: English
• Published: England 15-07-2004
• Subjects: Actins - metabolism; Animals; Base Sequence; Calcium - metabolism; Calmodulin - metabolism; Calmodulin-Binding Proteins - genetics; Calmodulin-Binding Proteins - metabolism; Cell Cycle; Cell Division; Cell Movement; CHO Cells; Cricetinae; DNA Primers; Humans; Myosins - antagonists & inhibitors; Point Mutation; Protein Binding
• ISSN: 0021-9533
• DOI: 10.1242/jcs.01216

Despite intensive in vitro studies, little is known about the regulation of caldesmon (CaD) by Ca(2+)-calmodulin (Ca(2+)-CaM) in vivo. To investigate this regulation, a mutant was generated of the C-terminal fragment of human fibroblast CaD, termed CaD39-AB, in which two crucial tryptophan residues involved in Ca(2+)-CaM binding were each replaced with alanine. The mutation abolished most CaD39-AB binding to Ca(2+)-CaM in vitro but had little effect on in vitro binding to actin filaments and the ability to inhibit actin/tropomyosin-activated heavy meromyosin ATPase. To study the functional consequences of these mutations in vivo, we transfected an expression plasmid carrying CaD39-AB cDNA into Chinese hamster ovary (CHO) cells and isolated several clones expressing various amounts of CaD39-AB. Immunofluorescence microscopy revealed that mutant CaD39-AB was distributed diffusely throughout the cytoplasm but also concentrated at membrane ruffle regions. Stable expression of CaD39-AB in CHO cells disrupted assembly of stress fibers and focal adhesions, altered cell morphology, and slowed cell cycle progression. Moreover, CaD39-AB-expressing cells exhibited motility defects in a wound-healing assay, in both velocity and the persistence of translocation, suggesting a role for CaD regulation by Ca(2+)-CaM in cell migration. Together, these results demonstrate that CaD plays a crucial role in mediating the effects of Ca(2+)-CaM on the dynamics of the actin cytoskeleton during cell migration.