title: Caldesmon mutant defective in Ca(2+)-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility.
abstract: 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.
authors.affiliation: Department of Biological Sciences, University of Iowa, 340 Biology Building East, Iowa City, IA 52242-1324, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Jenny L C
authors.initials: JL
authors.lastname: Lin
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Reiter
authors.forename: Karla
authors.initials: K
authors.lastname: Daniels
authors.forename: David R
authors.initials: DR
authors.lastname: Soll
authors.forename: Jim J C
authors.initials: JJ
authors.lastname: Lin
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D002455:Cell Division; D002465:Cell Movement; D006224:Cricetinae; D017931:DNA Primers; D006801:Humans; D009218:Myosins / Q000037:antagonists & inhibitors; D017354:Point Mutation; D011485:Protein Binding
chemical_list: D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D009218:Myosins; D002118:Calcium