Caldesmon mutant defective in Ca(2+)-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility.
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.
Li
Yan
Y
Department of Biological Sciences, University of Iowa, 340 Biology Building East, Iowa City, IA 52242-1324, USA.
Lin
Jenny L C
JL
Reiter
Rebecca S
RS
Daniels
Karla
K
Soll
David R
DR
Lin
Jim J C
JJ
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
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D009218:Myosins; D002118:Calcium