Caldesmon mutant defective in Ca2+-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 Ca2+-calmodulin (Ca2+-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 Ca2+-CaM binding were each replaced with alanine. The mutation abolished most CaD39-AB binding to Ca2+-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 Ca2+-CaM in cell migration. Together, these results demonstrate that CaD plays a crucial role in mediating the effects of Ca2+-CaM on the dynamics of the actin cytoskeleton during cell migration.

Comprehensive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment

“…Consistent with the plasticity of actin polymerization at this time point, we also observed decreases in levels of proteins (e.g. caldesmon, CapZA) that have predicted roles in the stabilization of actin filaments (67)(68)(69)(70)(71). Actin capping proteins are thus critical to direct local polymerization to facilitate lamellipodia formation and motility and cell velocity (72).…”

Sox4-mediated caldesmon expression facilitates skeletal myoblast differentiation

“…The cellular functions of CaD are controversial. There is evidence that h-CaD expression enhances stress fiber stability and reduces cell motility in non-muscle cells (Castellino et al, 1992;Castellino et al, 1995;Eppinga et al, 2006;Li et al, 2004;Mayanagi et al, 2008;Mirzapoiazova et al, 2005). Moreover, CaD expression induced by p53 suppresses Src-kinase-dependent podosome formation, which inhibits fibroblast and rat aortic SMC migration (Mukhopadhyay et al, 2009).…”

“…CaD is involved in actin-dependent cell morphology and motility in various cells (Castellino et al, 1995;Eppinga et al, 2006;Li et al, 2004;Mayanagi et al, 2008;Mirzapoiazova et al, 2005). Therefore, we examined the effect of depletion of CaD on the spreading of C2C12 cells.…”

Alternative Splicing in Colon, Bladder, and Prostate Cancer Identified by Exon Array Analysis

“…We identified variants of CALD1 (34), which links myosin and actin filaments; TPM1, an actin-binding tumor suppressor with apoptosis-promoting function (36); and ACTN1 and VCL, both encoding cytoskeletal elements that participate in the organization of the cytoskeleton by interacting with actin and with each other (35). Whether this group of proteins is more prone to splicing variation without functional effects or whether the malignant process selects for these variants is unknown.…”

“…3). CALD1, encoding an actin-and calmodulin-binding protein (34), displayed an identical splicing pattern in all three types of cancers. A long CALD1 isoform, including an extended form of exon 5 (via a downstream alternative 5Ј splice site) and exon 6, was absent or reduced in bladder, colon, and metastatic prostate cancer but also missing from two of five normal bladder samples.…”