Caldesmon Research Articles

Activation of Toll-like receptor 3 increases mouse aortic vascular smooth muscle cell contractility through ERK1/2 pathway.

Activation of Toll-like receptor 3 (TLR3), a pattern recognition receptor of the innate immune system, is associated with vascular complications. However, whether activation of TLR3 alters vascular contractility is unknown. We, therefore, hypothesized that TLR3 activation augments vascular contractility and activates vascular smooth muscle cell (VSMC) contractile apparatus proteins. Male mice were treated with polyinosinic-polycytidylic acid (Poly I:C group, 14days), a TLR3 agonist; control mice received saline (vehicle, 14days). At the end of protocol, blood pressure was measured by tail cuff method. Aortas were isolated and assessed for contractility experiments using a wire myograph. Aortic protein content was used to determine phosphorylated/total interferon regulatory factor 3 (IRF3), a downstream target of TLR3 signaling, and ERK1/2 using Western blot. We investigated the TLR3/IRF3/ERK1/2 signaling pathway and contractile-related proteins such as phosphorylated/total myosin light chain (MLC) and caldesmon (CaD) in aortic VSMC primary cultures. Poly I:C-treated mice exhibited (vs. vehicle-treated mice) (1) elevated systolic blood pressure. Moreover, Poly I:C treatment (2) enhanced aortic phenylephrine-induced maximum contraction, which was suppressed by PD98059 (ERK1/2 inhibitor), and (3) increased aortic levels of phosphorylated IRF3 and ERK1/2. Stimulation of mouse aortic VSMCs with Poly I:C resulted in increased phosphorylation of IRF3, ERK1/2, MLC, and CaD. Inhibition of ERK1/2 abolished Poly I:C-mediated phosphorylation of MLC and CaD. Our data provide functional evidence for the role of TLR3 in vascular contractile events, suggesting TLR3 as a potential new therapeutic target in vascular dysfunction and regulation of blood pressure.

The role of caldesmon and its phosphorylation by ERK on the binding force of unphosphorylated myosin to actin

BackgroundStudies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low Adenosine triphosphate (ATP) consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin. MethodsTo measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (Funb) in the absence or presence of caldesmon, extracellular signal-regulated kinase (ERK)-phosphorylated CaD, or CaD plus tropomyosin. ResultsFunb from unregulated actin (0.10±0.01pN) was significantly increased in the presence of CaD (0.17±0.02pN), tropomyosin (0.17±0.02pN) or both regulatory proteins (0.18±0.02pN). ERK phosphorylation of CaD significantly reduced the Funb (0.06±0.01pN). Inspection of the traces of the Funb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment. ConclusionsCaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels. General significanceThis study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state.

Effect of electroacupuncture stimulation at Zusanli acupoint (ST36) on gastric motility: possible through PKC and MAPK signal transduction pathways.

BackgroundElectroacupuncture (EA) stimulation has been shown to have a great therapeutic potential for treating gastrointestinal motility disorders. However, no evidence has clarified the mechanisms contributing to the effects of EA stimulation at the Zusanli acupoint (ST.36). This study was designed to investigate the regulative effect of EA stimulation at the ST.36 on gastric motility and to explore its possible mechanisms.MethodsThirty Sprague-Dawley rats were randomly divided into three groups: the ST.36 group, the non-acupoint group, and the control group. EA stimulation was set at 2 Hz, continuous mode, and 1 V for 30 min. The frequency and average peak amplitude of gastric motility were measured by electrogastrography. The protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) signaling pathways were assessed using real-time polymerase chain reactions. Caldesmon (CaD) and calponin (CaP) protein expression in the gastric antrum were detected on Western blots. A Computed Video Processing System was used to evaluate morphological changes in smooth muscle cells (SMCs) from the gastric antrum.ResultsEA stimulation at ST.36 had a dual effect on the frequency and average peak amplitude. Additionally, EA stimulation at ST.36 regulated the expression of some genes in the PKC and MAPK signaling pathways, and it regulated the expression of the CaD and CaP proteins. EA serum induced SMC contractility. Promotion of gastric motility may correlate with up-regulation of MAPK6 (ERK3), MAPK13, and Prostaglandin-endoperoxide synthase 2 (PTGS2) gene expression, and the down-regulation of the collagen, type I, alpha 1 (COL1A1) gene and CaD and CaP protein expression. Inhibition of gastric motility may correlate with down-regulation of the Interleukin-1 receptor type 2 (IL1R2) and Matrix metalloproteinase-9 (MMP9) genes, and up-regulation of CaD and CaP protein expression.ConclusionsEA stimulation at ST.36 regulated gastric motility, and the effects were both promoting and inhibiting in rats. The possible mechanisms may correlate with the PKC and MAPK signal transduction pathways.

Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle

Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

Overexpression of caldesmon is associated with lymph node metastasis and poorer prognosis in patients with oral cavity squamous cell carcinoma

A previous comparative tissue proteomics study by the authors of the current study led to the identification of caldesmon (CaD) as one of the proteins associated with cervical metastasis of oral cavity squamous cell carcinoma (OSCC). In the current investigation, the authors focused on the potential functions of CaD in patients with OSCC. CaD expression was examined in tissue samples from 155 patients using immunohistochemical analysis. The expression of CaD variants was determined by Western blot analysis and reverse transcriptase-polymerase chain reaction. In addition, the specific effects of CaD gene overexpression and silence were determined in OSCC cell lines. CaD expression was found to be significantly higher in tumor cells from metastatic lymph nodes compared with primary tumor cells, and was nearly absent in normal oral epithelia. Higher CaD expression was found to be correlated with positive N classification, poor differentiation, perineural invasion, and tumor depth (P = .001, P = .029, P = .001, and P = .031, respectively). In survival analyses, OSCC patients with higher CaD expression were found to have poorer prognosis with regard to disease-specific survival and disease-free survival (P = .003 and P = .014, respectively). Multivariate analyses further indicated that higher CaD expression was an independent predictor of disease-specific survival (P = .043). Serum CaD levels were found to be significantly higher in patients with OSCC, but this finding was not associated with clinicopathological manifestations. Data obtained from in vitro suppression, rescue, and overexpression of CaD in OEC-M1 cells indicated that CaD promotes migration and invasive processes in OSCC cells. The findings of the current study collectively suggest that the low-molecular-weight CaD expression in OSCC tumors is associated with tumor metastasis and patient survival.

Inverse interaction between tropomyosin and phosphorylated myosin in the presence or absence of caldesmon

In the present study, co-sedimentation assay, intrinsic fluorescence intensity measurement, and Mg²⁺-ATPase activity analysis were carried out to investigate the direct effect of tropomyosin (TM) on unphosphorylated myosin (UM) or phosphorylated myosin (PM) in the presence or absence of caldesmon (CaD). Results showed that TM significantly decreased the sedimentation, intrinsic fluorescence intensity, and the Mg²⁺-ATPase activity of PM, but not UM. In the presence of CaD, TM also significantly decreased these parameters irrespective of myosin phosphorylation, suggesting that the interaction between TM and CaD abolished the effects of TM on PM or UM and that there was an inverse interaction between TM and PM, characterized by the decreased PM sedimentation and intrinsic fluorescence intensity.

Caldesmon: New Insights for Diagnosing Endometriosis1

Considerable effort has been invested in searching for less invasive methods of diagnosing endometriosis. Previous studies have indicated altered levels of the CALD1 gene (encoding the protein caldesmon) in endometriosis. The aims of our study were to investigate whether average CALD1 expression and caldesmon protein levels are differentially altered in the endometrium and endometriotic lesions and to evaluate the performance of the CALD1 gene and caldesmon protein as potential biomarkers for endometriosis. Paired biopsies of endometrial tissue (eutopic endometrium) and endometriotic lesions (ectopic endometrium) were obtained from patients with endometriosis to evaluate CALD1 gene expression and caldesmon protein levels by real-time PCR and Western blot analysis, respectively. In addition, immunostaining for caldesmon to determine cellular localization was also performed. Endometrium from women without endometriosis was used as a control. Increased CALD1 expression and caldesmon levels were detected in the endometriotic lesions. The electrophoretic profile of caldesmon by Western blot analysis was clearly different between the control group (endometrium of women without endometriosis) and the group of women with endometriosis (eutopic endometrium and endometriotic lesions). Caldesmon expression as determined by immunostaining showed no variation among the cell types in endometriotic lesions and eutopic endometrium. Stromal cells marked positively in eutopic endometrium from control patients and in the endometriotic lesions. The presence of caldesmon in the endometrium of patients with and without endometriosis permitted diagnoses with 95% sensitivity (specificity 100%) and 100% sensitivity (specificity 100%) for the disease and for minimal to mild endometriosis in the proliferative phase of the menstrual cycle, respectively. In the secretory phase, minimal to mild endometriosis was detected with 90% sensitivity and 93.3% specificity. Caldesmon is a possible predictor of endometrial dysregulation in patients with endometriosis. A potential limitation of our study is the fact that other endometrial diseases were not excluded, and therefore prospective studies are needed to confirm the potential of caldesmon as a biomarker exclusively for endometriosis.

286 AMINO ACID MUTATIONS IN THE MOUSE CALDESMON C-TERMINAL FUNCTIONAL DOMAIN CAUSES DETRUSOR OVERACTIVITY DURING THE FILLING PHASE OF THE BLADDER

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology (I)1 Apr 2013286 AMINO ACID MUTATIONS IN THE MOUSE CALDESMON C-TERMINAL FUNCTIONAL DOMAIN CAUSES DETRUSOR OVERACTIVITY DURING THE FILLING PHASE OF THE BLADDER Maoxian Deng, Boopathi Ettickan, Joseph Hypolite, Tobias Raabe, Shaohua Chang, Stephen Zderic, and Samuel Chacko Maoxian DengMaoxian Deng Glenolden, PA More articles by this author , Boopathi EttickanBoopathi Ettickan Glenolden, PA More articles by this author , Joseph HypoliteJoseph Hypolite Glenolden, PA More articles by this author , Tobias RaabeTobias Raabe Philadelphia, PA More articles by this author , Shaohua ChangShaohua Chang Glenolden, PA More articles by this author , Stephen ZdericStephen Zderic Philadelphia, PA More articles by this author , and Samuel ChackoSamuel Chacko Glenolden, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1670AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Partial bladder outlet obstruction (PBOO) leads to remodeling of the detrusor smooth muscle (DSM) and hypertrophy. Hypertrophied DSM reveals alterations in the contractile and regulatory proteins and detrusor overactivity and overactive bladder. Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of CaD functional domain that inhibits the actomyosin ATPase leads to diminished CaD-induced inhibition of actin-actin-activated ATP hydrolysis by myosin. This study was conducted to determine if mutation of the ATPase inhibitory domain in the CaD alter the contractile characteristics of DSM during the resting phase of the bladder and during voiding contraction. METHODS Transgenic mice with mutations of 5 amino acid residues (Lys523 to Gln, Val524 to Leu, Ser526 to Thr, Pro527 to Cys, and Lys529 to Ser), which encompasses part of the tropomyosin-binding and ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. RESULTS The homozygous (−/−) animals did not develop, but heterozygous (−/+) mice, carrying the expected mutations, matured and reproduced normally. The bladder from −/+ mice were slightly hypertrophied compared to WT. The force produced by DSM strips from −/+ mice in response to KCl and electrical field stimulation was 40-60% more than that of the WT. Furthermore, −/+ mice revealed non-voiding bladder contractions during the bladder filling phase on awake cystometry, indicating that the CaD ATPase inhibitory site suppresses force generation during bladder filling and changing the amino acid sequence in 50% of the CaD partially releases the suppression of force generation. CONCLUSIONS Our data show for the first time a functional phenotype, at the tissue and organ level in vivo, following mutation of the CaD ATPase inhibitory domain. Furthermore, homozygosity for mutation of the ATPase inhibitory site appears to be lethal for embryonic development. The CaD mutant mouse line might serve as a model for detrusor overactivity and overactive bladder. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e116-e117 Peer Review Report Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Maoxian Deng Glenolden, PA More articles by this author Boopathi Ettickan Glenolden, PA More articles by this author Joseph Hypolite Glenolden, PA More articles by this author Tobias Raabe Philadelphia, PA More articles by this author Shaohua Chang Glenolden, PA More articles by this author Stephen Zderic Philadelphia, PA More articles by this author Samuel Chacko Glenolden, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

CGMP-dependent Protein Kinase Iβ Regulates Breast Cancer Cell Migration and Invasion via a Novel Interaction with the Actin/Myosin-associated Protein Caldesmon

The two isoforms of type I cGMP-dependent protein kinase (PKGIα and PKGIβ) differ in their first ∼100 amino acids, giving each isoform unique dimerization and autoinhibitory domains. The dimerization domains form coiled-coil structures and serve as platforms for isoform-specific protein-protein interactions. Using the PKGIβ dimerization domain as an affinity probe in a proteomic screen, we identified the actin/myosin-associated protein caldesmon (CaD) as a PKGIβ-specific binding protein. PKGIβ phosphorylated human CaD on serine 12 in vitro and in intact cells. Phosphorylation on serine 12 or mutation of serine 12 to glutamic acid (S12E) reduced the interaction between CaD and myosin IIA. Because CaD inhibits myosin ATPase activity and regulates cell motility, we examined the effects of PKGIβ and CaD on cell migration and invasion. Inhibition of the NO/cGMP/PKG pathway reduced migration and invasion of human breast cancer cells, whereas PKG activation enhanced their motility and invasion. siRNA-mediated knockdown of endogenous CaD had pro-migratory and pro-invasive effects in human breast cancer cells. Reconstituting cells with wild-type CaD slowed migration and invasion; however, CaD containing a phospho-mimetic S12E mutation failed to reverse the pro-migratory and pro-invasive activity of CaD depletion. Our data suggest that PKGIβ enhances breast cancer cell motility and invasive capacity, at least in part, by phosphorylating CaD. These findings identify a pro-migratory and pro-invasive function for PKGIβ in human breast cancer cells, suggesting that PKGIβ is a potential target for breast cancer treatment.

Sox4-mediated caldesmon expression facilitates skeletal myoblast differentiation

Caldesmon (CaD), which was originally identified as an actin-regulatory protein, is involved in the regulation of diverse actin-related signaling processes, including cell migration and proliferation, in various cells. The cellular function of CaD has been studied primarily in the smooth muscle system; nothing is known about its function in skeletal muscle differentiation. In this study, we found that the expression of CaD gradually increased as differentiation of C2C12 myoblasts progressed. Silencing of CaD inhibited cell spreading and migration, resulting in a decrease in myoblast differentiation. Promoter analysis of the caldesmon gene (Cald1) and gel mobility shift assays identified Sox4 as a major trans-acting factor for the regulation of Cald1 expression during myoblast differentiation. Silencing of Sox4 decreased not only CaD protein synthesis but also myoblast fusion in C2C12 cells and myofibril formation in mouse embryonic muscle. Overexpression of CaD in Sox4-silenced C2C12 cells rescued the differentiation process. These results clearly demonstrate that CaD, regulated by Sox4 transcriptional activity, contributes to skeletal muscle differentiation.

Molluscan smooth catch muscle contains calponin but not caldesmon

We isolated Ca(2+)-regulated thin filaments from the smooth muscle of the mussel Crenomytilus grayanus and studied the protein composition of different preparations from this muscle: whole muscle, heat-stable extract, fractions from heat-stable extract, thin filaments and intermediate stages of thin filaments purification. Among the protein components of the above-listed preparations, we did not find caldesmon (CaD), although two isoforms of a calponin-like (CaP-like) protein, which along with CaD is characteristic of vertebrate smooth muscle, were present in thin filaments. Thus, CaD is not Ca(2+)-regulator of thin filaments of this muscle. On the other hand, the mussel CaP-like protein is also not such Ca(2+)-regulator since we have shown that this protein can be selectively removed from isolated mussel thin filaments without loss of their Ca(2+)-sensitivity. We suggest that thin filaments in the smooth catch muscle possess other type of Ca(2+)-regulation, different from that in vertebrate smooth muscles.

Glucocorticoid Suppresses Dendritic Spine Development Mediated by Down-Regulation of Caldesmon Expression

Glucocorticoids (GCs) mediate the effects of stress to cause structural plasticity in brain regions such as the hippocampus, including simplification of dendrites and shrinkage of dendritic spines. However, the molecular mechanics linking stress and GCs to these effects remain largely unclear. Here, we demonstrated that corticosterone (CORT) reduces the expression levels of caldesmon (CaD), causing dendritic spines to become vulnerable. CaD regulates cell motility by modulating the actin-myosin system and actin filament stability. In cultured rat hippocampal neurons, CaD localized to dendritic spines by binding to filamentous actin (F-actin), and CaD expression levels increased during spine development. CaD stabilized the F-actin dynamics in spines, thereby enlarging the spine heads, whereas CaD knockdown decreased the spine-head size via destabilization of the F-actin dynamics. CaD was also required for chemical LTP-induced actin stabilization. The CaD expression levels were markedly decreased by exposure to CORT mediated by suppression of serum response factor-dependent transcription. High CORT levels reduced both the spine-head size and F-actin stability similarly to CaD knockdown, and overexpressing CaD abolished the detrimental effect of CORT on dendritic spine development. These results indicate that CaD enlarges the spine-head size by stabilizing F-actin dynamics, and that CaD is a critical target in the GC-induced detrimental effects on dendritic spine development.

Differential role of actin-binding proteins in controlling the adipogenic differentiation of human CD105-positive Wharton's Jelly cells

BackgroundWharton's Jelly cells (WJCs) can be differentiated into adipocytes by cytoskeletal reorganisation in association with changes in the mechanical properties of cells. MethodsWJCs subjected to adipocyte induction were observed changes in the cell morphology and alterations in actin filament formation. Transfection with either small interfering RNAs (siRNAs) against formin-2 (FMN-2), tropomyosin-1 (Tm-1), caldesmon (CaD), and profilin (Pro) or a pcDNA6-gelsolin (GSN)-constructed vector in WJCs was used to establish their regulatory roles in controlling adipogenesis. Phenotypic transformation of the cell shape and changes in cell surface adhesion force were determined in WJCs after transformation. ResultsThe levels of protein and mRNA expression of β-actin and several key actin binding ptoteins (ABPs) were decreased during the early stage of adipogenic induction but were recovered in the later induction. The siFMN-2, siTm-1, siCaD, and siPro gene knockdown in WJCs caused a widening of the cell shape, while WJCs overexpressing GSN retained a fibroblast cell shape. For both transformations, atomic force microscopy revealed alterations in the biomechanical signals on the cell surface. However, the adipogenic potency was increased after siFMN-2, siTm-1, siCaD, and siPro gene knockdown and decreased during GSN overexpression. ConclusionssiRNA gene knockdown of siFMN-2, siTm-1, siCaD, and siPro enhances the potency for WJCs commitment to adipocyte, while GSN overexpression modulates the PPAR-γ-independent pathway for the adipogenesis of WJCs. General significanceThe phenotypic changes associated with decreased ABP gene expression are critical for regulating the adipogenic differentiation of WJCs through the temporal control of actin filament organisation.

Smooth muscle caldesmon modulates peristalsis in the wild type and non‐innervated zebrafish intestine

The high molecular weight isoform of the actin-binding protein Caldesmon (h-CaD) regulates smooth muscle contractile function by modulating cross-bridge cycling of myosin heads. The normal inhibitory activity of h-CaD is regulated by the enteric nervous system; however, the role of h-CaD during intestinal peristalsis has never been studied. We identified a zebrafish paralog of the human CALD1 gene that encodes an h-CaD isoform expressed in intestinal smooth muscle. We examined the role of h-CaD during intestinal peristalsis in zebrafish larvae by knocking down the h-CaD protein using an antisense morpholino oligonucleotide. We also developed transgenic zebrafish that express inhibitory peptides derived from the h-CaD myosin and actin-binding domains, and examined their effect on peristalsis in wild-type zebrafish larvae and sox10 (colourless) mutant larvae that lack enteric nerves. Genomic analyses identified two zebrafish Caldesmon paralogs. The cald1a ortholog encoded a high molecular weight isoform generated by alternative splicing whose intestinal expression was restricted to smooth muscle. Propulsive intestinal peristalsis was increased in wild-type zebrafish larvae by h-CaD knockdown and by expression of transgenes encoding inhibitory myosin and actin-binding domain peptides. Peristalsis in the non-innervated intestine of sox10 (colourless) larvae was partially restored by h-CaD knockdown and expression of the myosin-binding peptide. Disruption of the normal inhibitory function of h-CaD enhances intestinal peristalsis in both wild-type zebrafish larvae and mutant larvae that lack enteric nerves, thus confirming a physiologic role for regulation of smooth muscle contraction at the actin filament.

Arp2/3 Exhibits Higher Affinity for Young than for Mature Actin Filaments

Actin undergoes a conformational transition during polymerization, known as maturation. Caldesmon (CaD) or its C-terminal fragment, H32K, when present before the transition, inhibits this process and arrests the actin filament at the pre-transitional “young” state, which is otherwise transient (Huang et al., J Biol Chem 285, 71-79, 2010). However, CaD in cells usually binds to stable actin filaments, except when it is phosphorylated and translocated to the cell leading edge where actin is being actively assembled (Kordowska et al., Exp Cell Res 312, 95-110, 2006); this may be the only occasion that CaD could interfere with actin maturation in vivo. We hypothesized that the young actin filaments stabilized by phosphorylated CaD are rendered more amenable to remodeling. Electron microscopic images indeed show that these young filaments have a rough and fuzzy morphology (Collins et al., BioArchitecture 1, 127-133, 2011). The question then arises: Could such an irregular appearance in fact provide more available docking sites for other actin-binding proteins, and thereby facilitate actin dynamics? To test this idea, we have performed binding experiments between Arp2/3 and actin filaments formed under the condition favoring either the young or the mature state. Actin filaments polymerized in the presence and absence of the CaD fragment H32K were incubated with the Arp2/3 complex and centrifuged; the amount of actin-bound Arp2/3 in the pellet was then analyzed by immuno-reactivity toward anti-Arp2 after serial dilutions. We found that the level of Arp2/3 cosedimented with actin filaments polymerized in the presence of H32K was higher than that bound to CaD-free F-actin, indicating stronger binding of Arp2/3 to the young actin filaments than to the mature filaments. These results appear to support our hypothesis. Supported by a grant from NIH.

Expression of genes encoding smooth muscle contractile proteins in vaginal tissue of women with and without pelvic organ prolapse

We hypothesize that the expression of genes encoding vaginal smooth muscle (SM) contractile proteins is altered in patients with pelvic organ prolapse (POP) and is influenced by age and menopausal status. We aim to analyze the expression of SM-myosin heavy chain (MHY11), caldesmon (CALD1), SM gamma-actin (ACTG2), and tropomyosin (TPM1), in premenopausal and postmenopausal women with advanced POP and asymptomatic controls. During total hysterectomy we collected anterior vaginal wall biopsy samples from 55 women, 37 premenopausal (23 patients and 14 controls), and 18 postmenopausal women (13 patients and 5 controls). Total mRNA from the tissues was quantified by real-time RT-PCR. MHY11 gene expression was down-regulated in premenopausal POP patients compared to premenopausal controls (fivefold, P = 0.002). In the postmenopausal groups, we observed a sixfold increase in the CALD1 gene expression in POP patients compared to asymptomatic controls (P = 0.03). The gene expression of CALD1, ACTG2, and TPM1 was significantly down-regulated in vaginal tissue of healthy women after menopause (P < 0.05). Dysregulation of the vaginal SM content in POP patients involves alteration of different cellular pathways according to age and menopausal status.

Caldesmon regulates actin dynamics to influence cranial neural crest migration inXenopus

Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells to migrate a significantly shorter distance, prevents their segregation into distinct migratory streams, and later results in severe defects in cartilage formation. Demonstrating specificity, these effects are rescued by adding back exogenous CaD. Interestingly, CaD proteins with mutations in the Ca(2+)-calmodulin-binding sites or ErK/Cdk1 phosphorylation sites fail to rescue the knockdown phenotypes, whereas mutation of the PAK phosphorylation site is able to rescue them. Analysis of neural crest explants reveals that CaD is required for the dynamic arrangements of actin and, thus, for cell shape changes and process formation. Taken together, these results suggest that the actin-modulating activity of CaD may underlie its critical function and is regulated by distinct signaling pathways during normal neural crest migration.

Interaction between myosin and a trace amount of caldesmon

Caldesmon (CaD) is known as an actin binding protein. In this study, we proposed that a trace amount of caldesmon (TACD) could highly, efficiently, interact with myosin by producing a 'domino-like cascade' and characterized that TACD (lowest caldesmon/myosin molar ratio: 1/10,000) significantly increased precipitations and intrinsic tryptophan fluorescence intensity of myosin in both phosphorylated and unphosphorylated states compared to the base controls (P < 0.01). Actin-blocked TACD-myosin interaction, suggesting that it functioned as a negative regulator. Since CaD is not an enzyme, the in vivo significance of the highly efficient TACD-myosin interaction needs further investigation.

The effect of l-arginine on bladder dysfunction following ovariectomy in a rabbit model

The present study was designed to investigate the effect of nitric oxide precursor, L: -arginine, on bladder function following ovariectomy. Twenty-eight New Zealand white female rabbits were separated into seven groups. Groups 1 to 6 underwent ovariectomy surgery. Among them, groups 1 and 2 received ovariectomy without treating with L-arginine. Groups 3, 4, 5, and 6 were given high L-arginine diet and were sacrificed 1, 3, 7, and 14 days after ovariectomy, respectively. Group 7 served as the control group. The effects of L: -arginine on the contractility of bladder tissues were determined in response to various stimulations. In addition, L-arginine effects on the expression of Rho kinase (ROK), protein kinase C potentiated inhibitor (CPI-17), caldesmon (CaD), and calponin (CaP) were studied by immunoblotting. Ovariectomy significantly decreases contractile response to all forms of stimulation. Feeding rabbits L: -arginine significantly increases contractile response at 1 day following ovariectomy, but the response decreases to the control level by 14 days. Ovariectomy increases the expressions of both isoforms of CaD, CaP, and CPI-17; L-arginine treatment induces ROK underexpression, while CaP is overexpressed in the early few days of ovariectomy but returns to the control level at 2 weeks after ovariectomy. Ovariectomy appreciably reduced bladder contractility. Treatment with L-arginine reversed the ovariectomy-induced bladder dysfunction. Decreased bladder contractile response was observed in the early days following ovariectomy.

Structural studies on maturing actin filaments

We have previously reported that actin undergoes a conformational transition (which we named "maturation") during polymerization, and that the actin-binding protein, caldesmon (CaD), when added at an early phase of polymerization, interferes with this process (Huang et al. J Biol Chem 2010; 285:71). The pre-transition filament is characterized by relatively low pyrene-fluorescence intensity when pyrene-labeled actin is used as a reporter of subunit assembly into filaments, whereas the mature filament emits a characteristic enhanced fluorescence. Previously reported co-sedimentation experiments suggest that filament formation is not inhibited by the presence of CaD, despite blocking the transition associated with filament maturation. In this study we visualized structural effects of CaD on the assembly of actin filaments by TIRF and electron microscopy. CaD-free actin forms "rough" filaments with irregular edges and indistinct subunit organization during the initial phase (∼20 min under our conditions) of polymerization as reported previously by others (Steinmetz et al. J Cell Biol 1997; 138:559; Galinska-Rakoczy et al. J Mol Biol 2009; 387:869), which most likely correspond to the pre-transition state preceding the maturation step. Later during the polymerization process "mature" filaments exhibit a smoother F-actin appearance with easily detectible double helically arranged actin subunits. While the inclusion of the actin-binding domain of CaD during actin polymerization does not affect the elongation rate, it is associated with a prolonged pre-transition phase, characterized by a delayed alteration (rough to smooth) of the appearance of filaments, consistent with a later onset of the maturation process.