Anillin binds nonmuscle myosin II and regulates the contractile ring

We demonstrate that the contractile ring protein anillin interacts directly with nonmuscle myosin II and that this interaction is regulated by myosin light chain phosphorylation. We show that despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. We propose a role for anillin in spatially regulating the contractile activity of myosin II during cytokinesis.     

Differential localization of unconventional myosin I and nonmuscle myosin II during B cell spreading

Cross-linking of CD44 in vitro promotes chemokinesis and actin-based dendrite formation in T and B cells. However, the mechanisms by which the adhesion molecule CD44 induces cytoskeleton activation in lymphocytes are still poorly understood. In this study, we have investigated whether myosin isoforms are involved in CD44-dependent dendrite formation in activated B cells. Pharmacological inhibition of myosin with 2,3-butanedione monoxime strongly affected spreading and dendrite formation, suggesting that these cellular motors may participate in these phenomena. Furthermore, immunofluorescence analysis showed differences in subcellular localization of class I and class II myosin during B cell spreading. In response to CD44 cross-linking, myosin-1c was polarized to lamellipodia, where F-actin was high. In contrast, the distribution of cytosplasmic nonmuscle class II myosin was not altered. Expressions of myosin-1c and II were also demonstrated in B cells by Western blot. Although the inhibition of PLCgamma, PI3K and MEK-1 activation affected the spreading and dendrite formation in activated B cells, only PLCgamma and MEK-1 inhibition correlated with absence of myosin-1c polarization. Additionally, myosin-1c polarization was observed upon cross-linking of other surface molecules, suggesting a common mechanism for B cell spreading. This work shows that class I and class II myosin are expressed in B cells, are differentially distributed, and may participate in the morphological changes of these cells.     

Native nonmuscle myosin II stability and light chain binding in Drosophila melanogaster

Native nonmuscle myosin IIs play essential roles in cellular and developmental processes throughout phylogeny. Individual motor molecules consist of a heterohexameric complex of three polypeptides which, when properly assembled, are capable of force generation. Here, we more completely characterize the properties, relationships and associations that each subunit has with one another in Drosophila melanogaster. All three native nonmuscle myosin II polypeptide subunits are expressed in close to constant stoichiometry to each other throughout development. We find that the stability of two subunits, the heavy chain and the regulatory light chain, depend on one another whereas the stability of the third subunit, the essential light chain, does not depend on either the heavy chain or regulatory light chain. We demonstrate that heavy chain aggregates, which form when regulatory light chain is lacking, associate with the essential light chain in vivo-thus showing that regulatory light chain association is required for heavy chain solubility. By immunodepletion we find that the majority of both light chains are associated with the nonmuscle myosin II heavy chain but pools of free light chain and/or light chain bound to other proteins are present. We identify four myosins (myosin II, myosin V, myosin VI and myosin VIIA) and a microtubule-associated protein (asp/Abnormal spindle) as binding partners for the essential light chain (but not the regulatory light chain) through mass spectrometry and co-precipitation. Using an in silico approach we identify six previously uncharacterized genes that contain IQ-motifs and may be essential light chain binding partners.     

Identification and characterization of nonmuscle myosin II-C, a new member of the myosin II family

A previously unrecognized nonmuscle myosin II heavy chain (NMHC II), which constitutes a distinct branch of the nonmuscle/smooth muscle myosin II family, has recently been revealed in genome data bases. We characterized the biochemical properties and expression patterns of this myosin. Using nucleotide probes and affinity-purified antibodies, we found that the distribution of NMHC II-C mRNA and protein (MYH14) is widespread in human and mouse organs but is quantitatively and qualitatively distinct from NMHC II-A and II-B. In contrast to NMHC II-A and II-B, the mRNA level in human fetal tissues is substantially lower than in adult tissues. Immunofluorescence microscopy showed distinct patterns of expression for all three NMHC isoforms. NMHC II-C contains an alternatively spliced exon of 24 nucleotides in loop I at a location analogous to where a spliced exon appears in NMHC II-B and in the smooth muscle myosin heavy chain. However, unlike neuron-specific expression of the NMHC II-B insert, the NMHC II-C inserted isoform has widespread tissue distribution. Baculovirus expression of noninserted and inserted NMHC II-C heavy meromyosin (HMM II-C/HMM II-C1) resulted in significant quantities of expressed protein (mg of protein) for HMM II-C1 but not for HMM II-C. Functional characterization of HMM II-C1 by actin-activated MgATPase activity demonstrated a V(max) of 0.55 + 0.18 s(-1), which was half-maximally activated at an actin concentration of 16.5 + 7.2 microm. HMM II-C1 translocated actin filaments at a rate of 0.05 + 0.011 microm/s in the absence of tropomyosin and at 0.072 + 0.019 microm/s in the presence of tropomyosin in an in vitro motility assay.     

Characterization and differential expression of human vascular smooth muscle myosin light chain 2 isoform in nonmuscle cells

The 20-kDa regulatory myosin light chain (MLC), also known as MLC-2, plays an important role in the regulation of both smooth muscle and nonmuscle cell contractile activity. Phosphorylation of MLC-2 by the enzyme MLC kinase increases the actin-activated myosin ATPase activity and thereby regulates the contractile activity. We have isolated and characterized an MLC-2 cDNA corresponding to the human vascular smooth muscle MLC-2 isoform from a cDNA library derived from umbilical artery RNA. The translation of the in vitro synthesized mRNA, corresponding to the cDNA insert, in a rabbit reticulocyte lysate results in the synthesis of a 20,000-dalton protein that is immunoreactive with antibodies raised against purified chicken gizzard MLC-2. The derived amino acid sequence of the putative human smooth muscle MLC-2 shows only three amino acid differences when compared to chicken gizzard MLC-2. However, comparison with the human cardiac isoform reveals only 48% homology. Blot hybridizations and S1 nuclease analysis indicate that the human smooth muscle MLC-2 isoform is expressed restrictively in smooth muscle tissues such as colon and uterus and in some, but not all, nonmuscle cell lines. Previously reported MLC-2 cDNA from rat aortic smooth muscle cells in culture is ubiquitously expressed in all muscle and nonmuscle cells, and it was suggested that both smooth muscle and nonmuscle MLC-2 proteins are identical and are probably encoded by the same gene. In contrast, the human smooth muscle MLC-2 cDNA that we have characterized from an intact smooth muscle tissue is not expressed in skeletal and cardiac muscles and also in a number of nonmuscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mammalian SEPT2 is required for scaffolding nonmuscle myosin II and its kinases

Mammalian septin SEPT2 belongs to a conserved family of filamentous GTPases that are associated with actin stress fibers in interphase cells and the contractile ring in dividing cells. Although SEPT2 is essential for cytokinesis, its role in this process remains undefined. Here, we report that SEPT2 directly binds nonmuscle myosin II (myosin II), and this association is important for fully activating myosin II in interphase and dividing cells. Inhibition of the SEPT2-myosin II interaction in interphase cells results in loss of stress fibers, while in dividing cells this causes instability of the ingressed cleavage furrow and dissociation of the myosin II from the Rho-activated myosin kinases ROCK and citron kinase. We propose that SEPT2-containing filaments provide a molecular platform for myosin II and its kinases to ensure the full activation of myosin II that is necessary for the final stages of cytokinesis.     

Correlation between the clinical phenotype of MYH9-related disease and tissue distribution of class II nonmuscle myosin heavy chains

Nonmuscle myosin heavy chain II-A is responsible for MYH9-related disease, which is characterized by macrothrombocytopenia, granulocyte inclusions, deafness, cataracts, and renal failure. Since another two highly conserved nonmuscle myosins, II-B and II-C, are known, an analysis of their tissue distribution is fundamental for the understanding of their biological roles. In mouse, we found that all forms are ubiquitously expressed. However, megakaryocytic and granulocytic lineages express only II-A, suggesting that congenital features, macrothrombocytopenia, and leukocyte inclusions correlate with its exclusive presence. In kidney, eye, and ear, where clinical manifestations have a late onset, as well as in other tissues apparently not affected in patients, II-A and at least one of the other two isoforms are expressed, suggesting that II-B and II-C can partially compensate for each other. We hypothesize that cells expressing only II-A manifest the congenital defects, while tissues expressing additional myosin II isoforms show either late onset of abnormalities or no pathological sign.     

Activation of Src mediates PDGF-induced Smad1 phosphorylation and contributes to the progression of glomerulosclerosis in glomerulonephritis

Platelet-derived growth factor (PDGF) plays critical roles in mesangial cell (MC) proliferation in mesangial proliferative glomerulonephritis. We showed previously that Smad1 contributes to PDGF-dependent proliferation of MCs, but the mechanism by which Smad1 is activated by PDGF is not precisely known. Here we examined the role of c-Src tyrosine kinase in the proliferative change of MCs. Experimental mesangial proliferative glomerulonephritis (Thy1 GN) was induced by a single intravenous injection of anti-rat Thy-1.1 monoclonal antibody. In Thy1 GN, MC proliferation and type IV collagen (Col4) expression peaked on day 6. Immunohistochemical staining for the expression of phospho-Src (pSrc), phospho-Smad1 (pSmad1), Col4, and smooth muscle α-actin (SMA) revealed that the activation of c-Src and Smad1 signals in glomeruli peaked on day 6, consistent with the peak of mesangial proliferation. When treated with PP2, a Src inhibitor, both mesangial proliferation and sclerosis were significantly reduced. PP2 administration also significantly reduced pSmad1, Col4, and SMA expression. PDGF induced Col4 synthesis in association with increased expression of pSrc and pSmad1 in cultured MCs. In addition, PP2 reduced Col4 synthesis along with decreased pSrc and pSmad1 protein expression in vitro. Moreover, the addition of siRNA against c-Src significantly reduced the phosphorylation of Smad1 and the overproduction of Col4. These results provide new evidence that the activation of Src/Smad1 signaling pathway plays a key role in the development of glomerulosclerosis in experimental glomerulonephritis.     

Inflammasome activation and metabolic disease progression

Innate pattern recognition receptors NLRs are cytosolic sensors that detect endogenous metabolic stress and form a multiprotein complex called the inflammasome, that recruits and activates caspase enzymes mediating the activation of the cytokines IL-1β and IL-18. The innate immune system and metabolic system are evolutionarily conserved, intimately integrated, and functionally dependent. In recent decades, obesity-associated metabolic diseases have been become a worldwide epidemic. Here we review recent evidence that demonstrates the important roles of NLRs and inflammasomes in response to metabolic stress in different tissues.     

MIF induces osteoclast differentiation and contributes to progression of periodontal disease in mice

Periodontal disease (PD) is a chronic inflammatory and alveolar bone destructive disease triggered by microorganisms from the oral biofilm. Oral inoculation of mice with the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) induces marked alveolar bone loss and local production of inflammatory mediators, including Macrophage Migration Inhibitory Factor (MIF). The role of MIF for alveolar bone resorption during PD is not known. In the present study, experimental PD was induced in BALB/c wild-type mice (WT) and MIF knockout mice (MIF^?/?) through oral inoculation of Aa. Despite enhanced number of bacteria, MIF^?/? mice had reduced infiltration of TRAP-positive cells and reduced alveolar bone loss. This was associated with decreased neutrophil accumulation and increased levels of IL-10 in periodontal tissues. TNF-α production was similar in both groups. In vitro, LPS from Aa enhanced osteoclastic activity in a MIF-dependent manner. In conclusion, MIF has role in controlling bacterial growth in the context of PD but contributes more significantly to the progression of bone loss during PD by directly affecting differentiation and activity of osteoclasts.     

Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos

The early Drosophila embryo undergoes two distinct membrane invagination events believed to be mechanistically related to cytokinesis: metaphase furrow formation and cellularization. Both involve actin cytoskeleton rearrangements, and both have myosin II at or near the forming furrow. Actin and myosin are thought to provide the force driving membrane invagination; however, membrane addition is also important. We have examined the role of myosin during these events in living embryos, with a fully functional myosin regulatory light-chain-GFP chimera. We find that furrow invagination during metaphase and cellularization occurs even when myosin activity has been experimentally perturbed. In contrast, the basal closure of the cellularization furrows and the first cytokinesis after cellularization are highly dependent on myosin. Strikingly, when ingression of the cellularization furrow is experimentally inhibited by colchicine treatment, basal closure still occurs at the appropriate time, suggesting that it is regulated independently of earlier cellularization events. We have also identified a previously unrecognized reservoir of particulate myosin that is recruited basally into the invaginating furrow in a microfilament-independent and microtubule-dependent manner. We suggest that cellularization can be divided into two distinct processes: furrow ingression, driven by microtubule mediated vesicle delivery, and basal closure, which is mediated by actin/myosin based constriction.     

Differential expression of transforming growth factor-beta receptors I and II and activation of Smad 3 in keloid fibroblasts

Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially collagen. However, the molecular mechanisms regulating this pathologic collagen deposition still remain to be elucidated. A previous study by this group demonstrated that transforming growth factor (TGF)-beta1 and -beta2 ligands were expressed at greater levels in keloid fibroblasts when compared with normal human dermal fibroblasts (NHDFs), suggesting that TGF-beta may play a fibrosis-promoting role in keloid pathogenesis.To explore the biomolecular mechanisms of TGF-beta in keloid formation, the authors first compared the expression levels of the type I and type II TGF-beta receptors in keloid fibroblasts and NHDFs. Next, they investigated the phosphorylation of Smad 3, an intracellular TGF-beta signaling molecule, in keloid fibroblasts and NHDFs. Finally, they examined the regulation of TGF-beta receptor II by TGF-beta1, TGF-beta2, and TGF-beta3 ligands.Our findings demonstrated an increased expression of TGF-beta receptors (types I and II) and increased phosphorylation of Smad 3 in keloid fibroblasts relative to NHDFs. These data support a possible role of TGF-beta and its receptors as fibrosis-inducing growth factors in keloids. In addition, all three isoforms of recombinant human TGF-beta proteins could further stimulate the expression of TGF-beta receptor II in both keloids and NHDFs. Taken together, these results substantiate the hypothesis that the elevated levels of TGF-beta ligands and receptors present in keloids may support increased signaling and a potential role for TGF-beta in keloid pathogenesis.     

Angiotensin II contributes to renal fibrosis independently of Notch pathway activation

Recent studies have described that the Notch signaling pathway is activated in a wide range of renal diseases. Angiotensin II (AngII) plays a key role in the progression of kidney diseases. AngII contributes to renal fibrosis by upregulation of profibrotic factors, induction of epithelial mesenchymal transition and accumulation of extracellular matrix proteins. In cultured human tubular epithelial cells the Notch activation by transforming growth factor-β1 (TGF-β1) has been involved in epithelial mesenchymal transition. AngII mimics many profibrotic actions of TGF-β1. For these reasons, our aim was to investigate whether AngII could regulate the Notch/Jagged system in the kidney, and its potential role in AngII-induced responses. In cultured human tubular epithelial cells, TGF-β1, but not AngII, increased the Notch pathway-related gene expression, Jagged-1 synthesis, and caused nuclear translocation of the activated Notch. In podocytes and renal fibroblasts, AngII did not modulate the Notch pathway. In tubular epithelial cells, pharmacological Notch inhibition did not modify AngII-induced changes in epithelial mesenchymal markers, profibrotic factors and extracellular matrix proteins. Systemic infusion of AngII into rats for 2 weeks caused tubulointerstitial fibrosis, but did not upregulate renal expression of activated Notch-1 or Jagged-1, as observed in spontaneously hypertensive rats. Moreover, the Notch/Jagged system was not modulated by AngII type I receptor blockade in the model of unilateral ureteral obstruction in mice. These data clearly indicate that AngII does not regulate the Notch/Jagged signaling system in the kidney, in vivo and in vitro. Our findings showing that the Notch pathway is not involved in AngII-induced fibrosis could provide important information to understand the complex role of Notch system in the regulation of renal regeneration vs damage progression.     

Antibody to thymus myosin: its immunological characterization and use for immunocytochemical localization of myosin in vertebrate nonmuscle cells

Thymus myosin differs immunologically from smooth muscle and striated muscle myosin isoenzymes. In the enzyme linked immunosorbent assay a moderate degree of cross reaction was observed between anti-thymus myosin and myosin from chicken gizzard (about 50% of the titer of the homologous reaction). In contrast, the cross reactivity between thymus myosin and antibodies to gizzard myosin was very low (about 5%) and no significant cross reaction was observed between thymus myosin and antibodies to striated muscle myosin and vice versa (below 1%). Antibodies to thymus myosin were further distinguished from antibodies to gizzard and striated muscle myosin by their reaction with both smooth muscle and a very broad spectrum of vertebrate nonmuscle cells. Nonmuscle cells reacting with anti-thymus myosin included (1) cell types which did not display any detectable affinity for anti-gizzard myosin (e.g. lymphocytes, polymorphonuclear leucocytes, vascular endothelium, adrenal chromaffine cells) and (2) cell types which reacted with anti-gizzard myosin as well (e.g. intestinal epithelial brush border, thymic epithelial cells, liver cells and stress fibres of cultured cells). These results illustrate, that anti-thymus myosin is a potent tool for investigating the intracellular localization of myosin in most if not all vertebrate nonmuscle cells. With respect to lymphatic tissue the present findings indicate that lymphocyte maturation appears to be accompanied by an increased level of expression of myosin and filamentous actin (the latter was visualized by labelled phalloidin). On the ultrastructural level, gold labelled antibodies to thymus myosin bound preferentially to the head region of in vitro assembled thymus myosin filaments. In cultured cells (PtK1) the antibodies showed a particular affinity for stress fibre densities, and in lymphocytes the anti-myosin label (immunoperoxidase) displayed a more or less diffuse distribution which was similar to the distribution of actin filaments (identified by decoration with heavy meromyosin).     

Chicken nonmuscle myosin heavy chains: differential expression of two mRNAs and evidence for two different polypeptides

Two different mRNAs encoding two different nonmuscle myosin heavy chains (MHCs) of approximately 200 kD have been identified in chicken nonmuscle cells, in agreement with the results of Katsuragawa et al. (Katsuragawa, Y., M. Yanagisawa, A. Inoue, and T. Masaki. 1989. Eur. J. Biochem. 184:611-616). In this paper, we quantitate the content of mRNA encoding the two MHCs in a number of different tissues using RNA blot analysis with two specific oligonucleotide probes. Our results show that the relative content of mRNA encoding MHC-A and MHC-B differs in a tissue-dependent manner. Thus the ratio of mRNA encoding MHC-A versus MHC-B varies from greater than 9:1 in spleen and intestinal epithelial cells, to 6:4 in kidney and 2:8 in brain. The effect of serum on MHC mRNA expression was studied in serum-starved cultures of chick embryo fibroblasts. Serum stimulation results in a threefold increase in the mRNA encoding MHC-A and a threefold decrease in mRNA encoding MHC-B. Using SDS polyacrylamide gels, we have separated two nonmuscle MHC isoforms (198 and 196 kD) that can be distinguished from each other by two-dimensional peptide mapping of chymotryptic digests. We provide preliminary evidence that the MHC-A mRNA encodes the 196-kD polypeptide and that the MHC-B mRNA encodes the 198-kD polypeptide.