3-D image reconstruction of reconstituted smooth muscle thin filaments containing calponin: visualization of interactions between F-actin and calponin

The ubiquity of biofilm and its classification as a microbial aggregate is discussed. Investigations into any microbial ecological problem operate at four levels: (i) in situ investigations, (ii) the use of microcosms, (iii) experimental model systems, and (iv) mathematical models. Each of these is defined and their use in biofilm research illustrated. It is concluded that all these approaches are valid and that scientific research in general and biofilm research in particular must profit by the use widely different methods if a complete understanding of a system is to be achieved.     

Electrostatic effects of smooth muscle calponin on actin assembly

The contribution of electrostatic interactions to the effects of chicken gizzard calponin on the kinetics of actin polymerization and the bundling of F-actin were characterized by a combination of fluorescence, light-scattering, co-sedimentation, and electron-microscopic methods. Stoichiometric amounts of calponin accelerate actin polymerization in low-ionic-strength solutions, but this effect is diminished at [KCI] = 150 mM. At low ionic strengths, micromolar concentrations of calponin induce the formation of large bundles of actin filaments, and lower concentrations of calponin quench the fluorescence of pyrene-labeled F-actin. The latter effect is related to binding of calponin to F-actin rather than to bundling of the filaments. The concentration of calponin required to bundle a fixed concentration of actin filaments increases with increasing ionic strength, as the average diameter of the bundles decreases. Millimolar concentrations of ATP, GTP or ITP are equally efficient at dispersing actin bundles to single filaments or smaller aggregates, even though a significant fraction of calponin remains bound to F-actin. Our findings show that the binding of calponin to actin is determined at least in part by electrostatic interactions, and that the polycationic nature of calponin is primarily responsible for the formation of F-actin bundles via its ability to reduce the electrostatic repulsion between the negatively charged actin filaments.     

Phosphorylation of smooth muscle myosin and myosin light chains

The most popular theory to account for the regulation of the contractile activity of smooth muscle, at the contractile protein level, is based on the phosphorylation and dephosphorylation of the myosin molecule. The enzymes involved are a myosin light chain kinase and a phosphatase, respectively. In this communication a method is given for the purification of the kinase. Using the purified kinase in combination with calmodulin, the pH dependence and rates of P transfer were examined. An Arrhenius plot of phosphorylation rates indicated that Q10 is approximately 2. The rates of P transfer to myosin light chains at 25 C and 37 C were about 15 and 34 mumol.min-1.mg-1 kinase, respectively. It is shown also that the rate of phosphorylation of isolated myosin light chains is significantly faster than the rate obtained when whole myosin is used as the phosphate acceptor, the latter being at least an order of magnitude slower. This difference in rates was not due entirely to the difference in physical states of the two substrates since at an increased ionic strength, where myosin was soluble, the rate of phosphorylation of the light chain fraction was still considerably faster than the rate of phosphorylation of whole myosin.     

Interaction of actin and ADP with the head domain of smooth muscle myosin: implications for strain-dependent ADP release in smooth muscle

Transient kinetic methods were used to study interactions between actin, MgADP, and smooth muscle (chicken gizzard) myosin subfragment 1 (smS1). The equilibrium dissociation constant (Kd) of actin for smS1 was 3.5 nM, tighter than that of skeletal S1 (skS1). Actin binding to smS1 was weakened 5-fold by saturation with ADP compared to 30-60-fold for skS1. The Kd of ADP for smS1 was increased from 1.2 to 5 microM by actin, whereas for skS1 values increased from 2 to 100 microM. Thus, coupling between ADP and actin binding is weaker for smS1. Previous studies show that release of ADP from actin.smS1.ADP produces a tilt of the regulatory domain [Whittaker, M., Wilson-Kubalek, E. M., Smith, J. E., Faust, L., Milligan, R. A., and Sweeney, H. L. (1995) Nature 378, 748-751]. This result was confirmed by independent structural methods; tilting was absent for skS1, and the Kd for ADP was in agreement with the values measured here [Gollub, J., Cremo, C. R., and Cooke, R. (1996) Nat. Struct. Biol. 3, 796-802; Poole, K. I. V., Lorenz, M., Ellison, P., Evans, G., Rosenbaum, G., Boesecke, P., Holmes, K. C., and Cremo, C. R. (1997) J. Muscle Res. Cell Motility 18, 264]. We discuss tilting upon ADP release with respect to our measurements, previous measurements with skS1, and nucleotide concentrations in smooth muscle. We propose that these data suggest a strain-dependent ADP release mechanism that may be accentuated in smooth muscles.     

Localization of protein regions involved in the interaction between calponin and myosin

Calponin is a 33-kDa smooth muscle-specific protein that has been suggested to play a role in muscle contractility. It has previously been shown to interact with actin, tropomyosin, and calmodulin. More recently we showed that calponin also interacts with myosin (Szymanski, P. T., and Tao, T. (1993) FEBS Lett. 331, 256-259). In the present study we used a combination of co-sedimentation and fluorescence assays to localize the regions in myosin and calponin that are involved in the interaction between these two proteins. We found that recombinant chicken gizzard alpha-calponin co-sediments with myosin rod and, to a lesser extent, with light meromyosin. Fluorescently labeled recombinant calponin shows interaction with heavy meromyosin and myosin subfragment 2 but not subfragment 1. A fragment comprising residues 7-182 and a synthetic peptide spanning residues 146-176 of calponin co-sediment with myosin, but fragments comprising residues 7-144 and 183-292 do not. Our results indicate that there are calponin binding sites in the subfragment 2 and light meromyosin regions of myosin, and that the region comprising residues 145-182 of calponin mediates its interaction with myosin.     

Interaction between growth factors and kinins in arterial smooth muscle cells

Bradykinin receptors are present on vascular smooth muscle cells; however, the regulation and biological function of these receptors is unclear. To address these questions the interaction between growth factors and kinins in cultured arterial smooth muscle cells has been examined. Based upon the data a hypothesis is presented that platelet-derived growth factor (PDGF) upregulates cell surface bradykinin B2 receptors on arterial smooth muscle cells. The biological effect of the increase in B2 receptors is currently unclear but under certain conditions they may enhance mitogenesis. These mitogenic effects however, are strongly opposed by the effects of bradykinin acting via a B1-type of receptor which mediates potent inhibition of growth factor-induced mitogenesis.     

Interaction of CArG elements and a GC-rich repressor element in transcriptional regulation of the smooth muscle myosin heavy chain gene in vascular smooth muscle cells

We have previously shown that maximal expression of the rat smooth muscle myosin heavy chain (SM-MHC) gene in cultured rat aortic smooth muscle cells (SMCs) required the presence of a highly conserved domain (nucleotides -1321 and -1095) that contained two positive-acting serum response factor (SRF) binding elements (CArG boxes 1 and 2) and a negative-acting GC-rich element that was recognized by Sp1 (Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997) J. Biol. Chem. 272, 6332-6340). In this study, to better understand the functional role of these three cis elements, we created a series of SM-MHC reporter-gene constructs in which each element was mutated either alone or in combination with each other and tested them for activity in transient transfection assays using primary cultured rat aortic SMCs. Results demonstrated that the most proximal SRF binding element (CArG-box1) was active in the absence of CArG-box2, but only upon removal of the GC-rich repressor. In contrast, regardless of sequence context, CArG-box2 was active only when CArG-box1 was present. We further demonstrated using electrophoretic mobility shift assays that Sp1 binding to the GC-rich repressor element did not prevent SRF binding to the adjacent CArG-box2. Thus, unlike other proteins reported to inhibit SRF activity, the repressor activity associated with the GC-rich element does not appear to function through direct inhibition of SRF binding. As a first step toward understanding the importance of these elements in vivo, we performed in vivo footprinting on the intact rat aorta. We demonstrated that both CArG boxes and the GC-rich element were bound by protein within the animal. Additionally, using the rat carotid injury model we showed that Sp1 protein was significantly increased in SMCs located within the myointimal lesion, suggesting that increased expression of this putative repressor factor may contribute to the decreased SM MHC expression within SMCs found in myointimal lesions.     

Isolation and identification of myosin I from porcine aorta media smooth muscle

A complex of 110-kDa heavy chain and calmodulin was isolated from porcine aorta media smooth muscle and identified as myosin I. The isolated myosin I consisted of equimolar amounts of 110-kDa heavy chain and calmodulin. The addition of exogenous calmodulin to the complex revealed that a maximum of two molecules of calmodulin could be bound to the heavy chain. Isolated complex bound to F-actin in an ATP-dependent manner and its Mg(2+)-ATPase activity was activated by F-actin. In addition, it bound to phospholipid, which is a characteristic property of myosin I. Calcium ions induced a structural change, which was revealed by a difference in the cleavage pattern and for rate of cleavage by alpha-chymotrypsin. This behavior was similar to that reported for brush border myosin I [L.M. Collins and A. Bretscher (1988) J. Cell. Biol. 106, 367-373]. Calcium-dependent structural change of a complex of 110-kDa heavy chain and calmodulin was found from its solubility change at various NaCl concentrations in the presence of ATP. A complex of 116-kDa heavy chain and calmodulin, possibly another type of myosin I, was also isolated. A polyclonal antibody against the complex of 110-kDa heavy chain and calmodulin did not recognize the 116-kDa heavy chain. This result suggests that at least two types of myosin Is may exist at the protein level in porcine aorta media smooth muscle.     

Effect of preincubation on smooth muscle myosin light chain kinase activity

Phosphorylation of myosin regulatory light chain (RLC) catalysed by myosin light chain kinase (MLCK) is a key reaction in the regulation of actin-myosin interaction in smooth muscle. The activation of MLCK by calmodulin (CaM) and Ca2+ was investigated over a wide range of the enzyme concentrations using myosin or its RLC with Mw = 20 kDa as substrates. Kinase activation by CaM (at saturating Ca2+ concentrations) was characterized by positive cooperativity even though noncooperative activation would be expected from the established 1:1 binding stoichiometry between MLCK and CaM. The activation of the kinase by Ca2+ was also cooperative but only at relatively low CaM levels. This cooperativity was shown to result from time dependent changes in MLCK that take place during its incubation with Ca2+ and CaM before substrate addition in phosphorylation assays. As a result the kinase activity as a function of its concentration at constant CaM level was biphasic: there was the activity optimum at 1:1 ratio of CaM to MLCK and almost complete inhibition at 3 to 7 molar excess of kinase over CaM. Such changes that take place during 10 to 15 min preincubation with Ca2+ and CaM may involve the kinase supramolecular structure formation or/and its conformational rearrangements.     

Molecular cloning and characterization of human non-smooth muscle calponin

cDNA clones encoding a calponin isoform with 309 amino acids have been isolated from human heart. The deduced amino acid polypeptide (M(r) 33,697) showed a neutral isoelectric point of 7.1. The mRNA, expressed in cultured smooth muscle cells as well as in fibroblasts, vascular endothelial cells, and keratinocytes, contains a 3' untranslated region of 1.2 kilobases that includes an Alu repetitive sequence in the antisense direction. On the basis of the nucleotide sequence identity to an expressed sequence tag, HUM21ES93 [Cheng, J.-F., Boyartchuk, V., and Zhu, Y. (1994) Genomics 23, 75-84], the human neutral calponin gene is assigned to chromosome 21q11.1. The amino acid sequence indicates that this protein is the human equivalent of mouse calponin-h2 (94.8% identity) [Strasser, P., Gimona, M., Moessler, H., Herzog, M., and Small, J.V. (1993) FEBS Lett. 330, 13-18]. Three tandem repeats of 29 amino acids, a Vav-homologous region and an actin-binding sequence, originally identified in the basic calponin isoform, are conserved. There are two consensus phosphorylation sites for tyrosine kinase. An immunoreactive form of the neutral calponin appears to be localized with vinculin in the cell-to-cell junctions of cardiomyocytes. Mouse calponin-h2 is also expressed in both embryonic and adult heart. These results indicate that the human neutral calponin is a non-smooth muscle isoform, and may play a physiological role in cytoskeletal organization.     

Actin binding to cross-linked 10 S smooth muscle myosin and 9 S heavy meromyosin

Unphosphorylated gizzard myosin and heavy meromyosin were cross linked in the 10 S and 9 S states, respectively, by the cleavable cross linker, 3,3'-dithiobis (sulfosuccinimidyl-propionate) (DTSSP). The 10 S to 6 S transition for cross-linked 10 S myosin appeared to cease; myosin appeared to remain in the 10 S state from measurements of viscosity and Mg(2+)-ATPase activity. The loss of the transition for cross-linked 9 S heavy meromyosin (HMM) was also indicated by Mg(2+)-ATPase activity. The cross links were cleaved by incubation with 50 mM dithiothreitol. From direct binding measurements, the estimated Kd's of actin to cross-linked and control heavy meromyosin were 167 and 16 microM, respectively. The binding affinity of cross-linked HMM to actin was restored to the control level by dithiothreitol.     

Mice lacking smooth muscle calponin display increased bone formation that is associated with enhancement of bone morphogenetic protein responses

BACKGROUND: Calponin is a calmodulin-and actin-binding protein expressed in smooth muscle. It promotes actin polymerization and inhibits actin-activated myosin ATPase activity. Despite the molecular and functional characterization of calponin in vitro, the physiological role of calponin in vivo has not been clarified. RESULTS: We investigated the in vivo function of smooth muscle calponin (also called basic calponin or calponin h1) by generating mice carrying a targeted mutation in both alleles of the calponin gene. Mice lacking basic calponin expression displayed enhanced ectopic bone formation in vivo, induced by recombinant human bone morphogenetic protein-2 (rhBMP-2), and an augmentation of the degree of osteoblastic differentiation of embryonic mesenchymal cells when they were stimulated by rhBMP-2. Basic calponin messenger RNA was shown to be expressed in developing and healing bone tissues, and in undifferentiated MC3T3-E1 osteoblasts. An examination of the skeletons of mutated mice showed an early onset of cartilage formation and ossification, and increased postnatal bone formation characterized by an increase in the number of activated periosteal osteoblasts. Bone fracture healing was accelerated in mutated mice. CONCLUSION: This is the first demonstration of animals with enhanced BMP responsiveness in host cells, suggesting that endogenous basic calponin may play a negative role in an osteogenic programme.     

Inhibitory effect of forskolin on myosin phosphorylation-dependent and independent contractions in bovine tracheal smooth muscle

In bovine tracheal smooth muscle, carbachol (CCh, 1 microM) and high K+ (72.7 mM) induced sustained increases in cytosolic Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction. Forskolin (FK, 1-10 microM) inhibited the CCh-induced increase in [Ca2+]i, MLC phosphorylation and force in parallel. In contrast, FK inhibited the high K(+)-induced contraction and MLC phosphorylation without changing [Ca2+]i. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), CCh (10 microM) and caffeine (20 mM) induced transient increase in [Ca2+]i and contractile force by releasing Ca2+ from cellular store. FK strongly inhibited the CCh-induced Ca2+ transient, but failed to inhibit the caffeine-induced Ca2+ transient. In the absence of external Ca2+, 12-deoxyphorbol 13-isobutylate (DPB, 1 microM) induced sustained contraction without increase in [Ca2+]i and MLC phosphorylation. FK inhibited this contraction without changing [Ca2+]i. In permeabilized muscle, Ca2+ induced contraction in a concentration-dependent manner. FK (10 microM) and cAMP (1-100 microM) shifted the Ca(2+)-force curve to the higher Ca2+ levels. CCh with GTP, GTP gamma S or DPB enhanced contraction in the presence of constant level of Ca2+. Forskolin and cAMP also inhibited the enhanced contractions in the permeabilized muscle. In the permeabilized, thiophosphorylated muscle, ATP induced contraction in the absence of Ca2+. cAMP (300 microM) had no effect on this contraction. These results suggest that forskolin inhibits agonist-induced contraction in tracheal smooth muscle by multiple mechanisms of action; 1) inhibition of MLC phosphorylation by reducing Ca2+ influx and Ca2+ release, 2) inhibition of MLC phosphorylation by changing the MLC kinase/phosphatase balance, and 3) inhibition of regulatory mechanism which is not dependent on MLC phosphorylation.     

Expression and epitopic conservation of calponin in different smooth muscles and during development

The synaptic organization of the saccade-related neuronal circuit between the superior colliculus (SC) and the brainstem saccade generator was examined in an awake monkey using a saccadic, midflight electrical-stimulation method. When microstimulation (50-100 microA, single pulse) was applied to the SC during a saccade, a small, conjugate contraversive eye movement was evoked with latencies much shorter than those obtained by conventional stimulation. Our results may be explained by the tonic inhibition of premotor burst neurons (BNs) by omnipause neurons that ceases during saccades to allow BNs to burst. Thus, during saccades, signals originating from the SC can be transmitted to motoneurons and seen in the saccade trajectory. Based on this hypothesis, we estimated the number of synapses intervening between the SC and motoneurons by applying midflight stimulation to the SC, the BN area, and the abducens nucleus. Eye position signals were electronically differentiated to produce eye velocity to aid in detecting small changes. The mean latencies of the stimulus-evoked eye movements were: 7.9 +/- 1.0 ms (SD; ipsilateral eye) and 7.8 +/- 0.9 ms (SD; contralateral eye) for SC stimulation; 4.8 +/- 0.5 ms (SD; ipsilateral eye) and 5.1 +/- 0.7 ms (SD; contralateral eye) for BN stimulation; and 3.6 +/- 0.4 ms (SD; ipsilateral eye) and 5.2 +/- 0.8 ms (SD; contralateral eye) for abducens nucleus stimulation. The time difference between SC- and BN-evoked eye movements (about 3 ms) was consistent with a disynaptic connection from the SC to the premotor BNs.     

Regulation of force and shortening velocity by calcium and myosin phosphorylation in chemically skinned smooth muscle

The phosphatase inhibitor okadaic acid (OA) was used to study the relationship between [Ca2+], rates of phosphorylation/dephosphorylation and the mechanical properties of smooth muscle fibres. Force/velocity relationships were determined with the isotonic quick release technique in chemically skinned guinea-pig taenia coli muscles at 22 degrees C. In the maximally thiophosphorylated muscle neither OA (10 microM) nor Ca2+ (increase from pCa 9.0 to pCa 4.5) influenced the force-velocity relationship. When the degree of activation was altered by varying [Ca2+] in the presence of 0.5 microM calmodulin, both force and the maximal shortening velocity (Vmax) were altered. At pCa 5.75, at which force was about 35% of the maximal at pCa 4.5, Vmax was 55% of the maximal value. When OA was introduced into fibres at pCa 6.0, force was increased from less than 5% to 100% of the maximal force obtained in pCa 4.5. The relationship between the degree of myosin light chain phosphorylation and force was similar in the two types of activation; varied [OA] at constant [Ca2+] and at varied [Ca2+]. The relation between force and Vmax when the degree of activation was altered with OA was almost identical to that obtained with varied [Ca2+]. The results show that Ca2+ and OA do not influence force or Vmax in the maximally phosphorylated state and suggest that the level of myosin light chain phosphorylation is the major factor determining Vmax. The finding that the relationship between force and Vmax was similar when activation was altered with OA and Ca2+ suggests, however, that alterations in the absolute rates of phosphorylation and dephosphorylation at a constant phosphorylation level do not influence the mechanical properties of the skinned smooth muscle fibres.     

Localization of 17-kDa myosin light chain isoforms in cultured aortic smooth muscle cells

Smooth muscle myosin II contains two 17-kDa essential light chain isoforms (LC17gi and LC17nm) of which the relative contents differ among myosins. To understand the roles of LC17 isoforms in the functions of myosin, we performed an immunofluorescence microscopic examination of their localization in primary cultured cells isolated from rat aortic smooth muscle. To identify the isoforms, rabbit polyclonal antibodies were prepared against C-terminal nonapeptides corresponding to either LC17gi or LC17nm from porcine aortic smooth muscle myosin. These isoforms differ in only 5 amino acid residues within the C-terminal 9 residues. These antibodies specifically recognize each LC17 isoform on urea-PAGE of total rat aortic cell lysates. Immediately after plating, the smooth muscle cells stained heterogeneously with each antibody, indicating differing contents of LC17 isoforms among cells. On double staining 1-2 d cultures with both antibodies, LC17nm was detected diffusely throughout the cytoplasm, whereas LC17gi was concentrated in specific regions such as the cell periphery and the base of cytoplasmic processes. These results support the suggestion that myosin containing LC17gi is essential for force-generation by aortic smooth muscle and that myosin containing LC17nm may play an important role in maintaining smooth muscle tension.     

Developmental and tissue distribution of expression of nonmuscle and smooth muscle isoforms of myosin light chain kinase

Using a combination of Northern and Western blotting and RT-PCR, we demonstrate the existence of a high molecular mass MLCK, which is expressed during chicken embryogenesis. It is expressed in developing smooth muscle containing tissues, and is detected at low concentrations in adult tissues. Direct sequencing of the RT-PCR product from embryonic tissue RNA revealed that the embryonic, high molecular mass MLCK is indeed the previously cloned "nonmuscle MLCK". Therefore, the high molecular mass MLCK should be termed embryonic/non-muscle MLCK isoform. Curiously, cultured embryonic gizzard and vascular smooth muscle cells express the lower molecular mass smMLCK protein, albeit at lower levels than in the in vivo tissues.     

Phosphorylation changes the spatial relationship between Glu124-Arg143 and Cys18 and Cys165 of the regulatory light chain in smooth muscle myosin

Regulatory light chain (RLC) mutants, RLC-C18 and RLC-C165, containing a single cysteine at positions 18 and 165 near the N and C terminus, respectively, were each labeled with benzophenone 4-iodoacetamide and exchanged into myosin in their phosphorylated or unphosphorylated forms and then photolyzed. SDS-PAGE showed that, for RLC-C18, the intrachain photo-cross-linking in myosin was inhibited by phosphorylation. For myosin containing RLC-C165, the yield of one intrachain cross-linked band decreased significantly whereas the other was unaffected by phosphorylation. Peptide mapping in conjunction with mass spectrometry showed that Cys165 was cross-linked to site(s) within Ala17-Lys34 independent of the phosphorylation of Ser19. This clearly demonstrates that the proximity between the N- and C-terminal regions of RLC is not affected by phosphorylation. In addition, Cys165 could also be cross-linked to the region of Phe133-Arg143; however, this type of cross-linking was inhibited in the phosphorylated state. For RLC-C18, the cross-linking took place with the region of Glu124-Arg132 or Phe133-Arg143, also only in the unphosphorylated state. Thus, phosphorylation changes the spatial relationship between the region of Glu124-Arg143 and Cys18 and Cys165. In scallop myosin, the region corresponding to Glu124-Arg143 is located at the interfaces between RLC and the essential light chain as well as the heavy chain [Xie, X. , et al. (1994) Nature 368, 306-312]. In light of that work, our results suggest that the region of Glu124-Arg143 is involved in the phosphorylation-dependent signaling and the change in its spatial relationship with respect to the N and C termini of RLC may underlie the activation of the smooth muscle myosin.     

A long, weakly charged actin-binding loop is required for phosphorylation-dependent regulation of smooth muscle myosin

Chimeric substitution of the weak actin-binding loop (ABL) from chicken skeletal muscle myosin for that of gizzard smooth muscle heavy meromyosin (HMM) causes activation of the dephosphorylated mutant (SABL HMM; Rovner, A. S., Freyzon, Y., and Trybus, K. M. (1995) J. Biol. Chem. 270, 30260-30263). The present study determined whether this loss of regulation is due to the greater positive charge density (5 versus 3 clustered lysine residues) or lesser length (14 versus 26 residues) of the mutant ABL. Charge augmentation had little effect on regulation of expressed mutants, but elimination of the 12 N-terminal amino acids from the wild-type ABL significantly increased actin-activated ATPase activity of the dephosphorylated relative to the phosphorylated molecule while conferring the ability to move actin filaments in vitro on the former. Addition of the same 12 residues to the SABL mutant increased the ratio of phosphorylated to dephosphorylated ATPase activity while imparting wild type-like regulation to motility. However, full actin activation of dephosphorylated ATPase activity required both the shorter length and greater positive charge density found in the SABL loop. These results demonstrate that, compared with skeletal, both the greater length and lesser positive charge density of the smooth muscle myosin ABL are required for proper phosphorylation-mediated regulation of the molecule.