Calponin and tropomyosin interactions.

The interaction between chicken gizzard calponin and tropomyosin was examined using viscosity, light scattering, electron microscopy and affinity chromatography. At neutral pH, 10 mM NaCl and in the absence of Mg2+, calponin induced tropomyosin filaments to form paracrystals thus decreasing the viscosity while increasing dramatically the light scattering of the tropomyosin solution. Electron micrographs of the uranyl acetate stained calponin-tropomyosin complex showed the presence of spindle shaped paracrystals with regular striation patterns and repeating units of about 400 A. Under similar conditions, smooth muscle caldesmon also induced tropomyosin to form paracrystals. To localize the calponin-binding site on tropomyosin, binding of fragments of tropomyosin, generated by chemical and mutational means, to a calponin-affinity column was studied. The COOH-terminal tropomyosin fragment Cn1B(142-281) and the NH2-terminal fragment CSM-beta(1/8/12-227) bound to a calponin-affinity column with an affinity similar to that of intact tropomyosin; while the NH2-terminal fragment, Cn1A(11-127), did not bind, indicating that the calponin-binding site(s) resides within residues 142-227 of tropomyosin. To determine the involvement in calponin binding of the area around Cys-190 of tropomyosin, fragments with cleavage sites near or at Cys-190 were used. Thus, while fragments Cy2(190-284) and CSM-beta(1/8/12-200) bound weakly to the calponin-affinity column, fragment Cy1(1-189) did not. These results demonstrate that calponin binds to tropomyosin between residues 142 and 227, and that the integrity of the region around Cys-190 of tropomyosin is important for strong interaction between the two proteins.     

Interaction of tropomyosin with F-actin-heavy meromyosin complex

The effect of phosphorylated and dephosphorylated heavy meromyosins (HMMs) saturated with Ca2+ or Mg2+ on the binding of tropomyosin to F-actin and on the conformational changes of tropomyosin on actin was investigated. The experimental data were analysed on the basis of th emodel of cooperative binding of tropomyosin to F-actin with overlapping binding sites. In general, attachment of both HMMs to F-actin increased around 100-fold the tropomyosin-binding affinity but concomittantly reduced the cooperatively of binding. In the presence of Ca2+ and in the absence of ATP the binding of tropomyosin to F-actin in a "doubly contiguous" manner was three-fold stronger for F-actin saturated with dephosphorylated HMM as compared to phosphorylated HMM. Under the same rigor conditions but in the absence of Ca2+ the reverse was true but the difference was about 1.5-fold. The binding stoichiometry of tropomyosin to actin was 7:1 in the presence of dephosphorylated HMM saturated with Ca2+ or phosphorylated-saturated with Mg2+ and tended to be about 6:1 for both after the exchange of the cation bound to myosin heads. Bound HMM was also found to influence the fluorescence polarization of 1,5-IAEDANS-labelled tropomyosin complexed with F-actin in muscle ghost fibres. In the presence of Ca2+, the amount of randomly arranged tropomyosin fluorophores decreased when dephosphorylated HMM was bound to ghost fibres, in contrast to an observed increase in the case of bound phosphorylated HMM. Thus HMM induced conformational changes of tropomyosin in the actin-tropomyosin complex that was reflected in an alteration of the geometrical arrangement between tropomyosin and actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caldesmon, calmodulin and tropomyosin interactions

Binary complex interactions between caldesmon and tropomyosin, and calmodulin and tropomyosin, and ternary complex interaction involving the three proteins were studied using viscosity, electron microscopy, fluorescence and affinity chromatography techniques. In 10 mM NaCl, caldesmon decreased the viscosity of chicken gizzard tropomyosin by 7-8 fold with a concomitant increase in turbidity (A330nm). Electron micrographs showed spindle-shaped particles in the tropomyosin-caldesmon samples. These results suggest side-by-side aggregation of tropomyosin polymers induced by caldesmon. Binding studies in 10 mM NaCl between caldesmon and chicken gizzard tropomyosin labelled with the fluorescent probe N-(1-anilinonaphthyl-4)maleimide (ANM) gave association constants from 5.3.10(6) to 7.9.10(6) M-1 and stoichiometry from 1.0 to 1.4 tropomyosin per caldesmon. Similar binding was observed for rabbit cardiac tropomyosin and caldesmon. Removal of 18 and 11 residues from the COOH ends of the gizzard and cardiac tropomyosin by carboxypeptidase A, respectively, had no significant effect on their binding to caldesmon. In the presence of Ca2+, chicken gizzard tropomyosin bound to a calmodulin-Sepharose-4B column and was eluted with a salt concentration of 140 mM. This interaction was weakened in the absence of Ca2+, and the bound tropomyosin was eluted by 65 mM KCl. ANM-labelled tropomyosin bound calmodulin in the presence of Ca2+ with a binding constant of 3.5.10(6) M-1 and a binding stoichiometry of 1 to 1.4 tropomyosin per calmodulin. In 10 mM NaCl, calmodulin reduced the specific viscosity of chicken gizzard tropomyosin in the presence of Ca2+ by 5 fold, while a 1.5-fold reduction in viscosity was observed in the absence of Ca2+. In either case, no significant increase in turbidity was observed suggesting that calmodulin reduced head-to-tail polymerization of tropomyosin. The interaction of caldesmon with the calmodulin-ANM-tropomyosin complex in the presence and absence of Ca2+ was also examined. The result is consistent with a model that in the absence of Ca2+, calmodulin binds weakly to either caldesmon or tropomyosin and has little effect on the tropomyosin-caldesmon interaction; whereas, Ca2(+)-calmodulin interacts with caldesmon and reduces its affinity to tropomyosin.     

Cardiac tropomyosin crystals and their interactions with troponin subunits

Crystals and paracrystals of bovine cardiac tropomyosin and their mixtures with different combinations of troponin subunits were examined in the electron microscope after negative staining. Although the cardiac proteins gave most of the same crystalline and paracrystalline patterns as observed previously with skeletal muscle tropomyosin and troponin, two important differences were noted. Cardiac troponin T was incapable of forming hexagonal networks with either skeletal or cardiac muscle tropomyosins, while skeletal troponin T readily associated in this manner with tropomyosins from either tissue source. Also the characteristic paracrystalline pattern seen with skeletal muscle tropomyosin, troponin T and troponin C only in the presence of calcium was consistently obtained with mixtures of the corresponding cardiac components when calcium was absent.     

alpha-Actinin localization in the junctional complex of intestinal epithelial cells

We have used antibody to chicken gizzard alpha-actinin to identify and localize this molecule in chicken intestinal epithelium. The antibody binds only to alpha-actinin when tested against a crude extract of chicken gizzard. Extracts of purified epithelial cells contain a molecule which has a subunit molecular weight of 100,000 on sodium dodecyl sulphate gels and which is able to inhibit the interaction of alpha-actinin antibody and 125I-labeled chicken gizzard alpha-actinin. By indirect immunofluorescence, alpha-actinin is localized in the apical portion of chicken intestinal epithelial cells. Ethanol-fixed cryostat sections of intestine taken through the apical portion of the epithelial cells and in a plane perpendicular to the long axis of the cells show that alpha-actinin is organized in a polygonal pattern which corresponds to the outlines of the polygonally packed epithelial cells. We interpret the data as indicating that alpha-actinin is a component of the tight junction (zonula occludens) and/or the belt desmosome (zonula adherens), both of which are membrane structures known to encircle the cell and to be confined to its apical portion.     

Troponin and its interactions with tropomyosin: An electron microscope study

Some new features of the troponin complex have been revealed by electron microscope study of rotary shadowed molecules. Our results demonstrate that the troponin complex has both a globular and a rod-like domain. The length of the entire complex is ~265 Å and that of the tail is ~ 160 Å. Isolated troponin T has a shape and dimensions that correspond closely to those of the tail, so that the troponin I and C subunits would comprise most of the globular region of the complex. Native and reconstituted troponin-tropomyosin complexes have also been visualized and show the globular portion of troponin bound at regular intervals along the tropomyosin filaments. These electron microscope results, together with recent biochemical studies, suggest that troponin subunits C and I, and part of subunit T bind near Cys190 of tropomyosin, about one-third of the way along the molecule, with the rest of subunit T extending toward the COOH terminus. This arrangement implies that tropomyosin filaments lie on the actin helix with their COOH termini toward the Z-line. The shape of the complex suggests that troponin may interact with tropomyosin over a considerable portion of its length, and may therefore be important in the dynamics of the switching process.     

Targeting of a tropomyosin isoform to short microfilaments associated with the Golgi complex

A growing body of evidence suggests that the Golgi complex contains an actin-based filament system. We have previously reported that one or more isoforms from the tropomyosin gene Tm5NM (also known as gamma-Tm), but not from either the alpha- or beta-Tm genes, are associated with Golgi-derived vesicles (Heimann et al., (1999). J. Biol. Chem. 274, 10743-10750). We now show that Tm5NM-2 is sorted specifically to the Golgi complex, whereas Tm5NM-1, which differs by a single alternatively spliced internal exon, is incorporated into stress fibers. Tm5NM-2 is localized to the Golgi complex consistently throughout the G1 phase of the cell cycle and it associates with Golgi membranes in a brefeldin A-sensitive and cytochalasin D-resistant manner. An actin antibody, which preferentially reacts with the ends of microfilaments, newly reveals a population of short actin filaments associated with the Golgi complex and particularly with Golgi-derived vesicles. Tm5NM-2 is also found on these short microfilaments. We conclude that an alternative splice choice can restrict the sorting of a tropomyosin isoform to short actin filaments associated with Golgi-derived vesicles. Our evidence points to a role for these Golgi-associated microfilaments in vesicle budding at the level of the Golgi complex.     

Pig platelet tropomyosin: interactions with the other thin-filament proteins

Pig platelet tropomyosin exhibits many of the functional activities of skeletal tropomyosin. At low ionic strength it forms end-to-end aggregates similar to those formed by skeletal tropomyosins. It forms a 1:1 complex with muscle troponin or with a troponin I-pig brain calmodulin complex, as well as a 1:6 association with platelet filamentous actin. Electron microscopy of paracrystals shows that the troponin binding site is slightly C-terminal of the unique cysteine, corresponding to position 190 of the rabbit skeletal alpha-tropomyosin sequence. The effect of a complex comprising platelet actin and tropomyosin on the ATPase activity of rabbit skeletal muscle myosin subfragment-1 was similar to that displayed by its skeletal muscle counterpart. Platelet tropomyosin decreased the activity by roughly half in a calcium-independent manner. Addition of troponin to the actin-tropomyosin in the absence of calcium results in further inhibition and allows the full activity of the complex to be restored by Ca2+. These results differ from those obtained by C?té & Smillie for horse platelet tropomyosin and this may reflect the different isomeric nature of pig platelet tropomyosin. These results suggest that the functional properties of non-muscle tropomyosins may differ when comparisons are made between proteins isolated from the same type of cell but in different species. Differences in self-association and actin-binding properties may be finely graded between different isoforms.     

Proteins of the human placental microvillar cytoskeleton. alpha-Actinin.

The human placental syncytiotrophoblast microvilli are supported by an underlying cytoskeleton consisting mainly of actin microfilaments. The major proteins associated with the actin have Mr values of 105 000, 80 000 and 68 000. The 105 000-Mr protein is recognized by an antibody preparation raised to purified chicken gizzard alpha-actinin. Electron microscopy has shown that the human placental protein has dimensions similar to those reported for muscle alpha-actinin. About half of the placental microvillar alpha-actinin is released from the cytoskeleton in the presence of Ca2+. This effect occurs at concentrations of Ca2+ greater than 0.3 muM and has been used as the basis of a method for the purification of the placental alpha-actinin. This sensitivity to Ca2+ is not affected by trifluoperazine and is therefore likely to be a property of the alpha-actinin as such rather than being mediated via calmodulin.     

Phosphorylation of an actin.tropomyosin.troponin complex from human skeletal muscle.

A human skeletal actin.tropomyosin.troponin complex was phosphorylated in the presence of [gamma-32 P]ATP, Mg2+, adenosine 3':5'-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 microM cyclic AMP. In the presence of 10(-7) M Ca2+ and protein kinase 0.1 mole of [32P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [32P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca2+, 5.10(-5) M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [32P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [32P]phosphate. Increasing the Ca2+ concentration did not significantly decrease the [32P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstitute human skeletal actomyosin made with the [32P] phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca2+.     

Concanavalin A interactions with asparagine-linked glycopeptides. The mechanisms of binding of oligomannose,bisected hybrid,and complex type carbohydrates

The affinity of concanavalin A (Con A) for simple saccharides has been known for over 50 years. However, the specificity of binding of Con A with cell-surface related carbohydrates has only recently been examined in detail. Brewer and coworkers [J Biol Chem (1986) 261:7306–10; J Biol Chem (1987) 262:1288–93; J Biol Chem (1987) 262:1294–99] have recently studied the binding interactions of a series of oligomannose and bisected hybrid type glycopeptides and complex type glycopeptides and oligosaccharides with Con A. The relative affinities of the carbohydrates were determined using hemagglutination inhibition measurements, and their modes of binding to the lectin examined by nuclear magnetic relaxation dispersion (NMRD) spectroscopy and quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves of Con A and the carbohydrates indicate that certain oligomannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, two protein binding sites on each glycopeptide have been identified and characterized. Certain bisected complex type oligosaccharides also bind and precipitate Con A, while the corresponding nonbisected analogs bind but do not precipitate the protein. The precipitation data indicate that the bisected complex type oligosaccharides are also bivalent for lectin binding, while the nonbisected analogs are univalent. The NMRD and precipitation data are consistent with different mechanisms of binding of nonbisected and bisected complex type carbohydrates to Con A, including different conformations of the bound saccharides.Abbreviations Con A Concanavalin A with unspecified metal ion content - CMPL Con A with Mn²⁺ and Ca²⁺ at the S1 and S2 sites respectively, in the locked conformation [12]; trisaccharide1, 3,6-di-O-(-d-mannopyranosyl)-d-mannose - -MDM methyl -d-mannopyranoside - NMRD nuclear magnetic relaxation dispersion, the magnetic field dependence of nuclear magnetic relaxation rates, in the present case, the longitudinal relaxation rate, 1/T₁, of solvent protons     

Interaction of tropomyosin with troponin components.

1. The TN-T and TN-I components of troponin both interact with tropomyosin and cause its precipitation in 0.1 M KC1 at neutral pH. The precipitate contains both end-to-end and side-by-side aggregates of tropomyosin molecules. 2. The TN-T and TN-I components change the band pattern of tropomyosin paracrystals formed in MgC1(2) solutions, although in different ways. TN-T causes the formation of hexagonal net structures, double-stranded net or paracrystals which result from the collapse of the double-stranded net. TN-I at pH 7.9 causes the formation of paracrystals with a 400 A periodic band pattern and a 200 A repeat. The same band pattern can also be seen in tropomyosin paracrystals formed at pH values below 6.0. 3. The TN-C component does not precipitate tropomyosin in 0.1 M KC1. The aggregates of tropomyosin obtained with either TN-T or TN-I can be solubilized by the addition of TN-C. No interaction of TN-C was observed with tropomyosin paracrystals formed in the presence of MgC12.     

Interaction of plasma gelsolin with tropomyosin.

Horse plasma gelsolin labelled with benzophenone-4-isothiocyanate can be photochemically cross-linked to rabbit cardiac tropomyosin. The cross-linking proceeds with greater efficiency in calcium-containing buffers. Further evidence for interaction between these proteins is provided by retention of fluorescently labelled gelsolin on tropomyosin-agarose affinity columns and by the ability of tropomyosin to cause an increase in the fluorescence intensity of gelsolin labelled with fluorescein-5-isothiocyanate. Both of these effects require the presence of calcium ions.     

Analysis of troponin-tropomyosin binding to actin. Troponin does not promote interactions between tropomyosin molecules.

The binding of tropomyosin to actin and troponin-tropomyosin to actin was analyzed according to a linear lattice model which quantifies two parameters: Ko, the affinity of the ligand for an isolated site on the actin filament, and gamma, the fold increase in affinity when binding is contiguous to an occupied site (cooperativity). Tropomyosin-actin binding is very cooperative (gamma = 90-137). Troponin strengthens tropomyosin-actin binding greatly but, surprisingly, does so solely by an 80-130-fold increase in Ko, while cooperativity actually decreases. Additionally, troponin complexes containing TnT subunits with deletions of either amino acids 1-69 (troponin70-259) or 1-158 (troponin159-259) were examined. Deletion of amino acids 1-69 had only small effects on Ko and y, despite this peptide's location spanning the joint between adjacent tropomyosins. Ca2+ reduced Ko by half for both troponin and troponin70-159 and had no detectable effect on cooperativity. Troponin159-259 had much weaker effects on tropomyosin-actin binding than did troponin70-259 and had no effect at all in the presence of Ca2+. This suggests the importance of Ca(2+)-insensitive interactions between tropomyosin and troponin T residues 70-159. Cooperativity was slightly lower for troponin159-259 than tropomyosin alone, suggesting that the globular head region of troponin affects tropomyosin-tropomyosin interactions along the thin filament.     

Hybridization of rabbit cardiac tropomyosin with nonpolymerizable tropomyosin species

Rabbit cardiac tropomyosin was hybridized with its nonpolymerizable form, produced by treatment with carboxypeptidase A, and with a naturally occurring nonpolymerizable tropomyosin from horse platelets. Hybridization was achieved by heating equimolar mixtures to 60 degrees C in the presence of 10 mM dithiothreitol, followed by recooling. Samples of intact and carboxypeptidase-truncated tropomyosins treated in this way show lower viscosities at low ionic strength than predicted assuming random reformation of the coiled coils, suggesting that hybrids formed with one intact COOH-terminus are unable to polymerize normally. Hybridization of cardiac and platelet tropomyosins was detected by observation of the fluorescence of pyrene groups attached to cysteine residues on platelet tropomyosin.     

Genetic interactions underlying hybrid male sterility in the Drosophila bipectinata species complex

Understanding genetic mechanisms underlying hybrid male sterility is one of the most challenging problems in evolutionary biology especially speciation. By using the interspecific hybridization method roles of Y chromosome, Major Hybrid Sterility (MHS) genes and cytoplasm in sterility of hybrid males have been investigated in a promising group, the Drosophila bipectinata species complex that consists of four closely related species: D. pseudoananassae, D. bipectinata, D. parabipectinata and D. malerkotliana. The interspecific introgression analyses show that neither cytoplasm nor MHS genes are involved but X-Y interactions may be playing major role in hybrid male sterility between D. pseudoananassae and the other three species. The results of interspecific introgression analyses also show considerable decrease in the number of males in the backcross offspring and all males have atrophied testes. There is a significant positive correlation between sex - ratio distortion and severity of sterility in backcross males. These findings provide evidence that D. pseudoananassae is remotely related with other three species of the D. bipectinata species complex.