Smooth muscle tropomyosin coiled-coil dimers. Subunit composition, assembly, and end-to-end interaction

Subunits of gizzard smooth muscle tropomyosin, dissociated by guanidinium chloride and reassociated by high salt dialysis, form a 1:1 mixture of the beta beta and gamma gamma homodimers (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The homodimers have now been separated by anion-exchange chromatography and native gel electrophoresis, enabling us to show that the native protein is composed of more than 90% heterodimer. The in vitro equilibrium distribution of heterodimer and homodimers, at close to physiological temperature and ionic conditions, was calculated from thermal unfolding profiles of separated homodimers and heterodimer, as monitored by circular dichroism. The results, for an equal proportion of beta and gamma chains, indicate a predominant formation of heterodimer via chain dissociation and chain exchange, although the proportion of heterodimer was much less than the 90-100% found in the native protein. However, the proportion of heterodimer for actin-bound tropomyosin, determined by analyzing tropomyosin sedimented with actin, was greater than 90%, which may provide a model for assembly in vivo. The end-to-end interactions of the homodimers are about the same but are much less than that of the native heterodimer, as determined by viscometry. The greater end-to-end interaction of heterodimers may lead to stronger binding to actin compared to homodimers and thus would further shift the equilibrium between heterodimer and homodimers toward heterodimer and possibly account for the almost exclusive population of heterodimer in the presence of actin. The greater end-to-end interaction of the heterodimer may also provide a functional advantage for its preferred assembly. This study also shows that the two-step thermal unfolding of the homodimer mixture is due to the formation of heterodimer via an intermediate which is a new type of tropomyosin species which forms a gel in low salt. This tropomyosin is also present in small amounts in native tropomyosin preparations.     

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.     

Gene-environment interactions differentially affect mouse strain behavioral parameters

Systematic phenotyping of mouse strains and mutants generated through genome-wide mutagenesis programs promises to deliver a wealth of functional genetic information. To this end, the appropriation of a standard series of phenotyping protocols is desirable to produce data sets that are consistent within and across laboratories and across time. Standard phenotyping protocols such as EMPReSS (European Mouse Phenotyping Resource for Standardised Screens) provide a series of protocols aimed at phenotyping multiple body systems that could realistically be adopted and/or reproduced in any laboratory. This includes a series of neurologic and behavioral screens, bearing in mind that this class of phenotype is well represented in targeted mutants and mutagenesis screens. Having cross-validated screening batteries in a number of laboratories and in a number of commonly used inbred strains, our group was interested in establishing whether subtle changes in cage environment could affect behavioral test outcome. Aside from unavoidable quantitative differences in test outcome, we identified significant and distinct genotype-environment-test interactions. For example, specific strain order in open-field center entries and total distance traveled can be reversed depending on the form of enrichment used, while prepulse inhibition of the acoustic startle response is, even quantitatively, unaffected by the enrichment condition. Our findings argue that unless systematically recorded, behavioral studies conducted under subtle variations in cage environment may lead to data misinterpretation, although this could be limited to particular behaviors. Further investigations into the extent and limits of genetic and environmental variables are critical for the realization of both behavioral and functional genomics endpoints.     

Changes in maternal investment in eggs can affect population dynamics

The way that mothers provision their offspring can have important consequences for their offspring's performance throughout life. Models suggest that maternally induced variation in life histories may have large population dynamical effects, even perhaps driving cycles such as those seen in forest Lepidoptera. The evidence for large maternal influences on population dynamics is unconvincing, principally because of the difficulty of conducting experiments at both the individual and population level. In the soil mite, Sancassania berlesei, we show that there is a trade-off between a female's fecundity and the per-egg provisioning of protein. The mother's position on this trade-off depends on her current food availability and her age. Populations initiated with 250 eggs of different mean sizes showed significant differences in the population dynamics, converging only after three generations. Differences in the growth, maturation and fecundity of the initial cohort caused differences in the competitive environment for the next generation, which, in turn, created differences in their growth and reproduction. Maternal effects in one generation can therefore lead to population dynamical consequences over many generations. Where animals live in environments that are temporally variable, we conjecture that maternal effects could result in long-term dynamical effects.     

Phosphorylation of tropomyosin in live frog muscle

Phosphate incorporation into tropomyosin was found in muscles of live frogs injected with [³²P]orthophosphate and left at room temperature for from 1 to 4 days. The labeled tropomyosin was purified to homogeneity as evidenced by amino acid analysis and analytical gel electrophoresis. Partial hydrolysis indicated that the incorporated ³²P was in the form of serine phosphate.     

In-register homodimers of smooth muscle tropomyosin

Gizzard smooth muscle tropomyosin dimer molecules were dissociated by guanidinium chloride and reassociated by dialysis against 1 M NaCl. Several properties of the protein were changed by this treatment. There was a large decrease in tropomyosin's low-salt viscosity, owing to reduced end-to-end polymerization, the helix unfolding profile changed from a one-step to a two-step process, and the ability to form intramolecular, interchain, disulfide-cross-linked homodimers increased dramatically. Thus, the native molecule, though to exist predominantly as by the beta gamma heterodimer which cannot form disulfide cross-links [Sanders, C., Burtnick, L.D., & Smillie, L. B. (1986) J. Biol. Chem. 261, 12774-12778], reassembles, after dissociation, to form predominantly parallel, in-register beta beta and gamma gamma homodimers able to form disulfide cross-links. This suggests that the physical properties, including the end-to-end interaction, of gizzard tropomyosin homodimers differ considerably from those of the heterodimer. This is a first step toward a molecular understanding of the end-to-end interaction of smooth muscle tropomyosin.     

Comparative dynamics of tropomyosin in vertebrates and invertebrates

Tropomyosin (Tpm) is an extended α-helical coiled-coil homodimer that regulates actinomyosin interactions in muscle. Molecular simulations of four Tpms, two from the vertebrate class Mammalia (rat and pig), and two from the invertebrate class Malacostraca (shrimp and lobster), showed that despite extensive sequence and structural homology across metazoans, dynamic behavior—particularly long-range structural fluctuations—were clearly distinct. Vertebrate Tpms were more flexible and sampled complex, multi-state conformational landscapes. Invertebrate Tpms were more rigid, sampling a highly constrained harmonic landscape. Filtering of trajectories by principle component analysis into essential subspaces showed significant overlap within but not between phyla. In vertebrate Tpms, hinge-regions decoupled long-range interhelical motions and suggested distinct domains. In contrast, crustacean Tpms did not exhibit long-range dynamic correlations—behaving more like a single rigid rod on the nanosecond time scale. These observations suggest there may be divergent mechanisms for Tpm binding to actin filaments, where conformational flexibility in mammalian Tpm allows a preorganized shape complementary to the filament surface, and where rigidity in the crustacean Tpm requires concerted bending and binding.     

Localization of tropomyosin in mouse embryo fibroblasts.

Antiserum to chick skeletal muscle tropomyosin was used to localize tropomyosin in mouse embryo fibroblasts by the indirect fluorescein labeled antibody technique. Specific staining was observed cytoplasmic fibers, which extended out into the cell processes. The staining pattern in these cells is similar to that previously described by others for actin. This observation suggests that in fibroblasts tropomyosin, like actin, is localized in fibers in the cytoplasm.     

Circular-dichroic studies on the conformational behaviour of troponin and tropomyosin from bovine cardiac muscle.

Bovine cardiac troponin is similar to rabbit skeletal troponin with respect to secondary structure, amino acid composition and molecular weight of the subunits, but differs slightly with respect to biological activity and surface charges of the subunits. Previous circular-dichroic studies of the subunits and recombination of subunits have indicated significant Ca2+-induced delocalized conformational changes. Present studies of the native troponin complex are not in accord with such changes. Furthermore the formation of the troponin-tropomyosin complex in vitro results in no delocalized conformational changes, nor does it sensitize troponin to Ca2+-induced changes. It is suggested that the troponin complex cannot be dissociated into subunits without significant and irreversible conformational perturbation.     

A model for the oxygen-paradox in mouse cardiac muscle

1. Mouse ventricle strips provide a good model system for studying cellular damage in mammalian cardiac muscle. 2. Anoxia rapidly causes destruction of the myofilament apparatus that is characteristic of calcium-triggered damage in muscle cells, and it is suggested that anoxia promotes release of calcium from the mitochondria. 3. Oxygen exacerbates this damage which is independent of extracellular calcium; it is suggested that it initiates myofilament damage by activation at an intracellular site, probably the sarcoplasmic reticulum.     

Blebbistatin specifically inhibits actin-myosin interaction in mouse cardiac muscle

Blebbistatin is a powerful inhibitor of actin-myosin interaction in isolated contractile proteins. To examine whether blebbistatin acts in a similar manner in the organized contractile system of striated muscle, the effects of blebbistatin on contraction of cardiac tissue from mouse were studied. The contraction of paced intact papillary muscle preparations and shortening of isolated cardiomyocytes were inhibited by blebbistatin with inhibitory constants in the micromolar range (1.3–2.8 µM). The inhibition constants are similar to those previously reported for isolated cardiac myosin subfragments showing that blebbistatin action is similar in filamentous myosin of the cardiac contractile apparatus and isolated proteins. The inhibition was not associated with alterations in action potential duration or decreased influx through L-type Ca²⁺ channels. Experiments on permeabilized cardiac muscle preparations showed that the inhibition was not due to alterations in Ca²⁺ sensitivity of the contractile filaments. The maximal shortening velocity was not affected by 1 µM blebbistatin. In conclusion, we show that blebbistatin is an inhibitor of the actin-myosin interaction in the organized contractile system of cardiac muscle and that its action is not due to effects on the Ca²⁺ influx and activation systems. heart; electrophysiology; permeabilized muscle     

Changes in the composition of cardiac muscle myosin light chains during cardiac diseases

In this paper our data of the study on composition of human cardiac myosin light chains in norm and its changes at different stages of dilated cardiomyopathy and heart valvular diseases are presented. Functional role and diagnostic value of these changes are discussed.     

Changes in the composition of cardiac muscle myosin light chains during cardiac diseases

In this paper our data of the study on composition of human cardiac myosin light chains in norm and its changes at different stages of dilated cardiomyopathy and heart valvular diseases are presented. Functional role and diagnostic value of these changes are discussed.     

On the electrotonic spread in cardiac muscle of the mouse

As an appropriate model which can simulate the cardiac working muscle with respect to the passive electrical spread, a lattice whose sides have linear cable properties is presented, and the passive potential spread on the model is mathematically analyzed in the fiber direction. Distribution of electrotonic potential in the fiber direction was measured with a pair of intracellular microelectrodes in the cardiac muscle fiber of mouse. By employing “pencil type” microelectrodes potential distribution in the transverse direction within a fiber was also measured. This transverse effect was differentiated from the longitudinal potential distribution. A tonically applied potential at any point of a cell interior spreads continuously in a manner described by a Bessel function. Using appropriate electrical and morphological parameters the experimental results proved to fit the curve obtained from numerical calculation on the model. The apparent length constant obtained for smaller distances (less than 100 μ) from the current source was 70 μ, and it increases as the distance becomes larger. At a point inside the fiber the resistance to the extracellular fluid ranged from 200 to 600 KΩ. The influence of coupling resistance between current and recording electrodes on the measurement of electrotonic potential was examined for small interelectrode distance.