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P Vibert

Publications and source records attributed to P Vibert.

At least 19 recordsLinked to original sources
PubMed1997

Visualization of caldesmon on smooth muscle thin filaments.

Caldesmon, a narrow, elongated actin-binding protein, is found in both nonmuscle and smooth muscle cells. It inhibits actomyosin ATPase and filament severing in vitro, and is thus a putative regulatory protein. To elucidate its function, we have used electron microscopy and three-dimensional image reconstruction to reveal the location of caldesmon on isolated smooth muscle thin filaments. Caldesmon density was clearly delineated in reconstructions and found to occur peripherally, on the extreme outer edge of actin subdomains-1 and 2, without making obvious contacts with tropomyosin strands on the inner domains of actin. When the reconstructions were fitted to the atomic model of F-actin, caldesmon appeared to cover potentially weak sites of myosin interaction with actin, while, together with tropomyosin, it flanked strong sites of myosin interaction, without covering them. These interactions are unlike those of troponin-tropomyosin and therefore inhibition of actomyosin ATPase by caldesmon-tropomyosin and by troponin-tropomyosin cannot occur in the same way. The location of caldesmon would allow it to compete with a number of cellular actin-binding proteins, including those known to sever or sequester actin.

Actins↗
PubMed1997

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

Calponin is a putative thin filament regulatory protein of smooth muscle that inhibits actomyosin ATPase in vitro. We have used electron microscopy and three-dimensional reconstruction to elucidate the structural organization of calponin on actin and actin-tropomyosin filaments. Calponin density was clearly delineated in the reconstructions and found to occur peripherally along the long-pitch actin-helix. The main calponin mass was located over sub-domain 2 of actin, and connected axially adjacent actin monomers by binding to the "upper" and "lower" edges of sub-domains 1 of each actin. When the reconstructions were fitted to the atomic model of F-actin, calponin appeared to contact actin near the N terminus and at residues 349 to 352 close to the C terminus of sub-domain 1 on one monomer. It also touched residues 92 to 95 of sub-domain 1 on the axially neighboring actin and continued up the side of this monomer as far as residues 43 to 48 of sub-domain 2. These positions are consensus binding sites for a number of actin-associated proteins and are also near to sites of weak myosin interaction. Calponin did not appear to block strong myosin binding sites on actin. In contrast to the calponin mass which appeared monomeric in reconstructions, tropomyosin formed a continuous strand of added density along F-actin. When added to tropomyosin-containing filaments, calponin caused a shift of tropomyosin away from sub-domain 1 towards sub-domain 3 of actin, exposing strong myosin-binding sites that were previously covered by tropomyosin. This structural effect is unlike that of troponin and therefore inhibition of actomyosin ATPase by calponin and troponin cannot be strictly analogous. The location of calponin would allow it to directly compete or interact with a number of actin-binding proteins.

Actins↗
PubMed1997

Steric-model for activation of muscle thin filaments.

The structural basis of thin filament-linked regulation of muscle contraction is not yet understood. Here we have used electron microscopy and three-dimensional image reconstruction to observe the effects of Ca2+ and myosin head binding on thin filament structure, especially on the position of tropomyosin. Thin filaments isolated in EGTA were treated with Ca2+ or myosin heads (S-1) and negatively stained. Tropomyosin strands were directly visualized in electron micrographs, and distinct EGTA, Ca2+ and S-1-dependent positions were apparent in reconstructions. By fitting reconstructions to the atomic model of F-actin, clusters of amino acids on actin lying beneath tropomyosin were defined under each set of conditions. In the presence of Ca2+, tropomyosin moved 25 degrees away from its low Ca2+ position, exposing most, but not all, of the previously blocked myosin-binding sites. Saturation of filaments with myosin heads produced a further 10 degrees shift in tropomyosin position, thereby exposing the entire myosin-binding site. Our results thus suggest that full switching-on of thin filaments by reversal of steric-blocking requires both Ca2+ and the binding of myosin heads, acting in sequence. By using filaments which were partially decorated with heads, tropomyosin movement was shown to be cooperative, and the size of the actin-tropomyosin cooperative unit was estimated directly. Our results provide direct structural support for previous models of thin filament activation based on kinetics of actin-myosin interaction.

Actins↗
PubMed1997

Three-dimensional image reconstruction of reconstituted smooth muscle thin filaments: effects of caldesmon.

Caldesmon inhibits actomyosin ATPase and filament sliding in vitro, and therefore may play a role in modulating smooth and non-muscle motile activities. A bacterially expressed caldesmon fragment, 606C, which consists of the C-terminal 150 amino acids of the intact molecule, possesses the same inhibitory properties as full-length caldesmon and was used in our structural studies to examine caldesmon function. Three-dimensional image reconstruction was carried out from electron micrographs of negatively stained, reconstituted thin filaments consisting of actin and smooth muscle tropomyosin both with and without added 606C. Helically arranged actin monomers and tropomyosin strands were observed in both cases. In the absence of 606C, tropomyosin adopted a position on the inner edge of the outer domain of actin monomers, with an apparent connection to sub-domain 1 of actin. In 606C-containing filaments that inhibited acto-HMM ATPase activity, tropomyosin was found in a different position, in association with the inner domain of actin, away from the majority of strong myosin binding sites. The effect of caldesmon on tropomyosin position therefore differs from that of troponin on skeletal muscle filaments, implying that caldesmon and troponin act by different structural mechanisms.

Actins↗
PubMed1996

Single-headed scallop myosin and regulation.

Single-headed scallop myosin (shM) was prepared by papain digestion of filamentous scallop myosin and purified by hydrophobic interaction chromatography. The shM preparation consisted of equimolar amounts of polypeptides corresponding to an intact heavy chain, rod chain, essential light chain, and regulatory light chain. In electron micrographs the shape of shM showed the presence of a single head domain to which a normal looking rod was attached. Myosin and shM bound Ca2+ with association constants of 5 x 10(6) and 11 x 10(6) M-1, respectively. The ATPase activity of shM was activated about 3-fold by Ca2+. Both heads of myosin and shM had comparable ATPase activities in the presence of Ca2+. The activation of the ATPase activity of single-headed scallop myosin by Ca2+ paralleled closely the Ca2+ binding, in sharp contrast to the activation of intact myosin by Ca2+, which is highly cooperative. Single turnover experiments of myosin with radioactive ATP gave a half-life for the ATPase cycle of approximately 3 min in the presence of EGTA, whereas that of single-headed myosin was shorter than approximately 30 s, which was the resolution time of these measurements. The results suggest that the presence of two heads, as well as the attachment of the head to the coiled coil rod, contribute to the regulation of scallop myosin by Ca2+.

Adenosine Triphosphatases↗
PubMed1995

Steric-blocking by tropomyosin visualized in relaxed vertebrate muscle thin filaments.

Although widely accepted, the steric-blocking model of vertebrate skeletal muscle regulation has not been confirmed. Previous attempts to directly visualize tropomyosin in relaxed skeletal muscle and demonstrate that it interferes with the crossbridge-thin filament contractile cycle were unsuccessful. In the work reported here, tropomyosin was resolved in electron micrographs of native thin filaments isolated from relaxed vertebrate striated muscle. Three-dimensional helical reconstructions of these filaments showed continuous narrow strands of density, representing tropomyosin, which followed the outer domains of successive actin monomers. The results obtained from fitting the atomic model of filamentous actin to these reconstructions illustrate, and are consistent with, the mechanism of steric-blocking, since tropomyosin was found to be positioned on the actin surface of thin filaments over clusters of identifiable amino acids required for myosin crossbridge docking.

Actin Cytoskeleton↗
PubMed1995

A minimal motor domain from chicken skeletal muscle myosin.

The myosin head (S1) consists of a wide, globular region that contains the actin- and nucleotide-binding sites and an alpha-helical, extended region that is stabilized by the presence of two classes of light chains. The essential light chain abuts the globular domain, whereas the regulatory light chain lies near the head-rod junction of myosin. Removal of the essential light chain by a mild denaturant exposes the underlying heavy chain to proteolysis by chymotrypsin. The cleaved fragment, or "motor domain" (MD), migrates as a single band on SDS-polyacrylamide gel electrophoresis, with a slightly greater mobility than S1 prepared by papain or chymotrypsin. Three-dimensional image analysis of actin filaments decorated with MD reveals a structure similar to S1, but shorter by an amount consistent with the absence of a light chain-binding domain. The actin-activated MgATPase activity of MD is similar to that of S1 in Vmax and Km. But the ability of MD to move actin filaments in a motility assay is considerably reduced relative to S1. We conclude that the globular, active site region of the myosin head is a stable, independently folded domain with intrinsic motor activity, but the coupling efficiency between ATP hydrolysis and movement declines markedly as the light chain binding region is truncated.

Actins↗
PubMed1994

Ca(2+)-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction.

The steric model of muscle regulation holds that tropomyosin strands running along thin filaments move away from myosin-binding sites on actin when muscle is activated. Exposing these sites would permit actomyosin interaction and contraction to proceed. This compelling and widely cited model is based on changes observed in X-ray diffraction patterns of skeletal muscle following activation. Although analysis of X-ray patterns can suggest models of filament structure, unambiguous interpretation is not possible. In contrast, three-dimensional reconstruction of thin-filament electron micrographs could, in principle, offer direct confirmation of the predicted tropomyosin movement, but so far tropomyosin in skeletal muscle has been resolved definitively only in the 'on' state but not in the 'off' state. Thin filaments from the arthropod Limulus have a similar composition to those from vertebrate skeletal muscle, and troponin-tropomyosin is distributed in both species with the same characteristic 38-nm periodicity. Limulus thin filaments activate skeletal muscle myosin ATPase at micromolar Ca2+ concentrations and confer a high calcium dependence on the enzyme. Arthropod and vertebrate troponin subunits form functional hybrids in vitro and the respective tropomyosins are functionally interchangeable, arguing for a common mechanism of thin-filament-linked regulation in the two phyla. Here we report that tropomyosin is readily resolved in native filaments of troponin-regulated Limulus muscle in both the 'on' and 'off' states, and demonstrate tropomyosin movement, providing support for the importance of steric effects in muscle activation.

Actin Cytoskeleton↗
PubMed1993

Mini-titins in striated and smooth molluscan muscles: structure, location and immunological crossreactivity.

Invertebrate mini-titins are members of a class of myosin-binding proteins belonging to the immunoglobulin superfamily that may have structural and/or regulatory properties. We have isolated mini-titins from three molluscan sources: the striated and smooth adductor muscles of the scallop, and the smooth catch muscles of the mussel. Electron microscopy reveals flexible rod-like molecules about 0.2 micron long and 30 A wide with a distinctive polarity. Antibodies to scallop mini-titin label the A-band and especially the A/I junction of scallop striated muscle myofibrils by indirect immunofluorescence and immuno-electron microscopy. This antibody crossreacts with mini-titins in scallop smooth and Mytilus catch muscles, as well as with proteins in striated muscles from Limulus, Lethocerus (asynchronous flight muscle), and crayfish. It labels the A/I junction (I-region in Lethocerus) in these striated muscles as well as in chicken skeletal muscle. Antibodies to the repetitive immunoglobulin-like regions and also to the kinase domain of nematode twitchin crossreact with scallop mini-titin and label the A-band of scallop myofibrils. Electron microscopy of single molecules shows that antibodies to twitchin kinase bind to scallop mini-titin near one end of the molecule, suggesting how the scallop structure might be aligned with the sequence of nematode twitchin.

Animals↗
PubMed1993

Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments.

Caldesmon is known to inhibit actomyosin ATPase and filament sliding in vitro, and may play a role in modulating smooth muscle contraction as well as in diverse cellular processes including cytokinesis and exocytosis. However, the structural basis of caldesmon action has not previously been apparent. We have recorded electron microscope images of negatively stained thin filaments containing caldesmon and tropomyosin which were isolated from chicken gizzard smooth muscle in EGTA. Three-dimensional helical reconstructions of these filaments show actin monomers whose bilobed shape and connectivity are very similar to those previously seen in reconstructions of frozen-hydrated skeletal muscle thin filaments. In addition, a continuous thin strand of density follows the long-pitch actin helices, in contact with the inner domain of each actin monomer. Gizzard thin filaments treated with Ca2+/calmodulin, which dissociated caldesmon but not tropomyosin, have also been reconstructed. Under these conditions, reconstructions also reveal a bilobed actin monomer, as well as a continuous surface strand that appears to have moved to a position closer to the outer domain of actin. The strands seen in both EGTA- and Ca2+/calmodulin-treated filaments thus presumably represent tropomyosin. It appears that caldesmon can fix tropomyosin in a particular position on actin in the absence of calcium. An influence of caldesmon on tropomyosin position might, in principle, account for caldesmon's ability to modulate actomyosin interaction in both smooth muscles and non-muscle cells.

Actin Cytoskeleton↗
PubMed1992

Helical reconstruction of frozen-hydrated scallop myosin filaments.

Native myosin filaments from scallop striated muscle that have been rapidly frozen in relaxing solutions appear to be well preserved in vitreous ice. Electron micrographs of samples at -177 degrees C were recorded with an electron dose of 10 e/A2 at 1.5 microns defocus. After filament images were straightened by spline-fitting, several transforms showed well-defined layer-lines arising from the helical structure of the filament. A set of 17 near-meridional layer-lines has been collected and corrected for background and for phase and amplitude contrast functions. Preliminary helical reconstructions from this still incomplete data set reveal aspects of structure that were not apparent from earlier analysis of negatively stained filaments from scallop muscle. Individual pear-shaped myosin heads now appear to be well resolved from each other and from the filament backbone. The two heads of each myosin molecule appear to be splayed apart axially. The reconstructions also reveal that the filament backbone has a polygonal shape in cross-section, and that it appears to contain seven peripherally located subfilaments.

Animals↗
PubMed1992

Location of paramyosin in relation to the subfilaments within the thick filaments of scallop striated muscle.

Myosin co-assembles with paramyosin in the thick filaments of invertebrate muscles. The molar ratio of the two proteins varies greatly but where sufficient paramyosin is present it forms the filament core with myosin arranged on its surface. In the fastest acting striated muscles, paramyosin is present in small amounts, and neither its location nor the nature of its interactions with myosin has previously been established. Antibodies to paramyosin have now been used in an attempt to locate the protein in thick filaments that have been isolated from the striated adductor muscle of the scallop and then frayed apart into their constituent subfilaments. Using a gold-conjugated secondary antibody, the location of paramyosin in relation to the subfilaments has been determined by electron microscopy of negatively stained samples. The labelling indicates that paramyosin extends throughout the length of the scallop filaments and appears to be associated with each subfilament, raising the possibility that in these filaments paramyosin may not be confined to a central core domain.

Actin Cytoskeleton↗
PubMed1989

Substructure and accessory proteins in scallop myosin filaments.

Native myosin filaments from scallop striated muscle fray into subfilaments of approximately 100-A diameter when exposed to solutions of low ionic strength. The number of subfilaments appears to be five to seven (close to the sevenfold rotational symmetry of the native filament), and the subfilaments probably coil around one another. Synthetic filaments assembled from purified scallop myosin at roughly physiological ionic strength have diameters similar to those of native filaments, but are much longer. They too can be frayed into subfilaments at low ionic strength. Synthetic filaments share what may be an important regulatory property with native filaments: an order-disorder transition in the helical arrangement of myosin cross-bridges that is induced on activation by calcium, removal of nucleotide, or modification of a myosin head sulfhydryl. Some native filaments from scallop striated muscle carry short "end filaments" protruding from their tips, comparable to the structures associated with vertebrate striated muscle myosin filaments. Gell electrophoresis of scallop muscle homogenates reveals the presence of high molecular weight proteins that may include the invertebrate counterpart of titin, a component of the vertebrate end filament. Although the myosin molecule itself may contain much of the information required to direct its assembly, other factors acting in vivo, including interactions with accessory proteins, probably contribute to the assembly of a precisely defined thick filament during myofibrillogenesis.

Actin Cytoskeleton↗
PubMed1987

Myosin binding to actin. Structural analysis using myosin fragments.

The actin-binding property of the myosin head 20 K (K = 10(3) Mr) fragment has been examined by a structural assay. A new fragment is produced by digestion of scallop myosin synthetic filaments with a lysine-specific protease. This fragment consists of the rod together with two "nubs" corresponding to the 20 K fragment, which retain both the regulatory and essential light chains. Myosin filaments, digested for different lengths of time, were mixed with F-actin and visualized by electron microscopy after negative staining. When the head is cleaved, but the head fragments remain associated, the filaments bind actin in an ATP-sensitive manner. Filaments made primarily of the nub-containing fragments, however, bind actin very poorly. In addition, electron microscopic characterization of actin-binding by the isolated tryptic 20 K fragment from chicken myosin indicates that binding of this fragment to actin is probably non-specific. These results suggest that interactions between the 20 K region and the other peptides in the head are essential for actin-binding.

Actins↗
PubMed1985

Dimer ribbons in the three-dimensional structure of sarcoplasmic reticulum.

The three-dimensional structure of scallop sarcoplasmic reticulum membranes has been determined from electron micrographs of two classes of stain-filled tubules by helical reconstruction methods. These structures are characterized by dimer ribbons of Ca2+-ATPase molecules running diagonally around the tube wall. Deep right-handed grooves separate the ribbons. The elongated, curved units of the dimer (approximately 95 A long in the radial direction; 60 to 70 A axially, and about 30 A wide) are displaced axially by approximately 34 A and are connected at their outer ends by a bridge running nearly parallel to the tube axis. The monomers make a second contact at their inner ends. Adjacent units with the same orientation form a strong contact that is responsible for the ribbon appearance. Comparison of tubules of different diameter shows that one set of connections between the dimer ribbons is conserved: the inner ends of axially displaced dimers appear to make contact along a left-handed path almost perpendicular to the major grooves. The lipid bilayer cannot be clearly identified. The two-dimensional map obtained from flattened tubules is consistent with the three-dimensional reconstruction in showing dimer ribbons connected by a weak contact across the grooves, strongly resembling the inter-dimer bond observed in three dimensions. The two-dimensional map shows a 2-fold axis relating units of the dimer, but the three-dimensional tubes show a slight axial polarity that may arise from the presence of proteins other than the Ca2+-ATPase.

Animals↗
PubMed1985

Electron microscopy of cross-linked scallop myosin.

The N-terminal regions of the regulatory light chains on the two heads of scallop myosin can be cross-linked to one another. Electron microscopy of cross-linked myosin molecules, and of dimers of myosin subfragment-1 produced by digesting them with papain, shows that the site of cross-linking is very close to the head-rod junction.

Animals↗