Biomedical subjects

W Lehman

Publications and source records attributed to W Lehman.

At least 19 recordsLinked to original sources

PubMed2001

Crossbridge and tropomyosin positions observed in native, interacting thick and thin filaments.

Tropomyosin movements on thin filaments are thought to sterically regulate muscle contraction, but have not been visualized during active filament sliding. In addition, although 3-D visualization of myosin crossbridges has been possible in rigor, it has been difficult for thick filaments actively interacting with thin filaments. In the current study, using three-dimensional reconstruction of electron micrographs of interacting filaments, we have been able to resolve not only tropomyosin, but also the docking sites for weak and strongly bound crossbridges on thin filaments. In relaxing conditions, tropomyosin was observed on the outer domain of actin, and thin filament interactions with thick filaments were rare. In contracting conditions, tropomyosin had moved to the inner domain of actin, and extra density, reflecting weakly bound, cycling myosin heads, was also detected, on the extreme periphery of actin. In rigor conditions, tropomyosin had moved further on to the inner domain of actin, and strongly bound myosin heads were now observed over the junction of the inner and outer domains. We conclude (1) that tropomyosin movements consistent with the steric model of muscle contraction occur in interacting thick and thin filaments, (2) that myosin-induced movement of tropomyosin in activated filaments requires strongly bound crossbridges, and (3) that crossbridges are bound to the periphery of actin, at a site distinct from the strong myosin binding site, at an early stage of the crossbridge cycle.

Actins↗

PubMed2001

Myosin light chain kinase binding to a unique site on F-actin revealed by three-dimensional image reconstruction.

Ca2+-calmodulin-dependent phosphorylation of myosin regulatory light chains by the catalytic COOH-terminal half of myosin light chain kinase (MLCK) activates myosin II in smooth and nonmuscle cells. In addition, MLCK binds to thin filaments in situ and F-actin in vitro via a specific repeat motif in its NH2 terminus at a stoichiometry of one MLCK per three actin monomers. We have investigated the structural basis of MLCK-actin interactions by negative staining and helical reconstruction. F-actin was decorated with a peptide containing the NH2-terminal 147 residues of MLCK (MLCK-147) that binds to F-actin with high affinity. MLCK-147 caused formation of F-actin rafts, and single filaments within rafts were used for structural analysis. Three-dimensional reconstructions showed MLCK density on the extreme periphery of subdomain-1 of each actin monomer forming a bridge to the periphery of subdomain-4 of the azimuthally adjacent actin. Fitting the reconstruction to the atomic model of F-actin revealed interaction of MLCK-147 close to the COOH terminus of the first actin and near residues 228-232 of the second. This unique location enables MLCK to bind to actin without interfering with the binding of any other key actin-binding proteins, including myosin, tropomyosin, caldesmon, and calponin.

Actins↗

PubMed2001

Troponin organization on relaxed and activated thin filaments revealed by electron microscopy and three-dimensional reconstruction.

The steric model of muscle regulation holds that at low Ca(2+) concentration, tropomyosin strands, running along thin filaments, are constrained by troponin in an inhibitory position that blocks myosin-binding sites on actin. Ca(2+) activation, releasing this constraint, allows tropomyosin movement, initiating actin-myosin interaction and contraction. Although the different positions of tropomyosin on the thin filament are well documented, corresponding information on troponin has been lacking and it has therefore not been possible to test the model structurally. Here, we show that troponin can be detected on thin filaments and demonstrate how its changing association with actin can control tropomyosin position in response to Ca(2+). To accomplish this, thin filaments were reconstituted with an engineered short tropomyosin, creating a favorable troponin stoichiometry and symmetry for three-dimensional analysis. We demonstrate that in the absence of Ca(2+), troponin bound to both tropomyosin and actin can act as a latch to constrain tropomyosin in a position on actin that inhibits actomyosin ATPase. In addition, we find that on Ca(2+) activation the actin-troponin connection is broken, allowing tropomyosin to assume a second position, initiating actomyosin ATPase and thus permitting contraction to proceed.

Actin Cytoskeleton↗

PubMed2001

Effects of a cardiomyopathy-causing troponin t mutation on thin filament function and structure.

Familial hypertrophic cardiomyopathy (FHC) is caused by missense or premature truncation mutations in proteins of the cardiac contractile apparatus. Mutant proteins are incorporated into the thin filament or thick filament and eventually produce cardiomyopathy. However, it has been unclear how the several, genetically identified defects in protein structure translate into impaired protein and muscle function. We have studied the basis of FHC caused by premature truncation of the most frequently implicated thin filament target, troponin T. Electron microscope observations showed that the thin filament undergoes normal structural changes in response to Ca(2+) binding. On the other hand, solution studies showed that the mutation alters and destabilizes troponin binding to the thin filament to different extents in different regulatory states, thereby affecting the transitions among states that regulate myosin binding and muscle contraction. Development of hypertrophic cardiomyopathy can thus be traced to a defect in the primary mechanism controlling cardiac contraction, switching between different conformations of the thin filament.

Actins↗

PubMed2000

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments.

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.

Actin Cytoskeleton↗

PubMed2000

An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin.

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+) to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca(2+) showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.

Actin Cytoskeleton↗

PubMed2000

Three-dimensional reconstruction of thin filaments containing mutant tropomyosin.

Interactions of the components of reconstituted thin filaments were investigated using a tropomyosin internal deletion mutant, D234, in which actin-binding pseudo-repeats 2, 3, and 4 are missing. D234 retains regions of tropomyosin that bind troponin and form end-to-end tropomyosin bonds, but has a length to span only four instead of seven actin monomers. It inhibits acto-myosin subfragment 1 ATPase (acto-S-1 ATPase) and filament sliding in vitro in both the presence and absence of Ca(2+) (, J. Biol. Chem. 272:14051-14056) and lowers the affinity of S-1.ADP for actin while increasing its cooperative binding. Electron microscopy and three-dimensional reconstruction of reconstituted thin filaments containing actin, troponin, and wild-type or D234 tropomyosin were carried out to determine if Ca(2+)-induced movement of D234 occurred in the filaments. In the presence and absence of Ca(2+), the D234 position was indistinguishable from that of the wild-type tropomyosin, demonstrating that the mutation did not affect normal tropomyosin movement induced by Ca(2+) and troponin. These results suggested that, in the presence of Ca(2+) and troponin, D234 tropomyosin was trapped on filaments in the Ca(2+)-induced position and was unable to undergo a transition to a completely activated position. By adding small amounts of rigor-bonded N-ethyl-maleimide-treated S-1 to mutant thin filaments, thus mimicking the myosin-induced "open" state, inhibition could be overcome and full activation restored. This myosin requirement for full activation provides support for the existence of three functionally distinct thin filament states (off, Ca(2+)-induced, myosin-induced; cf.;, J. Mol. Biol. 266:8-14). We propose a further refinement of the three-state model in which the binding of myosin to actin causes allosteric changes in actin that promote the binding of tropomyosin in an otherwise energetically unfavorable "open" state.

Actin Cytoskeleton↗

PubMed1999

Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy.

Past attempts to detect tropomyosin in electron micrograph images of frozen-hydrated troponin-regulated thin filaments under relaxing conditions have not been successful. This raised the possibility that tropomyosin may be disordered on filaments in the off-state, a possibility at odds with the steric blocking model of muscle regulation. By using cryoelectron microscopy and helical image reconstruction we have now resolved the location of tropomyosin in both relaxing and activating conditions. In the off-state, tropomyosin adopts a position on the outer domain of actin with a binding site virtually identical to that determined previously by negative staining, although at a radius of 3.8 nm, slightly higher than found in stained filaments. Molecular fitting to the atomic model of F-actin shows that tropomyosin is localized over sites on actin subdomain 1 required for myosin binding. Restricting access to these sites would inhibit the myosin-cross-bridge cycle, and hence contraction. Under high Ca(2+) activating conditions, tropomyosin moved azimuthally, away from its blocking position to the same site on the inner domain of actin previously determined by negative staining, also at 3.8 nm radius. These results provide strong support for operation of the steric mechanism of muscle regulation under near-native solution conditions and also validate the use of negative staining in investigations of muscle thin filament structure.

Actins↗

PubMed1998

An atomic model of fimbrin binding to F-actin and its implications for filament crosslinking and regulation.

Using a new procedure that combines electron-density correlation with biochemical information, we have fitted the crystal structure of the N-terminal actin-binding domain of human T-fimbrin to helical reconstructions of fimbrin-decorated actin filaments. The map locates the N-terminal calcium-binding domain and identifies actin-binding site residues on the two calponin-homology domains of fimbrin. Based on this map, we propose a model of a fimbrin crosslink in an actin bundle and its regulation by calcium.

Actins↗

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↗

PubMed1997

Salter-Harris type III fracture-dislocation of the proximal humerus.

Salter-Harris type III fractures of the proximal humerus are rare injuries. We report a Salter-Harris type III anterior fracture-dislocation of the proximal humerus in a 10-year-old boy that was open reduced and internally stabilized. A bone scan performed during the initial hospitalization and at 2-year follow-up revealed devascularization and subsequent revascularization of the humeral head. At 2-year follow-up, the patient had full motion of the shoulder, no pain, and arm strength equal to that of the contralateral side. Four cases of Salter-Harris type III fractures of the proximal humerus have been previously reported; good early clinical outcomes were obtained in all. Despite devascularization of the epiphyseal fragment, excellent clinical outcomes may result.

Child↗

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↗

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

The caldesmon content of vertebrate smooth muscle.

Caldesmon and tropomyosin can be selectively and quantitatively extracted from vascular and visceral smooth muscle following heat treatment; all other smooth muscle proteins are precipitated by this procedure. Estimates of the caldesmon/tropomyosin molar ratio in heat-extracts determined by SDS-PAGE densitometry are 1 caldesmon:5.1-5.3 tropomyosin for rabbit and sheep aorta, and 1 caldesmon:5.9 tropomyosin for rabbit stomach and chicken gizzard. If the assumption is made that tropomyosin serves as a true reference of thin-filament content in intact muscle, it follows that the relative caldesmon contents in the above tissues are similar to each other. Caldesmon in heat extracts was identified by Western blotting, by its anomalous migration on several different SDS-PAGE systems and by its position on two-dimensional PAGE. Values of caldesmon contents in unfractionated total tissue homogenates were found to be similar to those cited above. Smooth muscles contain different thin-filament classes and only one type appears to possess caldesmon. By comparing values for the molar composition of caldesmon-specific filaments (1 caldesmon:2 tropomyosin:14 actin) with the values above determined for intact tissue, we conclude that the caldesmon filaments account for approx. 35-45% of the total thin-filament pool in arterial smooth muscle and slightly less in visceral muscles.

Animals↗

PubMed1993

Disulphide cross-linking of smooth-muscle and non-muscle caldesmon to the C-terminus of actin in reconstituted and native thin filaments.

It was reported that chicken gizzard smooth-muscle caldesmon Cys-580 can be disulphide-cross-linked to the C-terminal pen-ultimate residue (Cys-374) of actin, indicating that these residues are close in the protein complex [Graceffa, P. and Jancso, A. (1991) J. Biol. Chem. 266, 20305-20310]. Since the possibility that the cross-link involves a cysteine residue other than actin Cys-374 was not absolutely excluded, more direct evidence was sought for the identify of the cysteine residues involved in the cross-link. We show here that caldesmon could not be disulphide-cross-linked to actin which had Cys-374 removed by carboxypeptidase A digestion, providing direct support for the participation of actin Cys-374 in the cross-link to caldesmon. In order to assign the caldesmon cysteine residue involved in the cross-link, use was made of caldesmon from porcine stomach muscle, which is shown to contain one cysteine residue close to, or at, position 580, in contrast with chicken gizzard caldesmon, which has an additional cysteine residue at position 153. The porcine stomach caldesmon also formed a disulphide-cross-link to actin, further supporting the original conclusion that Cys-580 of the chicken gizzard caldesmon had been cross-linked to actin. Disulphide-cross-linking with similar yield was also observed in native chicken gizzard muscle thin filaments, indicating that the interaction between actin and the C-terminal domain of caldesmon is the same in native and reconstituted thin filaments. The much smaller non-muscle isoform of caldesmon, from rabbit liver, could be similarly cross-linked to actin, consistent with the sequence similarity between the C-terminal domain of muscle and non-muscle caldesmon. The ability to cross-link caldesmon Cys-580 to actin Cys-374 suggests the possibility that the Cys-580 region of caldesmon and the C-terminus of actin form part of the actin-caldesmon binding interface.

Actins↗