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.

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.

Bench to Bedside: The Effectiveness of a Professional Development Program Focused on Biomedical Sciences and Action Research.

A three-year, National Institutes of Health-funded residential project at a southeastern research university immersed 83 secondary science teachers in a summer institute called "Bench to Bedside." Teachers were provided with knowledge, skills, experiences, and incentives to improve their science teaching and increase their awareness of scientific processes, technologies, and careers by examining the translational medicine continuum of basic to clinical research. This was done with the help of medical school researchers, clinical personnel, biotechnology entrepreneurs, program mentors, and prior year participants. A critical component of the institute was the preparation and implementation of an action research project that reflected teachers' newly acquired knowledge and skills. Action research proposals were critiqued by project team members and feedback provided prior to action research implementation in schools during the following year. Teachers shared their action research with colleagues and project team at a symposium and online as a critical step in networking the teachers. Results of a mixed methods program evaluation strategy indicate that the program produced significant gains in teachers' confidence to explain advanced biosciences topics, development of action research skills, and formation of a statewide biosciences network of key stakeholders. Constraints of time, variation in teacher content and action research background, technology availability, and school-related variables, among others, are discussed.

Caldesmon association with smooth muscle thin filaments isolated in the presence and absence of calcium.

Thin filaments isolated from chicken gizzard smooth muscle in either the presence or the absence of Ca2+ possess identical caldesmon contents. Hence, a 'flip-flop' mechanism, involving Ca2+-dependent association and dissociation of caldesmon and thin filaments, does not appear to operate in vivo and is an unlikely model for caldesmon function.

The ionic requirements for regulation by molluscan thin filaments.

Initial studies on molluscan muscle regulation indicated that thin filaments do not confer Ca2+-dependence on vertebrate myosin ATPase, and hence that molluscan muscles do not possess thin filament-linked regulatory systems. Subsequently it was shown that molluscan thin filaments do, in fact, impart Ca2+-sensitivity but only at Mg2+ concentrations greater than those used in the earlier studies. In the present study it is shown that Mg2+ prevents significant dissociation of tropomyosin and troponin subunits from thin filaments at the low monovalent ion concentrations typically employed to assay actomyosin ATPase; as a result Mg2+ allows expression of the molluscan thin filament regulatory system under these conditions.

35 kDa proteins are not components of vertebrate smooth muscle thin filaments.

A 35 kDa protein present in vertebrate smooth muscle and capable of binding to purified actin does not appear to be a constituent of smooth-muscle thin filaments in vivo; instead, it is more likely to be a component easily solubilized from particulate material which then spuriously interacts with actin.

Diversity in smooth muscle thin filament composition.

Two classes of smooth muscle thin filament can be identified and separated based on their interaction with antibodies specific either to filamin or to caldesmon. One type is composed of actin, tropomyosin and filamin and the other of actin, tropomyosin and caldesmon.

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.

The stoichiometry of the components of arthropod thin filaments.

Limulus thin filaments confer calcium sensitivity on calcium-independent myosins and contain in addition to actin and tropomyosin, three troponin components. The molar ratio of actin:tropomyosin:troponin sub-unit T (TN-T): troponin sub-unit C (TN-C) is approximately 7:1:1:1, as in vertebrates, but twice the amount of the troponin sub-unit I (TN-I) may be present. Arthropod troponin binds approximately 1 mole Ca/mol troponin, a significantly smaller amount than bound by vertebrate troponin.

Activation of the adenosine triphosphatase of Limulus polyphemus actomyosin by tropomyosin.

Purified actin does not stimulate the adenosine triphosphatase (ATPase) activity of Limulus myosin greatly. The ATPase activity of such reconstituted preparations is only about one-fourth the ATPase of myofibrils or of natural actomyosin. Actin preparations containing tropomyosin, however, activate Limulus myosin fully. Both the tropomyosin and the actin preparations appear to be pure when tested by different techniques. Tropomyosin combines with actin but not with myosin and full activation is reached at a tropomyosin-to-actin ratio likely to be present in muscle. Tropomyosin and actin of several different animals stimulate the ATPase of Limulus myosin. Tropomyosin, however, is not required for the ATPases of scallop and rabbit myosin which are fully activated by pure actin alone. Evidence is presented that Limulus myosin, in the presence of ATP at low ionic strength, has a higher affinity for actin modified by tropomyosin than for pure actin.

Regulation of muscular contraction. Distribution of actin control and myosin control in the animal kingdom.

The control systems regulating muscle contraction in approximately 100 organisms have been categorized. Both myosin control and actin control operate simultaneously in the majority of invertebrates tested. These include insects, chelicerates, most crustaceans, annelids, priapulids, nematodes, and some sipunculids. Single myosin control is present in the muscles of molluscs, brachiopods, echinoderms, echiuroids, and nemertine worms. Single actin control was found in the fast muscles of decapods, in mysidacea, in a single sipunculid species, and in vertebrate striated muscles. Classification is based on functional tests that include measurements of the calcium dependence of the actomyosin ATPase activity in the presence and the absence of purified rabbit actin and myosin. In addition, isolated thin filaments and myosins were also analyzed. Molluscs lack actin control since troponin is not present in sufficient quantities. Even though the functional tests indicate the complete lack of myosin control in vertebrate striated muscle, it is difficult to exclude unambiguously the in vivo existence of this regulation. Both control systems have been found in animals from phyla which evolved early. We cannot ascribe any simple correlation between ATPase activity, muscle structure, and regulatory mechanisms.

Calcium-dependent myosin from insect flight muscles.

Calcium regulation of the insect actomyosin ATPase is associated with the thin filaments as in vertebrate muscles, and also with the myosin molecule as in mollusks. This dual regulation is demonstrated using combinations of locust thin filaments with rabbit myosin and locust myosin with rabbit actin; in each case the ATPase of the hybrid actomyosin is calcium dependent. The two regulatory systems are synergistic, the calcium dependency of the locust actomyosin ATPase being at least 10 times that of the hybrid actomyosins described above. Likewise Lethocerus myosin also contains regulatory proteins. The ATPase activity of Lethocerus myosin is labile and is stabilized by the presence of rabbit actin. Tropomyosin activates the ATPase of insect actomyosin and the activation occurs irrespective of whether the myosin is calcium dependent or rendered independent of calcium.

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.

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.

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.

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.

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.

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.

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.