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.     

A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fibre diagrams from non-overlap muscle

The regulation of striated muscle contraction involves changes in the interactions of troponin and tropomyosin with actin thin filaments. In resting muscle, myosin-binding sites on actin are thought to be blocked by the coiled-coil protein tropomyosin. During muscle activation, Ca2+ binding to troponin alters the tropomyosin position on actin, resulting in cyclic actin-myosin interactions that accompany muscle contraction. Evidence for this steric regulation by troponin-tropomyosin comes from X-ray data [Haselgrove, J.C., 1972. X-ray evidence for a conformational change in the actin-containing filaments of verterbrate striated muscle. Cold Spring Habor Symp. Quant. Biol. 37, 341-352; Huxley, H.E., 1972. Structural changes in actin and myosin-containing filaments during contraction. Cold Spring Habor Symp. Quant. Biol. 37, 361-376; Parry, D.A., Squire, J.M., 1973. Structural role of tropomyosin in muscle regulation: analysis of the X-ray diffraction patterns from relaxed and contracting muscles. J. Mol. Biol. 75, 33-55] and electron microscope (EM) data [Spudich, J.A., Huxley, H.E., Finch, J., 1972. Regulation of skeletal muscle contraction. II. Structural studies of the interaction of the tropomyosin-troponin complex with actin. J. Mol. Biol. 72, 619-632; O'Brien, E.J., Gillis, J.M., Couch, J., 1975. Symmetry and molecular arrangement in paracrystals of reconstituted muscle thin filaments. J. Mol. Biol. 99, 461-475; Lehman, W., Craig, R., Vibert, P., 1994. Ca2+-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature 368, 65-67] each with its own particular strengths and limitations. Here we bring together some of the latest information from EM analysis of single thin filaments from Pirani et al. [Pirani, A., Xu, C., Hatch, V., Craig, R., Tobacman, L.S., Lehman, W. (2005). Single particle analysis of relaxed and activated muscle thin filaments. J. Mol. Biol. 346, 761-772], with synchrotron X-ray data from non-overlapped muscle fibres to refine the models of the striated muscle thin filament. This was done by incorporating current atomic-resolution structures of actin, tropomyosin, troponin and myosin subfragment-1. Fitting these atomic coordinates to EM reconstructions, we present atomic models of the thin filament that are entirely consistent with a steric regulatory mechanism. Furthermore, fitting the atomic models against diffraction data from skinned muscle fibres, stretched to non-overlap to preclude crossbridge binding, produced very similar results, including a large Ca2+-induced shift in tropomyosin azimuthal location but little change in the actin structure or apparent alteration in troponin position.     

Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction

Recent developments in the field of myofibrillar proteins will be reviewed. Consideration will be given to the proteins that participate in the contractile process itself as well as to those involved in Ca-dependent regulation of striated (skeletal and cardiac) and smooth muscle. The relation of protein structure to function will be emphasized and the relation of various physiologically and histochemically defined fiber types to the proteins found in them will be discussed.     

Role of actin C-terminus in regulation of striated muscle thin filament

In striated muscle, regulation of actin-myosin interactions depends on a series of conformational changes within the thin filament that result in a shifting of the tropomyosin-troponin complex between distinct locations on actin. The major factors activating the filament are Ca²⁺ and strongly bound myosin heads. Many lines of evidence also point to an active role of actin in the regulation. Involvement of the actin C-terminus in binding of tropomyosin-troponin in different activation states and the regulation of actin-myosin interactions were examined using actin modified by proteolytic removal of three C-terminal amino acids. Actin C-terminal modification has no effect on the binding of tropomyosin or tropomyosin-troponin + Ca²⁺, but it reduces tropomyosin-troponin affinity in the absence of Ca²⁺. In contrast, myosin S1 induces binding of tropomyosin to truncated actin more readily than to native actin. The rate of actin-activated myosin S1 ATPase activity is reduced by actin truncation both in the absence and presence of tropomyosin. The Ca²⁺-dependent regulation of the ATPase activity is preserved. Without Ca²⁺ the ATPase activity is fully inhibited, but in the presence of Ca²⁺ the activation does not reach the level observed for native actin. The results suggest that through long-range allosteric interactions the actin C-terminus participates in the thin filament regulation.     

Calcium regulation of skeletal muscle thin filament motility in vitro.

Using an in vitro motility assay, we have investigated Ca2+ regulation of individual, regulated thin filaments reconstituted from rabbit fast skeletal actin, troponin, and tropomyosin. Rhodamine-phalloidin labeling was used to visualize the filaments by epifluorescence, and assays were conducted at 30 degrees C and at ionic strengths near the physiological range. Regulated thin filaments exhibited well-regulated behavior when tropomyosin and troponin were added to the motility solutions because there was no directed motion in the absence of Ca2+. Unlike F-actin, the speed increased in a graded manner with increasing [Ca2+], whereas the number of regulated thin filaments moving was more steeply regulated. With increased ionic strength, Ca2+ sensitivity of both the number of filaments moving and their speed was shifted toward higher [Ca2+] and was steepest at the highest ionic strength studied (0.14 M gamma/2). Methylcellulose concentration (0.4% versus 0.7%) had no effect on the Ca2+ dependence of speed or number of filaments moving. These conclusions hold for five different methods used to analyze the data, indicating that the conclusions are robust. The force-pCa relationship (pCa = -log10[Ca2+]) for rabbit psoas skinned fibers taken under similar conditions of temperature and solution composition (0.14 M gamma/2) paralleled the speed-pCa relationship for the regulated filaments in the in vitro motility assay. Comparison of motility results with the force-pCa relationship in fibers suggests that relatively few cross-bridges are needed to make filaments move, but many have to be cycling to make the regulated filament move at maximum speed.     

Early mineral deposition in calcifying tendon characterized by high voltage electron microscopy and three-dimensional graphic imaging.

Extracellular matrix organization and the spatial relationship between collagen fibrils, vesicular structures, and the first deposits of mineral in the calcifying leg tendon from the domestic turkey, Meleagris gallopavo, have been investigated by high voltage electron microscopy and three-dimensional computer graphic imaging of serial thick tissue sections. The work demonstrates that the tendon extracellular matrix is a complex assembly of somewhat flexible, highly aligned collagen fibrils with different diameters and occasionally opposite directionality. Smaller collagen fibrils appear to branch from larger fibrils or to aggregate to form those of greater size. While the matrices are dominated by fibrils, space exists between adjacent packed fibrils. The three-dimensional perspective indicates that approximately 60% of the total tendon volume is extrafibrillar over the regions examined. The first observable mineral in this tissue is extrafibrillar and appears to derive from vesicles. This view of three-dimensional matrix-mineral spatial relations supports earlier two-dimensional results that mineral is initially associated with membrane-invested vesicles and is deposited between collagen fibrils, but it is distinct in showing the mineral at different depths in the matrix rather than at a single depth as deduced from two-dimensional conventional electron microscopy. These results are important in the onset and development of tendon calcification in that they suggest, first, that collagen fibrils appear to be aligned three-dimensionally such that their hole zones are in contiguous arrangement. This situation may create channels or grooves within the collagen volume to accommodate extensive mineral deposition in association with the fibrils. Second, the results indicate that there are widely dispersed sites of vesicle-mediated mineralization in the tendon matrix, that the bulk of mineralization in this tissue is collagen-mediated, and that, while vesicles may possibly exert some local influence temporally on mineralization of neighboring collagen, vesicle- and collagen-mediated mineralization arise at spatially and structurally distinct sites by independent nucleation phenomena. Such concepts are fundamental in considerations of possible mechanisms of mineralization of tendon and potentially of other normally calcifying vertebrate tissues in general.     

Sutural mineralization of rat calvaria characterized by atomic-force microscopy and transmission electron microscopy

The application of transmission electron microscopy (TEM) and atomic-force microscopy (AFM) aid the acquisition of detailed structural information on the process of hard tissue formation. The sutural mineralization of rat calvaria is taken as a model for a collagen-related mineralization system. After cryofixation or chemical fixation an anhydrous tissue preparation technique with no staining procedures is used. The atomic-force microscope and the transmission electron microscope are used for structural analysis of the mineralizing region of the sutural tissue. With the application of AFM the collagen macroperiod is shown to be well represented in the unmineralized sutural tissue. At the mineralization front the collagen fibrils are found to be thickened and to change to a characteristic stacked platelet structure. Using TEM the macroperiod is faintly visible before mineral crystallites have formed and is more prominent after the apatite crystallization has started in the fibrils. In this step a needle-like structure of the newly formed apatitic crystals is visible.     

Structure and microtubule-nucleation activity of isolated Drosophila embryo centrosomes characterized by whole mount scanning and transmission electron microscopy

Experimental approaches in Drosophila melanogaster over the last 20 years have played a fundamental role in elucidating the function, structure and molecular composition of the centrosome. However, quantitative data on the structure and function of the Drosophila centrosome are still lacking. This study uses, for the first time, whole mount electron microscopy in combination with negative staining on isolated centrosomes from the early Drosophila embryos to analyze its dimensions, structure and capacity to nucleate microtubules in vitro. We show that these organelles are on average 0.75 μm in diameter and have abundant pericentriolar material which often appears fibrillar and with bulbous protrusions. Corresponding to the abundant pericentriolar material, extensive microtubule nucleation occurs. Quantification of the number of microtubules nucleated showed that 50–300 active nucleation sites are present. We examined via electron microscopy immunogold labeling the distribution of γ-tubulin, CNN, Asp and the MPM-2 epitopes that are phosphorylated through Polo and the Cdk1 kinase. The distribution of these proteins is homogeneous, with the MPM-2 epitopes exhibiting the highest density. In contrast, centrosomal subdomains are identified using a centriole marker to relate centrosome size to the centriole number by electron microscopy. In conclusion, we present a clear-cut technique assaying and quantifying the microtubule nucleation capacity and antigen distribution complementing molecular studies on centrosome protein complexes, cell organelle assembly and protein composition.     

TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy

Background  

Transmission electron tomography is an increasingly common three-dimensional electron microscopy approach that can provide new insights into the structure of subcellular components. Transmission electron tomography fills the gap between high resolution structural methods (X-ray diffraction or nuclear magnetic resonance) and optical microscopy. We developed new software for transmission electron tomography, TomoJ. TomoJ is a plug-in for the now standard image analysis and processing software for optical microscopy, ImageJ.     

The S-layer of Caulobacter crescentus: three-dimensional image reconstruction and structure analysis by electron microscopy.

The regular surface protein structure (S-layer) of Caulobacter crescentus was analyzed by electron microscopy and three-dimensional image reconstruction to a resolution of 2 nm. Projections showed that the S-layer is an array of ring structures, each composed of six subunits that are arranged on a lattice with p6 symmetry. Three-dimensional reconstructions showed that the ring subunits were approximately rod-shaped structures and were perpendicular to the plane of the array, with a linker arm emanating from approximately the middle of the rod, accounting for the connections between the rings. The calculated subunit mass was ca. 100 kDa, very close to the size of RsaA (the protein known to be at least the predominant species in the S-layer) predicted from the DNA sequence of the rsaA gene. The core region of the rings creates an open pore 2.5 to 3.5 nm in diameter. The size of the gaps between the neighboring unit cells is in the same range, suggesting a uniform porosity predicted to exclude molecules larger than ca. 17 kDa. Attempts to remove membrane material from S-layer preparations with detergents revealed that the structure spontaneously rearranged into a mirror-image double layer. Negative-stain and thin-section electron microscopy examination of colonies of C. crescentus strains with a mutation in a surface molecule involved in the attachment of the S-layer showed that shed RsaA protein organized into large sheets. The sheets in turn organized into stacks that tended to accumulate near the upper surface of the colony. Image reconstruction indicated that these sheets were also precise mirror-image double layers, and thickness measurements obtained from thin sections were consistent with this finding. The sheets were absent when these mutant strains were grown without calcium, supporting other data that calcium is involved in attachment of the S-layer to a surface molecule and perhaps in subunit-subunit interactions. We propose that when the membrane is removed from S-layer fragments by detergents or the attachment-related surface molecule is absent, the attachment sites of the S-layer align precisely to form a double layer via a calcium interaction.     

Isolation, electron microscopy and 3D reconstruction of invertebrate muscle myofilaments

Understanding the molecular mechanism of muscle contraction and its regulation has been greatly influenced and aided by studies of myofilament structure in invertebrate muscles. Invertebrates are easily obtained and cover a broad spectrum of species and functional specializations. The thick (myosin-containing) filaments from some invertebrates are especially stable and simple in structure and thus much more amenable to structural analysis than those of vertebrates. Comparative studies of invertebrate filaments by electron microscopy and image processing have provided important generalizations of muscle molecular structure and function. This article reviews methods for preparing thick and thin filaments from invertebrate muscle, for imaging filaments by electron microscopy, and for determining their three dimensional structure by image processing. It also highlights some of the key insights into filament function that have come from these studies.     

Molecular determination by electron microscopy of the actin filament end structure

In eukaryotic cells, actin filaments play various crucial roles by altering their spatial and temporal distributions in the cell. The distribution of actin filaments is regulated by the binding of end-binding proteins, including capping protein (CapZ in muscle), the Arp2/3 complex, gelsolin, formin and tropomodulin, to the end of the actin filament. In order to determine the nature of these regulations, structural elucidations of actin filament-end-binding protein complexes are crucially important. Here, we have developed new procedures on the basis of single-particle analysis to determine the structure of the end of actin filaments from electron micrographs. In these procedures, the polarity of the actin filament image, as well as the azimuth orientation and the axial position of each actin protomer within a short stretch near the filament end, were determined accurately. This improved both the stability and accuracy of the structural determination dramatically. We tested our procedures by reconstructing structures from simulated filament images, which were obtained from 24 model structures for the actin-CapZ complex. These model structures were generated by random docking of the atomic structure of CapZ to the barbed end of an atomic model of the actin filament. Of the 24 model structures, 23 were recovered correctly by the present procedures. We found that our analysis was robust against local aberrations of the helical twist near the end of the actin filament. Finally, the procedures were applied successfully to determine the structure of the actin-CapZ complex from real cryo-electron micrographs of the complex. This is the first method for elucidating the detailed 3D structures at the end of the actin filament.     

High-resolution three-dimensional reconstruction of a whole yeast cell using focused-ion beam scanning electron microscopy

We developed an approach for focused gallium-ion beam scanning electron microscopy with energy filtered detection of backscattered electrons to create near isometric voxels for high-resolution whole cell visualization. Specifically, this method allowed us to create three-dimensional volumes of high-pressure frozen, freeze-substituted Saccharomyces cerevisiae yeast cells with pixel resolutions down to 3 nm/pixel in x, y, and z, supported by both empirical data and Monte Carlo simulations. As a result, we were able to segment and quantify data sets of numerous targeted subcellular structures/organelles at high-resolution, including the volume, volume percentage, and surface area of the endoplasmic reticulum, cell wall, vacuoles, and mitochondria from an entire cell. Sites of mitochondrial and endoplasmic reticulum interconnectivity were readily identified in rendered data sets. The ability to visualize, segment, and quantify entire eukaryotic cells at high-resolution (potentially sub-5 nanometers isotropic voxels) will provide new perspectives and insights of the inner workings of cells.     

Electron microscopy and three-dimensional reconstruction of native thin filaments reveal species-specific differences in regulatory strand densities

Throughout the animal kingdom striated muscle contraction is regulated by the thin filament troponin-tropomyosin complex. Homologous regulatory components are shared among vertebrate and arthropod muscles; however, unique protein extensions and/or components characterize the latter. The Troponin T (TnT) isoforms of Drosophila indirect flight and tarantula femur muscle for example contain distinct C-terminal extensions and are ∼20% larger overall than their vertebrate counterpart. Using electron microscopy and three-dimensional helical reconstruction of native Drosophila, tarantula and frog muscle thin filaments we have identified species-specific differences in tropomyosin regulatory strand densities. The strands on the arthropod thin filaments were significantly larger in diameter than those from vertebrates, although not significantly different from each other. These findings reflect differences in the regulatory troponin-tropomyosin complex, which are likely due to the larger TnT molecules aligning and extending along much of the tropomyosin strands’ length. Such an arrangement potentially alters the physical properties of the regulatory strands and may help establish contractile characteristics unique to certain arthropod muscles.     

Calcium binding of arterial tropomyosin: involvement in the thin filament regulation of smooth muscle

Bovine aortic tropomyosin has been isolated by DEAE-Sepharose chromatography following isoelectric precipitation and ammonium sulfate fractionation. A single polypeptide [Mr 36 000 on a sodium dodecyl sulfate (SDS)-polyacrylamide gel] was obtained under different electrophoretic conditions. The amino acid composition of bovine tropomyosin was very similar to that of rabbit skeletal muscle; the amino-terminal residue is blocked. The molecular weight of the native tropomyosin (76 000), which is twice that calculated from the SDS-polyacrylamide gel, suggests that the molecule is a dimer. The diffusion coefficient of 3.4 X 10(-7) cm2 s-1 and the frictional coefficient of 1.7 indicate that the molecule is asymmetric. Comparative high-pressure liquid chromatography peptide mapping of rabbit skeletal and bovine aortic tropomyosins shows primary structure variation. Bovine aortic tropomyosin binds calcium under physiological conditions of pH and ionic strength (22 mol of Ca2+/mol of tropomyosin with a Kd of 1.4 mM). Such a property is not shared by skeletal tropomyosin. In low Mg2+ concentration, both skeletal and aortic actin activations of the skeletal myosin ATPase activity are calcium independent. Addition of aortic tropomyosin to a hybrid actomyosin (aortic actin, skeletal myosin) yields an enhancement of the actin activation of the myosin ATPase activity, but the addition of skeletal tropomyosin yields a decrease of this activity. However, both the enhancement and decrease are calcium dependent. Addition of skeletal or aortic tropomyosin to an actomyosin system, where both actin and myosin come from skeletal muscle, yields only an enhancement of the actin activation of the myosin ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visualization of monoclonal antibody binding to tropomyosin on native smooth muscle thin filaments by electron microscopy

We have used three different monoclonal antibodies (LCK16, JLH2 and JLF15) to tropomyosin for the localization of tropomyosin molecules within smooth muscle thin filaments. Thin filaments were incubated with monoclonal antibodies and visualized by negative staining electron microscopy. All three monoclonal antibodies caused the aggregation of thin filaments into ordered bundles, which displayed cross-striations with a periodicity of 37 ± 1 nm. In contrast, conventional rabbit antiserum to tropomyosin distorted and aggregated the thin filaments without generating cross-striations. Therefore, monoclonal antibodies to tropomyosin allow us, for the first time, to observe directly the distribution of tropomyosin molecules along the thin filaments of smooth muscle cells. The binding sites of the antibodies to skeletal muscle tropomyosin were examined by decorating tropomyosin paracrystals with monoclonal antibodies. The LCK16 monoclonal antibody binds the narrow band of tropomyosin paracrystals, whereas the JLF15 antibody binds the wide band of tropomyosin paracrystals.     

A kinetic model of troponin dissociation in relation to thin filament regulation in striated muscle

The apparent rate of troponin (Tn) dissociation from myofibrils has been used as a method to study thin filament regulation in striated muscle. The rate is dependent upon calcium and strong crossbridges and supports the three-state model for thin filament regulation. The dissociation rate of Tn is extremely low so it is not intuitively clear that such a slow process would probe thin filament regulation. We have investigated this issue by developing a simple kinetic model to explain the Tn dissociation rate measured by labeled Tn exchange in the myofibrils. Tn is composed of three interacting subunits, TnC, TnI and TnT. In our model, TnI’s regulatory domain switches from actin-tropomyosin to TnC followed by TnT dissociation from actin-tropomyosin. This TnI regulatory domain switching is linked to the transition of the thin filament from the blocked state to the closed state. It is calcium dependent and several orders of magnitude faster than TnT dissociation from actin-tropomyosin. By integrating the dimensionless rate equations of this model, we have computed the time course of each of the various components. In our numerical simulations, the rate constant for TnI switching from actin-tropomyosin to TnC was varied from 10 s^?1 to 1000 s^?1 to simulate the low calcium, blocked state to high calcium, closed state. The computed progress curves for labeled Tn exchange into the myofibrils and the derived intensity ratio between the non-overlap and overlap regions well explains the intensity ratio progress curves observed experimentally. These numerical simulations and experimental observations reveal that the apparent rate of Tn dissociation probes the blocked state to closed state equilibrium of the myofibrillar thin filament.