X-ray diffraction studies on muscle regulation

Using the intensity of the outer part of the second actin layer line as an indicator of thin filament conformation in vertebrate muscle we were able to identify the four different states of rest, and the three states induced by the presence of Ca²⁺ ions, rigor bridge attachment and actively cycling bridges, respectively. These findings are in qualitative agreement with a number of biochemical studies by Eisenberg and Greene and others, indicating that activation of the thin filament depends both on Ca²⁺ ions and crossbridge binding. Yet quantitatively, the biochemical data and our structural data are contradictory. Whereas the biochemical studies suggest a strong coupling between structural changes of the thin filament and the ATPase activity, the structural studies indicate that this is not necessarily the case.Troponin molecules also change their conformation upon activation depending on both Ca²⁺ ions and crossbridge binding as demonstrated by the early part of the time course of the thin filament meridional reflections in contracting frog muscle.Low ionic strength which has been shown by Brenner and collaborators to increase weakly binding crossbridges in relaxed rabbit psoas muscle does not influence the intensity of the second actin layer line in this muscle. Yet in contracting frog muscle the increase of the second actin layer line increases very rapidly in one step, suggesting that weak binding bridges which are attached to actin prior to force production may indeed influence the thin filament conformation. It therefore appears that weakly bound bridges in the low ionic strength state do not have the same effect on the thin filament conformation as weakly bound bridges in an actively contracting muscle.Arthropod muscles like the thin filament regulated lobster muscles differ from vertebrate muscle in not showing an increase of the second layer line during contraction, which may have to do with differences in crossbridge attachment. The myosin-regulated molluscan muscle ABRM shows a large increase on the second actin layer line upon phasic contraction and a much smaller increase in catch or rigor, indicating that actively cycling bridges influence the thin filament conformation differently than catch or rigor bridges.Several pieces of evidence which we have briefly outlined in this paper suggest that the thin filament conformational changes we have observed do not arise solely from tropomyosin movements and that conformational changes of actin domains should be considered.     

Direct modeling of x-ray diffraction pattern from skeletal muscle in rigor

Available high-resolution structures of F-actin, myosin subfragment 1 (S1), and their complex, actin-S1, were used to calculate a 2D x-ray diffraction pattern from skeletal muscle in rigor. Actin sites occupied by myosin heads were chosen using a "principle of minimal elastic distortion energy" so that the 3D actin labeling pattern in the A-band of a sarcomere was determined by a single parameter. Computer calculations demonstrate that the total off-meridional intensity of a layer line does not depend on disorder of the filament lattice. The intensity of the first actin layer A1 line is independent of tilting of the "lever arm" region of the myosin heads. Myosin-based modulation of actin labeling pattern leads not only to the appearance of the myosin and "beating" actin-myosin layer lines in rigor diffraction patterns, but also to changes in the intensities of some actin layer lines compared to random labeling. Results of the modeling were compared to experimental data obtained from small bundles of rabbit muscle fibers. A good fit of the data was obtained without recourse to global parameter search. The approach developed here provides a background for quantitative interpretation of the x-ray diffraction data from contracting muscle and understanding structural changes underlying muscle contraction.     

An atomic model of the thin filament in the relaxed and Ca2+-activated states

Contraction of striated muscles is regulated by tropomyosin strands that run continuously along actin-containing thin filaments. Tropomyosin blocks myosin-binding sites on actin in resting muscle and unblocks them during Ca2+-activation. This steric effect controls myosin-crossbridge cycling on actin that drives contraction. Troponin, bound to the thin filaments, couples Ca2+-concentration changes to the movement of tropomyosin. Ca2+-free troponin is thought to trap tropomyosin in the myosin-blocking position, while this constraint is released after Ca2+-binding. Although the location and movements of tropomyosin are well known, the structural organization of troponin on thin filaments is not. Its mechanism of action therefore remains uncertain. To determine the organization of troponin on the thin filament, we have constructed atomic models of low and high-Ca2+ states based on crystal structures of actin, tropomyosin and the "core domain" of troponin, and constrained by distances between filament components and by their location in electron microscopy (EM) reconstructions. Alternative models were also built where troponin was systematically repositioned or reoriented on actin. The accuracy of the different models was evaluated by determining how well they corresponded to EM images. While the initial low and high-Ca2+ models fitted the data precisely, the alternatives did not, suggesting that the starting models best represented the correct structures. Thin filament reconstructions were generated from the EM data using these starting models as references. In addition to showing the core domain of troponin, the reconstructions showed additional detail not present in the starting models. We attribute this to an extension of TnI linking the troponin core domain to actin at low (but not at high) Ca2+, thereby trapping tropomyosin in the OFF-state. The bulk of the core domain of troponin appears not to move significantly on actin, regardless of Ca2+ level. Our observations suggest a simple model for muscle regulation in which troponin affects the charge balance on actin and hence tropomyosin position.     

Structure of insect fibrillar flight muscle in the presence and absence of ATP

Low-angle X-ray diffraction pictures were taken of Lethocerus flight muscle in one or other of its two inactive states, relaxation and rigor. They showed more detail than previous pictures and the parameters of the actin and myosin helices could be deduced from the spacings of the observed layer lines. Subunits consisting of actin monomers and of myosin heads were arranged on helices so as to conform to these parameters, and the diffraction patterns from these models were calculated. When these model systems resembled the structures observed in the electron microscope, the calculated diffraction patterns had layer Unes similar in radial distribution and relative intensity to those observed, with certain exceptions. The fit was quite critical, in that variation of the size and orientation of the subunits affected it considerably. The results support the idea that in rigor (i.e. without ATP) all of the myosin heads attach to actin monomers in a regular angled configuration. In contrast, on relaxation (i.e. on addition of ATP but no Ca²⁺) most or all of the myosin heads detach from the actin and are arranged with a specific symmetry around the myosin filament. It is not necessary, however, to assume that they change shape or move far from the actin filament in this process. Features of the X-ray diffraction pattern which remained unaccounted for by this model can be explained on the basis of an arrangement of actin helices on a further helix around a thick filament. Types of extended lattices containing actin helices in statistical axial or azimuthal positions are discussed.     

A structural origin of latency relaxation in frog skeletal muscle

A time-resolved x-ray diffraction study at a time resolution of 0.53 ms was made to investigate the structural origin of latency relaxation (LR) in frog skeletal muscle. Intensity and spacing measurements were made on meridional reflections from the Ca-binding protein troponin and the thick filament and on layer lines from the thin filament. At 16 degrees C, the intensity and spacing of all reflections started to change at 4 ms, simultaneously with the LR. At 0 degrees C, the intensity of the troponin reflection and the layer lines from the thin filament and the spacing of the 14.3-nm myosin meridional reflection, but not the spacing of other myosin meridional reflections, began to change at approximately 15 ms, when the LR also started. Intensity of myosin-based reflections started to change later. When the muscle was stretched to non-overlap length, the intensity and spacing changes of the myosin reflections disappeared. The simultaneous spacing change of the 14.3-nm myosin meridional reflection with the LR suggests that detachment of myosin heads that are bound to actin in the resting muscle is the cause of the LR.     

LOCALIZATION OF MYOSIN FILAMENTS IN SMOOTH MUSCLE

Thick myosin filaments, in addition to actin filaments, were found in sections of glycerinated chicken gizzard smooth muscle when fixed at a pH below 6.6. The thick filaments were often grouped into bundles and run in the longitudinal axis of the smooth muscle cell. Each thick filament was surrounded by a number of thin filaments, giving the filament arrangement a rosette appearance in cross-section. The exact ratio of thick filaments to thin filaments could not be determined since most arrays were not so regular as those commonly found in striated muscle. Some rosettes had seven or eight thin filaments surrounding a single thick filament. Homogenates of smooth muscle of chicken gizzard also showed both thick and thin filaments when the isolation was carried out at a pH below 6.6, but only thin filaments were found at pH 7.4. No Z or M lines were observed in chicken gizzard muscle containing both thick and thin filaments. The lack of these organizing structures may allow smooth muscle myosin to disaggregate readily at pH 7.4.     

Structural studies of arthrin: monoubiquitinated actin

Here, we report on the structure and in situ location of arthrin (monoubiquitinated actin). Labelling of insect muscle thin filaments with a ubiquitin antibody reveals that every seventh subunit along the filament long-pitch helices is ubiquitinated. A three-dimensional reconstruction of frozen-hydrated arthrin filaments was produced. This was based on a novel algorithm that divides filament images into short segments that are used for single-particle image processing. Difference maps with an actin filament reconstruction locate ubiquitin at the side of actin sub-domain 1 opposite where myosin binds. Consistent with the reconstructions, peptide mapping places the ubiquitin linkage on lysine 118 in actin. Molecular modelling was used to generate arthrin monomers from ubiquitin and actin crystal structures. Filament models constructed from these monomers were compared with the arthrin reconstruction. The reconstruction suggests ubiquitin attached to Lys118 adopts one or a few conformers, stabilized by a small interface with actin. The function of actin ubiquitination is not known, but may involve regulation of muscle contractile activity.     

Non-specific termination of simian virus 40 DNA replication.

Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å.     

Tropomodulins: life at the slow end

Dynamic exchange of actin monomers at filament ends is crucial for the functional architecture of many cytoskeletal-dependent processes. Recent evidence indicates that tropomodulins (Tmods), a conserved family of actin-capping proteins that bind to the pointed (slow-growing) end of actin filaments, regulate a variety of actin structures, including dynamic actin networks found in some motile cells. Actin structures that are more stable, such as sarcomeric thin filaments, require capping by Tmods to specify filament lengths and to provide filament stability. Here, we discuss the functional differences between the capping of pointed and barbed ends within the context of these actin-filament systems, and how Tmods uniquely contribute to their regulation and organization.     

The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments

Strict regulation of actin thin filament length is critical for the proper functioning of sarcomeres, the basic contractile units of myofibrils. It has been hypothesized that a molecular template works with actin filament capping proteins to regulate thin filament lengths. Nebulin is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. Tropomodulin (Tmod), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod as demonstrated by blot overlay, bead binding, and solid phase binding assays. These data demonstrate that the N terminus of the nebulin molecule extends to the extreme end of the thin filament and also establish a novel biochemical function for this end. Two Tmod isoforms, erythrocyte Tmod (E-Tmod), expressed in embryonic and slow skeletal muscle, and skeletal Tmod (Sk-Tmod), expressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal nebulin fragments. Sk-Tmod binds nebulin with higher affinity than E-Tmod does, suggesting that the Tmod/nebulin interaction exhibits isoform specificity. These data provide evidence that Tmod and nebulin may work together as a linked mechanism to control thin filament lengths in skeletal muscle.     

The state of the filament

Movement is a defining characteristic of life. Macroscopic motion is driven by the dynamic interactions of myosin with actin filaments in muscle. Directed polymerization of actin behind the advancing membrane of a eukaryotic cell generates microscopic movement. Despite the fundamental importance of actin in these processes, the structure of the actin filament remains unknown. The Holmes model of the actin filament was published 15 years ago, and although it has been widely accepted, no high-resolution structural data have yet confirmed its veracity. Here, we review the implications of recently determined structures of F-actin-binding proteins for the structure of the actin filament and suggest a series of in silico tests for actin-filament models. We also review the significance of these structures for the arp2/3-mediated branched filament.     

Nebulin regulates the assembly and lengths of the thin filaments in striated muscle

In many tissues, actin monomers polymerize into actin (thin) filaments of precise lengths. Although the exact mechanisms involved remain unresolved, it is proposed that "molecular rulers" dictate the lengths of the actin filaments. The giant nebulin molecule is a prime candidate for specifying thin filament lengths in striated muscle, but this idea has never been proven. To test this hypothesis, we used RNA interference technology in rat cardiac myocytes. Live cell imaging and triple staining revealed a dramatic elongation of the preexisting thin filaments from their pointed ends upon nebulin knockdown, demonstrating its role in length maintenance; the barbed ends were unaffected. When the thin filaments were depolymerized with latrunculin B, myocytes with decreased nebulin levels reassembled them to unrestricted lengths, demonstrating its importance in length specification. Finally, knockdown of nebulin in skeletal myotubes revealed its involvement in myofibrillogenesis. These data are consistent with nebulin functioning as a thin filament ruler and provide insight into mechanisms dictating macromolecular assembly.     

Structure and periodicities of cross-bridges in relaxation, in rigor, and during contractions initiated by photolysis of caged Ca2+.

Ultra-rapid freezing and electron microscopy were used to directly observe structural details of frog muscle fibers in rigor, in relaxation, and during force development initiated by laser photolysis of DM-nitrophen (a caged Ca2+). Longitudinal sections from relaxed fibers show helical tracks of the myosin heads on the surface of the thick filaments. Fibers frozen at approximately 13, approximately 34, and approximately 220 ms after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridge dispositions. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridge densities have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z line. This highly variable distribution is established very early during development of contraction. Cross-bridge density across the interfilament space is more uniform than in rigor, wherein the cross-bridges are more dense near the thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze periodicities of structures within the overlap regions of the sarcomeres. Most aspects of these patterns are consistent with time resolved x-ray diffraction data from the corresponding states of intact muscle, but some features are different, presumably reflecting different origins of contrast between the two methods and possible alterations in the structure of the electron microscopy samples during processing. In relaxed fibers, OD patterns show strong meridional spots and layer lines up to the sixth order of the 43-nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, layer lines at 18, 24, and 36 nm indicate cross-bridge attachment along the thin filament helix. After activation by photorelease of Ca2+, the 14.3-nm meridional spot is present, but the second-order meridional spot (22 nm) disappears. The myosin 43-nm layer line becomes less intense, and higher orders of 43-nm layer lines disappear. A 36-nm layer line is apparent by 13 ms and becomes progressively stronger while moving laterally away from the meridian of the pattern at later times, indicating cross-bridges labeling the actin helix at decreasing radius.     

The actin filament content of hair cells of the bird cochlea is nearly constant even though the length, width, and number of stereocilia vary depending on the hair cell location

By direct counts off scanning electron micrographs, we determined the number of stereocilia per hair cell of the chicken cochlea as a function of the position of the hair cell on the cochlea. Micrographs of thin cross sections of stereociliary bundles located at known positions on the cochlea were enlarged and the total number of actin filaments per stereocilium was counted and recorded. By comparing the counts of filament number with measurements of actin filament bundle width of the same stereocilium, we were able to relate actin filament bundle width to filament number with an error margin (r2) of 16%. Combining this data with data already published or in the process of publication from our laboratory on the length and width of stereocilia, we were able to calculate the total length of actin filaments present in stereociliary bundles of hair cells located at a variety of positions on the cochlea. We found that stereociliary bundles of hair cells contain 80,000-98,000 micron of actin filament, i.e., the concentration of actin is constant in all hair cells with a range of values that is less than our error in measurement and/or biological variation, the greatest variation being in relating the diameters of the stereocilia to filament number. We also calculated the membrane surface needed to cover the stereocilia of hair cells located throughout the cochlea. The values (172-192 micron 2) are also constant. The implications of our observation that the total amount of actin is constant even though the length, width, and number of stereocilia per hair cell vary are 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.     

Electron microscopic study of actin polymerization in airway smooth muscle

Actin polymerization as part of the normal smooth muscle response to various stimuli has been reported. The actin dynamics are believed to be necessary for cytoskeletal remodeling in smooth muscle in its adaptation to external stress and strain and for maintenance of optimal contractility. We have shown in our previous studies in airway smooth muscle that myosins polymerized in response to contractile activation as well as to adaptation at longer cell lengths. We postulated that the same response could be elicited from actins under the same conditions. In the present study, actin filament formation was quantified electron microscopically in cell cross sections. Nanometer resolution allowed us to examine regional distribution of filaments in a cell cross section. Airway smooth muscle bundles were fixed in relaxed and activated states at two lengths; muscle preparations were also fixed after a period of oscillatory strain, a condition known to cause depolymerization of myosin filaments. The results indicate that contractile activation and increased cell length nonsynergistically enhanced actin polymerization; the extent of actin polymerization was substantially less than that of myosin polymerization. Oscillatory strain increased thin filament formation. Although thin filament density was found higher in cytoplasmic areas near dense bodies, contractile activation did not preferentially enhance actin polymerization in these areas. It is concluded that actin thin filaments are dynamic structures whose length and number are regulated by the cell in response to changes in extracellular environment and that polymerization and depolymerization of thin filaments occur uniformly across the whole cell cross section.     

Troponin Revealed: Uncovering the Structure of the Thin Filament On-Off Switch in Striated Muscle

Recently, our understanding of the structural basis of troponin-tropomyosin’s Ca²⁺-triggered regulation of striated muscle contraction has advanced greatly, particularly via cryo-electron microscopy data. Compelling atomic models of troponin-tropomyosin-actin were published for both apo- and Ca²⁺-saturated states of the cardiac thin filament. Subsequent electron microscopy and computational analyses have supported and further elaborated the findings. Per cryo-electron microscopy, each troponin is highly extended and contacts both tropomyosin strands, which lie on opposite sides of the actin filament. In the apo-state characteristic of relaxed muscle, troponin and tropomyosin hinder strong myosin-actin binding in several different ways, apparently barricading the actin more substantially than does tropomyosin alone. The troponin core domain, the C-terminal third of TnI, and tropomyosin under the influence of a 64-residue helix of TnT located at the overlap of adjacent tropomyosins are all in positions that would hinder strong myosin binding to actin. In the Ca²⁺-saturated state, the TnI C-terminus dissociates from actin and binds in part to TnC; the core domain pivots significantly; the N-lobe of TnC binds specifically to actin and tropomyosin; and tropomyosin rotates partially away from myosin’s binding site on actin. At the overlap domain, Ca²⁺ causes much less tropomyosin movement, so a more inhibitory orientation persists. In the myosin-saturated state of the thin filament, there is a large additional shift in tropomyosin, with molecular interactions now identified between tropomyosin and both actin and myosin. A new era has arrived for investigation of the thin filament and for functional understandings that increasingly accommodate the recent structural results.     

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.