Michael K. Reedy

Tomographic 3D Reconstruction of Quick-Frozen, Ca2+-Activated Contracting Insect Flight Muscle

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayments rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.

Thick myofilament mass determination by electron scattering measurements with the scanning transmission electron microscope

SummaryAn accurate value for mass/length of thick myofilaments is required to establish a limit for the maximum number of myosin molecules per crossbridge repeat. The mass/length of the crossbridge regions of desalted thick myofilaments from insect flight muscle (Lethocerus andMusca) and rabbit psoas has been measured with a computer-linked STEM by comparing the electron scattering signal per unit length of unstained thick filaments with that from TMV particles in the same image. Filament preparation was aided by limited digestion of myofibrils to remove Z bands using calcium-activated factor (CAF) from rabbit skeletal muscle; SDS gels showed that this selective protease spared myosin and tended to spare paramyosin but removed C protein.Lethocerus filaments prepared by the CAF procedure were 20–25% heavier per unit length than those prepared by conventional (simple) shearing, and retained a clear and generally uniform 14.5 nm crossbridge repeat by negative staining.We have expressed mass/length of thick filaments as myosin equivalents (mol. wt 0.470 × 106) per crown (that is, the 14.5 nm insect or 14.3 nm vertebrate repeat along thick filaments). TMV standards, calculated to weigh 0.1304×106 daltons nm−1 and thus equivalent to 4.02 myosins per 14.5 nm, were uniform to ±3%s.d. for 73 particles after normalizing means for each different image field. After subtracting the known paramyosin content from insect measurements (11% for waterbug, 2% for the housefly), but making no C protein correction to rabbit measurements, the following results were obtained:Lethocerus (all) 4.19±0.50 (243 filaments);Lethocerus (CAF prepns) 4.40±0.44 (145 filaments);Musca (all CAF) 4.14±0.37 (57 filaments); rabbit (all CAF) 2.86±0.34 (75 filaments).These values favour the lowest integral number of myosins per crown among currently competing models of thick filament structure. The rabbit value agrees with several previous estimates, including the STEM measurements of Lamvik, which indicated three rather than four myosins/crown. The insect flight myofilament value of four forces re-evaluation of previous estimates by quantitative gels and quantitative microscopy of whole fibrils which had favoured six myosins/crown.

X-ray diffraction observations of chemically skinned frog skeletal muscle processed by an improved method

Whole frog sartorius muscles can be chemically skinned in approximately 2 h by relaxing solutions containing 0.5% Triton X-100. The intensity and order of the X-ray diffraction pattern from living muscle is largely retained after such skinning, indicating good retention of native structure in fibrils and filaments. Best X-ray results were obtained using a solution with (mM): 75 K acetate; 5 Mg acetate; 5 ATP; 5 EGTA; 15 K phosphate, 2% PVP, pH 7.0. Equatorial X-ray patterns showed that myofibrils swell after detergent skinning, as also observed after mechanical skinning. This swelling could be reversed by adding high molecular weight colloids (PVP or dextran) to the extracting solution. By finding the colloid osmotic pressure needed to restore the in vivo interfilament spacing (3% PVP, 4 X 10(4) mol wt) the swelling pressure was estimated as 35 Torr in a standard KCl-based relaxing solution. The swelling pressure and the extent of swelling were less than acetate replaced chloride as the major anion. Detergent-skinned muscle lost the constant-volume relation between sarcomere length and lattice spacing seen in intact muscle. Changes in A band spacing were paralleled by changes in I and band-Z line spacing at a constant sarcomere length. After detergent skinning, I1,0 rose while I1,1 fell, a change in the relaxing direction. Since raising the calcium ion concentrations from pCa 9 to PCa 6.7 was without effect on equatorial or axial X-ray patterns, we concluded that these intensity changes were not due to calcium-dependent cross-bridge movement but rather to disordering of thin filaments in the A band.

Molecular Modeling of Averaged Rigor Crossbridges from Tomograms of Insect Flight Muscle

Electron tomography, correspondence analysis, molecular model building, and real-space refinement provide detailed 3-D structures for in situ myosin crossbridges in the nucleotide-free state (rigor), thought to represent the end of the power stroke. Unaveraged tomograms from a 25-nm longitudinal section of insect flight muscle preserved native structural variation. Recurring crossbridge motifs that repeat every 38.7 nm along the actin filament were extracted from the tomogram and classified by correspondence analysis into 25 class averages, which improved the signal to noise ratio. Models based on the atomic structures of actin and of myosin subfragment 1 were rebuilt to fit 11 class averages. A real-space refinement procedure was applied to quantitatively fit the reconstructions and to minimize steric clashes between domains introduced during the fitting. These combined procedures show that no single myosin head structure can fit all the in situ crossbridges. The validity of the approach is supported by agreement of these atomic models with fluorescent probe data from vertebrate muscle as well as with data from regulatory light chain crosslinking between heads of smooth muscle heavy meromyosin when bound to actin.

Myosin Head Configuration in Relaxed Insect Flight Muscle: X-Ray Modeled Resting Cross-Bridges in a Pre-Powerstroke State Are Poised for Actin Binding

Low-angle x-ray diffraction patterns from relaxed insect flight muscle recorded on the BioCAT beamline at the Argonne APS have been modeled to 6.5 nm resolution (R-factor 9.7%, 65 reflections) using the known myosin head atomic coordinates, a hinge between the motor (catalytic) domain and the light chain-binding (neck) region (lever arm), together with a simulated annealing procedure. The best head conformation angles around the hinge gave a head shape that was close to that typical of relaxed M*ADP*Pi heads, a head shape never before demonstrated in intact muscle. The best packing constrained the eight heads per crown within a compact crown shelf projecting at approximately 90 degrees to the filament axis. The two heads of each myosin molecule assume nonequivalent positions, one head projecting outward while the other curves round the thick filament surface to nose against the proximal neck of the projecting head of the neighboring molecule. The projecting heads immediately suggest a possible cross-bridge cycle. The relaxed projecting head, oriented almost as needed for actin attachment, will attach, then release Pi followed by ADP, as the lever arm with a purely axial change in tilt drives approximately 10 nm of actin filament sliding on the way to the nucleotide-free limit of its working stroke. The overall arrangement appears well designed to support precision cycling for the myogenic oscillatory mode of contraction with its enhanced stretch-activation response used in flight by insects equipped with asynchronous fibrillar flight muscles.

Studies on α-actinin-like proteins liberated during trypsin digestion of α-actinin and of myofibrils

Abstract When the Z-band region of isolated myofibrils is digested away by trypsin, a protein with α-actinin activity becomes solubilized (Z-protein). Prolonged digestion of α-actinin itself with trypsin leads to the appearance of a separate protein fraction with most of the activity (A-protein). Both A- and Z-protein are more active than α-actinin in various tests: the two proteins are very similar. During the digestion of myofibrils, specific changes visible in the electron microscope occur in the Z-band region. The results are compatible with the hypothesis that actinin is a constituent of the Z-band and of adjacent regions of the I-filaments of the myofibril.

X-ray diffraction evidence for myosin-troponin connections and tropomyosin movement during stretch activation of insect flight muscle

Stretch activation is important in the mechanical properties of vertebrate cardiac muscle and essential to the flight muscles of most insects. Despite decades of investigation, the underlying molecular mechanism of stretch activation is unknown. We investigated the role of recently observed connections between myosin and troponin, called “troponin bridges,” by analyzing real-time X-ray diffraction “movies” from sinusoidally stretch-activated Lethocerus muscles. Observed changes in X-ray reflections arising from myosin heads, actin filaments, troponin, and tropomyosin were consistent with the hypothesis that troponin bridges are the key agent of mechanical signal transduction. The time-resolved sequence of molecular changes suggests a mechanism for stretch activation, in which troponin bridges mechanically tug tropomyosin aside to relieve tropomyosin’s steric blocking of myosin–actin binding. This enables subsequent force production, with cross-bridge targeting further enhanced by stretch-induced lattice compression and thick-filament twisting. Similar linkages may operate in other muscle systems, such as mammalian cardiac muscle, where stretch activation is thought to aid in cardiac ejection.

X-Ray Diffraction Indicates That Active Cross-Bridges Bind to Actin Target Zones in Insect Flight Muscle

We report the first time-resolved study of the two-dimensional x-ray diffraction pattern during active contraction in insect flight muscle (IFM). Activation of demembranated Lethocerus IFM was triggered by 1.5-2.5% step stretches (risetime 10 ms; held for 1.5 s) giving delayed active tension that peaked at 100-200 ms. Bundles of 8-12 fibers were stretch-activated on SRS synchrotron x-ray beamline 16.1, and time-resolved changes in diffraction were monitored with a SRS 2-D multiwire detector. As active tension rose, the 14.5- and 7.2-nm meridionals fell, the first row line dropped at the 38.7 nm layer line while gaining a new peak at 19.3 nm, and three outer peaks on the 38.7-nm layer line rose. The first row line changes suggest restricted binding of active myosin heads to the helically preferred region in each actin target zone, where, in rigor, two-headed lead bridges bind, midway between troponin bulges that repeat every 38.7 nm. Halving this troponin repeat by binding of single active heads explains the intensity rise at 19.3 nm being coupled to a loss at 38.7 nm. The meridional changes signal movement of at least 30% of all myosin heads away from their axially ordered positions on the myosin helix. The 38.7- and 19.3-nm layer line changes signal stereoselective attachment of 7-23% of the myosin heads to the actin helix, although with too little ordering at 6-nm resolution to affect the 5.9-nm actin layer line. We conclude that stretch-activated tension of IFM is produced by cross-bridges that bind to rigors lead-bridge target zones, comprising < or = 1/3 of the 75-80% that attach in rigor.

Co-ordinated electron microscopy and X-ray studies of glycerinated insect flight muscle. I. X-ray diffraction monitoring during preparation for electron microscopy of muscle fibres fixed in rigor, in ATP and in AMPPNP

SummarySynchrotron radiation was used for low-angle X-ray diffraction to monitor structural changes produced in insect flight muscle during fixation, dehydration and embedding for electron microscopy of thin sections. Fibre bundles were fixed by cold glutaraldehyde in one of three states, namely rigor, ATP or AMPPNP, followed by additional cross-linking treatment. No heavy metals were used before embedding. During fixation-embedding, all specimens lost the continuous actin layer lines of spacing 11–5 nm, shrank 18–21% in lattice spacing, shrank 0.5–2.5% in axial spacings and showed equatorial intensity changes which were similar for all three states, while the well-sampled inner layer lines (39–13 nm) were preserved with different fidelity in each state, highest for rigor and lowest for ATP. In different AMPPNP bundles, these layer lines indicated different degrees of unexplained shift (from slight to total) towards the structure of muscle fixed in ATP. Fixation in ATP caused obvious gain of intensity on 39, 19 and 13 nm layer lines, which can be interpreted as trapping of myosin crossbridge attachments to actin; this artifact was unchanged by seven variations in fixation conditions. Fixation in rigor gave no indication of crossbridge detachment nor of the presence or alteration of any significant population of non-bridging myosin heads. X-ray monitoring allowed selection of best-preserved samples for subsequent electron microscopy. The rapid pattern-recording possible with synchrotron X-ray intensity allowed us to complete and compare experiments with many fibre bundles from a single glycerinatedLethocerus muscle.

Co-ordinated electron microscopy and X-ray studies of glycerinated insect flight muscle. II. Electron microscopy and image reconstruction of muscle fibres fixed in rigor, in ATP and in AMPPNP.

SummaryThis paper presents electron microscopy, supported by optical diffraction and filtering of images from 100 nm and 25 nm sections, to complement the companion report of X-ray diffraction monitoring (immediately preceding this article) performed on the same insect flight muscle specimens during fixation, dehydration and embedding. GlycerinatedLethocerus fibre bundles initially fixed in rigor, in ATP relaxing buffer, or in 1mM AMPPNP at 2° C, gave thin-section images from each state whose optical transforms match the distinctive X-ray diffraction patterns from the embedded samples. For rigor and relaxed states, this extends and confirms a long-known correlation between X-ray patterns and EM image regularities. For the AMPPNP state, such correlation is here fully developed for the first time, and involves a new and distinctive EM image pattern of the crossbridge array, clearly different from a previously reported structure in AMPPNP-treated muscles that appears identical to fixed relaxed muscle. We found this latter artifact of ‘AMPPNP-relaxed structure’ in many fibres from our best AMPPNP specimen, but could identify other fibres which retained the distinctive AMPPNP structure, known to be dominant in this specimen from the X-ray pattern. The true AMPPNP structure shows features of both the ATP-relaxed and rigor crossbridge patterns, not as separate patches, but hybridized uniformly along each filament and throughout each affected sarcomere and fibre. It presents a 14.5 nm repeat of striping and lateral projections along thick filaments, together with variously angled crossbridge attachments to actin that form a 38.7 nm repeat of diffuse chevrons or deltoids replacing the more clearly delinated rigor double chevrons. The associated optical transform has the typical AMPPNP features, that is, it has in common with rigor a strong 19.3 nm layer line and strong second to fourth row line sampling on the 38.7 nm layer line, it has in common with relaxed patterns a strong 14.5 nm meridional and layer line, but it uniquely shows no intensity at the first row line on the 38.7 nm layer line (the 10.3 X-ray reflection), where rigor and relaxed transforms always show high intensity. The processing artifacts which intensify the 10.3 reflection, and produce the weak 19.3 nm layer line (a gain of intensity for ATP but a loss for the AMPPNP state), throughout ATP specimens and in those analogue-treated fibres showing AMPPNP-relaxed structure, might indicate trapping and accumulation of minority populations within the native equilibrium distribution of crossbridge conformations in each nucleotide state.