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M K Reedy

Publications and source records attributed to M K Reedy.

At least 37 records · Page 2Linked to original sources
PubMed1988

Two attached non-rigor crossbridge forms in insect flight muscle.

We have performed thin-section electron microscopy on muscle fibers fixed in different mechanically monitored states, in order to identify structural changes in myosin crossbridges associated with force production and maintenance. Tension and stiffness of fibers from glycerinated Lethocerus flight muscle were monitored during a sequence of conditions using AMPPNP and then AMPPNP plus increasing concentrations of ethylene glycol, which brought fibers through a graded sequence from rigor relaxation. Two intermediate crossbridge forms distinct from the rigor or relaxed forms were observed. The first was produced by AMPPNP at 20 degrees C, which reduced isometric tension 60 to 70% below rigor level without reducing rigor stiffness. Electron microscopy of these fibers showed that, in spite of the drop in tension, no obvious change from the 45 degrees crossbridge angle characteristic of rigor occurred. However, the thick filament ends of the crossbridges were altered from their rigor positions, so that they now marked a 14.5 nm repeat, and formed four separate origins at each crossbridge level. The bridges were also less slewed and bent than rigor bridges, as seen in transverse sections. The second crossbridge form was seen in glycol-AMPPNP at 4 degrees C, just below the glycol concentration that produced mechanical relaxation. These fibers retained 90% of rigor stiffness at 40 Hz oscillation, but would not bear sustained tension. Stiffness was also high in the presence of calcium at room temperature under similar conditions. Electron microscopy showed crossbridges projecting from the thick filaments at an angle that centered around 90 degrees, rather than the 45 degree angle familiar from rigor. This coupling of relaxed appearance with persistent stiffness suggests that the 90 degree form may represent a weakly attached crossbridge state like that proposed to precede force development in current models of the crossbridge power stroke.

Adenosine Triphosphate↗
PubMed1988

Two attached non-rigor crossbridge forms.

It is possible to produce a graded progression from rigor toward relaxation using MgAMPPNP and substituting ethylene glycol for part of the solvent water. Fibers have been brought through this progression to various stages while measuring isometric force and stiffness, then fixed for thin-section electron microscopy. Distinct state-dependent crossbridge forms were observed in thin cross and longitudinal sections. When MgAMPPNP was added to rigor fibers at 23 degrees C, the tension dropped to about one-third of its original value, but crossbridge angle remained at 45 degrees. Distinct changes were seen in crossbridge shape and angle close to the thick filament, presumably in the S2 region of myosin. Adding 30% glycol in the presence of AMPPNP reduced tension to nearly zero while stiffness remained high, provided either calcium was present or the muscle was kept cold. Under these conditions, the crossbridges were oriented at approximately 90 degrees to the filaments, and in cross-section appeared straight and joined the thick filament at separate azimuths. Raising the glycol concentration to 40% or the temperature to 23 degrees C in the absence of calcium lowered the stiffness to a value slightly above that of MgATP relaxed muscle. The 90 degrees crossbridge forms seen in stiff versus relaxed fibers were closely similar but the distribution of bridges and the optical transforms suggested more bridge attachments when stiffness was high. The 90 degrees crossbridges appear structurally distinct from the rigor form and may in the stiff fibers represent a stable but weak binding state of the actomyosin contact.

Actins↗
PubMed1987

The structure of insect flight muscle in the presence of AMPPNP.

Glycerinated insect (Lethocerus) flight muscle in the presence of the non-hydrolysable ATP-analogue AMPPNP (1 mM at 4 degrees C) has been prepared for electron microscopy using X-ray diffraction monitoring during fixation and embedding. Superior preservation of the original structure has been achieved through use of a fixative which included tannic acid and excess Mg2+. New features have been recognized in single filament layers (myac and actin) and 15 nm cross sections. As previously shown, some aspects of relaxed structure (14.5 nm shelves along thick filaments) and of rigor (38.7 nm angled bridges along thin filaments) are retained in a modified form. New observations include: (1) In 15 nm cross sections that show single 14.5 nm levels: (a) The flared X structure characteristic of rigor is replaced by a straight-X figure in which the crossbridge density is aligned along the myosin-actin plane, rather than skewed across it as in rigor. (b) In AMPPNP, each crossbridge appears to have a separate origin from the thick filament, rather than bifurcating two from one stem as in the flared X of rigor. The separation of crossbridge origins is also evidenced by the loss of 'ladder rungs' in actin layers. (2) A halving (19.3 nm) of the 38.7 nm axial repeat along actin, rather than a thirding (12.9 nm) as in rigor, indicates redistribution of bridge attachments in cold AMPPNP. (3) In AMPPNP, the 14.5 nm shelves of density around the thick filament shaft are thicker but extend to smaller radius than similar shelves in ATP-relaxed muscle. This is shown by a lack of 14.5 nm periodicity and diffraction in actin layers of AMPPNP, in contrast to ATP-relaxed actin layers, in which the 14.5 nm period is present. Our results suggest that attached crossbridges are modified by AMPPNP and that ordered features of the analogue state are not accounted for solely by detached myosin heads or by a mixture of relaxed and rigor crossbridges. A two-domain model for the crossbridge is proposed. Domain 1 binds to the thin filament, and while bound, maintains a constant stereospecificity to actin at low resolution, independent of the type or presence of nucleotide at the myosin active site. Domain 2 is proximal to the thick filament and can exist in two characteristic states. In the absence of nucleotide (rigor), Domain 2 adopts a variable relationship to the thick filament, to accommodate the actin-bound end of the bridge.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1986

Image reconstruction using electron micrographs of insect flight muscle. Use of thick transverse sections to supplement data from tilted thin longitudinal sections.

Three-dimensional reconstruction using electron micrographs of thin sections is a powerful technique for determining cross-bridge structure. Tilt restrictions in the electron microscope prevent data collection beyond tilt angles of 60 degrees, giving rise to a "missing cone" of transform data. We show here how much of this data can be obtained using micrographs of thick transverse sections, and the effect this data has on reconstructed images of the insect flight muscle MYAC layer. As a byproduct, the analysis showed that section thinning resulting from prolonged electron irradiation had occurred in the thin longitudinal section used for the previously published MYAC layer reconstruction (Taylor et al., 1984). Comparison of projection density maps calculated from the thin longitudinal section reconstruction and the thick section data show that the data within the missing cone that is not accessible by tilting sharpens the boundaries of the components, flattens the density profile across the thick filament, and enlarges the molecular envelope of the thin filament. We conclude that the reconstructed images of the MYAC layer provide a picture of the structural principles underlying the system but that transform data within the missing cone are necessary to describe accurately the envelopes and profiles of these structural elements.

Actin Cytoskeleton↗
PubMed1985

Rigor crossbridge structure in tilted single filament layers and flared-X formations from insect flight muscle.

The averaged structure of rigor crossbridges in insect flight muscle has been studied in filtered images. Their three-dimensional structure has been deduced by relating tilt views of single filament layers in 25 nm longitudinal sections (myac layers and actin layers) to the flared-X appearance in 15 nm cross-sections showing single crossbridge levels. Tilting myac or actin layers around the filament axis makes crossbridges show one of two patterns. Beadlike densities appear either singly over thin filaments ("center-beading") or doubled and flanking thin filaments ("straddle-beading"). These express two different projections from the crossbridge-actin complexes as seen end-on in flared-X formations. Tannic acid/glutaraldehyde fixation gave improved actin preservation, showing, in 15 nm cross-sections, the long-pitch helical strands as "two-dot" profiles of consistent azimuth in the gaps between double chevrons. The azimuth in the flared-X arms was then inferred from lattice relationships, since it was not seen directly. The tangential attachment of comma-shaped crossbridges to the inferred actin dyad fits the binding geometry in recent actin-subfragment 1 complex reconstructions. However, averaged crossbridge structure differs between lead and rear members of double chevrons, unlike the uniform heads on decorated actin. In filtered images of myac layers, the lead bridges are dense and steeply angled; the rear chevron is seen as a dense bead over the thin filament with faint, less angled bars extending laterally. Actin layer images also suggest that rear and lead bridges differ in angle. Left and right flared-X arms are end-on views of lead and rear chevron bridges, respectively, and differ in shape. Improved fixation with tannic acid/glutaraldehyde allows us to distinguish three crossbridge domains in flared-X arms: (1) a dense bulb-like head merged into the thin filament; (2) a dense but thinner neck tangential to actin; and (3) a faint thin stem joining the necks to myosin filaments. Shape differences in lead and rear members between the head-neck-actin complexes are indicated by the names "L sigmoid" and "R dogleg". Within crossbridges, internal angles between the head-neck axis and the head-actin-head axis differ between sigmoid and dogleg by about 30 degrees, implying a flexible junction between bridge-head and bridge-neck. Lead and rear bridges are axially at least 13 nm apart on actin; the expected 60 degrees difference in azimuth is expressed by head-neck portions, but the head-actin-head axis rotates by only 30 degrees.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1985

Binding of myosin subfragment 1 to glycerinated insect flight muscle in the rigor state.

The binding of rabbit muscle myosin subfragment 1 (S1) to glycerinated insect flight muscle fibers has been studied by low-angle x-ray diffraction, quantitative sodium dodecyl sulfate gel electrophoresis, quantitative interference microscopy, and electron microscopy. Changes induced in the rigor x-ray diffraction pattern are consistent with the idea that vacant myosin-binding sites on thin filaments are filled by exogenous S1. Electron microscopy indicates that S1 permeates and labels fibers and fibrils completely. Electron micrographs also show that cross-bridges are not displaced by exogenous S1 under the conditions used, and this is supported by the unchanged mechanical stiffness of the S1-labeled fibers. The amount of bound S1, as measured by gel electrophoresis and interference microscopy, together with the magnitude of the intensity changes in the x-ray diffraction pattern, is consistent with a thick filament structure that contains four molecules of endogenous myosin per 14.5 nm of its length, but does not agree well with earlier estimates of six myosins per crown. Lack of information on possible inhibition of S1-binding by factors other than the presence of cross-bridges, e.g., troponin, render uncertain calculations of the number of attached cross-bridges in the rigor state. However, it appears that at least 75% of the endogenous myosin heads are attached. Occupancy of binding sites on thin filaments after incubation with S1 is high, probably greater than 85%, so that x-ray scattering from those parts of the structure that adhere to the symmetry of the thin filaments can be treated as diffraction from S1-decorated thin filaments. In addition, we show in thin flared X cross sections that exo-S1 heads bind to actin with the geometry described in decorated actin by Taylor, K.A., and L.A. Amos.

Actins↗
PubMed1984

A-band mass exceeds mass of its filament components by 30-45%.

We have measured filament lattice spacing in fibrils using X-ray diffraction, and find that STEM-determined mass/length values reported for myofilaments should give 13% w/v as the filament protein concentration in the lattice of the AO-band (filament overlap zone) of both insect flight muscle (IFM) and vertebrate skeletal muscle ( VSkM ). This is well below the actual mass concentration of AO-bands as measured by immersion refractometry of detergent-washed rigor myofibrils under the phase microscope. This technique identifies an immersion fluid of suitable refractive index (RI) for matching out all image contrast between background and the selected cross-band. We used RI fluids in which the effective RI matching component is a large-particle solute (Percoll or hemocyanin) excluded by the filament lattice. The measured RI indicates that protein concentration in AO-bands is 16-17% in Lethocerus and other IFM fibrils including CAF-digested fibrils, and is 18-19% in rabbit VSkM fibrils. On IFM fibrils we also measured absolute buoyant density in Percoll as greater than or equal to 1.042; this supports the value for mass concentration as determined by RI. The mass discrepancy between fibrils and filaments does not seem to arise from faults in the methods used. We therefore accept the STEM-determined mass/length values for thick filaments which indicate 4 myosins/crown in IFM and 3 in VSkM , and we believe there is considerable extra nonfilament material (concentration: 30-50 mg/ml) between the filaments in fibrils. In stretched VSkM , it is the H-bands, not the I-bands, which have an excess over filament mass content. The extra mass has not been identified.

Animals↗
PubMed1984

Geometrical constraints affecting crossbridge formation in insect flight muscle.

Computer-modelling studies have explored how rigor crossbridge interactions in insect flight muscle are affected by using a four-stranded helical thick filament and by restricting each myosin to forming one crossbridge with only one actin filament. Crossbridges searching over an axial range of +/- 7.2 nm, and within an azimuthal range around actin of +/- 45 degrees, can simulate the actin-labelling patterns observed in thin electron microscope sections well. However, the number of crossbridges attached between any myosin filament and an adjacent actin filament depends on their relative axial and azimuthal positions, and can vary by a factor of two. The relative position that maximized the number of attached bridges also produced the best modelling of the 'double chevron' appearance of two crossbridge pairs attaching within target zones every 38.6 nm, as seen in thin longitudinal sections, and the 'flared X' of crossbridges extending to four out of six surrounding actins at each crossbridge level seen in thin cross-sections. Micrographs show that excellent lattice register of rigor crossbridges in longitudinal sections does not depend on lateral register of thick or thin filament ends. Our modelling suggests how the crossbridge lattice may be generated by filaments becoming mutually annealed to the axial and azimuthal positions at which most crossbridges can attach, at which time the actin filaments are arranged at the diad positions on the P64 crystalline lattice. When the actin filaments are so oriented, in a P64 lattice, two crossbridges on adjacent actin filaments will slew toward the same point on the myosin filament, producing the flared X appearance of origin from a common stem and a single myosin, even if they originate from distinct points and separate molecules.

Actins↗
PubMed1983

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.

Synchrotron 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 glycerinated Lethocerus muscle.

Adenosine Triphosphate↗
PubMed1983

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.

This 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. Glycerinated Lethocerus fibre bundles initially fixed in rigor, in ATP relaxing buffer, or in 1 mM AMPPNP at 2 degrees 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.

Adenosine Triphosphate↗
PubMed1980

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.

Animals↗
PubMed1978

Modeling rigor cross-bridge patterns in muscle I. Initial studies of the rigor lattice of insect flight muscle.

We have undertaken some computer modeling studies of the cross-bridge observed by Reedy in insect flight muscle so that we investigate the geometric parameters that influence the attachment patterns of cross-bridges to actin filaments. We find that the appearance of double chevrons along an actin filament indicates that the cross-bridges are able to reach 10--14 nm axially, and about 90 degrees around the actin filament. Between three and five actin monomers are therefore available along each turn of one strand of actin helix for labeling by cross-bridges from an adjacent myosin filament. Reedy's flared X of four bridges, which appears rotated 60 degrees at successive levels on the thick filament, depends on the orientation of the actin filaments in the whole lattice as well as on the range of movement in each cross-bridge. Fairly accurate chevrons and flared X groupings can be modeled with a six-stranded myosin surface lattice. The 116-nm long repeat appears in our models as "beating" of the 14.5-nm myosin repeat and the 38.5-nm actin period. Fourier transforms of the labeled actin filaments indicate that the cross-bridges attach to each actin filament on average of 14.5 nm apart. The transform is sensitive to changes in the ease with which the cross-bridge can be distorted in different directions.

Actins↗