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H E Huxley

Publications and source records attributed to H E Huxley.

At least 19 recordsLinked to original sources
PubMed1994

Ultrastructure of skeletal muscle fibers studied by a plunge quick freezing method: myofilament lengths.

We have set up a system to rapidly freeze muscle fibers during contraction to investigate by electron microscopy the ultrastructure of active muscles. Glycerinated fiber bundles of rabbit psoas muscles were frozen in conditions of rigor, relaxation, isometric contraction, and active shortening. Freezing was carried out by plunging the bundles into liquid ethane. The frozen bundles were then freeze-substituted, plastic-embedded, and sectioned for electron microscopic observation. X-ray diffraction patterns of the embedded bundles and optical diffraction patterns of the micrographs resemble the x-ray diffraction patterns of unfixed muscles, showing the ability of the method to preserve the muscle ultrastructure. In the optical diffraction patterns layer lines up to 1/5.9 nm-1 were observed. Using this method we have investigated the myofilament lengths and concluded that there are no major changes in length in either the actin or the myosin filaments under any of the conditions explored.

Actin Cytoskeleton↗
PubMed1994

X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle.

We have used a small angle scattering system assembled on the high flux multipole wiggler beam line at CHESS (Cornell) to make very accurate spacing measurements of certain meridional and layer-line reflections from contracting muscles. During isometric contraction, the actin 27.3 A reflection increases in spacing from its resting value by approximately 0.3%, and other actin reflections, including the 59 and 51 A off-meridional reflections, show corresponding changes in spacing. When tension is augmented or diminished by applying moderate speed length changes to a contracting muscle, changes in spacing in the range of 0.19-0.24% (when scaled to full isometric tension) can be seen. The larger difference between the resting and isometric spacings suggests either nonlinearity at low tension levels or the presence of a component related to activation itself. Myosin filaments also show similar increases in axial period during slow stretch, in addition to the well known larger change associated with activation. An actin spacing change of 0.25-0.3% can also be measured during a 2 ms time frame immediately after a quick release, showing that the elastic behavior is rapid. These observations of filament extensions totaling 2-3 nm per half-sarcomere may necessitate some significant revision of the interpretation of a number of mechanical experiments in muscle, in which it has usually been assumed that virtually all of the elasticity resides in the cross-bridges.

Actins↗
PubMed1990

A stopped-flow/rapid-freezing machine with millisecond time resolution to prepare intermediates in biochemical reactions for electron microscopy.

We have developed an instrument capable of freezing transient intermediates in rapid biochemical reactions for subsequent freeze-fracturing, replication, and viewing by transmission electron microscopy. The machine combines a rapid mixing unit similar to one widely used in chemical kinetics (Johnson, 1986) with a propane jet freezing unit previously used to prepare static samples for freeze-fracturing (Gilkey and Staehelin, 1986). The key element in the system is a unique thin-walled flow cell of copper that allows for injection and aging of the sample, followed by rapid freezing. During freeze-fracturing, a tangential cut is made along the wall of the flow cell to expose the sample for etching and replication. The dead time required for mixing and injection of the reactants into the flow cell is less than 5 ms. Electronic controls allow one to specify, on a millisecond time scale, any time above 5 ms between initiation of the reaction and quenching by rapid freezing.

Actins↗
PubMed1988

X-ray diffraction studies on muscle during rapid shortening and their implications concerning crossbridge behaviour.

In isometric contraction, a high proportion of crossbridges are always in the attached state and crossbridge cycling is slow. During shortening, crossbridges must be entering the detached state at a higher rate, as they come to the end of their working strokes. The size of the population of detached crossbridges will then depend on the re-attachment rate and it is therefore of some interest to find out whether a significant detached population can be detected. Observations on the equatorial X-ray diffraction pattern indicate that this is the case at higher speeds of shortening, for example at a speed where the detachment rate must be of the order of 200 per second. In a muscle under these conditions, the 59 A and 51 A actin layer line reflections decrease in intensity compared to their values during isometric contraction. This decrease does not appear to be associated with a change in structure of the actin-troponin-tropomyosin complex, since the second actin layer-line reflection remains virtually unchanged in intensity. Thus the change is likely to arise from either a different total number of attached crossbridges, or a different number of attached crossbridges in the tension generating state. The result provides some further evidence for specific helical labelling of the actin structure by crossbridges during contraction, as do some recent electronmicroscope studies of rapidly frozen contracting muscle.

Actins↗
PubMed1987

X-ray diffraction studies of the structural state of crossbridges in skinned frog sartorius muscle at low ionic strength.

Low-angle X-ray diffraction diagrams were obtained from chemically skinned frog sartorius muscles under low ionic strength relaxing conditions. Experiments on single muscle fibres from rabbit muscle and on muscle proteins in solution have suggested the presence of a 'low ionic strength attached state' of the myosin crossbridges to actin, in which the overall ATP splitting and force-generating cycle is still blocked. This opened up the possibility that structural information about one of the intermediate states in the crossbridge cycle might be obtained under these conditions. Using synchrotron radiation as a high intensity X-ray source we were able to record the appropriate diffraction diagrams with short exposure times and were able to compare the same muscles at normal and at low ionic strength. Changes in the intensities of the equatorial reflections an increase in the 143 A meridional intensity can be interpreted in a similar way. However, these attached bridges do not give rise to changes in the actin-based layer line reflections, nor is their presence associated with a weakening of the myosin layer line pattern. These results provide further evidence for the existence of bound states of crossbridges, in which their orientation relative to actin is not sharply defined.

Actins↗
PubMed1986

Structural changes during activation of frog muscle studied by time-resolved X-ray diffraction.

The pattern given by contracting frog muscle can be followed with high time resolution using synchrotron radiation as a high-intensity X-ray source. We have studied the behaviour of the second actin layer-line (axial spacing of approximately 179 A) at an off-meridional spacing of approximately 0.023 A-1, a region of the diagram that is sensitive to the position of tropomyosin in the thin filaments. In confirmation of earlier work, we find that there is a substantial increase in the intensity of this part of the pattern during contraction. We find that the reflection reaches half its final intensity about 17 milliseconds after the stimulus at 6 degrees C. The changes in the equatorial reflections, which arise from movement of crossbridges towards the thin filaments, occur with a delay of about 12 to 17 milliseconds relative to this change in the actin pattern. In over-stretched muscle, where thick and thin filaments no longer overlap, the changes in the actin second layer-line still take place upon stimulation with a time course and intensity similar to that observed at full overlap. This indicates that tropomyosin movement, in response to calcium binding to troponin, is the first structural step in muscular contraction, and is the prerequisite for myosin binding. A change in intensity similar to that found in contracting muscle is seen in rigor, where tropomyosin is probably locked in the active position. During relaxation the earlier stages in the decrease in intensity of the second actin layer-line take place significantly sooner after the last stimulus than tension decay. In over-stretched muscles the intensity decay is appreciably faster than in the same muscles at rest length, where attached crossbridges may interfere with the return of tropomyosin to its resting position.

Actins↗
PubMed1985

Crossbridge behaviour during muscle contraction.

A number of recent observations by probe and X-ray methods on the behaviour of crossbridges during contraction is considered in relation to the energetics of the process. It is shown that a self-consistent picture of the crossbridge cycle, compatible with these observations and involving strongly and weakly attached crossbridges, can be obtained providing that the tension-generating part of the crossbridge stroke is only about 40 A i.e. about one-third of the usually accepted value. The myosin head subunits in the tension-generating bridges could have a configuration close to that of rigor. A mechanism is suggested whereby rapid tension recovery after quick releases up to 120 A could still be produced by such a system.

Actins↗
PubMed1985

The crossbridge mechanism of muscular contraction and its implications.

The basic features of the sliding-filament crossbridge mechanism are reviewed briefly, and some recent objections involving supposed changes in A-filament lengths are discussed. X-ray diffraction studies on live muscles show no sign of a decrease in axial spacing during contraction, and it is unlikely that a stepwise shortening or depolymerization of A-filaments would provide a plausible contraction mechanism. Thus electron microscope observations which occasionally are reported to show such length changes probably arise from experimental artefact, of which there are many sources. The factors which govern tension and speed in muscle contraction are described. Since all vertebrate striated muscles which have been studied have A-bands of at least approximately the same length, they are likely to have rather similar maximum isometric tensions. The design probably matches this tension to the strength of the filaments themselves. The large variations in shortening speeds between different muscles and different animals arise because of corresponding variations in the rates of particular steps in the crossbridge cycle and in the rate of ATP splitting by the actin-myosin complex involved. Questions concerning the nature and the speed of the activation mechanism are also discussed.

Animals↗
PubMed1984

Time-resolved X-ray diffraction studies of cross-bridge movement and their interpretation.

The purpose of these studies has been to obtain information about the structural behaviour of the cross-bridges during contraction. Since there are so few reflections still present in the part of the X-ray diagram produced by cross-bridges in a contracting muscle they cannot on their own give a detailed picture. However, they can give information of a more general nature - much in the same way as measurements of tension may do, for example - and the patterns can also tell us what structural regularities are no longer present during contraction. The experiments which I will describe have been carried out in nearly all cases on frog sartorius muscles using synchrotron radiation as an intense X-ray source. The necessary facilities were provided by the European Molecular Biology Laboratory Outstation on the storage ring DORIS at DESY Hamburg. The results to which I will refer have in many cases already been described in papers published or in press (Huxley, 1979; Huxley, Faruqi , Bordas , Koch and Milch , 1980; Huxley, Simmons, Faruqi , Kress , Bordas and Koch, 1981; Huxley, Faruqi , Kress , Bordas and Koch, 1982), to which reference may also be made for experimental details.

Animals↗
PubMed1984

The effect of the ATP analogue AMPPNP on the structure of crossbridges in vertebrate skeletal muscles: X-ray diffraction and mechanical studies.

Adenylylimidodiphosphate (AMPPNP), a nonhydrolysable analogue of ATP, has been used to arrest the crossbridge cycle of muscular contraction in one of its hypothetical intermediate states. Whole frog sartorius muscles were chemically demembranated, and it was found possible to cycle such skinned muscles reversibly between the relaxed and rigor states. The effect of binding of AMPPNP on the structure and spatial arrangement of the crossbridges of such muscles was studied using low-angle X-ray diffraction, with simultaneous recording of the mechanical effects, starting from the rigor state. Saturating concentrations of MgAMPPNP produce a characteristic decrease of about 50% in the original rigor isometric tension with a concomitant increase in muscle length by 0.13%. The equatorial X-ray diffraction pattern is modified in the following way: the lattice dimensions and the intensity of the (10) equatorial reflection do not change, while the intensity of the (11) equatorial reflection increases slightly. These observations of very small equatorial changes could be explained by assuming that in these muscles (as distinct from others such as rabbit psoas) the analogue does not produce a significant degree of detachment of crossbridges; that is, there are only AMPPNP-modified attached ones. The changes in the meridional X-ray diffraction pattern are more pronounced: the meridional reflection at 14.5 nm decreases in intensity, and the meridional reflection at 7.2 nm increases considerably: the intensity of all the actin-based off-meridional layer-lines decreases. There are no signs of the characteristic relaxed layer-lines, and the changes in the layer-line intensities are probably due to there being a single population of AMPPNP-modified attached crossbridges, rather than a mixture of attached and detached crossbridges. Thus the AMPPNP X-ray pattern, both equatorially and meridionally, is somewhat similar to the rigor one, indicating that most of the crossbridges remain attached. On the other hand, the fact that there are some changes in the layer-line intensities of the AMPPNP frog pattern, without the appearance of any signs of a relaxed equatorial pattern, indicates that the attached crossbridges are in a structural state that is different from rigor, one is not seeing, apparently, simply a mixture of rigor and relaxed states. Our tentative interpretation of this result is that there may be a structural change in the crossbridge near to the junction with S2, with less significant changes occurring in the parts of the crossbridge close to actin.

Adenosine Triphosphate↗
PubMed1983

Changes in the X-ray reflections from contracting muscle during rapid mechanical transients and their structural implications.

During normal contractions of vertebrate striated muscle, it is believed that the cross-bridges which produce the sliding force undergo asynchronous cyclical changes in their structure. Thus, an X-ray diffraction diagram from a muscle under these conditions will give structural information averaged over the whole range of cross-bridge states. Such diagrams show characteristic and informative differences from those given by relaxed muscle, but can give little information about changes in the configuration of the cross-bridges at different stages of their working stroke. However, it is possible to effect a partial synchronization of these changes by applying very rapid changes in length, completed in less than one millisecond to an otherwise isometrically contracting muscle. If the amplitude of these length changes is comparable to the length of the cross-bridge stroke (say 100 A per half-sarcomere), then it should bring about a transient but significant redistribution of cross-bridge states, which would show up in the X-ray diagram. We have made use of synchrotron radiation as a high intensity X-ray source in order to record such patterns with the necessary time resolution (1 ms or less) and have found major changes in the intensity of the 143 A meridional reflection accompanying the rapid length changes of the muscle. These changes appear to arise from specific configurational changes in the cross-bridges during the working stroke. A model is suggested in which the 143 A meridional intensity in a contracting muscle arises mainly from attached cross-bridges and is generated by the part of the myosin head near the S1-S2 junction. During normal contraction, cross-bridges go through their structural cycle asynchronously with each other, since they start at different times, but if the S2 changes in length rather little, then the configurational changes in the myosin heads are synchronized with the actin filament movement in such a way that the S1-S2 junction remains relatively fixed in its axial position. In a quick release, it is suggested that bringing many S1 heads simultaneously to the end of their working strokes on actin disrupts the 143 A axial repeat of their distal ends near S2, and brings about the large decrease of the 143 A meridional reflection. This model therefore involves a large change in the position of part of the myosin head structure relative to actin during the working stroke of the cross-bridge.

Animals↗
PubMed1981

Millisecond time-resolved changes in x-ray reflections from contracting muscle during rapid mechanical transients, recorded using synchrotron radiation.

Low-angle x-ray diffraction diagrams have been recorded from frog sartorius muscles by using synchrotron radiation as a high-intensity x-ray source. This has enabled changes in some of the principal reflections of interest to be followed with a time resolution of 1 ms, during small but very rapid length changes imposed on a contracting muscle. The 143-A meridional reflection, which is believed to arise from a repeating pattern of myosin cross-bridges along the length of the muscle, shows large changes in intensity in these circumstances. During both rapid releases and rapid stretches, by amounts that produce a translation of actin and myosin filaments past each other by about 100 A and that are completed in about a millisecond (i.e., before significant cross-bridge detachment would be expected), an almost synchronous decrease in 143-A intensity occurs, by 50% or more. This is followed, in the case of quick releases, by a rapid partial recovery of intensity lasting 5--6 ms (which may represent cross-bridge release and reattachment) and then by a more gradual return to the normal isometric value. Quick stretches show only the slower return of intensity. Immediately after the length change, the initial drop in 143-A intensity can be reversed if the release (or stretch) is reversed. These changes provide evidence of a more direct kind than has hitherto been available that the active sliding of actin filaments past myosin filaments during contraction is produced by longitudinal movement of attached cross-bridges.

Actins↗
PubMed1980

The use of synchrotron radiation in time-resolved X-ray diffraction studies of myosin layer-line reflections during muscle contraction.

Experiments on striated muscle have been carried out at the EMBL Outstation at DESY, Hamburg, using the electron-positron storage ring DORIS as a high-intensity X-ray source. The low-angle reflections from the myosin cross-bridges could be recorded more than 1,000 times more rapidly than with the best conventional X-ray sources, and could be followed during contraction with a time resolution of 10 ms.

Animals↗