Applications of multiwire proportional chambers to time resolved X-ray studies on muscle

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Nuclear Instruments and Methods in Physics Research A269 (1988) 362-368 North-Holland, Amsterdam

APPLICATIONS OF MULTIWIRE PROPORTIONAL CHAMBERS TO TIME RESOLVED X-RAY STUDIES ON MUSCLE A.R . FARUQI

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England

Multiwire proportional chambers have been used for making time resolved X-ray diffraction measurements from skeletal muscle. The most useful properties are low noise, high efficiency and fast readout. A number of applications from measurements on skeletal muscle are described. 1. Introduction

tile proteins which form the major part of the "ordered" structure in skeletal muscle . The proteins are arranged on interdigitating filaments: myosin is the major part of the thick filament and actin, along with regulatory proteins, is located in the thin filaments shown schematically in fig. 1. It is believed that during contraction the periodic "projections" from the thick filaments, or cross-bridges, form attachments with actin on the thin filament and go through a conformational change which results in a relative sliding movement between the filaments and the consequent generation of force [1]. The myosin cross-bridges are arranged in an approximately helical manner on the thick filament which gives rise to a characteristic layer line pattern shown in fig. 2 and is discussed in greater detail in section 4.1 . As cross-bridges occur in groups separated axially by 143 Â along the thick filament, there is a strong meridional reflection at 143 Â which is discussed further in section 4.2 . The actin layer lines at 59 and 51 f1 are relatively weaker than the myosin layer lines but get stronger in contracting muscle ; a difference pattern between con-

Multiwire proportional chambers (MWPC) have proved to be of great value in obtaining time resolved X-ray diffraction data from muscle which allows a dynamic physiological process to be studied at a molecular level on a millisecond time scale. The main aim of these studies is to understand the basic molecular mechanisms responsible for force generation during muscular contraction. A short discussion regarding X-ray diffraction from muscle is presented to outline the technical requirements of the detector system. The main properties of MWPCs are discussed with particular reference to muscle studies. Finally, a few results are given to illustrate the use of the detectors. 2. X-ray diffraction from muscle The X-ray pattern from resting muscle provides a wealth of structural information regarding the contracA

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Fig. 1 . Schematic diagram showing the interdigitating thick and thin filaments. The projections from the thick filaments are arranged regularly along the length of the filament in groups which occur with an axial period of 143 Â. 0168-9002/88/$03 .50 O Elsevier Science Publishers B.V . (North-Holland Physics Publishing Division)

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A.R . Faruqi / Time resolved X-ray studies on muscle

Fig. 3. A difference pattern between contracting and resting muscle showing the actin layer lines.

lution of interest is - 10 -4 [4]. Since diffraction arises only from the "ordered" part of a structure, one nor-

mally strips the "background", except in some special

cases, where this "background" has also been used for

PSD SET FOR LAYER RECORDNG Fig. 2. The X-ray pattern from resting muscle is dominated by diffraction from the cross-bridges arranged with a pseudohelical repeat of 429 A along the thick filament. The first layer line, at 429 A, is the strongest in intensity and its change during contraction is shown in fig. 4.

obtaining structural information [5]. A typical HuxleyHolmes Camera, with monochromator optics on a laboratory source, provides about 2 x 10 8 photons/s on the muscle specimen resulting in - 2 x 10 4 photons/s in the scattered pattern. Multiwire detectors required to

operate at these rates do not pose a serious problem.

However, a synchrotron camera, such as the small angle camera at EMBL, Hamburg [6], provides

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tracting and resting muscle, which shows the 59 and 51 fA layer lines, is shown in fig. 3 [2]. The second actin

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layer line, with an axial spacing of - 180 A is absent in resting muscle and only appears during contraction [3].

As seen in projection, the filaments are arranged on a hexagonal lattice with the thin filaments occupying the trigonal positions; this gives rise to strong diffraction in the equatorial plane as shown in fig. 4. The inner reflection, which can be indexed as (1, 0), arises mainly from the thick filaments, while the (1, 1) arises from both thick and thin filament contributions.

Major changes occur in the X-ray pattern as the

muscle goes through a contraction, and time resolved

X-ray diffraction aims to interpret the changes in pattern during contraction in terms of molecular changes,

which are responsible for force generation. The typical time constants are - 1 ms requiring time resolutions of

the same order to be able to follow the structural changes with adequate resolution. The total amount of scattering from the ordered and disordered part of the muscle structure within the reso-

- 10 11 pho-

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Fig. 4. Equatorial pattern from resting muscle recorded as a linear position sensitive detector [18] . The two main reflections on the equator, the (1, 1) and (1, 0) are shown with a Gaussian fitted to the peaks and a fourth order polynomial to the background .

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A.R . Faruqi / Time resolved X-ray studies on muscle

tor. The delay line based area detectors are capable of a maximum count rate of 200-300 kHz, and they can only be used by attenuating the central part of the pattern [2,7]. Linear detectors, based on parallel readout from the anode wires [8,9] can operate at very high fluxes since they consist of essentially - 100 separate proportional counters. The rather limited spatial resolution, - 1 mm, is not a problem due to the present large size of beam in the synchrotron cameras. 3. Relevance of wire chambers for muscle studies Multiwire chambers offer a number of very important properties which make them eminently suitable for recording small angle X-ray patterns and time resolved patterns from contracting muscle [10] ; we discuss various aspects of the requirements, and how wire chambers are capable of fulfilling those requirements . (a) One of the most important requirements of the detector system for time resolved studies is that system for time resolved studies is that it has to have a fairly fast readout; the detector has to image the dynamic diffraction pattern, i.e . it has to record, read out and "clear" in times significantly smaller than the required experimental time resolution . Since the readout times of MWPCs are _ 10 -e s they are suitable for dynamic imaging, provided suitable timing electronics is available. Integrating detectors with relatively slow readout are less suitable, since the typical readout times for television type detectors are tens of milliseconds for the "fast" systems and several tens of seconds for the "slow" systems; however, such detectors are useful for the "slower" type of muscle experiments, where one requires say, only 50 ms time resolution [II] . X-ray film can also be used effectively for recording static patterns or patterns with a time resolution of - 1 s (reviewed in ref. [1]) . We have not used charge coupled devices (CCDs) for recording X-ray patterns as yet, but it appears that for "slower" experiments a fast readout CCD based detector [12] could be very useful . We have also used another integrating detector, the Fuji Imaging Plate [5] for slower, i.e . 50 ms time resolution experiments; with some technical improvements to the method of recording and readout it may be possible to improve the time resolution . (b) Since we are dealing with a specimen which is highly radiation sensitive, it is essential to record the X-ray patterns in as short an exposure as possible . Information from fresh, unfatigued muscle also gives a closer representation to in vivo conditions . This requires a high efficiency and low noise readout detector, capable of recording the pattern in a short time frame, which may be as short as 0.5 ms . Even with the highest intensity sources it is not possible to obtain adequate statistics in one cycle of contraction and it is therefore

repeated 50-100 times or more, to obtain adequate statistics . The signal-to-noise ratio in MWPCs is very high, since there is virtually no noise, making them very suitable . (c) The spatial resolution in MWPCs (typically - 250 Am in a 100 mm detector for 8 keV photons) is adequate to cope with most of the sampled reflections and layer lines in muscle patterns obtained on synchrotron cameras. As most of the pattern is recorded for small angles of up to only 2 °-3 ° , the parallax problem arising from oblique incidence of the incident photons is not very serious [13] . (d) The dynamic range is limited essentially by the size of the storage memory and can be made sufficiently large to cope with the experiment . (e) The integral and differential linearity are normally about a few percent ; the latter, representing the uniformity of response, can be corrected using a uniform pattern obtained with a radioactive source . (f) Lastly, we come to a less than adequate match to requirements . As mentioned earlier, the parallel readout detector is able to cope with the counting rates in any linear slice of the X-ray pattern, but area detectors are not yet able to count at - 10 MHz, the rates in the whole pattern, but several developments may lead to working systems in the future . 4. Time resolved studies on muscle 4.1 . Myosin layer lines

The X-ray pattern from resting muscle is dominated by the layer lines generated by the helical array of myosin cross-bridges on the thick filament, which have approximately a helical repeat of 429 f1 and occur in groups which are separated axially by 143 Â . A typical diffraction pattern [4] shows the myosin layer lines which occur at 429 f1 spacing with a strongly sampled meridional reflection at 143 t1 in fig. 2. As the layer lines are produced by myosin cross-bridges it is expected that during contraction they would give information about cross-bridge behaviour. Early work (reviewed in ref. [1]) was carried out using film as recording medium and conventional X-ray generators as a source of X-rays . It was found that there was a large decrease in the layer line intensities during contraction . These experiments were difficult to perform because of the long exposure times, and it was difficult to use muscle specimens which were fresh and unfatigued over the whole experiment. Further, it was not possible to follow the detailed time course of the change in intensities because of the relatively poor time resolution of the experiments . When higher flux cameras became available using synchrotron radiation, the myosin layer lines were

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Fig. 5. Time course of the 429 f1 myosin layer line during an isometric twitch . The tension generated by the muscle is shown as stars. studied at much higher time resolution (5 ms), with a linear detector set at right angles to the layer lines [14] as indicated in fig. 2. The time course of the first five layer lines was recorded during isometric contraction at three temperatures : 2, 5 and 10 ° C. The general behaviour of the layer lines was qualitatively similar at the three temperatures though, as one would expect, peak tension was slowed down at lower temperatures. The time course of the 429 Â layer line is shown in fig. 5 for experiments at 2 ° C. The decrease in the layer line intensity, measured at half maximum change, is 10-30 ms ahead of tension while the recovery of intensity is 50-60 ms behind tension decay. It is believed that as cross-bridges make attachment with the thin filament, they lose their "helical" periodicity, which, in turn, reduces the layer line intensities. The delay between the drop in layer line intensity and generation of tension is probably due to an intermediate step in the cross-bridge cycle which occurs after crossbridge attachment and prior to tension generation . At the end of contraction the cross-bridges return to the thick filament and the 429 A layer line recovers in intensity. 4 .2 . Quick release experiments on the 143 4 meridional reflection

Although the changes in the intensity of the myosin layer lines show very clearly the disruption in the helical periodicity of the cross-bridges during contraction, more direct evidence can be obtained by measuring the

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changes in the 143 Â meridional reflection . In particular, the changes in this reflection during small but very rapid length changes imposed on an isometrically contracting muscle are especially informative [15] . A small length step, - 1% of the muscle length, corresponds to a relative movement of about 100 f1 between the filaments, similar to the cross-bridge movement during a cycle. As the cross-bridges are abruptly forced to the end of their working stroke, they stop developing tension which drops to near zero . It is interesting to look at the cross-bridges with such length steps as they may become "synchronized", which would show up clearly in the X-ray pattern. During a quick release of - 1% the intensity of the 143 A meridional reflection also drops abruptly [15], closely following the drop in tension by - 0.5 ms, as shown in fig. 6. The intensity drops to about a third of the value at the beginning of the release, but the recovery of the intensity is fairly quick, taking about 6 ms to recover to about 65% followed by a much longer term recovery . It is believed that the 143 f1 meridional reflection is generated by the myosin heads in resting muscle, and during contraction it becomes stronger in intensity even though the off-meridional layer lines become considerably weaker . This is because, although the helical order may be disrupted during cross-bridge attachment, the axial order of the myosin heads actually improves . When the muscle is suddenly shortened by - 1% of its length, the axial order is disrupted with a consequent drop in the intensity. Cross-bridges detach and reattach to start another cycle of contraction producing the 1000 900 200 700

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Fig. 6. Time course of the 143 Â meridional reflection during 1% length shortening step (quick release) imposed on an isometrically contracting muscle . Tension is shown as stars.

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A .R . Faruqi / Time resolved X-ray studies on muscle 1000 Counts

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Fig . 7 . A quick release followed by a restretch 2 ms later almost restores the 143 .4 meridional intensity to its starting value . Tension shown as stars.

tension recovery and the recovery in the 143 Â intensity . If the cross-bridges had not attached to the thin filament there would be no disruption in the cross-bridge positions during a quick-release and there would be no change in the 143 f1 intensity. This experiment thus provides direct evidence for cross-bridge attachment during contraction. Further evidence regarding the behaviour of crossbridges is provided by experiments in which the release is followed by a restretch to the original length after time intervals ranging from 1 to 20 ms [15] . If the restretch is made within 1-2 ms after the original release, the 143 ?t, intensity almost recovers to the initial value as shown in fig. 7 ; if, however, the restretch is made about 20 ms after the original release, when the intensity has recovered to a large extent, there is a further drop in the intensity, as shown in fig. 8 . If the restretch is applied very rapidly, i.e. within 1-2 ms, myosin heads do not have a chance of detaching and the restretch restores the structure and the intensity . If the myosin heads have detached, however, and reattached at new sites generating tension, then the restretch disrupts the cycle and there is a further fall in the 143 A intensity and tension. 4.3 . Equator: isometric and fast shortening experiments The relative distribution of mass between the thick and thin filaments, i.e. the projection of density orthogonal to the fibre axis (fig. 1), are reflected in the

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Fig . 8 . A quick release followed by a restretch 20 ms later produces a second drop in intensity of the 143 f1 meridional reflection which had recovered towards the starting values. Tension is shown as stars .

equatorial pattern and the details of the motion of the cross-bridges between the thick and thin filaments can also be studied by following the time course of the equatorial pattern . As cross-bridges are made during contraction, there is a transfer of mass from the thick to the thin filament, which results in a drop in intensity of the (1, 0) and an increase in intensity of the (1, 1) intensity. An example of the time course of the (1, 1) intensity during an isometric twitch is shown in fig. 9, along with the tension generated. The changes in the (1, 1) intensity are 10-15 ms ahead of the tension changes, measured at half-maximum values, which suggests again [16] that cross-bridges go through a rate limiting step after attachment and before tension is generated . The delay in tension generation is most probably not due to internal shortening of the sarcomeres ; this was established by measuring the time course of the sarcomere length during an isometric contraction by imaging the first order optical diffraction pattern generated by the sarcomeres on a linear CCD array [2] . During the early phase of contraction, the internal changes in the sarcomere length take place very much earlier than tension making it unlikely that tension generation is delayed due to internal shortening in the muscle . In another series of experiments [17] the intensity of the (1, 1) reflection was measured while the contracting muscle was shortened at different speeds to investigate the number of cross-bridges as a function of shortening speed . Preliminary results indicate that for relatively slow shortening there is no significant detachment but for faster speeds (typically 5`Y shortening in 50 ms)

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A.R . Faruqi / Time resolved X-ray studies on muscle 75eee . 71500 . 68000 . 64500 . 61000 . 57500 . 54000 . 50500 . 47000 . 43500 . 40000 .

Fig. 9. Time course of the (1, 1) equatorial reflection during an isometric twitch . The tension generated, shown as stars, lags by 15 ms behind the intensity change during the initial phase.

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Milliseconds Fig. 10. Time course of the (1, 1) equatorial reflection during a fast shortening step imposed on an isometrically contracting muscle . The muscle length was changed by 5% in 50 ms . there is a significant fraction of detached cross-bridges as seen in fig. 10 . References [1] H.E . Huxley and A.R . Faruqi, Ann. Rev. Biophys. Bioeng. 12 (1983) 381 .

[2] A.R . Faruqi, H.E. Huxley and M. Kress, Nucl . Instr. and Meth. A252 (1986) 234. [31 M. Kress, H.E. Huxley, A.R . Faruqi and J. Hendrix, J. Mol. Biol . 188 (1986) 325. [4] A.R. Faruqi and H.E . Huxley, A Review of Techniques for Time Resolved X-ray Studies on Muscle, Scattering Techniques applied to Supramolecular and Nonequi-

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[5] [6] [7]

[8] [9] [10] [ll] [12]

A.R . Faruqi / Time resolved X-ray studies on muscle librium Systems, eds. SH . Chen, B. Chu and R. Nossal (Plenum, New York, 1981) p. 201. J. Lowy, F.R. Poulsen, R.M . Simmons and A.R. Faruqi, in preparation . J. Hendrix, M.H .J . Koch and J. Bordas, J. Appl . Cryst. 12 (1979) 467. J.R. Helliwell, G. Hughes, M.M . Przybylski, P. Ridley, 1. Sumner, J.E. Bateman, J. Connolly and R. Stephenson, Nucl. Instr. and Meth . 201 (1982) 175 . A.R . Faruqi and C.C. Bond, Nucl. Instr. and Meth . 201 (1982) 125 . J. Hendrix, B.H . Fuerst, B. Hartfiel and D. Dainton, Nucl . Instr. and Meth . 201 (1982) 139. A.R . Faruqi, Nucl . Instr. and Meth . 217 (1983) 19 . H.E. Huxley, A.R . Faruqi, J. Bordas, M.H .J . Koch and J.R . Milch, Nature 284 (1980) 140. C.J.S . Damerell, Vertex Detectors, RAL 86-077, Rutherford Laboratory Reprint (1986) .

[13] A.R . Faruqi, in : The Rotation Method in Crystallography, eds. U.W . Arndt and A.J . Wonacott (North-Holland, 1977) p. 227. [14] H.E . Huxley, A.R . Faruqi, M. Kress, J. Bordas and M.H .J . Koch, J. Mol. Biol . 158 (1982) 637. [15] H.E . Huxley, R.M . Simmons, A.R . Faruqi, M. Kress, J. Bordas and M.H .J . Koch, J. Mol. Biol . 169 (1983) 469. [161 H.E. Huxley, Cross-bridge Mechanism in Muscle Contraction, eds. H. Sugi and G.H . Pollack (University of Tokyo Press, Tokyo, 1979) p. 391 . [17] H.E. Huxley, M. Kress, A.R . Faruqi and R. Simmons, Molecular Mechanisms of Muscle Contraction, eds. H. Sugi and G.H. Pollack, in press. [18] A.R. Faruqi and H.E. Huxley, J. Appl . Cryst . 11 (1978) 449.