Journal of Muscle Research and Cell Motility 11, 1-11 (1990)
Cryo-electron microscopic studies of relaxed striated muscle thick filaments J. F. M E N E T R E T * , R. R. S C H R O D E R
a n d W. H O F M A N N
Max Planck Institute for Medical Research, Department of Biophysics, Jahnstrasse 29, D-6900 Heidelberg, West Germany Received 21 February 1989 and in revised form 12 July 1989
Summary Electron micrograph images of rapidly frozen suspensions of thick filaments from four different muscle types are presented. Their optical and computer transforms are compared with images and diffraction patterns of negatively stained filaments and with X-ray data from the same muscles. We conclude that myosin head arrangement can be preserved on rapid freezing and that the images produced can be analysed by image processing techniques to give new information on thick filament structure.
Introduction The m e c h a n i s m of muscle contraction at the molecular level is still not u n d e r s t o o d . More experimental evidence is n e e d e d to u n d e r s t a n d the f u n d a m e n t a l problem of transformation of chemical e n e r g y into mechanical work. Contraction involves the splitting of ATP coupled to the interaction of thick myosincontaining and thin actin-containing filaments. Electron microscope techniques have b e e n widely used to visualize the structure and interaction of thick and thin filaments u n d e r various conditions, either by sectioning fixed, stained and e m b e d d e d tissue (Reedy et al., 1965; Huxley, 1967; Reedy et al., 1983; Bennett et al., 1986) or b y examining the structural behaviour of isolated filaments (Kensler & Levine, 1982; Knight & Trinick, 1984; Stewart et al., 1985; Clarke et al., 1986). Cyro-electron microscopy is a technique which allows the examination of biological material in its natural e n v i r o n m e n t (Adrian et al., 1984; D u b o c h e t et al., 1985; r e v i e w e d by Stewart & Virges, 1986). Using this m e t h o d , it has b e e n s h o w n that structure can be well p r e s e r v e d in frozen h y d r a t e d sections of insect muscle fibres (McDowall et al., 1984) and that thick filaments from insect flight muscle in s u s p e n s i o n retain their regular structure w h e n rapidly frozen and v i e w e d in the electron microscope u n d e r liquid nitrogen conditions (Menetret et aL, 1988). We have used cryo-electron microscopy and c o m p u t e r analysis of the electron micrograph images to s t u d y the structure of muscle thick filaments in the presence of ATP in a fully h y d r a t e d , unstained, unfixed and u n s u p p o r t e d state. Here we *To whom correspondence should be addresssed. 0142-4319/90 $03.00 + .12 9 1990 Chapman and Hall Ltd.
describe results o n thick filaments from striated muscles of Limulus (horseshoe crab), rabbit and the insects Lethocerus a n d Drosophila. A preliminary analysis of the images using c o m p u t e r processing is described.
Materials and methods
Preparation offilaments Limulus telson muscle filaments were prepared according to Kensler & Levine (1982). Only animals which appeared healthy were used. Glycerinated material was also used. Small muscle bundles were dissected and tied to plastic rods at 4~ The bundles were stirred at 4~ in a 50% glycerol/relaxing solution (5mM ATP, 70mM K acetate, 2 mM EGTA, i mM DTT, 20 mM MOPS, 10 mM Mg acetate, pli 7.0) for 2-4 days and stored at -20 ~C. Filaments from this stock were prepared using the procedures described below for Lethocerus filaments and by Menetret and coworkers (1988). Filaments obtained from stored muscles were similar in quality to those from fresh muscle. Rabbit psoas muscle was freshly dissected, washed in relaxing buffer (7mM ATP, 70mM K propionate, 5mM EGTA, 6 mM MOPS, 8 mM Mg acetate) and slowly cooled to 4~ in the same buffer. Smaller bundles were skinned for 3-4 h in 0.5% Brij/or Triton/relaxing solution at 4~ (Wray, personal communication). After washing for about 3h in relaxing solution at 4~ the bundles were left in 50% glycerol/relaxing solution for 2-20h. After extensive washing in relaxing solution, the bundles were used for filament preparation using the same brief enzymatic digestion as described for Lethocerus filaments.
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Lethocerus thoraces were dissected, washed in rigor buffer (5mM Mg acetate, 100mM K acetate, 2mM EGTA, 1mM DTT, 20mM MOPS p l i 6.8) and, after several changes of a rigor/50% glycerol buffer at 4~ stored at -20~ Best preservation of the material was in the first four months. Fibrils were prepared by standard methods (Reedy et al., 1981) and digested (c. 5 min) using elastase (SIGMA, Type 4: from Porcine Pancreas) and trypsin inhibitor in rigor at 4~ (according to Magid, personal communication). The enzymatic reaction was stopped by washing several times with buffer. Fibrils were transferred into relaxing solution (2mM ATP in rigor buffer) and thick and thin filaments were obtained after homogenization with a hand homogenizer. Thorax muscles from c. 30 Drosophila melanogaster (wild type) were dissected, washed in rigor buffer (5 mM Mg acetate, 100 mM K acetate, 0.2 mM EGTA, I mM DTT, 20 mM MOPS pli 6.8) and glycerinated in 50% glycerol/rigor solution for 15-30 min at 4~ The small bundles were washed in rigor buffer and digested for c. 5 min using elastase. After several washes with rigor buffer, the bundles were incubated in relaxing solution (2 mM ATP in rigor buffer) at 4~ and thick and thin filaments were obtained after homogenization. The filament suspension presumably contained an u n k n o w n number of filaments from muscles other than the indirect flight muscles. Before freezing, each preparation was tested for structural quality and suitable filament distribution using negative staining techniques. Preparation of the grids Grids coated with a perforated carbon film were prepared as described by Fukami and Adachi (1963). The filament suspension was applied to these grids according to the method of Adrian and colleagues (1984). Best preserved filaments were obtained when the relative humidity was above 70%. This minimized changes in the ionic strength of the buffer due to evaporation. To avoid temperature changes, the copper grid, the forceps, and the blotting paper were adjusted to the desired temperature (c. 4 ~C for
MENETRET, S C H R O D E R a n d H O F M A N N
Limulus, Lethocerus and Drosophila, c. 26~
for rabbit). Effects of additional temperature changes due to evaporation from the grid were not observed. Best results were obtained w h e n the grid was frozen immediately (K 0.5 s) after blotting.
Electron microscopy Pictures were taken on a Philips 400 T transmission microscope at 80kV with 3~m condensor aperture and 100~m objective aperture. The electron dose on the specimen was reduced to a value of c. 330 e - per nm 2 using the Philips low dose kit. Only one picture of each area was taken. The grids were viewed using a Philips or Gatan cryo~holder at liquid nitrogen temperature. Depending on the degree of defocussing (4-6 ~m underfocus) the nominal resolution is limited to 4 - 5 n m (Hanszen, 1971; Frank, 1973). Film plates (Kodak SO-163) were developed for double the recommended time in full strength developer.
Image processing Film plates were examined on an optical diffraction bench as described by Erickson and coworkers. (1978) and selected with respect to astigmatism, defocus and structural features. Images of filaments showing periodic structures corresponding to those seen by X-ray diffraction were processed further. The negatives were scanned on an Optronics P-1000 with 256 grey levels and a spot size of 25 ~m, yielding a nominal resolution of about 1.7 nm for an electron migrograph taken at a magnification of 28700 (calibrated with images of catalase crystals; Dorset & Parsons (1975)). Ail image processing was done with a new software package to be described in detail elsewhere (Schr6der et al., in preparation). Pictures of filament areas were selected and floated on background arrays having a grey level value equal to the average of the perimeter values of the filament area. The computer programmes run on a CONVEX Computer Corp. C1 computer. Images and diffraction patterns were displayed on a PCS Corp. colour graphics workstation.
Fig. 1. Gallery of relaxed Limulus telson muscle thick filaments frozen, unstained, unsupported, fully hydrated and embedded in a layer of vitreous buffer. Their corresponding optical diffraction patterns are shown below. The periodicity of the crossbridges can be best seen by sighting along the filament axis. (Scale bar = exact helical repeat, 43.8 nm). The second filament will be further analysed in Fig. 4. Optical diffraction patterns are marked with arrows pointing to the approximate position of layer-lines 1, 3 and 4 (measured 11.2 + / - 0 . 5 nm) of 4 stranded 12/1 helix, E = equator. Fig. 2. Relaxed rabbit psoas muscle thick filaments. (Scale bar = 100 nm) (a) Relaxed rabbit psoas muscle thick filaments kept at c. 26~C before freezing. The optical diffraction patterns of the filaments A and B are different although the filaments have been imaged in the same conditions. A possible explanation is that the order-disorder transition is so rapid (Rapp et al., 1989) that a partial transition occurs during cooling, leading to a distribution of different degrees of order amongst individual filaments. The optical diffraction pattern of the filament A shows the first and fifth layer lines (measured 8.8 + / - 0 . 5 nm) and a meridional reflection on the third layer line of a 3 stranded 9/1 helix; it will be further analysed in Fig. 5. The optical diffraction pattern of the filament B shows a reflection on the first layer-line. (b) Relaxed rabbit psoas muscle thick filaments kept at 4 ~C before freezing. These filaments do not show obvious order of their crossbridges, t = tip and b = bare zone of the filaments. Fig. 3. Insect flight muscle thick filaments. (Scale bar = 100 nm) (a) Lethocerus flight muscle relaxed thick filaments. Typical axial periodicity of 14.5 nm, corresponding to the so-called 'crowns' of crossbridges can be seen along the filament axis. The optical diffraction pattern shows on its meridian the first, second and third order of the axial repeat (14.5 nm). Indications of helical arrangement of the crossbridges can be seen as off-meridional reflections at a spacing of about 9 nm. (see Fig. 6 for a detailed analysis). (b) Drosophila melanogaster in relaxed state. (scale bar = 100nm) No regular crossbridge order can be detected.
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MENETRET, SCHRODER and HOFMANN
Cryo-electron microscopic studies
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6 Results and discussion
Preservation, offilaments The main purpose of this work was to establish procedures by which thick filaments of muscle can be routinely observed by rapid freezing in suspension, i.e. in conditions which depart from the native only in the lack of a lattice structure. Myosin filaments are highly ordered structures, and the state of preservation can be monitored by comparison with conventional negatively stained electron micrographs and above all by comparing optical or computer transforms with X-ray diffraction data. In order to retain the structure of the myosin filaments after preparation, and especially after freezing, it had to be considered whether the arrangement of the crossbridges in isolated thick filaments might be sensitive to the surrounding conditions (temperature, ionic strength, contact of myosin heads to surfaces such as solid supports of the grid). To avoid mechanical interference with the filaments, no supporting carbon film was used. In our experience, evaporation of water from the grid, which results in an increase of the ionic strength and a decrease in temperature, can influence the appearance of the filaments. Since the effects of evaporation are proportional to the surface to volume ratio a n excess of suspension was applied to the grid and frozen immediately after blotting. Furthermore, it often helped to work in a humid atmosphere, although this practice was not adopted routinely. In all four muscle types studied, it was possible to observe thick filaments in vitreous buffer. Filaments from Limulus telson muscle (Fig. 1), rabbit psoas muscle (Fig. 2) and Lethocerus flight muscle (Fig. 3a) show periodic organization of myosin heads, whereas filaments from Drosophila melanogaster (wild type) do not appear to do so (Fig. 3b). The thick filaments from Limulus have been most extensively studied previously by conventional methods. This muscle is a good test specimen for our work, particularly since the filaments are known to undergo structural changes w h e n the ionic strength increases (Wray et al., 1974). Negative staining studies showing preservation of order in native mammalian thick filaments have not been published, and X-ray diffraction studies suggested that the filaments are susceptible to disorder (Rome, 1972a). Patterns from living rabbit muscles (Rome, 1972b) nevertheless suggest that strong layer lines comparable to those of frog muscle can be obtained. A crucial factor may be the apparent sensitivity to temperature (Wray, 1987; Wakabayashi et al., 1988). In the present work best-ordered filaments were obtained w h e n the temperature was c. 26~ before freezing (Fig. 2a). Filaments which were
MENETRET, SCHRODER and HOFMANN at about 4~ before freezing gave no indication of ordered myosin heads (Fig. 2b). The filament from Lethocerus (Fig. 3a) shows, as previously described, clear 14.5nm periodicities (Menetret et al., 1988). The crowns of crossbridges show obvious demarcation into globular units. In addition, the optical diffraction pattern (insert Fig. 3a) of the filament contains indications of a helical ordering of its crossbridges (see Fig. 5 for more detailed analysis). In Drosophila, the filaments appeared to be well preserved with an average length of about 2.4 ~m and an overall diameter of about 30nm (Fig. 3b). However, myosin heads appeared randomly distributed without periodic structure. Electron micrographs of Drosophila filaments have not previously been published. However, Reedy and colleagues (1981) noted that filaments from another fly, Musca, also lacked longitudinal periodicity. Unless both types of filaments have been damaged or modified during isolation from the lattice, it appears that fly filaments depart from the Lethocerus stereotype.
Image analysis In the cases of Limulus, rabbit and Lethocerus thick filaments, we performed some preliminary image processing. When processing electron microscope images, the factors leading to formation of the images in the transmission electron microscope (TEM) must be considered. Vitrified specimens are weak phase objects yielding very little amplitude contrast. The contrast seen in the electron micrographs is phase contrast produced by a certain degree of defocussing. However, this also affects the transmittance of spatial frequencies in the back focal plane of the objective lens, expressed mathematically as the 'phase contrast transfer function' (PCTF; Hanszen, 1971, Frank, 1973, reviewed by Glaeser, 1982). This function has a complex form: it modulates transmittance sinusoidally as a function of spatial frequency and additionally suppresses very low and very high frequencies, depending on the degree of defocus (Erickson & King, 1971, Frank, 1973). It corresponds to the obvious decrease in background intensity at higher frequencies in the spectra presented (Figs 4b, 5b, 6b). In the present study, these effects were not corrected for, so the filtered images show projected filament structures under the influence of the PCTF. Images with an underlying periodicity were filtered in the usual way (Klug & DeRosier, 1966, reviewed by Stewart, 1988). Since images obtained by cyromicroscopy should be perfect projections of an object, muscle thick filaments should theoretically give projections of a helically ordered structure. Strong reflections in the spectra could be assigned to layer lines expected for a simple helical system. The
Cryo-electron microscopic studies
7
Fig. 4. Limulus telson muscle relaxed thick filaments. (The numbers in the Fourier power spectra correspond to possible layer lines in a diffraction pattern of a helical structure respective to the matching layer lines spacing.) (a) Scanned filament area (c. 400 nm long), corresponding to the filament in the middle of Fig. 1. (b) Computed Fourier power spectrum of the embedded filament Fig. 4a. Four off-meridional and one meridonal reflections can be seen. Those spots fit to a layer line system (4 stranded 12/1 helix) of 43.8 nm together with a 14.6 nm axial repeat which is clearly shown in the filtered spectrum (4c). E = equator. (c) Computed filtered Fourier power spectrum (Gaussian filter for corresponding layer lines). (d) Fi|tered image using the corresponding Fourier data of (4c). The arrowheads indicate the 14.6 nm repeat, arrows mark the 43.8 nm helical repeat. corresponding filter consisted of Gaussian shaped lines, i.e. ai1 data on the supposed layer lines were multiplied by a factor of 1, all other data by a factor < 1 inversely proportional to their distance from the layer line. In all cases, possible layer line systems were indexed at higher spatial frequencies and the matching to reflections at lower frequencies was used as a consistency test. No evidence of a possible layer line splitting was seen in the images processed. This possibility must be considered since it is seen in X-ray diffraction pattems from frog muscle (Huxley & Brown, 1967). The images of Limulus filaments (Fig. 1) gave layer lines 1, 3, and 4 of a helical structure (4 stranded 12/1 (12 units, 1 turn for the exact repeat), Stewart et al., 1985) with an expected repeat of 43.8 nm (helical) and
14.6 nm (axial). The absence of higher orders may be due to the effect of the PCTF or to structural disorder in the filaments. In the filtered image (Fig. 4d) the arrows indicate the expected periodicity of 14.6nm and arrowheads mark the helical repeat of 43.8nm. The comparison of the filtered image (Fig. 4d) of this filament is in good agreement with the electron density map of the Limulus reconstruction from Stewart and coworkers (!981). The myosin heads of rabbit psoas muscle thick filaments show a temperature dependance of their structural arrangement (Wray, 1987; Wakabayashi, et al., 1988). Filaments frozen rapidly from a starting temperature of 4~ showed disorganized crossbridges (Fig. 2b), whereas filaments initially at c. 26~C h a d an ordered arrangement of the myosin heads
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MENETRET, SCHRODER a n d HOFMANN
Fig. 5. Rabbit psoas muscle relaxed thick filament kept at c. 25~ before freezing. (The numbers in the Fourier power spectra correspond to possible layer lines in a diffraction pattern of a helical structure respective to the matching layer line spacing.) (a) Shows a c. 400nm long area of the scanned filament. (b) Computed Fourier power spectrum of Fig. 5a. E = equator. The meridional reflections fit to a layer line system of 42.9 nm axial repeat with a meridional repeat of 14.3 nm. (c) Computed filtered Fourier power spectrum (Gaussian filter for corresponding layer lines). (d) Filtered image using the corresponding Fourier data of Fig. 5c. The periodic structure is marked with arrows indicating the long 42.9 nm helical repeat while the arrowheads point to the 14.3 nm axial repeat.
(Fig. 2a). However, the optical diffraction patterns s h o w e d that the organisation of the myosin heads along the filament axis differed from filament to filament. The micrograph in Fig. 2a shows that filaments having different structures, even t h o u g h they were imaged u n d e r the same conditions. A possible explanation is that the order-disorder transition is so rapid that a partial transition occurs during cooling, leading to a distribution of different degrees of order amongst individual filaments. The micrograph s h o w n in Fig. 2a was taken closer to focus than those of Limulus filaments in Fig. 1. Therefore, it shows more intensity at higher spatial frequencies (see power spectrum (Fig. 5b) of the filament A, Fig. 2a). The power spectrum (Fig. 5b) shows strong reflections up to a resolution of about
7 n m fitting well with a periodicity of 42.9nm together with clear indications of helical ordering (3 stranded 9/1, analogous to data from frog muscle. Stewart & Kensler, 1986) from layer lines 1 and 5 (off-meridional reflections) and layer line 3 (meridional reflection). Images from Lethocerus filaments (Fig. 3a) s h o w a completely different crossbridge appearance. The identification of the underlying structure is difficult. In the spectrum (Fig. 6b) one can easily identify three meridional reflections corresponding to an axial periodicity of 14.5nm. Helical ordering is not conspicuous. However, the two strong spots (symmetrical to the meridian), at a position corresponding to about 8.8 nm, suggest a possible w a y of indexing layer lines. The reflections at 8.8 n m were taken as a
Cryo-electron microscopic studies
9
Fig. 6. Lethocerus flight muscle thick filament at c. 4~C. (The numbers in the Fourier power spectra correspond to possible layer lines in a diffraction pattern of a helical structure respective to the matching layer line spacing.) (a) Area of c. 400 nm of the scanned filament. (b) The computed Fourier power spectrum of Fig. 8a. E = equator. The meridional reflections correspond to an axial repeat of 14.5 nm, while the strong off-meridional reflections at 8.8 nm are indications of a possible helical structure (4 stranded 32/3 helix). (c) The computed filtered spectrum of Fig. 8b (Gaussian filter for corresponding layer lines: 3, 5, 8, etc.). (d) The filtered image using the corresponding Fourier data of Fig. 8c. Arrows mark the theoretical exact helical repeat of 116.0nm and the arrowheads indicated the 14.5 nm axial repeat.
sign of a helical arrangement of the myosin heads indicating a possible 4 stranded 32/3 helix. In Fig. 6c. we show the corresponding filtered power spectrum in accordance with helical layer lines 3 (38.5 nm) and 5 (23.5 nm) with off-meridional spots and layer line n u m b e r 8 (14.5 nm) with a meridonal spot. In the filtered image (Fig. 6d) the arrowheads indicate the theoretically expected exact repeat of 116.0nm, the arrows indicate the 14.5nm axial repeat. Conclusion
In this paper we show that rapid freezing and electron microscopic imaging of the thick filaments in
an unstained, u n s u p p o r t e d a n d fully h y d r a t e d state is a viable m e t h o d of obtaining structural information on myosin head organisation. On the basis of the results presented, it is likely that 3-dimensional reconstructions of the filaments can be calculated. In the case of Limulus we expect a structural resolution comparable to that obtained from negatively stained filaments. More detailed studies on the rabbit filaments m u s t still be done, paying special attention to temperature a n d ionic strength effects. The rapid freezing involved in the cryo-electron microscope m e t h o d should allow the identification of transient states and the s t u d y of structural dynamics following perturbation of conditions using, for example, temperature j u m p or flash photolysis techniques.
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MENETRET, S C H R O D E R a n d H O F M A N N
Acknowle dgements W e t h a n k R. S. G o o d y , J. S. W r a y , J. L e p a u l t a n d K. C. H o l m e s for advice, s u p p o r t a n d h e l p f u l diScussions. T h e w o r k w a s s u p p o r t e d b y the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t (grant H o 481-13-2). O n e of
the a u t h o r s (R. R. S c h r 6 d e r ) w o u l d like to t h a n k the B o e h r i n g e r I n g e l h e i m F o n d Stuttgart, W e s t G e r m a n y for a p o s t d o c t o r a l fellowship.
References ADRIAN, M., DUBOCHET, J. LEPAULT, J. & McDOWALLr A. W. (1984) Cryo-electron microscopy of viruses. Nature 308, 32-6. BENNETT, P., CRAIG, R., STARR, R. & OFFER, G. (1986) The ultrastructural location of C-protein and H-protein in rabbit muscle. 1. Musc. Res. Cell Motility 7, 550-67. CLARKE, M. L., HOFMANN, W. & WRAY, J. S. (1986)ATP Binding and crossbridge Structure in Muscle. J. molec. Biol. 191, 581-85. DORSET, D. L. & PARSON, D. F. (1975) Electron diffraction from single, fully-hydrated ox-liver catalase microcrystals. Acta Cryst A31, 210-15. DUBOCHE% J. ADRIANr M., LEPAULT~ J. & MCDOWALLr A. W. (1985) Cryo-electron microscopy of biological specimens. TIBS 10, 143-6. ERICKSON, H. P. & KLUG, A. (1971) Measurement and comparison of defocusing and aberration by Fourier processing of electron micrographs. Philos. Trans. R. Soc. London, Ser. B261, 105-18. ERICKSON, H. P., VETER, W. A. & LEONARD, K. (1978) Image reconstruction in electron microscopy: enhancement of periodic structure by optical filtering. Methods in Enzymology ELIX, 39-63. FRANK, J. (1973) The envelope of electron microscopic transfer functions for partially coherent illumination. Optik 38, 519-36. FUKAMI, A. & ADACHI, K. (1963) A new method of Preparation of self-perforated micro plastic grid and its application. J. elec. microscopy 14, 112-18. GLAESER, R. M. (1982) Electron Microscopy. Meth. Exp. Physics, 20, 391-444. HANSZEN, K.-J. (1971) The optical transfer theory of the electron microscope: Fundamental principles and applications. Adv. Opt. Electron. Micros., 4, 1-84. HUXLEY, H. E. (1967) Recent X-ray diffraction and electron microscopy studies of striated muscle. J. gen. Physiol., 50, 71-84. HUXLEY, H. E. & BROWN, W. (1967) The low-angle X-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor. J. Mol. Biol., 30, 383-434. KENSLER, R. W. & LEVINE, R. J. C. (1982) An electron microscopic and optical diffraction analysis of the structure of Limulus telson muscle thick filaments. J. Cell Biol. 92, 443-51. KLUG, A. & DeROSIER, D. J. (1966) Optical filtering of electron micrographs: Reconstruction of one-sided images. Nature 212, 29-32. KNIGHT, P. & TRINICK, J. (1984) Structure of the myosin projections on native thick filaments from vertebrate skeletal muscle. J. Mol. Biol. 177, 461-82. MCDOWALL, A.r HOFMANN~ W., LEPAULT r J.~ ADRIAN, M. & DUBOCHET, J. (1984) Cryo-electron microscopy of vitrified insect muscle. J. Moi. Biol. 178, 105-111.
MENETRET, J.-F., HOFMANN, W. & LEPAULT, J. (1988) Cyro-electron microscopy of insect flight muscle thick filaments. An approach to dynamic electron microscope studies. J. Mol. Biol. 202, 175-78. RAPP, G.r POOLE, K. J. V., MAEDAr Y., KAPLAN/ J. H. I McCRAY, J. & GOODY, R. S. (1989) Lasers and flashlamps in research on the mechanism of muscle contraction. Berichte der Bunsen Gesellschaft fiir physikalische Chemie, 93, 410-15. REEDY, M. K., HOLMES, K. C. & TREGEAR, R. T. (1965) Induced changes in orientation of cross-bridges of glycerinated insect flight muscle. Nature 207, 1276-80. REEDYr M. K. r LEONARDr K.r FREEMAN, R. W. & ARAD, T. (1981) Thick filament mass determination by electron scattering measurements with the scanning transmission electron microscope. J. Musc. Res. Cell Motility, 2, 45-64. REEDY, M. C., REEDY, M. K. & GOODY, R. S. (1983) Coordinated EM and X-ray studies of glycerinated insect flight muscle. II. Electron microscopy and image reconstruction of muscle fibres fixed rigor, in ATP and in AMPPNP. J. Musc. Res. Cell Motility, 4, 55-81. ROME, E. (1972a) Relaxation of glycerinated muscle: Low angle X-ray diffraction studies. J. Moi. Biol., 65, 331-45. ROME, E. (1972b) Structural studies by X-ray diffraction of striated muscle permeated with certain ions and proteins. Cold Spring Harbor Symp. Quant. Biol. 37, 331-39. STEWART, M. (1988) Computer image processing of electron micrographs of biological structures with helical symmetry. J Electron Microsc. Tech. 9, 325-58. STEWART, M., KENSLER, R. W. LEVINE, R- J. C. (1981) Structure of Limulus telson muscle thick filaments. J. Mol. Biol., 153, 790-781. STEWART, M., KENSLER, R. W. & LEVINE, R. J. C. (1985) Three-dimensional reconstruction of thick filaments from Limulus and Scorpion muscle. J. Cell. Biol., 101, 402-11. STEWART, M. & KENSLER, R. W. (1986) Arrangement of myosin heads in relaxed thick filaments from frog skeletal muscle. J. Mol. Biol., 192, 831-51. STEWART, M. & VIRGES, G. (1986) Electron microscopy of frozen hydrated biological material. Nature 319, 631-36. WAKABAYASHIr T./ AKIBA~ T.r HIROSE, K. r TOMIOKA, A., TOKUNAGA, M., SUZUKI~ M., TOYOSHIBAr C.~ SUTOH, K.~ YAMAMOTO, K., SAEKI, K. & AMEMIYA~ Y. (1988) Temperature-induced change of thick filament and location of the functional sites of myosin. In Molecular mechanism of muscle contraction (EDITEDBY SUGI, H. & POLLACK, G. H.) pp. 39-46, Plenum, New York (1988).
Cryo-electron microscopic studies WRAY, J. S. (1987) Structure of relaxed myosin filaments in relation to nucleotide state in vertebrate skeletal muscle. J. Mus. Res. Cell Motility, 8, 62.
11 WRAY, J. S., VIBERT, P. J. & COHEN, C. (1974) Cross-bridge arrangements in Limulus muscle. J. Mol. Biol. 88, 343-48.
Cryo-electron microscopic studies of relaxed striated muscle thick filaments J. F. M E N E T R E T * , R. R. S C H R O D E R
a n d W. H O F M A N N
Max Planck Institute for Medical Research, Department of Biophysics, Jahnstrasse 29, D-6900 Heidelberg, West Germany Received 21 February 1989 and in revised form 12 July 1989
Summary Electron micrograph images of rapidly frozen suspensions of thick filaments from four different muscle types are presented. Their optical and computer transforms are compared with images and diffraction patterns of negatively stained filaments and with X-ray data from the same muscles. We conclude that myosin head arrangement can be preserved on rapid freezing and that the images produced can be analysed by image processing techniques to give new information on thick filament structure.
Introduction The m e c h a n i s m of muscle contraction at the molecular level is still not u n d e r s t o o d . More experimental evidence is n e e d e d to u n d e r s t a n d the f u n d a m e n t a l problem of transformation of chemical e n e r g y into mechanical work. Contraction involves the splitting of ATP coupled to the interaction of thick myosincontaining and thin actin-containing filaments. Electron microscope techniques have b e e n widely used to visualize the structure and interaction of thick and thin filaments u n d e r various conditions, either by sectioning fixed, stained and e m b e d d e d tissue (Reedy et al., 1965; Huxley, 1967; Reedy et al., 1983; Bennett et al., 1986) or b y examining the structural behaviour of isolated filaments (Kensler & Levine, 1982; Knight & Trinick, 1984; Stewart et al., 1985; Clarke et al., 1986). Cyro-electron microscopy is a technique which allows the examination of biological material in its natural e n v i r o n m e n t (Adrian et al., 1984; D u b o c h e t et al., 1985; r e v i e w e d by Stewart & Virges, 1986). Using this m e t h o d , it has b e e n s h o w n that structure can be well p r e s e r v e d in frozen h y d r a t e d sections of insect muscle fibres (McDowall et al., 1984) and that thick filaments from insect flight muscle in s u s p e n s i o n retain their regular structure w h e n rapidly frozen and v i e w e d in the electron microscope u n d e r liquid nitrogen conditions (Menetret et aL, 1988). We have used cryo-electron microscopy and c o m p u t e r analysis of the electron micrograph images to s t u d y the structure of muscle thick filaments in the presence of ATP in a fully h y d r a t e d , unstained, unfixed and u n s u p p o r t e d state. Here we *To whom correspondence should be addresssed. 0142-4319/90 $03.00 + .12 9 1990 Chapman and Hall Ltd.
describe results o n thick filaments from striated muscles of Limulus (horseshoe crab), rabbit and the insects Lethocerus a n d Drosophila. A preliminary analysis of the images using c o m p u t e r processing is described.
Materials and methods
Preparation offilaments Limulus telson muscle filaments were prepared according to Kensler & Levine (1982). Only animals which appeared healthy were used. Glycerinated material was also used. Small muscle bundles were dissected and tied to plastic rods at 4~ The bundles were stirred at 4~ in a 50% glycerol/relaxing solution (5mM ATP, 70mM K acetate, 2 mM EGTA, i mM DTT, 20 mM MOPS, 10 mM Mg acetate, pli 7.0) for 2-4 days and stored at -20 ~C. Filaments from this stock were prepared using the procedures described below for Lethocerus filaments and by Menetret and coworkers (1988). Filaments obtained from stored muscles were similar in quality to those from fresh muscle. Rabbit psoas muscle was freshly dissected, washed in relaxing buffer (7mM ATP, 70mM K propionate, 5mM EGTA, 6 mM MOPS, 8 mM Mg acetate) and slowly cooled to 4~ in the same buffer. Smaller bundles were skinned for 3-4 h in 0.5% Brij/or Triton/relaxing solution at 4~ (Wray, personal communication). After washing for about 3h in relaxing solution at 4~ the bundles were left in 50% glycerol/relaxing solution for 2-20h. After extensive washing in relaxing solution, the bundles were used for filament preparation using the same brief enzymatic digestion as described for Lethocerus filaments.
2
Lethocerus thoraces were dissected, washed in rigor buffer (5mM Mg acetate, 100mM K acetate, 2mM EGTA, 1mM DTT, 20mM MOPS p l i 6.8) and, after several changes of a rigor/50% glycerol buffer at 4~ stored at -20~ Best preservation of the material was in the first four months. Fibrils were prepared by standard methods (Reedy et al., 1981) and digested (c. 5 min) using elastase (SIGMA, Type 4: from Porcine Pancreas) and trypsin inhibitor in rigor at 4~ (according to Magid, personal communication). The enzymatic reaction was stopped by washing several times with buffer. Fibrils were transferred into relaxing solution (2mM ATP in rigor buffer) and thick and thin filaments were obtained after homogenization with a hand homogenizer. Thorax muscles from c. 30 Drosophila melanogaster (wild type) were dissected, washed in rigor buffer (5 mM Mg acetate, 100 mM K acetate, 0.2 mM EGTA, I mM DTT, 20 mM MOPS pli 6.8) and glycerinated in 50% glycerol/rigor solution for 15-30 min at 4~ The small bundles were washed in rigor buffer and digested for c. 5 min using elastase. After several washes with rigor buffer, the bundles were incubated in relaxing solution (2 mM ATP in rigor buffer) at 4~ and thick and thin filaments were obtained after homogenization. The filament suspension presumably contained an u n k n o w n number of filaments from muscles other than the indirect flight muscles. Before freezing, each preparation was tested for structural quality and suitable filament distribution using negative staining techniques. Preparation of the grids Grids coated with a perforated carbon film were prepared as described by Fukami and Adachi (1963). The filament suspension was applied to these grids according to the method of Adrian and colleagues (1984). Best preserved filaments were obtained when the relative humidity was above 70%. This minimized changes in the ionic strength of the buffer due to evaporation. To avoid temperature changes, the copper grid, the forceps, and the blotting paper were adjusted to the desired temperature (c. 4 ~C for
MENETRET, S C H R O D E R a n d H O F M A N N
Limulus, Lethocerus and Drosophila, c. 26~
for rabbit). Effects of additional temperature changes due to evaporation from the grid were not observed. Best results were obtained w h e n the grid was frozen immediately (K 0.5 s) after blotting.
Electron microscopy Pictures were taken on a Philips 400 T transmission microscope at 80kV with 3~m condensor aperture and 100~m objective aperture. The electron dose on the specimen was reduced to a value of c. 330 e - per nm 2 using the Philips low dose kit. Only one picture of each area was taken. The grids were viewed using a Philips or Gatan cryo~holder at liquid nitrogen temperature. Depending on the degree of defocussing (4-6 ~m underfocus) the nominal resolution is limited to 4 - 5 n m (Hanszen, 1971; Frank, 1973). Film plates (Kodak SO-163) were developed for double the recommended time in full strength developer.
Image processing Film plates were examined on an optical diffraction bench as described by Erickson and coworkers. (1978) and selected with respect to astigmatism, defocus and structural features. Images of filaments showing periodic structures corresponding to those seen by X-ray diffraction were processed further. The negatives were scanned on an Optronics P-1000 with 256 grey levels and a spot size of 25 ~m, yielding a nominal resolution of about 1.7 nm for an electron migrograph taken at a magnification of 28700 (calibrated with images of catalase crystals; Dorset & Parsons (1975)). Ail image processing was done with a new software package to be described in detail elsewhere (Schr6der et al., in preparation). Pictures of filament areas were selected and floated on background arrays having a grey level value equal to the average of the perimeter values of the filament area. The computer programmes run on a CONVEX Computer Corp. C1 computer. Images and diffraction patterns were displayed on a PCS Corp. colour graphics workstation.
Fig. 1. Gallery of relaxed Limulus telson muscle thick filaments frozen, unstained, unsupported, fully hydrated and embedded in a layer of vitreous buffer. Their corresponding optical diffraction patterns are shown below. The periodicity of the crossbridges can be best seen by sighting along the filament axis. (Scale bar = exact helical repeat, 43.8 nm). The second filament will be further analysed in Fig. 4. Optical diffraction patterns are marked with arrows pointing to the approximate position of layer-lines 1, 3 and 4 (measured 11.2 + / - 0 . 5 nm) of 4 stranded 12/1 helix, E = equator. Fig. 2. Relaxed rabbit psoas muscle thick filaments. (Scale bar = 100 nm) (a) Relaxed rabbit psoas muscle thick filaments kept at c. 26~C before freezing. The optical diffraction patterns of the filaments A and B are different although the filaments have been imaged in the same conditions. A possible explanation is that the order-disorder transition is so rapid (Rapp et al., 1989) that a partial transition occurs during cooling, leading to a distribution of different degrees of order amongst individual filaments. The optical diffraction pattern of the filament A shows the first and fifth layer lines (measured 8.8 + / - 0 . 5 nm) and a meridional reflection on the third layer line of a 3 stranded 9/1 helix; it will be further analysed in Fig. 5. The optical diffraction pattern of the filament B shows a reflection on the first layer-line. (b) Relaxed rabbit psoas muscle thick filaments kept at 4 ~C before freezing. These filaments do not show obvious order of their crossbridges, t = tip and b = bare zone of the filaments. Fig. 3. Insect flight muscle thick filaments. (Scale bar = 100 nm) (a) Lethocerus flight muscle relaxed thick filaments. Typical axial periodicity of 14.5 nm, corresponding to the so-called 'crowns' of crossbridges can be seen along the filament axis. The optical diffraction pattern shows on its meridian the first, second and third order of the axial repeat (14.5 nm). Indications of helical arrangement of the crossbridges can be seen as off-meridional reflections at a spacing of about 9 nm. (see Fig. 6 for a detailed analysis). (b) Drosophila melanogaster in relaxed state. (scale bar = 100nm) No regular crossbridge order can be detected.
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MENETRET, SCHRODER and HOFMANN
Cryo-electron microscopic studies
5
6 Results and discussion
Preservation, offilaments The main purpose of this work was to establish procedures by which thick filaments of muscle can be routinely observed by rapid freezing in suspension, i.e. in conditions which depart from the native only in the lack of a lattice structure. Myosin filaments are highly ordered structures, and the state of preservation can be monitored by comparison with conventional negatively stained electron micrographs and above all by comparing optical or computer transforms with X-ray diffraction data. In order to retain the structure of the myosin filaments after preparation, and especially after freezing, it had to be considered whether the arrangement of the crossbridges in isolated thick filaments might be sensitive to the surrounding conditions (temperature, ionic strength, contact of myosin heads to surfaces such as solid supports of the grid). To avoid mechanical interference with the filaments, no supporting carbon film was used. In our experience, evaporation of water from the grid, which results in an increase of the ionic strength and a decrease in temperature, can influence the appearance of the filaments. Since the effects of evaporation are proportional to the surface to volume ratio a n excess of suspension was applied to the grid and frozen immediately after blotting. Furthermore, it often helped to work in a humid atmosphere, although this practice was not adopted routinely. In all four muscle types studied, it was possible to observe thick filaments in vitreous buffer. Filaments from Limulus telson muscle (Fig. 1), rabbit psoas muscle (Fig. 2) and Lethocerus flight muscle (Fig. 3a) show periodic organization of myosin heads, whereas filaments from Drosophila melanogaster (wild type) do not appear to do so (Fig. 3b). The thick filaments from Limulus have been most extensively studied previously by conventional methods. This muscle is a good test specimen for our work, particularly since the filaments are known to undergo structural changes w h e n the ionic strength increases (Wray et al., 1974). Negative staining studies showing preservation of order in native mammalian thick filaments have not been published, and X-ray diffraction studies suggested that the filaments are susceptible to disorder (Rome, 1972a). Patterns from living rabbit muscles (Rome, 1972b) nevertheless suggest that strong layer lines comparable to those of frog muscle can be obtained. A crucial factor may be the apparent sensitivity to temperature (Wray, 1987; Wakabayashi et al., 1988). In the present work best-ordered filaments were obtained w h e n the temperature was c. 26~ before freezing (Fig. 2a). Filaments which were
MENETRET, SCHRODER and HOFMANN at about 4~ before freezing gave no indication of ordered myosin heads (Fig. 2b). The filament from Lethocerus (Fig. 3a) shows, as previously described, clear 14.5nm periodicities (Menetret et al., 1988). The crowns of crossbridges show obvious demarcation into globular units. In addition, the optical diffraction pattern (insert Fig. 3a) of the filament contains indications of a helical ordering of its crossbridges (see Fig. 5 for more detailed analysis). In Drosophila, the filaments appeared to be well preserved with an average length of about 2.4 ~m and an overall diameter of about 30nm (Fig. 3b). However, myosin heads appeared randomly distributed without periodic structure. Electron micrographs of Drosophila filaments have not previously been published. However, Reedy and colleagues (1981) noted that filaments from another fly, Musca, also lacked longitudinal periodicity. Unless both types of filaments have been damaged or modified during isolation from the lattice, it appears that fly filaments depart from the Lethocerus stereotype.
Image analysis In the cases of Limulus, rabbit and Lethocerus thick filaments, we performed some preliminary image processing. When processing electron microscope images, the factors leading to formation of the images in the transmission electron microscope (TEM) must be considered. Vitrified specimens are weak phase objects yielding very little amplitude contrast. The contrast seen in the electron micrographs is phase contrast produced by a certain degree of defocussing. However, this also affects the transmittance of spatial frequencies in the back focal plane of the objective lens, expressed mathematically as the 'phase contrast transfer function' (PCTF; Hanszen, 1971, Frank, 1973, reviewed by Glaeser, 1982). This function has a complex form: it modulates transmittance sinusoidally as a function of spatial frequency and additionally suppresses very low and very high frequencies, depending on the degree of defocus (Erickson & King, 1971, Frank, 1973). It corresponds to the obvious decrease in background intensity at higher frequencies in the spectra presented (Figs 4b, 5b, 6b). In the present study, these effects were not corrected for, so the filtered images show projected filament structures under the influence of the PCTF. Images with an underlying periodicity were filtered in the usual way (Klug & DeRosier, 1966, reviewed by Stewart, 1988). Since images obtained by cyromicroscopy should be perfect projections of an object, muscle thick filaments should theoretically give projections of a helically ordered structure. Strong reflections in the spectra could be assigned to layer lines expected for a simple helical system. The
Cryo-electron microscopic studies
7
Fig. 4. Limulus telson muscle relaxed thick filaments. (The numbers in the Fourier power spectra correspond to possible layer lines in a diffraction pattern of a helical structure respective to the matching layer lines spacing.) (a) Scanned filament area (c. 400 nm long), corresponding to the filament in the middle of Fig. 1. (b) Computed Fourier power spectrum of the embedded filament Fig. 4a. Four off-meridional and one meridonal reflections can be seen. Those spots fit to a layer line system (4 stranded 12/1 helix) of 43.8 nm together with a 14.6 nm axial repeat which is clearly shown in the filtered spectrum (4c). E = equator. (c) Computed filtered Fourier power spectrum (Gaussian filter for corresponding layer lines). (d) Fi|tered image using the corresponding Fourier data of (4c). The arrowheads indicate the 14.6 nm repeat, arrows mark the 43.8 nm helical repeat. corresponding filter consisted of Gaussian shaped lines, i.e. ai1 data on the supposed layer lines were multiplied by a factor of 1, all other data by a factor < 1 inversely proportional to their distance from the layer line. In all cases, possible layer line systems were indexed at higher spatial frequencies and the matching to reflections at lower frequencies was used as a consistency test. No evidence of a possible layer line splitting was seen in the images processed. This possibility must be considered since it is seen in X-ray diffraction pattems from frog muscle (Huxley & Brown, 1967). The images of Limulus filaments (Fig. 1) gave layer lines 1, 3, and 4 of a helical structure (4 stranded 12/1 (12 units, 1 turn for the exact repeat), Stewart et al., 1985) with an expected repeat of 43.8 nm (helical) and
14.6 nm (axial). The absence of higher orders may be due to the effect of the PCTF or to structural disorder in the filaments. In the filtered image (Fig. 4d) the arrows indicate the expected periodicity of 14.6nm and arrowheads mark the helical repeat of 43.8nm. The comparison of the filtered image (Fig. 4d) of this filament is in good agreement with the electron density map of the Limulus reconstruction from Stewart and coworkers (!981). The myosin heads of rabbit psoas muscle thick filaments show a temperature dependance of their structural arrangement (Wray, 1987; Wakabayashi, et al., 1988). Filaments frozen rapidly from a starting temperature of 4~ showed disorganized crossbridges (Fig. 2b), whereas filaments initially at c. 26~C h a d an ordered arrangement of the myosin heads
8
MENETRET, SCHRODER a n d HOFMANN
Fig. 5. Rabbit psoas muscle relaxed thick filament kept at c. 25~ before freezing. (The numbers in the Fourier power spectra correspond to possible layer lines in a diffraction pattern of a helical structure respective to the matching layer line spacing.) (a) Shows a c. 400nm long area of the scanned filament. (b) Computed Fourier power spectrum of Fig. 5a. E = equator. The meridional reflections fit to a layer line system of 42.9 nm axial repeat with a meridional repeat of 14.3 nm. (c) Computed filtered Fourier power spectrum (Gaussian filter for corresponding layer lines). (d) Filtered image using the corresponding Fourier data of Fig. 5c. The periodic structure is marked with arrows indicating the long 42.9 nm helical repeat while the arrowheads point to the 14.3 nm axial repeat.
(Fig. 2a). However, the optical diffraction patterns s h o w e d that the organisation of the myosin heads along the filament axis differed from filament to filament. The micrograph in Fig. 2a shows that filaments having different structures, even t h o u g h they were imaged u n d e r the same conditions. A possible explanation is that the order-disorder transition is so rapid that a partial transition occurs during cooling, leading to a distribution of different degrees of order amongst individual filaments. The micrograph s h o w n in Fig. 2a was taken closer to focus than those of Limulus filaments in Fig. 1. Therefore, it shows more intensity at higher spatial frequencies (see power spectrum (Fig. 5b) of the filament A, Fig. 2a). The power spectrum (Fig. 5b) shows strong reflections up to a resolution of about
7 n m fitting well with a periodicity of 42.9nm together with clear indications of helical ordering (3 stranded 9/1, analogous to data from frog muscle. Stewart & Kensler, 1986) from layer lines 1 and 5 (off-meridional reflections) and layer line 3 (meridional reflection). Images from Lethocerus filaments (Fig. 3a) s h o w a completely different crossbridge appearance. The identification of the underlying structure is difficult. In the spectrum (Fig. 6b) one can easily identify three meridional reflections corresponding to an axial periodicity of 14.5nm. Helical ordering is not conspicuous. However, the two strong spots (symmetrical to the meridian), at a position corresponding to about 8.8 nm, suggest a possible w a y of indexing layer lines. The reflections at 8.8 n m were taken as a
Cryo-electron microscopic studies
9
Fig. 6. Lethocerus flight muscle thick filament at c. 4~C. (The numbers in the Fourier power spectra correspond to possible layer lines in a diffraction pattern of a helical structure respective to the matching layer line spacing.) (a) Area of c. 400 nm of the scanned filament. (b) The computed Fourier power spectrum of Fig. 8a. E = equator. The meridional reflections correspond to an axial repeat of 14.5 nm, while the strong off-meridional reflections at 8.8 nm are indications of a possible helical structure (4 stranded 32/3 helix). (c) The computed filtered spectrum of Fig. 8b (Gaussian filter for corresponding layer lines: 3, 5, 8, etc.). (d) The filtered image using the corresponding Fourier data of Fig. 8c. Arrows mark the theoretical exact helical repeat of 116.0nm and the arrowheads indicated the 14.5 nm axial repeat.
sign of a helical arrangement of the myosin heads indicating a possible 4 stranded 32/3 helix. In Fig. 6c. we show the corresponding filtered power spectrum in accordance with helical layer lines 3 (38.5 nm) and 5 (23.5 nm) with off-meridional spots and layer line n u m b e r 8 (14.5 nm) with a meridonal spot. In the filtered image (Fig. 6d) the arrowheads indicate the theoretically expected exact repeat of 116.0nm, the arrows indicate the 14.5nm axial repeat. Conclusion
In this paper we show that rapid freezing and electron microscopic imaging of the thick filaments in
an unstained, u n s u p p o r t e d a n d fully h y d r a t e d state is a viable m e t h o d of obtaining structural information on myosin head organisation. On the basis of the results presented, it is likely that 3-dimensional reconstructions of the filaments can be calculated. In the case of Limulus we expect a structural resolution comparable to that obtained from negatively stained filaments. More detailed studies on the rabbit filaments m u s t still be done, paying special attention to temperature a n d ionic strength effects. The rapid freezing involved in the cryo-electron microscope m e t h o d should allow the identification of transient states and the s t u d y of structural dynamics following perturbation of conditions using, for example, temperature j u m p or flash photolysis techniques.
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MENETRET, S C H R O D E R a n d H O F M A N N
Acknowle dgements W e t h a n k R. S. G o o d y , J. S. W r a y , J. L e p a u l t a n d K. C. H o l m e s for advice, s u p p o r t a n d h e l p f u l diScussions. T h e w o r k w a s s u p p o r t e d b y the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t (grant H o 481-13-2). O n e of
the a u t h o r s (R. R. S c h r 6 d e r ) w o u l d like to t h a n k the B o e h r i n g e r I n g e l h e i m F o n d Stuttgart, W e s t G e r m a n y for a p o s t d o c t o r a l fellowship.
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MENETRET, J.-F., HOFMANN, W. & LEPAULT, J. (1988) Cyro-electron microscopy of insect flight muscle thick filaments. An approach to dynamic electron microscope studies. J. Mol. Biol. 202, 175-78. RAPP, G.r POOLE, K. J. V., MAEDAr Y., KAPLAN/ J. H. I McCRAY, J. & GOODY, R. S. (1989) Lasers and flashlamps in research on the mechanism of muscle contraction. Berichte der Bunsen Gesellschaft fiir physikalische Chemie, 93, 410-15. REEDY, M. K., HOLMES, K. C. & TREGEAR, R. T. (1965) Induced changes in orientation of cross-bridges of glycerinated insect flight muscle. Nature 207, 1276-80. REEDYr M. K. r LEONARDr K.r FREEMAN, R. W. & ARAD, T. (1981) Thick filament mass determination by electron scattering measurements with the scanning transmission electron microscope. J. Musc. Res. Cell Motility, 2, 45-64. REEDY, M. C., REEDY, M. K. & GOODY, R. S. (1983) Coordinated EM and X-ray studies of glycerinated insect flight muscle. II. Electron microscopy and image reconstruction of muscle fibres fixed rigor, in ATP and in AMPPNP. J. Musc. Res. Cell Motility, 4, 55-81. ROME, E. (1972a) Relaxation of glycerinated muscle: Low angle X-ray diffraction studies. J. Moi. Biol., 65, 331-45. ROME, E. (1972b) Structural studies by X-ray diffraction of striated muscle permeated with certain ions and proteins. Cold Spring Harbor Symp. Quant. Biol. 37, 331-39. STEWART, M. (1988) Computer image processing of electron micrographs of biological structures with helical symmetry. J Electron Microsc. Tech. 9, 325-58. STEWART, M., KENSLER, R. W. LEVINE, R- J. C. (1981) Structure of Limulus telson muscle thick filaments. J. Mol. Biol., 153, 790-781. STEWART, M., KENSLER, R. W. & LEVINE, R. J. C. (1985) Three-dimensional reconstruction of thick filaments from Limulus and Scorpion muscle. J. Cell. Biol., 101, 402-11. STEWART, M. & KENSLER, R. W. (1986) Arrangement of myosin heads in relaxed thick filaments from frog skeletal muscle. J. Mol. Biol., 192, 831-51. STEWART, M. & VIRGES, G. (1986) Electron microscopy of frozen hydrated biological material. Nature 319, 631-36. WAKABAYASHIr T./ AKIBA~ T.r HIROSE, K. r TOMIOKA, A., TOKUNAGA, M., SUZUKI~ M., TOYOSHIBAr C.~ SUTOH, K.~ YAMAMOTO, K., SAEKI, K. & AMEMIYA~ Y. (1988) Temperature-induced change of thick filament and location of the functional sites of myosin. In Molecular mechanism of muscle contraction (EDITEDBY SUGI, H. & POLLACK, G. H.) pp. 39-46, Plenum, New York (1988).
Cryo-electron microscopic studies WRAY, J. S. (1987) Structure of relaxed myosin filaments in relation to nucleotide state in vertebrate skeletal muscle. J. Mus. Res. Cell Motility, 8, 62.
11 WRAY, J. S., VIBERT, P. J. & COHEN, C. (1974) Cross-bridge arrangements in Limulus muscle. J. Mol. Biol. 88, 343-48.
