J. Hoi.
BioZ. (1977) 113, 199-218
The Myosin Filament V.+ Intermediate Voltage Electron Microscopy and Optical Diffraction Studies of the Substructure FRANK A.~ PEPE AND PETER DOWBEN Department of Anatomy ,!edical Xchool, University of Pennsylvaka Philadelphia, Pa 19174, U.X.A. (Received 27 July
1976, and in revised form 3 January
1977)
Using a 200 kV electron microscope (JEM FOOA), thick (up to 0.4 pm) crosssections of the myosin filaments of vertebrate striated muscle were studied. It was found that: (a) with increasing section thickness the cross-sectional profiles of the shaft of the filament were increasingly more triangular and in sections 0.4 pm thick each apex of the triangle was clearly blunted. This unique crosssectiorial profile is predicted by the model proposed by Pepe (1966,1967) in which 12 parallel structural units are packed to form a triangular profile with a structural unit missing at each apex of the triangle. (b) With increasing section thickness the substructure of the myosin filament was enhanced, with the best substructure visible in sections 0.2 pm to 0.3 pm thick. This strongly supports parallel alignment of structural units in the shaft of the filament as proposed by Pepe (1966,1967). (c) The substructure spacing, determined by optical diffraction from electron micrographs of cross-sections of individual myosin filaments or groups of filaments is about 4 nm. (d) The different optical diffraction patterns observed from individual myosin filaments can be explained if the projection of each structural unit in the plane of the section has an elongated profile. With a substructure spacing of 4 nm an elongated cross-sectional profile could be produced by having two myosin molecules per structural unit. Models drawn with two myosin molecules per structural unit in the model proposed by Pepe (1966,1967) gave optical diffraction patterns similar to those observed from individual filaments. (e) The different optical diffraction patterns observed from individual myosin filaments can be explained if the elongated profiles in each structural unit are similarly oriented but with the orientation changing along the length of the filament. The change in orientation per unit length of the filament must be small enough to maintain an elongated profile for the projection of the structural unit in the plane of the sections 0.3 pm thick. All of these observations and conclusions strongly support the model for the myosin frla.ment proposed by Pepe (1966,1967).
1. Introduction Studies of the detailed structure of vertebrate muscle myosin filament’s have generally been limited to the possible arrangement of myosin cross-bridges on the surface of the filament deduced from X-ray diffraction data (Huxley & Brown, 1967). Three t Paper IV in this series is Pepe & Drucker (1972). 199
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possible helical arrangements have been suggested (Squire, 1973) which are all consistent with this data. It can be shown, from X-ray diffraction that there are myosin cross-bridges at intervals of 14 nm along the filament with a helical repeat of 43 nm (Huxley & Brown, 1967). This indicates that staggers of I4 nm and 43 nm occur between myosin molecules in the filament but provides no information about the packing arrangement of the molecules. From electron microscope studies of the growth of synthetic myosin filaments from myosin solution, Huxley (1963) has concluded that the myosin molecules in one-half of the filament are all oriented opposite to those in the other half of the filament, but again no further information about the packing arrangement could be obtained from these studies. The light meromyosin fragment of myosin aggregates to form paracrystals in which presumably the packing arrangement should have some relation to that in the myosin filament. Light meromyosin paracrystals show axial repeats of 14 nm or 43 nm (Szent-Gyorgyi et al., 1960; Huxley, 1963; Lowey et al., 1967; King & Young, 1970; Chowrashi & Pepe, 1971; Nakamura et al., 1971; Sreter et al., 1972; Moos, 1972; Katsura I%Noda, 1973) indicating 14 nm or 43 nm stagger relations between molecules consistent with the X-ray diffraction data from myosin filaments, but likewise giving no further information about the packing of the molecules. Two different models have been proposed for the detailed packing of myosin molecules in vertebrate muscle myosin filaments (Pepe, 1966,1967; Squire, 1973). One of the models (Squire, 1973) was deduced from a general packing scheme in which all the myosin molecules are equivalent. The other (Pepe, 1966,1967) was deduced by relating the general structural characteristics of the A-band and of myosin filaments. The two models are clearly different. The basic distinctive features of the two models are as follows : Pepe (1966,1967) proposes t’hat the myosin filament is built up of structural units which can consist of one or more than one myosin molecule. The structural units in this model are all parallel to the long axis of the filament. In the shaft of the filament a cross-section will pass through 12 structural units packed to give a triangular profile with a structura~l unit missing at each apex. Squire (1973) proposes that the structural unit for the myosin filament is a single myosin molecule. The myosin molecules in this model are tilted both with respect to the long axis of the myosin filament and around the long axis of the filament, the angles of tilt being about 3”. In order to test the two models with respect to these distinctly different characteristics we have used an intermediate voltage (200 kV) electron microscope to observe the cross-sectional profiles in thick cross-sections of the myosin filaments. We have analyzed the electron micrographs by optical diffraction for evidence of substructure in the shaft of the filament. In Squire’s model (1973), since the myosin molecules are tilted both away from and around the long axis of the filament, increasing the section thickness (up to 0.4 pm) should lead to obliteration of visible substructure in the shaft of the filament with the appearance of increasingly more circular cross-sectional profiles. Also, the substructure spacing, if observable in thin sections, should be about 2 nm which is the packing distance of coiled-coils (Elliott et al., 1968) of which the myosin rod is an example. In Pepe’s model (1966,1967) exactly the opposite results should be observed. Since the structural units in this model are all parallel to the long axis of the filament, increasing the thickness (up to 0.4 pm) of a crosssection should lead to more observable substructure in the shaft of the filament. Also, the triangular cross-sectional profile of the shaft of the filament should be
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enhanced with increasing section thickness. In this model, the substructure spacing can be larger than 2 nm if there is more than one myosin moIecule per structural Unit. We ha.ve found that: (a) as the thickness of the cross-section increases (up to 0.4 ,~,m)the cross-sectional profile of the filament becomes increasingly more clearly t,riangular. In addition, each apex of the triangular profile is blunt’ed as predicted by the model (Pepe, 1966,1967), where 12 parallel structural units are hexagonally packed to give a triangular profile with one structural unit missing at each apex. (b) As the section thickness increases, there is an enchancement of the substructure in the shaft of the myosin filament observable by optical diffraction. The substructure spacing is about 4 nm, suggesting that there is more than one myosin molecule per structural unit. (c) The optical diffraction patterns obtained from images of crosssections of individual fiIaments suggest that the structural units have elongated profiles (as might be expected for two myosin molecules side by side) all similarly oriented and that the oreintation of these elongated profiles changes along the length of the filament, These observations strongly support the model proposed by Pepe (1966#:1967)and are not compatible with the model proposed by Squire (1973).
2. Materials and Methods All of this work was done using the lateral muscles of the fresh water killifish, Fundulua diaphccnus. The fresh muscle was fixed at 2 to 4°C for a minimum of 2 h in 6% buffered glutaraldehyde solution containing 0.1 M-KCl, 1 m&r-MgCl, and 10 m&%-phosphate at $1 7-O. The muscle was dissected into small fiber segments or bundles. This was followed by fixation in 1% 0~0, buffered in the same solution, and it was then embedded in Araldite. Sections were obtained with thicknesses ranging from 0.06 pm to 0.4 pm. These were picked up on uncoated grids. The grids were then immersed for 3 h in 10% uranyl acetate solution in methanol and followed by 4 brief rinses in methanol, each rinse being carried out in a separate container. They were then immersed for 3 h in a 0.5% solution of lead each rinse carried out in a citrate in 50% ethanol, followed by 4 rinses in 5076 ethanol, separate container. This cycle of staining was carried out a second and third time (Pepe, B975). Sections about 1~5 pm thick were stained in this way using one cycle of alcoholic uranyl acid and lead citrate. These were then embedded in Araldite and sectioned transversely to observe penetration of the stain. The heaviest staining occurred to a depth of about 0.5 pm from the surface of the section (G. Tabas, A. Reingold & F. Pepe, unpublished resuhs). Therefore 3 cycles of staining are more than sufficient to ensure penetration of the stain from both sides of sections O-4 pm or Iess in thickness. The sections (thicknesses ranging from 0.06 pm to 0.4 pm) were all observed in a JEM 200 A electron microscope at a.n accelerating voltage of 200 kY using a 50 pm objective aperture. Before observation the sections were coated with a carbon film. Since observation of substructure in the shaft of the filament was the object of these studies, crosssections of the myofibrils were always used. The sections were tilted until the best visual alignment of the filament, perpendicular to the plane of the section was obtained. In some cases several pictures were taken of the same area at several angles of tilt differing by I” or 2”. The side entry goniometer on the instrument permitted tilting over 360” rotation of the image. The micrographs were obtained on Kodak 4489 electron microscope film (3-5 in x 4 in) and were developed in full-strength Kodak D-19 developer for 4 min at 20°C. Relatively dense electron micrographs were used because it was found that these were best suited for optical diffraction. Optical diffraction patterns were obtained using a preassembled unit, Electron Micrograph Optical Diffractometer ESOOO, purchased from Polaron Equipment Limited, England. For optical diffraction from the myosin filaments, the actin filaments were
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painted out with black paint. The area to be diffracted was limited by using parallel strips of tape as a mask. In general the background of the negative was opaque enough so t,hat a mask function was not observed in the optical diffraction pattern. Areas containing 6 to 12 myosin filaments were used in addition to single myosin filaments. Optical diffraction patterns were adso obtained from actin filaments as a control on the results obtained with the myosin filaments. When the actin filaments were studied the myosin filaments were painted out with black paint. Optical diffraction was attempted from actin filaments only in areas of the electron micrograph where the myosin filaments gave good diffraction patterns.
3. Results (a) Observations
of cross-sectional projlles of myosin filaments function of section thickness
as a
Cross-sections of the A-band with thickness in the range of 0.06 ,um to O-4 pm were observed. The purpose of this was specifically to observe if the cross-sectional profiles become more distinctly circular or more distinctly triangular with increasing section thickness. In the thinner sections the cross-sectional profiles of the thick filaments were generally irregular in shape although occasional triangular profiles were observed (Fig. l(a)). As the section thickness was increased, more clearly triangular crosssectional profiles were observed (Fig. l(b)) and at a section thickness of about O-4 pm the cross-sectional profiles were unmistakably triangular with each apex of the triangle blunt’ed (Fig. l(c)). In making these observations, cross-sections occurring through a short region of the filament immediately adjacent to each side of the M-band were avoided. In this region of the filament triangular profiles are clearly observed in thin sections (Pepe, 1966,1967,1971). In the thickest sections (O-4 pm) it was not always possible to be sure that these portions of the filament were not included in the section. However, if they were included they would represent a maximum of about 0.08 pm of the length of the filament or only about one-fifth of the total length of the filament in the O-4 pm thick section, and therefore would not contribute significantly to determining the cross-sectional profile observed. (b) Optical diflraction patterns from myosin filaments as a function of section thickness Optical diffraction patterns were obtained from the electron micrographs after painting out the actin filaments. In general 6 to 12 filaments were included in the area of diffraction. In sections 0.1 pm or fess in thickness, it wa.s difficult to find any areas in which spacings other than the interfilament distance were observed. The clearest observations of substructure spacing were made with sections that were about 0.2 pm or more in thickness. Optical diffraction patterns similar to those in Figure 2 were observed. In general, sections about 0.2 pm to 0.3 pm in t’hickness gave optical diffraction patterns showing substructure in either two or three directions (Fig. 2). With sections about 0.4 pm thick the substructure spacing was observed predominantly in one direction. In the thickest sections even very small angles of tilt will result in decrease or elimination of spacings in directions other than that perpendicular to the direction of tilt: giving substructure spacing in only one direction. A general feature of the optical diffraction patterns was that the intensity of the substructure spacings observed in different directions was not the same (Fig. 2).
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FIG. 1. Cross-sectional profiles of myosin filaments as a function of section thickness. Muscle is from the fresh water k&fish, P. d&-q&znus. (a) Section about 0.09 pm thick. Most of the myosin filaments have roughly circular and solid. cross-sectional profiles. Occasional filaments appear triangular. (b) Section about 0.25 pm thick. Many of the myoain filament,s have triangular cross-sectional prOfileS.
(0) iieot,ion about 0.4 pm thick. Almost all of the myosin filaments are clearly Gang&r with each apex of the triangle blunted. (d) The cross-sectional profiIe of the shaft of the myosin filament expected from the model proposed by Pepe (1966,1967) in which 12 structural units are packed parallel to the long axis of the filament to give a triangular profile with a structural unit missing at each apex of the wiangle as shown in (e). This Figure is a modification of Fig. 2 appearing in Pepe (1975).
Although this may be explainable in terms of very slight tilt, a similar effect could be produced if the structural units themselves that make up the filament have elongated cross-sectional profiles. The substructure spacings measured in sections 0.2 pm, 0.3 pm and 0.4 pm thick are summarized in Table 1. It was found that the spacings were Iarger in the thinner sections. The reason for this may be that thin sections are more likely to spread out, on the trough liquid during sectioning and also during staining of the sections. Comparing tire distribution of substructure spacings measured at different section thicknesses and by applying t-statistics it was found that the difference between the 0.2 ,urn and 0.3 ,urn sections is highly significant (P 0.5). This suggests
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(b) FIG. 2. Optical diffraction patterns obtained from eiectron micrographs of cross-sections of myosin filaments. (a) Three myosin filaments were included in the area of diffraction. The spacing between the small dots represents the interfilament spacing. The spacing between the large spots corresponds to the sFa:ing of the substructure in the shaft of the myosin filament, which measures about 4 nm. The substructure spacing is observable in 3 directions and the intensity in different dire&Ions is not the same. (b) From 6 to 12 myosin filaments were included in the area of diffraction. This pattern is similar to that in (a) except that the substructure spacing is seen clearly in only 2 directions.
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that jn sections 0.3 pm to 0.4 ,am thick there is minimal increase in spacing of the substructure due to the handling procedure. Assuming that even at these section thicknesses this has not been entirely eliminated, the best estimate for the substructure spacing is about 4 nm (Table 1). TABLE
1
Xubstructure spacing as a function of section thickness measured from optical diffraction patterns obtained from groups of 6 to 12 myosin Jilaments Section thickness (Pm) 0.4 0.3 0.2
Mean spacing*s.D. (nm)
No. of measurements
4.4hl.O 4.750.8 53*0.7
17 74 130
(c) Optical diffraction patterns from individual
myosin j&me&s
A typical example of the electron micrographs used to obtain optical diffraction patterns from either groups of myosin filaments (Fig. 2) or individual myosin filaments (Fig. 3) is shown in Figure 4. This Figure was printed to bring out the substructure as clearly as possible, In the negative from which this micrograph was printed considerably more detail is present which cannot be reproduced in the print. The substructure spacing in many of the individual filaments is considerably clearer in one direction and this direction is different for different filaments. Examples of the three types of optical diffraction patterns obtained from crosssections of individual myosin filaments are shown in Figure 3. The substructure spacing was observed in either three directions (Fig. 3(a)), two directions (Fig. 3(b)) or only in one direction (Fig. 3(c)). Generally when the substructure spacing was observed in three directions, one direction was less intense than the other two. The possibility that these different patterns might be due to different degrees of tilt of the filaments was excluded by the fact that three neighboring filaments with different orientations of the substructure spacing in one direction could be observed (Fig. 5). If t,he single direction observed for each filament in Figure 5 was due to tilt, then neighboring filaments having the same direction of tilt should show substructure spacing in the same direction. Therefore, the substructure spacings observed in one direction in the optical diffraction patterns from the filaments in Figure 5 must reflect some aspect of the internal structure of the filament. The difference in relative orientation bet.ween the three filaments in Figure 5 suggests the presence of a superlattice similar to that suggested by Huxley & Brown (1967). Five plates were observed in which such a superlattice arrangement was clear. The number of filaments that could be identified as fitting this superlattice in the five plates was 9, 8, 5, 5 and 4 filaments in ea#chof the plates, respectively. In Figure 6(a), the frequency distribution of substructure spacing observed from individual myosin filaments is shown These include the best observations made over the entire range of section thicknesses studied. It is noteworthy that on applying
F.
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(b)
(Cl
FIG . 3. Optit Cal difl indivi’ dual mya sub&r 7rcture in (4 A filamel in one : direction (b) A filame~ (0) A filamer
of c.toss-se< :tion; 9 of action patterns obtained from electron micrographs Isir L fila nents. The spacing between the spots corresponds to the spacin g of the filament which is about 4 nm. tlx mesh ,ft of the myosin -it whit I gave substructure spacing in 3 directions. The inter x&y of the spar :ing L is less ,han that in the other 2 directions. showed substructure spacing in 2 directions. nt whit 31I clearly 1t 1whio h L showed substructure spacing in only 1 direction.
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FIG. 4. Typical cross-section used for optical diffraction. ‘This microgrsph has been printed to bring out its much of the substructure in the myosin filaments as possible. There is considerably more information in the negative that cannot be reproduced in the print,. The substructure in many individual fikments is predominantly in one direction, the direction being different in different filaments.
(b)
Cd) FIG. 5. The relative orientation of neighboring filaments showing substructure spacing in only one direction. The 3 optical diffraction patterns in (a), (b) and (c) obtained from the corresponding filaments outlined in (d). The relative orientation of the diffraction patterns and the filaments has been maintained. The fact that the direction of the substructure spacing is different in neighboring filaments each showing substructure spacing in one direction eliminates the possibility that the single direction is due to tilt of the filaments. Neighboring filaments therefore have different orientations on the long axis of the filament indicating the presenoe of a superlattice.
THE 20 -
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(a)
IO-
& E E 2
ii “0
(b) IO 0
t.lALdL 3.0
4.0
Substructure
spacirtqs from optical dlffraction
(nm)
FIG. 6. Frequency distribution of spacings observed in optical diffraction patterns from myosin filaments. (a) Measurements obtained from the best patterns observed from individual myosin filaments. Sections of different thicknesses were studied from about 25 different, electron micrographs. The mean spacing is 4.8kO.8 nm (S.D. from mean with 90 measurements). (b) Measurements obtained using sections 0.3 pm thick, and groups of 6 to 12 filaments. About 10 different micrographs were used. The mean spacing (Table 1) is 4.7kO.8 nm (S.D. from mean with 74 measurements). (c) Measurements obtained using sections 0.2 pm thick, and groups of 6 to 12 filaments. About 10 different micrographs were used. The mean spacing (Table 1) is 5.3hO.7 nm (E.D. from mean with 130 measurements). Applying t-statistics the distribution of spacings observed for 0.4 pm and 0.3 pm se&ions (see Table 1 and (b) above) are not significantly different, (P > 0x5), and those for 0.3 ym sections (b) and individual filaments (a) are also not significantly different (P > 0.5). The distribution of spacings observed for 0.3 pm sections (b) and 0.2 pm sections (c) are significantly different (P > 0.50) between this distribution (Fig. 6(a)) and that obtained from groups of filaments in sections 0.3 pm thick (Fig. 6(b)). This supports our conclusion that the structural integrity of the filament is minimally disrupted in the thick sections. Some difference was observed in the spacings in different directions from individual myosin filaments. In Table 2 the average of the spacings observed in the same direction for different filaments in the same section is shown. It is clear that the average spacing in the three directions is the same, eliminating the possibility that compression in the direction of sectioning is responsible for the different spacings observed from indivual filaments.
(d) Optical diffraction patterns
from
models
If the structural elements in the shaft of the filament (a) have circular crosssectional profiles, (b) are spaced about 4 nm apart, and (c) are hexagonally packed (Fig. 7(a)), the optical diffraction pattern should give spacings of equal intensity in three directions (Fig. 7(b) and (c)). This was not observed either from groups of filaments (Fig. 2) or from individual filaments (Fig. 3). The optical diffraction pattern obtained from the cross-sectional profile of the filaments without substructure as in Figure l(d), is shown in Figure 7(d). The spacing of the hexagonal bands corresponds to the diameter of the profiles. This pattern was not observed with the typical electron micrographs (Fig. 4) used in this work. The observed difference in optical diffraction patterns from individual myosin filaments (Fig. 3) could result from elongated rather than circular cross-sectional profiles for the parallel structura.1 units in the shaft of the myosin filament. Two myosin molecules per structural unit would produce such an elongated crosssectional profile. In the model in Figure 8(c) each of the structural units spaced 4 nm apart in Figure 7(a) has been replaced by two myosin molecules. The optical diffraction pattern from this model gives the 4 nm spacing only in one direction. In Figure S(b) each structural unit has been rotated on its axis by 15" relative to its position in Figure S(c). Now the optical diffraction pattern gives spacings in all three directions with one direction being strongest. In Figure 8(a) each structural unit similarly has been rotated on its axis by 30” and now the optical diffraction pattern shows two strong directions with the third direction somewhat weaker. In all of these only the first-order reflections would be expected to be observed from actual filaments because these would be less highly ordered than the models. These patterns closely approximate the different patterns observed with individual myosin filaments (Fig. 3).
(e) Optical diffraction patterns from the a&in jilaments Optical diffraction patterns were obtained from groups of actin filaments as well as individual actin filaments. The areas chosen for diffraction were determined by first finding areas in which the myosin filaments alone gave good patterns and then obtaining diffraction patterns from the actin filaments in the same areas. It, was easy to find 15 different areas (with 4 myosin filaments in each area) which gave good diffraction patterns from the myosin filaments alone. Only seven of these areas gave measurable spacings from the actin filaments alone (8 to 12 actin filaments in each area). The patterns that were measurable from the groups of actin filaments alone
(b)
FIG.
proposed
7. Optical diffraction patterns from cross-sections of the model for the myosin iilameot by Pepe (1966,1967) in which each structural unit has a circular cross-sectional profile.
(a) A cross-section
through
the shaft of the filament
model proposed by Pepe (1966,1967).
(b) Optical diffraction pattern obtained from (a). Only the first-order spots would be expected to be observed from actual filaments since the substructure would be less well ordered than in the model. The intensity of the spots in all 3 directions is the same. (6) Optical diffraction pattern from a group of the filament mod& in (a). The spacing between the small dots represents the interfilament spacing. The larger spots represent the spacing of the substructure which is the same as that in (b). (d) Optical diffraction pattern from a group of the filament models in (a) but with the substructure obliterated to give solid profiles as shown in Fig. l(d). The spacing between the small dots represents the interfilament spacing and the spacing of the hexagonally arranged bright bands corresponds to the diameter of the solid profile. No evidence of substructure spacing is present.
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*.a*(b)
l ..fl*** . . . . . .
FIG. 8. Optical diffraction patterns from cross-sections of the model for the myosin filament proposed by Pepe (1966,1967) in which each structural unit consists of 2 myosin molecules producing an elongated cross-sectional profile for the structural unit. (a) Each structural unit in the model is positioned so that it is tilted by 30” from the horizontal. The optical diffraction pattern is shown on the right. Only the first-order spacings would be likely to be observed from actual filaments. The intensity of the spacing in one direction is weaker than in the other 2 directions. (b) Each structural unit in the model is tilted by 15’ from the horizontal. The optical diffraction pattern shows substructure spacing predominantly in one direction among the first-order spacings. (c) Each structural unit in the model is oriented horizontally. The optical diffraction pattern shows substructure spacing clearly in only one direction. These 3 patterns approximate those observed from indiviudal myosin filaments (Fig. 3).
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were extremely poor in quality compared to the patterns obtained from the groups of myosin filaments alone, in the same area. Similarly individual myosin and actin filaments in the same area were diffracted. Three different plates were used where the section thickness in two plates was about 0.25 ,um and in the third about 0.3 pm, Areas which gave good diffraction patterns from groups of myosin filaments were chosen and all of the myosin and actin filaments in these areas were studied individually. Totals of 54 myosin filaments and 87 actin filaments in these areas were studied. Another 27 actin filaments, which were along the edges of the areas were not conveniently located and were therefore not included. Of the 54 myosin filaments 46 gave measurable substructure spacings
0
3.0
I .I.1 4-o
I S-0
III,IIl
Substructure
6-O
I 7.0
I
II. 8.0
spacings from optical diffraction
I I 9-o
I IO.0
I I. I i-0
(nm)
Fro. 9. Frequency distribution of spacings observed in optical diffraction patterns from iridividuai myoain (a) and actin (b) filaments in the same area. (a) Individual myosin filaments. The measurements come from all of the filaments in specified areas, without selection for the best optical diffraction patterns from individual filaments as was done in Fig. l(a). The mean spacing is 5.010.7 nm (S.D. from the mean with 77 measurements). (b) Individual a&in filaments in the same area used to obtain the measurements in (a.) The mean spacing is 6.OjI 1.5 nm (S.D. from the mean with 85 measurements). Applying t-statist,ics, the difference between these 2 distributions obtained from the same areas of the electron micrographs is highly significant (P < 0.001).
(59 f 0.7 nm, S.D. from the mean with 77 measurements). Of the 87 actin filaments 56 showed some spacings though of very poor quality (6.0 5 1.5 nm, S.D. from the mean with 85 measurements). The distribution of spacings for the individual myosin and actin filaments in the same area is shown in Figure 9(a) and (b), respectively. Applying t-statistics the difference between these two distributions is highly significant (P 05) was found in the distribution of spacings obtained from individual filaments (Fig. 6(a)) and f som groups of filaments in sections 0.3 pm thick (Fig. 6(b)). Since the best patterns from individual filaments must come from the best preserved filaments this is consistent with filament structure being best preserved in the thick sections.
(c) Xuperlattice
of$laments
in the A-band
Three different optical diffraction patterns were observed from cross-sections of individual myosin filaments (Fig. 3). One of these was characterized by substructure spacing in only one direction (Fig. 3(c)). It is ’ p ossible that such a pattern could be obtained by slightly tilting the filaments along one of the lattice planes for substructure packing. However, as is observed in Figure 5, neighboring filaments each showing substructure spacing in a different direction could be found, which eliminates the possibility that tilt is responsible. This means that neighboring filaments in the A-band are rotated on their long axis relative to one another giving a superlattice arrangement. On the basis of X-ray diffraction patterns arising from the myosin cross-bridges Huxley & Brown (1967) proposed a superlattice arrangement for the myosin filaments in the A-band, in which each filament is rotated on its long axis by 120” relative to each neighboring filament. In this study, the necessity to satisfy both of the conditions required to observe the superlattice, i.e. individual filaments with substructure spacing in only one direction and no significant tilt (less than 3/4”), as well as having to satisfy these over a large enough area is extremely difficult. In five electron micrographs these conditions were satisfied in areas including 9,8,5,5 and 4 filaments, respectively. The direction of substructure spacing in neighboring filaments approximated that expected by the superlattice arrangement described by Huxley & Brown (1967); in that neighboring filaments had different directions and that equivalent filaments in the superlattice had closely similar directions.
(d) Number of myosin molecdes per structural
unit
With a substructure spacing of 4 nm it is likely that each structural unit is made up of more than one myosin molecule. There is controversy in the literature about the number of myosin molecules in the myosin filament (Tregear & Squire, 1973; Morimoto & Harrington, 1974) and about the number of myosin cross-bridges on the surface of the filament, i.e. the helical arrangement of myosin cross-bridges (Pepe, 1966,1967; Huxley & Brown, 1967; Squire, 1973). Both of these are related to the question of the number of myosin molecules per structural unit. The three different optical diffraction patterns observed from individual filaments (Fig. 3) give us some information about the cross-sectional profiles of the structural units. These three different patterns mean that the cross-sectional profile of the structural unit, cannot be circular, since if this were so, substructure spacings of equal intensity in all three directions would aIways be observed (Fig. 7). An elongated cross-sectional profile for each structural unit could be responsibIe for these patterns (Fig. 3). With an elongated
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profile and 4 nm spacing between structural units only two myosin molecules can be accommodated per structural unit. Assuming two myosin molecules per structural unit in the model proposed by Pepe (1966,1967), models were drawn for a cross-section through the shaft of the filament, and optical diffraction patterns were obtained from them (Fig. 8). In each of the three cases shown the structural units are all similarly oriented but each unit is rotated in intervals of 15” about the point of contact between the two molecules. The optical diffraction patterns (Fig. 8) are clearly similar to those observed from individual filaments (Fig. 3) in that a single direction is observed in Figure 8(c) and three directions are observed in Figure 8(a) with a pattern intermediate between these ic Figure 8(b). The observed pattern of two directions (Fig. 3(b)) most likely corresponds to the interval between Figures 8(a) and (b). The similarity between the observed patterns (Fig. 3) and those obtained from the model (Fig. 8) strongIy supports an elongated cross-sectional profile for each structural unit and therefore is a strong argument in favor of two myosin molecules per structural unit. It has been shown that even at high ionic strength myosin exists in solution as a dimer with a stagger of 43 nm between the molecules (Harrington $ Burke, 1972; Burke $ Harrington, 1972). Each of the parallel linear aggregates in the model proposed by Pepe (1966,1967) can be built by the linear aggregation of these dimers, thus making two myosin molecules the basic building block or the structural unit for the myosin filament, consistent with the observations made in this work. It is likely that the three different patterns observed from individual myosin fila,ments (Fig. 3) represent sampling along different portions of the myosin filament rather than different filaments with different relative orientation of elongated substructural profiles. If this is so, then there must be a twist of the structural unit about the line of contact between the two myosin molecules. Since patterns consistent with the presence of elongated cross-sectional profiles are observed in cross-sections O-2 pm to O-3 pm thick, the angle of twist over this length of the filament must be small enough not to significantly round out the projection of the structural unit in the plane of the cross-section. We are presently obtaining serial cross-sections through individual filaments for observation of the optical diffraction patterns from different, portions of the same filament. It should be possible to observe the three patterns in Figure 3 from different portions of the same filament and this will give us information about the pitch of the helical twist.
This work was supported We are grateful to Barbara parts of this work.
by grant, HL-15835 to the Pennsylvania Muscle Institute. Drucker, Karen Hsia and Mary Stevens for assistance in
REFERENCES Baceetti, B. (1965). J. Ultrastruct. Res. 13, 245-256. Baccetti, B. (1966). Bol. Xoc. Ital. Biol. &per. 42, 1181-1184. Burke, M. & Harrington, W. F. (1972). Biochemistry, 11, 1456-1462. Chowrashi, P. K. & Pepe, F. A. (1971). Proc. .EYirst Europ. Biophys. Congr. (Baden, Austria) (Broda, E., Locker, A. & Springer-Lederer, eds), pp. 429-436, Verlag dar Wiener Medizinischen Akademie, Vienna. E!ljott, A., Lowy, J., Parry, D.A. D. & Vibert, P. J. (1968). ~Vuture (LondoFon), 218,656-659. Gilev, V. P. (1966a). Biochim. Biophys. Acta, 112, 340-345.
218 Gilev,
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V. P. (19668). In Electron Microscopy (Uyeda, R., ed.), vol. 2, pp. 689-690, Maruzen Co. Ltd, Tokyo. Harrington, W. F. & Burke, M. (1972). Biochemistry, 11, 1448-1455. Huxley, H. E. (1963). J. Mol. BioZ. 7, 281-308. Huxley, H. E. & Brown, W. (1967). J. Mol. BioZ. 30, 383-434. Katsura, I. & Noda, H. (1973). J. &o&em. 73, 257-268. King, M. V. & Young, M. (1970). J. Mol. Biol. 50, 491-507. Lowey, S., Goldstein, L., Cohen, C. 85 Luck, S. M. (1967). J. Mol. BioZ. 23, 287-304. Markham, R., Frey, S. & Hills, G. H. (1963). Virology, 20, 88-102. Moos, C. (1972). Cold Spring Harbor Symp. Qua&. Biol. 37, 93-95. Morimoto, K. & Harrington, W. F. (1974). J. Mol. BioZ. 83, 83-97. Nakamura, A., Sreter, F. & Gergely, J. (1971). J. Cell Biol. 49> 883-898. Pepe, F. A. (1966). In Electron Microscopy (Uyeda, R., ed.), vol. 2, pp. 53-54, Maruzen Co. Ltd, Tokyo. Pepe, F. A. (1967). J. Mol. Biol. 27, 203-225. Pepe, F. A. (1971). Progr. Biophys. Mol. Biol. 22, 77-96. Pepe, F. A. (1975). J. Histochem. Cytochem. 23, 543-562. Pepe, F. A. & Drucker, B. (1972). J. Cell BioZ. 52, 255-260. Squire, J. (1973). J. Mol. Biol. 77, 291-323. Sreter, F., Holtzer, S., Gergely, J. & Holtzer, H. (1972). J. Cell BioZ. 55, 586-594. Szent-Gyorgyi, A. G., Cohen, C. & Philpot, D. E. (1960). J. Mol. BioZ. 2, 133-142. Tregear, R. T. & Squire, J. M. (1973). J. Mol. Biol. 77, 279-290.
BioZ. (1977) 113, 199-218
The Myosin Filament V.+ Intermediate Voltage Electron Microscopy and Optical Diffraction Studies of the Substructure FRANK A.~ PEPE AND PETER DOWBEN Department of Anatomy ,!edical Xchool, University of Pennsylvaka Philadelphia, Pa 19174, U.X.A. (Received 27 July
1976, and in revised form 3 January
1977)
Using a 200 kV electron microscope (JEM FOOA), thick (up to 0.4 pm) crosssections of the myosin filaments of vertebrate striated muscle were studied. It was found that: (a) with increasing section thickness the cross-sectional profiles of the shaft of the filament were increasingly more triangular and in sections 0.4 pm thick each apex of the triangle was clearly blunted. This unique crosssectiorial profile is predicted by the model proposed by Pepe (1966,1967) in which 12 parallel structural units are packed to form a triangular profile with a structural unit missing at each apex of the triangle. (b) With increasing section thickness the substructure of the myosin filament was enhanced, with the best substructure visible in sections 0.2 pm to 0.3 pm thick. This strongly supports parallel alignment of structural units in the shaft of the filament as proposed by Pepe (1966,1967). (c) The substructure spacing, determined by optical diffraction from electron micrographs of cross-sections of individual myosin filaments or groups of filaments is about 4 nm. (d) The different optical diffraction patterns observed from individual myosin filaments can be explained if the projection of each structural unit in the plane of the section has an elongated profile. With a substructure spacing of 4 nm an elongated cross-sectional profile could be produced by having two myosin molecules per structural unit. Models drawn with two myosin molecules per structural unit in the model proposed by Pepe (1966,1967) gave optical diffraction patterns similar to those observed from individual filaments. (e) The different optical diffraction patterns observed from individual myosin filaments can be explained if the elongated profiles in each structural unit are similarly oriented but with the orientation changing along the length of the filament. The change in orientation per unit length of the filament must be small enough to maintain an elongated profile for the projection of the structural unit in the plane of the sections 0.3 pm thick. All of these observations and conclusions strongly support the model for the myosin frla.ment proposed by Pepe (1966,1967).
1. Introduction Studies of the detailed structure of vertebrate muscle myosin filament’s have generally been limited to the possible arrangement of myosin cross-bridges on the surface of the filament deduced from X-ray diffraction data (Huxley & Brown, 1967). Three t Paper IV in this series is Pepe & Drucker (1972). 199
200
F. A. PEPE
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possible helical arrangements have been suggested (Squire, 1973) which are all consistent with this data. It can be shown, from X-ray diffraction that there are myosin cross-bridges at intervals of 14 nm along the filament with a helical repeat of 43 nm (Huxley & Brown, 1967). This indicates that staggers of I4 nm and 43 nm occur between myosin molecules in the filament but provides no information about the packing arrangement of the molecules. From electron microscope studies of the growth of synthetic myosin filaments from myosin solution, Huxley (1963) has concluded that the myosin molecules in one-half of the filament are all oriented opposite to those in the other half of the filament, but again no further information about the packing arrangement could be obtained from these studies. The light meromyosin fragment of myosin aggregates to form paracrystals in which presumably the packing arrangement should have some relation to that in the myosin filament. Light meromyosin paracrystals show axial repeats of 14 nm or 43 nm (Szent-Gyorgyi et al., 1960; Huxley, 1963; Lowey et al., 1967; King & Young, 1970; Chowrashi & Pepe, 1971; Nakamura et al., 1971; Sreter et al., 1972; Moos, 1972; Katsura I%Noda, 1973) indicating 14 nm or 43 nm stagger relations between molecules consistent with the X-ray diffraction data from myosin filaments, but likewise giving no further information about the packing of the molecules. Two different models have been proposed for the detailed packing of myosin molecules in vertebrate muscle myosin filaments (Pepe, 1966,1967; Squire, 1973). One of the models (Squire, 1973) was deduced from a general packing scheme in which all the myosin molecules are equivalent. The other (Pepe, 1966,1967) was deduced by relating the general structural characteristics of the A-band and of myosin filaments. The two models are clearly different. The basic distinctive features of the two models are as follows : Pepe (1966,1967) proposes t’hat the myosin filament is built up of structural units which can consist of one or more than one myosin molecule. The structural units in this model are all parallel to the long axis of the filament. In the shaft of the filament a cross-section will pass through 12 structural units packed to give a triangular profile with a structura~l unit missing at each apex. Squire (1973) proposes that the structural unit for the myosin filament is a single myosin molecule. The myosin molecules in this model are tilted both with respect to the long axis of the myosin filament and around the long axis of the filament, the angles of tilt being about 3”. In order to test the two models with respect to these distinctly different characteristics we have used an intermediate voltage (200 kV) electron microscope to observe the cross-sectional profiles in thick cross-sections of the myosin filaments. We have analyzed the electron micrographs by optical diffraction for evidence of substructure in the shaft of the filament. In Squire’s model (1973), since the myosin molecules are tilted both away from and around the long axis of the filament, increasing the section thickness (up to 0.4 pm) should lead to obliteration of visible substructure in the shaft of the filament with the appearance of increasingly more circular cross-sectional profiles. Also, the substructure spacing, if observable in thin sections, should be about 2 nm which is the packing distance of coiled-coils (Elliott et al., 1968) of which the myosin rod is an example. In Pepe’s model (1966,1967) exactly the opposite results should be observed. Since the structural units in this model are all parallel to the long axis of the filament, increasing the thickness (up to 0.4 pm) of a crosssection should lead to more observable substructure in the shaft of the filament. Also, the triangular cross-sectional profile of the shaft of the filament should be
THE
MYOSIN
FILAMENT.
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20;
enhanced with increasing section thickness. In this model, the substructure spacing can be larger than 2 nm if there is more than one myosin moIecule per structural Unit. We ha.ve found that: (a) as the thickness of the cross-section increases (up to 0.4 ,~,m)the cross-sectional profile of the filament becomes increasingly more clearly t,riangular. In addition, each apex of the triangular profile is blunt’ed as predicted by the model (Pepe, 1966,1967), where 12 parallel structural units are hexagonally packed to give a triangular profile with one structural unit missing at each apex. (b) As the section thickness increases, there is an enchancement of the substructure in the shaft of the myosin filament observable by optical diffraction. The substructure spacing is about 4 nm, suggesting that there is more than one myosin molecule per structural unit. (c) The optical diffraction patterns obtained from images of crosssections of individual fiIaments suggest that the structural units have elongated profiles (as might be expected for two myosin molecules side by side) all similarly oriented and that the oreintation of these elongated profiles changes along the length of the filament, These observations strongly support the model proposed by Pepe (1966#:1967)and are not compatible with the model proposed by Squire (1973).
2. Materials and Methods All of this work was done using the lateral muscles of the fresh water killifish, Fundulua diaphccnus. The fresh muscle was fixed at 2 to 4°C for a minimum of 2 h in 6% buffered glutaraldehyde solution containing 0.1 M-KCl, 1 m&r-MgCl, and 10 m&%-phosphate at $1 7-O. The muscle was dissected into small fiber segments or bundles. This was followed by fixation in 1% 0~0, buffered in the same solution, and it was then embedded in Araldite. Sections were obtained with thicknesses ranging from 0.06 pm to 0.4 pm. These were picked up on uncoated grids. The grids were then immersed for 3 h in 10% uranyl acetate solution in methanol and followed by 4 brief rinses in methanol, each rinse being carried out in a separate container. They were then immersed for 3 h in a 0.5% solution of lead each rinse carried out in a citrate in 50% ethanol, followed by 4 rinses in 5076 ethanol, separate container. This cycle of staining was carried out a second and third time (Pepe, B975). Sections about 1~5 pm thick were stained in this way using one cycle of alcoholic uranyl acid and lead citrate. These were then embedded in Araldite and sectioned transversely to observe penetration of the stain. The heaviest staining occurred to a depth of about 0.5 pm from the surface of the section (G. Tabas, A. Reingold & F. Pepe, unpublished resuhs). Therefore 3 cycles of staining are more than sufficient to ensure penetration of the stain from both sides of sections O-4 pm or Iess in thickness. The sections (thicknesses ranging from 0.06 pm to 0.4 pm) were all observed in a JEM 200 A electron microscope at a.n accelerating voltage of 200 kY using a 50 pm objective aperture. Before observation the sections were coated with a carbon film. Since observation of substructure in the shaft of the filament was the object of these studies, crosssections of the myofibrils were always used. The sections were tilted until the best visual alignment of the filament, perpendicular to the plane of the section was obtained. In some cases several pictures were taken of the same area at several angles of tilt differing by I” or 2”. The side entry goniometer on the instrument permitted tilting over 360” rotation of the image. The micrographs were obtained on Kodak 4489 electron microscope film (3-5 in x 4 in) and were developed in full-strength Kodak D-19 developer for 4 min at 20°C. Relatively dense electron micrographs were used because it was found that these were best suited for optical diffraction. Optical diffraction patterns were obtained using a preassembled unit, Electron Micrograph Optical Diffractometer ESOOO, purchased from Polaron Equipment Limited, England. For optical diffraction from the myosin filaments, the actin filaments were
202
P. A. PEPE
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painted out with black paint. The area to be diffracted was limited by using parallel strips of tape as a mask. In general the background of the negative was opaque enough so t,hat a mask function was not observed in the optical diffraction pattern. Areas containing 6 to 12 myosin filaments were used in addition to single myosin filaments. Optical diffraction patterns were adso obtained from actin filaments as a control on the results obtained with the myosin filaments. When the actin filaments were studied the myosin filaments were painted out with black paint. Optical diffraction was attempted from actin filaments only in areas of the electron micrograph where the myosin filaments gave good diffraction patterns.
3. Results (a) Observations
of cross-sectional projlles of myosin filaments function of section thickness
as a
Cross-sections of the A-band with thickness in the range of 0.06 ,um to O-4 pm were observed. The purpose of this was specifically to observe if the cross-sectional profiles become more distinctly circular or more distinctly triangular with increasing section thickness. In the thinner sections the cross-sectional profiles of the thick filaments were generally irregular in shape although occasional triangular profiles were observed (Fig. l(a)). As the section thickness was increased, more clearly triangular crosssectional profiles were observed (Fig. l(b)) and at a section thickness of about O-4 pm the cross-sectional profiles were unmistakably triangular with each apex of the triangle blunt’ed (Fig. l(c)). In making these observations, cross-sections occurring through a short region of the filament immediately adjacent to each side of the M-band were avoided. In this region of the filament triangular profiles are clearly observed in thin sections (Pepe, 1966,1967,1971). In the thickest sections (O-4 pm) it was not always possible to be sure that these portions of the filament were not included in the section. However, if they were included they would represent a maximum of about 0.08 pm of the length of the filament or only about one-fifth of the total length of the filament in the O-4 pm thick section, and therefore would not contribute significantly to determining the cross-sectional profile observed. (b) Optical diflraction patterns from myosin filaments as a function of section thickness Optical diffraction patterns were obtained from the electron micrographs after painting out the actin filaments. In general 6 to 12 filaments were included in the area of diffraction. In sections 0.1 pm or fess in thickness, it wa.s difficult to find any areas in which spacings other than the interfilament distance were observed. The clearest observations of substructure spacing were made with sections that were about 0.2 pm or more in thickness. Optical diffraction patterns similar to those in Figure 2 were observed. In general, sections about 0.2 pm to 0.3 pm in t’hickness gave optical diffraction patterns showing substructure in either two or three directions (Fig. 2). With sections about 0.4 pm thick the substructure spacing was observed predominantly in one direction. In the thickest sections even very small angles of tilt will result in decrease or elimination of spacings in directions other than that perpendicular to the direction of tilt: giving substructure spacing in only one direction. A general feature of the optical diffraction patterns was that the intensity of the substructure spacings observed in different directions was not the same (Fig. 2).
THE
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FIG. 1. Cross-sectional profiles of myosin filaments as a function of section thickness. Muscle is from the fresh water k&fish, P. d&-q&znus. (a) Section about 0.09 pm thick. Most of the myosin filaments have roughly circular and solid. cross-sectional profiles. Occasional filaments appear triangular. (b) Section about 0.25 pm thick. Many of the myoain filament,s have triangular cross-sectional prOfileS.
(0) iieot,ion about 0.4 pm thick. Almost all of the myosin filaments are clearly Gang&r with each apex of the triangle blunted. (d) The cross-sectional profiIe of the shaft of the myosin filament expected from the model proposed by Pepe (1966,1967) in which 12 structural units are packed parallel to the long axis of the filament to give a triangular profile with a structural unit missing at each apex of the wiangle as shown in (e). This Figure is a modification of Fig. 2 appearing in Pepe (1975).
Although this may be explainable in terms of very slight tilt, a similar effect could be produced if the structural units themselves that make up the filament have elongated cross-sectional profiles. The substructure spacings measured in sections 0.2 pm, 0.3 pm and 0.4 pm thick are summarized in Table 1. It was found that the spacings were Iarger in the thinner sections. The reason for this may be that thin sections are more likely to spread out, on the trough liquid during sectioning and also during staining of the sections. Comparing tire distribution of substructure spacings measured at different section thicknesses and by applying t-statistics it was found that the difference between the 0.2 ,urn and 0.3 ,urn sections is highly significant (P 0.5). This suggests
204
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(b) FIG. 2. Optical diffraction patterns obtained from eiectron micrographs of cross-sections of myosin filaments. (a) Three myosin filaments were included in the area of diffraction. The spacing between the small dots represents the interfilament spacing. The spacing between the large spots corresponds to the sFa:ing of the substructure in the shaft of the myosin filament, which measures about 4 nm. The substructure spacing is observable in 3 directions and the intensity in different dire&Ions is not the same. (b) From 6 to 12 myosin filaments were included in the area of diffraction. This pattern is similar to that in (a) except that the substructure spacing is seen clearly in only 2 directions.
THE
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205
that jn sections 0.3 pm to 0.4 ,am thick there is minimal increase in spacing of the substructure due to the handling procedure. Assuming that even at these section thicknesses this has not been entirely eliminated, the best estimate for the substructure spacing is about 4 nm (Table 1). TABLE
1
Xubstructure spacing as a function of section thickness measured from optical diffraction patterns obtained from groups of 6 to 12 myosin Jilaments Section thickness (Pm) 0.4 0.3 0.2
Mean spacing*s.D. (nm)
No. of measurements
4.4hl.O 4.750.8 53*0.7
17 74 130
(c) Optical diffraction patterns from individual
myosin j&me&s
A typical example of the electron micrographs used to obtain optical diffraction patterns from either groups of myosin filaments (Fig. 2) or individual myosin filaments (Fig. 3) is shown in Figure 4. This Figure was printed to bring out the substructure as clearly as possible, In the negative from which this micrograph was printed considerably more detail is present which cannot be reproduced in the print. The substructure spacing in many of the individual filaments is considerably clearer in one direction and this direction is different for different filaments. Examples of the three types of optical diffraction patterns obtained from crosssections of individual myosin filaments are shown in Figure 3. The substructure spacing was observed in either three directions (Fig. 3(a)), two directions (Fig. 3(b)) or only in one direction (Fig. 3(c)). Generally when the substructure spacing was observed in three directions, one direction was less intense than the other two. The possibility that these different patterns might be due to different degrees of tilt of the filaments was excluded by the fact that three neighboring filaments with different orientations of the substructure spacing in one direction could be observed (Fig. 5). If t,he single direction observed for each filament in Figure 5 was due to tilt, then neighboring filaments having the same direction of tilt should show substructure spacing in the same direction. Therefore, the substructure spacings observed in one direction in the optical diffraction patterns from the filaments in Figure 5 must reflect some aspect of the internal structure of the filament. The difference in relative orientation bet.ween the three filaments in Figure 5 suggests the presence of a superlattice similar to that suggested by Huxley & Brown (1967). Five plates were observed in which such a superlattice arrangement was clear. The number of filaments that could be identified as fitting this superlattice in the five plates was 9, 8, 5, 5 and 4 filaments in ea#chof the plates, respectively. In Figure 6(a), the frequency distribution of substructure spacing observed from individual myosin filaments is shown These include the best observations made over the entire range of section thicknesses studied. It is noteworthy that on applying
F.
206
A.
PEPE
AND
P.
DOWBEN
(b)
(Cl
FIG . 3. Optit Cal difl indivi’ dual mya sub&r 7rcture in (4 A filamel in one : direction (b) A filame~ (0) A filamer
of c.toss-se< :tion; 9 of action patterns obtained from electron micrographs Isir L fila nents. The spacing between the spots corresponds to the spacin g of the filament which is about 4 nm. tlx mesh ,ft of the myosin -it whit I gave substructure spacing in 3 directions. The inter x&y of the spar :ing L is less ,han that in the other 2 directions. showed substructure spacing in 2 directions. nt whit 31I clearly 1t 1whio h L showed substructure spacing in only 1 direction.
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FIG. 4. Typical cross-section used for optical diffraction. ‘This microgrsph has been printed to bring out its much of the substructure in the myosin filaments as possible. There is considerably more information in the negative that cannot be reproduced in the print,. The substructure in many individual fikments is predominantly in one direction, the direction being different in different filaments.
(b)
Cd) FIG. 5. The relative orientation of neighboring filaments showing substructure spacing in only one direction. The 3 optical diffraction patterns in (a), (b) and (c) obtained from the corresponding filaments outlined in (d). The relative orientation of the diffraction patterns and the filaments has been maintained. The fact that the direction of the substructure spacing is different in neighboring filaments each showing substructure spacing in one direction eliminates the possibility that the single direction is due to tilt of the filaments. Neighboring filaments therefore have different orientations on the long axis of the filament indicating the presenoe of a superlattice.
THE 20 -
MYOSIN
FILAMENT.
V
209 -
(a)
IO-
& E E 2
ii “0
(b) IO 0
t.lALdL 3.0
4.0
Substructure
spacirtqs from optical dlffraction
(nm)
FIG. 6. Frequency distribution of spacings observed in optical diffraction patterns from myosin filaments. (a) Measurements obtained from the best patterns observed from individual myosin filaments. Sections of different thicknesses were studied from about 25 different, electron micrographs. The mean spacing is 4.8kO.8 nm (S.D. from mean with 90 measurements). (b) Measurements obtained using sections 0.3 pm thick, and groups of 6 to 12 filaments. About 10 different micrographs were used. The mean spacing (Table 1) is 4.7kO.8 nm (S.D. from mean with 74 measurements). (c) Measurements obtained using sections 0.2 pm thick, and groups of 6 to 12 filaments. About 10 different micrographs were used. The mean spacing (Table 1) is 5.3hO.7 nm (E.D. from mean with 130 measurements). Applying t-statistics the distribution of spacings observed for 0.4 pm and 0.3 pm se&ions (see Table 1 and (b) above) are not significantly different, (P > 0x5), and those for 0.3 ym sections (b) and individual filaments (a) are also not significantly different (P > 0.5). The distribution of spacings observed for 0.3 pm sections (b) and 0.2 pm sections (c) are significantly different (P > 0.50) between this distribution (Fig. 6(a)) and that obtained from groups of filaments in sections 0.3 pm thick (Fig. 6(b)). This supports our conclusion that the structural integrity of the filament is minimally disrupted in the thick sections. Some difference was observed in the spacings in different directions from individual myosin filaments. In Table 2 the average of the spacings observed in the same direction for different filaments in the same section is shown. It is clear that the average spacing in the three directions is the same, eliminating the possibility that compression in the direction of sectioning is responsible for the different spacings observed from indivual filaments.
(d) Optical diffraction patterns
from
models
If the structural elements in the shaft of the filament (a) have circular crosssectional profiles, (b) are spaced about 4 nm apart, and (c) are hexagonally packed (Fig. 7(a)), the optical diffraction pattern should give spacings of equal intensity in three directions (Fig. 7(b) and (c)). This was not observed either from groups of filaments (Fig. 2) or from individual filaments (Fig. 3). The optical diffraction pattern obtained from the cross-sectional profile of the filaments without substructure as in Figure l(d), is shown in Figure 7(d). The spacing of the hexagonal bands corresponds to the diameter of the profiles. This pattern was not observed with the typical electron micrographs (Fig. 4) used in this work. The observed difference in optical diffraction patterns from individual myosin filaments (Fig. 3) could result from elongated rather than circular cross-sectional profiles for the parallel structura.1 units in the shaft of the myosin filament. Two myosin molecules per structural unit would produce such an elongated crosssectional profile. In the model in Figure 8(c) each of the structural units spaced 4 nm apart in Figure 7(a) has been replaced by two myosin molecules. The optical diffraction pattern from this model gives the 4 nm spacing only in one direction. In Figure S(b) each structural unit has been rotated on its axis by 15" relative to its position in Figure S(c). Now the optical diffraction pattern gives spacings in all three directions with one direction being strongest. In Figure 8(a) each structural unit similarly has been rotated on its axis by 30” and now the optical diffraction pattern shows two strong directions with the third direction somewhat weaker. In all of these only the first-order reflections would be expected to be observed from actual filaments because these would be less highly ordered than the models. These patterns closely approximate the different patterns observed with individual myosin filaments (Fig. 3).
(e) Optical diffraction patterns from the a&in jilaments Optical diffraction patterns were obtained from groups of actin filaments as well as individual actin filaments. The areas chosen for diffraction were determined by first finding areas in which the myosin filaments alone gave good patterns and then obtaining diffraction patterns from the actin filaments in the same areas. It, was easy to find 15 different areas (with 4 myosin filaments in each area) which gave good diffraction patterns from the myosin filaments alone. Only seven of these areas gave measurable spacings from the actin filaments alone (8 to 12 actin filaments in each area). The patterns that were measurable from the groups of actin filaments alone
(b)
FIG.
proposed
7. Optical diffraction patterns from cross-sections of the model for the myosin iilameot by Pepe (1966,1967) in which each structural unit has a circular cross-sectional profile.
(a) A cross-section
through
the shaft of the filament
model proposed by Pepe (1966,1967).
(b) Optical diffraction pattern obtained from (a). Only the first-order spots would be expected to be observed from actual filaments since the substructure would be less well ordered than in the model. The intensity of the spots in all 3 directions is the same. (6) Optical diffraction pattern from a group of the filament mod& in (a). The spacing between the small dots represents the interfilament spacing. The larger spots represent the spacing of the substructure which is the same as that in (b). (d) Optical diffraction pattern from a group of the filament models in (a) but with the substructure obliterated to give solid profiles as shown in Fig. l(d). The spacing between the small dots represents the interfilament spacing and the spacing of the hexagonally arranged bright bands corresponds to the diameter of the solid profile. No evidence of substructure spacing is present.
212
F. A. PEPE
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*.a*(b)
l ..fl*** . . . . . .
FIG. 8. Optical diffraction patterns from cross-sections of the model for the myosin filament proposed by Pepe (1966,1967) in which each structural unit consists of 2 myosin molecules producing an elongated cross-sectional profile for the structural unit. (a) Each structural unit in the model is positioned so that it is tilted by 30” from the horizontal. The optical diffraction pattern is shown on the right. Only the first-order spacings would be likely to be observed from actual filaments. The intensity of the spacing in one direction is weaker than in the other 2 directions. (b) Each structural unit in the model is tilted by 15’ from the horizontal. The optical diffraction pattern shows substructure spacing predominantly in one direction among the first-order spacings. (c) Each structural unit in the model is oriented horizontally. The optical diffraction pattern shows substructure spacing clearly in only one direction. These 3 patterns approximate those observed from indiviudal myosin filaments (Fig. 3).
THE
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FILAMENT.
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were extremely poor in quality compared to the patterns obtained from the groups of myosin filaments alone, in the same area. Similarly individual myosin and actin filaments in the same area were diffracted. Three different plates were used where the section thickness in two plates was about 0.25 ,um and in the third about 0.3 pm, Areas which gave good diffraction patterns from groups of myosin filaments were chosen and all of the myosin and actin filaments in these areas were studied individually. Totals of 54 myosin filaments and 87 actin filaments in these areas were studied. Another 27 actin filaments, which were along the edges of the areas were not conveniently located and were therefore not included. Of the 54 myosin filaments 46 gave measurable substructure spacings
0
3.0
I .I.1 4-o
I S-0
III,IIl
Substructure
6-O
I 7.0
I
II. 8.0
spacings from optical diffraction
I I 9-o
I IO.0
I I. I i-0
(nm)
Fro. 9. Frequency distribution of spacings observed in optical diffraction patterns from iridividuai myoain (a) and actin (b) filaments in the same area. (a) Individual myosin filaments. The measurements come from all of the filaments in specified areas, without selection for the best optical diffraction patterns from individual filaments as was done in Fig. l(a). The mean spacing is 5.010.7 nm (S.D. from the mean with 77 measurements). (b) Individual a&in filaments in the same area used to obtain the measurements in (a.) The mean spacing is 6.OjI 1.5 nm (S.D. from the mean with 85 measurements). Applying t-statist,ics, the difference between these 2 distributions obtained from the same areas of the electron micrographs is highly significant (P < 0.001).
(59 f 0.7 nm, S.D. from the mean with 77 measurements). Of the 87 actin filaments 56 showed some spacings though of very poor quality (6.0 5 1.5 nm, S.D. from the mean with 85 measurements). The distribution of spacings for the individual myosin and actin filaments in the same area is shown in Figure 9(a) and (b), respectively. Applying t-statistics the difference between these two distributions is highly significant (P 05) was found in the distribution of spacings obtained from individual filaments (Fig. 6(a)) and f som groups of filaments in sections 0.3 pm thick (Fig. 6(b)). Since the best patterns from individual filaments must come from the best preserved filaments this is consistent with filament structure being best preserved in the thick sections.
(c) Xuperlattice
of$laments
in the A-band
Three different optical diffraction patterns were observed from cross-sections of individual myosin filaments (Fig. 3). One of these was characterized by substructure spacing in only one direction (Fig. 3(c)). It is ’ p ossible that such a pattern could be obtained by slightly tilting the filaments along one of the lattice planes for substructure packing. However, as is observed in Figure 5, neighboring filaments each showing substructure spacing in a different direction could be found, which eliminates the possibility that tilt is responsible. This means that neighboring filaments in the A-band are rotated on their long axis relative to one another giving a superlattice arrangement. On the basis of X-ray diffraction patterns arising from the myosin cross-bridges Huxley & Brown (1967) proposed a superlattice arrangement for the myosin filaments in the A-band, in which each filament is rotated on its long axis by 120” relative to each neighboring filament. In this study, the necessity to satisfy both of the conditions required to observe the superlattice, i.e. individual filaments with substructure spacing in only one direction and no significant tilt (less than 3/4”), as well as having to satisfy these over a large enough area is extremely difficult. In five electron micrographs these conditions were satisfied in areas including 9,8,5,5 and 4 filaments, respectively. The direction of substructure spacing in neighboring filaments approximated that expected by the superlattice arrangement described by Huxley & Brown (1967); in that neighboring filaments had different directions and that equivalent filaments in the superlattice had closely similar directions.
(d) Number of myosin molecdes per structural
unit
With a substructure spacing of 4 nm it is likely that each structural unit is made up of more than one myosin molecule. There is controversy in the literature about the number of myosin molecules in the myosin filament (Tregear & Squire, 1973; Morimoto & Harrington, 1974) and about the number of myosin cross-bridges on the surface of the filament, i.e. the helical arrangement of myosin cross-bridges (Pepe, 1966,1967; Huxley & Brown, 1967; Squire, 1973). Both of these are related to the question of the number of myosin molecules per structural unit. The three different optical diffraction patterns observed from individual filaments (Fig. 3) give us some information about the cross-sectional profiles of the structural units. These three different patterns mean that the cross-sectional profile of the structural unit, cannot be circular, since if this were so, substructure spacings of equal intensity in all three directions would aIways be observed (Fig. 7). An elongated cross-sectional profile for each structural unit could be responsibIe for these patterns (Fig. 3). With an elongated
THE
MYOSIN
FILAMENT.
V
21 I
profile and 4 nm spacing between structural units only two myosin molecules can be accommodated per structural unit. Assuming two myosin molecules per structural unit in the model proposed by Pepe (1966,1967), models were drawn for a cross-section through the shaft of the filament, and optical diffraction patterns were obtained from them (Fig. 8). In each of the three cases shown the structural units are all similarly oriented but each unit is rotated in intervals of 15” about the point of contact between the two molecules. The optical diffraction patterns (Fig. 8) are clearly similar to those observed from individual filaments (Fig. 3) in that a single direction is observed in Figure 8(c) and three directions are observed in Figure 8(a) with a pattern intermediate between these ic Figure 8(b). The observed pattern of two directions (Fig. 3(b)) most likely corresponds to the interval between Figures 8(a) and (b). The similarity between the observed patterns (Fig. 3) and those obtained from the model (Fig. 8) strongIy supports an elongated cross-sectional profile for each structural unit and therefore is a strong argument in favor of two myosin molecules per structural unit. It has been shown that even at high ionic strength myosin exists in solution as a dimer with a stagger of 43 nm between the molecules (Harrington $ Burke, 1972; Burke $ Harrington, 1972). Each of the parallel linear aggregates in the model proposed by Pepe (1966,1967) can be built by the linear aggregation of these dimers, thus making two myosin molecules the basic building block or the structural unit for the myosin filament, consistent with the observations made in this work. It is likely that the three different patterns observed from individual myosin fila,ments (Fig. 3) represent sampling along different portions of the myosin filament rather than different filaments with different relative orientation of elongated substructural profiles. If this is so, then there must be a twist of the structural unit about the line of contact between the two myosin molecules. Since patterns consistent with the presence of elongated cross-sectional profiles are observed in cross-sections O-2 pm to O-3 pm thick, the angle of twist over this length of the filament must be small enough not to significantly round out the projection of the structural unit in the plane of the cross-section. We are presently obtaining serial cross-sections through individual filaments for observation of the optical diffraction patterns from different, portions of the same filament. It should be possible to observe the three patterns in Figure 3 from different portions of the same filament and this will give us information about the pitch of the helical twist.
This work was supported We are grateful to Barbara parts of this work.
by grant, HL-15835 to the Pennsylvania Muscle Institute. Drucker, Karen Hsia and Mary Stevens for assistance in
REFERENCES Baceetti, B. (1965). J. Ultrastruct. Res. 13, 245-256. Baccetti, B. (1966). Bol. Xoc. Ital. Biol. &per. 42, 1181-1184. Burke, M. & Harrington, W. F. (1972). Biochemistry, 11, 1456-1462. Chowrashi, P. K. & Pepe, F. A. (1971). Proc. .EYirst Europ. Biophys. Congr. (Baden, Austria) (Broda, E., Locker, A. & Springer-Lederer, eds), pp. 429-436, Verlag dar Wiener Medizinischen Akademie, Vienna. E!ljott, A., Lowy, J., Parry, D.A. D. & Vibert, P. J. (1968). ~Vuture (LondoFon), 218,656-659. Gilev, V. P. (1966a). Biochim. Biophys. Acta, 112, 340-345.
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