Electron microscopy of side-by-side arrays of myosin and light meromyosin-C

J. Mol.

Biol. (1972) 63, 539-555

Electron Microscopy of Side-by-side Arrays of Myosin and Light Meromyosin-C MURRAYVERNON KING ANDMICHAELYOUNC Departments of Biological Chemistry and Medicine Harvard Medical School and the Massachusetts General Hospital, Boston, Mass. 02114, U.X.A.

(Received11 May 1971, and in a revised form 23 September

1971)

l3oth myosin and light meromyosin-C, a fragment prepared by cyanogen bromide cleavage, yield, under suitable solvent conditions, a variety of aggregates that have the form of side-by-side linear arrays. These resemble the segment structures obtained from myosin rods and light meromyosin derived from tryptic digestion (Cohen, Lowey, Harrison, Kendrick-Jones & Szent-Gyorgyi, 1970) and show that alternating antiparallel lateral packing is a common mode of aggregation among molecules containing the tail region of myosin. The aggregates exhibit a variety of overlap distances in response to changes in conditions and show that several patterns of molecular contacts are nearly equivalent energetically. Light meromyosin prepared by cyanogen bromidecleavage yields three segment phaseswhich, when measured, give a molecular length of about 1025 A. These phases differ in overlap length and striation pattern. One of them is a helically twisted ribbonlike structure, and another may be a bilayer. Myosin yields aligned segments, in which the molecular tails are in exact register only at high concentrations of alkaline-earth metals. These structures give a length of 1654 + 58 A for the myosin molecule. At lower concentrations of calcium or magnesiumions, myosin forms disordered segments and crossed segments in which overcrowding of the myosin heads is partially relieved by a random stagger in alignment of the tails. This process is facilitated by the ease with which myosin adopts alternative packing arrangements, and it throws some light on the morphogenesis of the thick filament of muscle.

1. Introduction One of the most prominent features of the structure of the thick filament in striated muscle is its bipolar symmetry. In existing models of the sarcomere, this type of symmetry is essential in explaining the contractile process. To describe the thick filament more fully, it possesses a bare central region containing the light meromyosin tails of myosin molecules, symmetrically flanked by regions in which the heavy meromyosin heads of these molecules protrude in different directions from a shank composedof LMMt tails (Huxley, 1963; Huxley & Brown, 1967). The symmetry of this

distinctive

structure

led to the idea that the central

region

(spanning

the M-line

and pseudo-H zone in intact muscle) contains myosin molecules oriented in both of the opposite fiber directions, whereas the myosin moleculesin either of the two terminal regions probably all point in the same direction. Huxley & Brown (1967) have t Abbreviations by digestion with

used: LMM, light cyanogen bromide.

meromyosin;

LMM-C, 539

LMM

fragment

of myosin

prepared

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M. V. KING

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proposed a model of the thick filament in which opposing pairs of HMN heads of the myosin moleculesprotrude from the filament every 143 A, with a 120’ rotation angle between successivesets. There is also someexperimental evidence on the periodicity of the thick filament in a recent study by Hanson, O’Brien & Bennett (1971), who have found a period of 420 & 15 A in intact A-segments. Most likely, the periodicity of the filament is established by the pattern of contacts made among the LMM tails of myosin molecules. Thus, any information that can be obtained about possible variants of parallel, or especially antiparallel, modes of packing of myosin may throw light on the structure and morphogenesisof the thick filament. Moreover, any information on the existence of alternative patterns of contact and on factors that influence what pattern the molecules adopt in forming aggregates under specific conditions will help in explaining the complexity of this structure. Segment structures have been prepared from myosin itself and from a fragment of myosin prepared by cyanogen bromide digestion of chicken myosin (Harrison, Lowey & Cohen, 1971), aswell as from myosin rods prepared by papain digestion and LMM prepared by tryptic digestion (Cohen, Lowey, Harrison, Kendrick-Jones & Szent-Gyorgyi, 1970). The latter authors also showed that whole myosin molecules can be incorporated into segment structures prepared from myosin rods. These structures have the form of ribbons, or planar arrays of strictly limited width, in which the moleculeslie transverse to the direction of growth of the ribbon. The most prevalent segment structures that they observed exhibited bipolar symmetry and thus resembled the thick filament itself. Characteristically, the bipolar segment structures possesseda central overlap zone formed by lateral contact of molecules that were alternately oriented in opposing directions. This central overlap zone was flanked on either side by a fringe, in which free ends of molecules protruded into the surrounding medium. We report here the preparation and study of analogoussegment phasesof myosin itself, as well as of a non-enzymically prepared fragment, LMM-C, which is produced by cyanogen bromide digestion of myosin (Young, Blanchard & Brown, 1968). This fragment has a length intermediate between those of the myosin rod and tryptic LMM, and has the favorable distinctive feature t’hat it contains no internal peptidebond cleavages. Examination of these structures by electron microscopy, as outlined below, has made it possibleto demonstrate alternative modesof antiparallel packing having differing overlap lengths, and to measure more accurately the length of the LMN-C molecule. Further, studies under a range of preparative conditions have allowed us to identify some of the factors which influence the choice of different modes of packing that the molecules adopt.

2. Materials

and Methods

(a) Materials Glass-distilled water and reagent-grade chemicals were used in all preparations. Light meromyosin-C was prepared as described by Young et al. (1968). Rabbit skeletal myosin was isolated as described by Kielley & Bradley (1956), and either used directly, or purified further by the chromatographic procedure of Richards, Chung, Menzel 85 Olcott (1967). All dialysis tubing (Visking) was boiled for 10 mm in 0.1 M-NaHCOa , then boiled for 10 min in 0.1 M- acetic acid, and finally washed exhaustively with glass-distilled water. Protein concentrations were measured with a Zeiss PMQII spectrophotometer at a wavelength of 280 nm and extinction coefficients of 0.54 ml./mg cm for myosin and 0.300 ml./mg cm for LMM-C (Young et al., 1968).

ARRAYS

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Preparation

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MEROMYOSIN-C

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of Sam&es

The several aggregated forms of myosin and LMM-C were prepared by the following methods. (1) Direct dialysis against (a) 0.5 M-ammonium acetate or (b) 0.225 M-CdCiUm acetate. (2) Dialysis against a solution in which the protein remained soluble, followed by gradual dilution of the dialysate with small portions of distilled water over a period of 1.5 to 4 months to a point slightly past incipient precipitation. The specific dialysis solvents used were: (a) 1 M-ammonium acetate alone; (b) 1 M-ammonium acetate containing varying amounts (O-01 to 0.1 M) of calcium acetate or magnesium chloride, occasionally with addition of KI (0.01 M) or KSCN (0.01 to 0.05 M) ; (c) 1 M-ammonium acetate containing 0.01 M-EDTA; (d) 1 M-ammOniUm acetate containing 0.12 m-Mgcls and 0.02 M-EDTA; (e) 0.5 M-barium acetate, calcium acetate or magnesium acetate; (f) a mixture containing 0.5 M-KCl, 0.1 M-calcium acetate, 0.01 M-Tris.HCl, pH 8.18.

(c) Electron

microscopy

Protein suspensions were diluted with their respective dialysis solvents to a protein concentration of about 0.05 mg/ml., and were deposited on carbon-coated 400-mesh copper grids that had been exposed to a glow discharge at 2 x 10-s Torr pressure to improve their wettability (Huxley & Zubay, 1960; Holland, 1961; Reissig & Orrell, 1970). Specimens were washed free of salts with 30 PM-KHCO, and fixed with 1 y0 glutaraldehyde adjusted to neutrality with NaHCOc. Preparations were then stained with uranyl oxalate, or ammonium molybdate. To improve many1 acetate, potassium phosphotungstate, further the wetting of specimens with the uranyl stains, these stains were modified by inclusion of 0.0025% Brij-35, a non-ionic surfactant (cf. Mordoh, Leloir & Krisman, 1965). This additive did not improve the behavior of the other stains, and therefore was omitted from them. Electron micrographs were taken with an RCA model EMU3-G instrument operated at 50 kV. The condenser and objective lenses contained a 200-pm and a 50-pm aperture, respectively. Magni6cation factors were established from a set of micrographs of a diffraction-grating replica (54,864 lines/in), and 15 to 20 measurements were taken per magnification tap. Alternatively, magnification factors were estimated by measurements on images of the tmtoid phase of LMM-C having a 143 A periodicity (phase B in King & Young, 1970). The day-by-day variation in magnification was less than 5%. Enlargement factors were determined from the image of a millimeter scale on a spectrometer plate that was printed concurrently with each set of micrographs.

3. Results (a) Segmentphasesof light meromyosin-C The various segment phasesof LMM-C were generally obtained by method (2)(a) (2)(f) from media containing no deliberately added alkaline-earth salts. Deliberate addition of the latter to the ammonium acetate dialysis solvent in most casesgave rise to other, non-segment, phases. On the other hand, addition of EDTA to the dialysis medium suppressedtheir appearance. Table 1 lists the observed segment phases of LMM-C, with the details of their preparation, overlap lengths (widths of the densely packed center strips), fringe widths, and molecular lengths derived therefrom. or

(i) Phase P Plate I shows a specimen of phase I? of LMM-C. This preparation was grown b;y method (2)(a). The salient features of this phase are: (1) There is a well-defined central strip of width 890 A, with a standard deviation of 17 A (i.e. a variation explainable by variation in magnification of the microscope alone).

043

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V.

KING

AND TABLE

M.

YOUNG

1

The segment phases of LMM-C Phase symbol

Description

F

Fringed

ribbon

L

Fringed

ribbon

N

Twisted

festoon

Growth medium and preparative method 0.59 M-ammonium acetate (method (2)(a)) 0.59 M-ammonium acetate (method (2)(a) or (l)(a)) 0.33 M-KCl, 0.066 calcium acetate (method (2)(f))

Dimensions Center

M-

(A)

strip, 890 Fringe, 119 Sum, 1009 Center strip, 830 Fringe, 220 sum, 1050 Width, 1029 Center strip, 252

f 17 f 44 f 47

f 43 3 19

(2) The fringes on both sides of the ribbon have a somewhat more ragged appearance than the central strip. This supports the idea that they represent regions in which the protruding ends of molecules are not supported by neighbors. The width of the fringes is 119 A, with a standard deviation of 44 A. (3) If we make the natural assumption that each molecule in the aggregate extends from the edge of one fringe through that fringe and the center strip to the opposite boundary of the center strip (but does not penetrate into the opposite fringe), then the molecular length must be the sum of the widths of one fringe and the central strip. This width is 1009 A, with a standard deviation of47 A, and it lies within the range of lengths (1000 1160 A) measured from the images of shadowed single molecules (King, O’Hara, Molberg & Young, 1970) ; but it has considerably superior precision. Presumably, the advantage of measuring the molecular length in specimens of an aggregated phase stems from the fact t,hat the greater part of the length of each molecule is embedded in the aggregate, and thus cannot bend. Also, the ends of the molecules are better delineated in the negatively stained specimens than in the shadowed specimensof single molecules. (4) Specimens of phase F appear to be bipolar in symmetry. In no specimen did the two fringes appear to be asymmetric in length or character, although in some local regions one fringe appeared to be more deeply embedded in stain than the opposite fringe was. (5) Specimensof this phase stained with uranyl compounds were striated with a pattern of fine lines running in the direction of growth. They also revealed transverse striations that indicated that the moleculeslay normal to the growth direction. The most intense striations lie along the midline of the center strip, and at positions 225 A from the midline on either side. They are interspersed with a set of fainter parallel lines that are roughly equally spaced (56 to 63 A), and which extend throughout the width of the center strip. In all cases,the striation pattern was symmetrical about the midline. No striations could be seenin the fringe areas. (ii) Phase L PhaseL of LMM-C is a segmentphasewhich can be grown under the same conditions as phase F (method (2)(a)), but from a different preparation of LMM-C, which has proved to give this phase consistently in lieu of F. This m-C preparation also yielded phase L when treated by method (l)(a).

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543

Plate 11 shows a specimen of phase L, together with tactoids plus a square net. This illustrates a common feature of most of these preparations of aggregates, in that the segment phases of LMM-C practically never appear alone, but are accompanied by other phases having non-segment structures. (Inclusion of the tactoid phase in the same picture made it possible to determine the magnification factor accurately.) The width of the overlap zone is 830 A, and that of the fringe is 220 A. By the same argument advanced for phase F, the molecular length should then be 1050 A, a value within one standard deviation of that obtained from phase F. While the central zone is characteristically narrower in phase L than in F, and the fringe correspondingly broader, this is not the most evident trait distinguishing these phases, for the differences are barely outside the limits of error. Rather, they differ distinctly in striation pattern. The pattern in phase L is dominated by light bands, interspersed with dark lines that do not show a regular periodicity, but are spaced about 54 A apart on the average. Thus, they have about the same spacing as the fainter lines in phase F, but are somewhat bolder in contrast than the latter. Another striking feature in this striation pattern is that the arrangement does not remain constant from end-to-end of the object, as it does in phase F. Rather, the lines tend to merge or separate in a manner resembling moire fringes. Perhaps the simplest explanation is that they are indeed moire fringes that arise from superposition of two like objects in slight disregister. This would then imply that phase L may be a bilayer structure composed of two superimposed ribbons similar, if not identical, to phase F. (iii) Phase N The last of the segment phasesof LMM-C that we have observed is phase N, which has thus far been prepared only by method (2)(f). These ribbons have a width of 1029 A, with a standard deviation of 43 8. A striking feature of phase N is that the ribbons are not flat, except in short terminal regions, or in very short specimens,but are helically twisted in a manner resembling crepe-paper festoons. The pitch of the helices varies somewhat, probably becauseof the strains involved in specimenpreparation, but it was found to average about 2550 A in the least deformed areas of the cluster of festoons pictured in Plate III. The objects seenhere appear to be all twisted in the same sense. Some of the micrographs show faint longitudinal striations with an average spacing of about 43 A. These are usually symmetrical about the midline, although one specimen appeared to be asymmetric. Thus, phase N also exhibits faint longitudinal striations exhibiting no exact periodicity, but with an average spacing in the range 40 to 65 A. Some of the more heavily stained specimensof phase N show a light center strip with a width of 252 A (standard deviation = 19 A). If we try to interpret this feature by the sametype of model as for phasesF and L, we get the extremely short molecular length of 640 A. Yet, the fact that this phase is the major constituent in an LlKM-C preparation that has otherwise yielded the normally expected phases renders this explanation unlikely. More likely, in view of the helical twist, we can assume thai; the center strip is a pivot area in which each molecule adheres to its neighbors on either side, while the ends are splayed out slightly by the helical twist, and do not make lateral contact with one another. For a model, see Figure 1. Note, however, that the width of the pivot area probably depends on an intricate pattern of sidechain contacts that establishes a volume from which stain is excluded. Thus, a structure of the type depicted in Figure 1 should show a center strip when stained

544

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AND

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__.-- PlVOf OXIS

I-

-

+ x Rfch

FIQ. 1. A schematic illustration of the postulated molecular packing in phase N of LMM-C. The angle of twist between consecutive molecules has been exaggerated from the actual values, which FSEI believed to be in the vicinity of 3’ (of. Plates III and IV).

negatively, although the width of this strip cannot be predicted from present knowledge. Therefore, the molecular length of LMM-C is most probably identical to the width (1029 + 43 d) of these structures. This interpretation is supported by the somewhat lacy appearance of the festoons, which again suggests that the molecules contact their neighbors only over a limited fraction of their lengths. Thus, we can describe phase N either as a segment phase having no fringe, in that no molecule extends out on either end appreciably beyond its neighbors, or as a phase that is mostly fringe, in that most of the length of each molecule is unsupported by its neighbors. Such a structure would arise if the lines of contact of adjacent molecules are slightly skewed with respect to the molecular axes. If we assume a molecular diameter of 20 A (Zobel & Carlsen, 1963), the skew angle must be about 3” to give the observed pitch of2550A. Plate IV, which shows phase N at higher magnification, shows the striations and the center strip, both in the long, well-developed festoon and in the shorter fragments. Note that some of the latter have lengths shorter than the 1029 d width, i.e. shorter than LMM-C molecules are. This is the most direct proof that the molecules lie transverse to the direction of growth of phase N. Occasionally, strands can be seen protruding from the ends of pieces of phase N, or extending across the gap between fragments of a broken aggregate. When they occur, such strands most often lie along the center strip, but they appear only sporadically, and they vary in width, unlike the center strips themselves. Thus, we do not consider protruding strands to represent an essential part of the st)ructure of phase N. Rather, they are probably molecules or aggregates of LMM-C that have adventitiously attached themselves to pieces of phase N during growth or during mounting. (iv) Comparison

of the segment phuses of LMH-C

One point to be established in all of these phases is whether the molecules are truly perpendicular to the direction of growth of the ribbons. The appearance of the micrographs suggests this in all cases, for they generally show fine transverse striations normal to the length, in addition to the above-described longitudinal striations. These striations are near the limit of resolution, and no transverse features on any coarser

ARRAYS

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AND

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MEROMYOSIN-C

545

scale can be seen. This suggests that the molecules actually run in this direction. However, we cannot rule out bilayer structures in which the molecules in each layer are slightly tipped from the normal to the growth direction, and the directions in the two layers are opposite. However, existence of small angles of tipping would have relatively little effect on estimates of the molecular length. Gross tipping should reveal itself in discernible features that we have not detected, with the possible exception of the moire appearance of phase L. Thus, the results for phases F, L and N alike are consistent with models for the segment phases consisting of linear arrays of the rodlike molecules. We also note that all three of these phases yield closely similar estimates of the molecular length of LMM-C in the vicinity of 1025 A. The question of the role of alkaline-earth cations in the formation of these phases is of interest, in line with the experience of Cohen et al. (1970) who obtained analogous segment phases from preparations of other myosin fragments to which calcium 01 magnesium had been added. In contrast, phases F and L of LMM-C have consistently appeared in various preparations having no added alkaline earths. Nevertheless, inclusion of EDTA in the dialysis media (method (2)(c)) completely suppressed for.. mation of these phases. This would suggest that traces of alkaline earths (or other metals) may suffice to induce segment growth. Such contaminants could have been carried in the LMM-C preparations themselves. However, traces of alkaline earths present in the reagent-grade chemicals might be effective in inducing segment growth. In support of this argument, we note that the specifications for analytical reagents established by the American Chemical Society (1968) permit amounts of calcium and magnesium that are often at a level of 10v4 mole fraction of the main component. (b) Xegment structures

of myosin

Comparison of myosin with its tail fragment (LMM-C) showed several marked contrasts in behavior. First, while both proteins could be converted into segment structures, the optimum conditions for growth of these structures differed substantially for myosin and LMM-C. Further, the segment structures of myosin cannot readily be classified into distinct phases. Indeed, the term phase is perhaps properly applicable only to the well-ordered aligned segments that are described in detail below, since the other aggregates of myosin seem to form a continuous series of objects varying in regularity of packing of the LMM tails of the myosin molecules within the overlap zone. Finally, myosin shows a strong tendency to form crossed segments, which are composites, each made of two distinct segment structures, one lying on top of the other with perpendicular orientations. On the other hand, none of the fragments of myosin has ever been observed to form comparable structures, (i) Aligned segment structures Plat,e V shows a typical view of these aggregates. Each of these objects has a wellmarked, transversely striated central zone. Outside this central zone, molecules (or bundles of molecules) extend out on either side, splaying out as they go. They finally terminate in a set of knobs that are densely clustered at the margins of the aggregates. We consider it natural to interpret the central zones as regions of overlap of the distal portions of the tails of myosin molecules inserted from both sides. In 6h.e lateral zones, then, the molecular shanks are less densely packed, as they are not supported by neighbors belonging to the antiparallel set of molecules. Thus, the molecules can flex readily in this region. Also, the heads of the myosin molecules

546

M. V. KING

AND

M. YOUNG

are considerably wider than the tails. Nevertheless, the exact register of the molecular tails in the overlap zone constrains the heads to lie at a certain dist,ance from that zone. Thus, since the heads are poorly accommodated within the width that the molecular tails occupy, they force the margins of the aggregat,es to splay out, and flex the unsupported portions of the tails. These aggregates could be obtained only at high alkaline-earth concentrations, preferably by gradual dilution (method (2)(e)) from 0.5 M-barium, calcium, or magnesium acetate to a concentration slightly below the point of incipient precipitation The final concentrations were from 0.22 to 0.36 M-calcium acetate or 0.30 M-magnesium acetate or 0.24 M-barium acetate. The aggregates obtained showed no variation in appearance over this range of concentrations, nor over a range of initial myosin concentrations from 0.046 mg/ml. to 2.3 mg/ml. Chromatographically purified myosin gave aggregates indistinguishable from those obtained from the ordinary myosin stock. Thus, the impurities removed by column chromatography have no role in the formation of these segments. Preparation by the much more abrupt procedure of direct dialysis against 0.225 M-calcium acetate gave a mixture of aggregates in which some typical aligned segments could be seen, but many of the aggregates in this preparation were disordered segments looking much like those obtained at low calcium concentrations (as described below). Apparently, the growth of aligned segments is favored by very slow precipitation. The average length of the overlap zone of the aligned segments from 37 measurements was 832 A, with a standard deviation of 22 A. The over-all head-to-head spans of the segments varied considerably more widely, as 14 observations gave a mean span of 2476 A, with a standard deviation of 115 A. This would suggest that t,he flexibility of the cephalad regions of the myosin tails permits some of them to buckle under the stresses arising from overcrowding of the heads. Nevertheless, since the Ootal span should be determined primarily by the positions of the heads rest,ing on the straighter stalks, we can thus obtain a good estimate of the total end-to-end length of a myosin molecule from the expression (L + a)/2, where I; is the mean overall span, and a is the mean overlap length. This estimate is 1654 A, with a standard deviation of 58 A. It may be compared with the values of 1100 A (Rice, 1961); or 1590*165 A (Zobel & Carlson, 1963); or 1420 A, which is t,he sum of the mean rod lengths and head diameters obtained by Lowey, Slayter, Weeds & Baker (1969); or the value 1520 A measured by Huxley (1963). (However, Huxley states bhat his best fields gave a greater mean length of 1680 A.) In contrast to the overlap lengths, the widths of the central shafts of the segment structures varied much more widely, with a mean of 393 A, and a standard deviation of 132 A (23 observations). Evident’ly, while the length of the overlap zone is an invariant structural feature of these aggregates (reflecting a highly regular antiparallel packing of the myosin tails), the widths vary. Nevertheless, it is remarkable that preparations grown over a 50-fold range of initial myosin concentrations give particles having the same range of widths. While the width direction of these aggregates is presumably structurally analogous to the growth direction of LMM-C segments,the myosin segmentsactually grow very little in this direction, as compared with the great lengths attained by LMM-C segments. Presumably, the overcrowding of the myosin heads may act as a growt’h-limiting feat,ure to prevent formation of long ribbons.

PLATE II. Phase L of LMM-C prepared by direct, dialysis against 0.5 w-ammonium accxtatv, stained with uranyl oxalata containing Brij-35. It is accompanied by tactoids and a square net. The arrows show the margins separating the overlap zone (840 il wide) from the fringes (220 x wide) on either side. A region in which some of the bright lines appear to merge is seen at, J\. Bar = 1000 A.

III. Phase E of LMM-C, prepared by dialysis with gradual dilution to a final concentraof 0.33 AI-KCl, 66 mM-cdcium metate, pH 8.18; stained with uranyl oxalate containing Rrij-35. Sate the helical twist of the ribbons. and also the demarcat.ion of each ribbon into a rc~utral acme (252 A v ide), flanked hy fringes on either side. Thv total widt,h of thP ribbons is 102!+ x. khw ~~~ I0,000 A. I'LATE

tivn

PLATE IV. Phase N of LMWC, prr:paw~L and st~ainetl as iu Plate III. at’ higher magnikxtion. Note the coexistence of a long, helically-twisted ribbon, t,ogethrr with many shorter fragments of this phase. which also show the charactwistic center strip an11 longitudinal striation pattern. Her 1001) .L

PLATE V. Aligned segments of myosin prepared by dialysis with gradual dilution to a final concentration of 0.36 M-calcium acetate, stained with uranyl acetate containing Brij-35. in thl: marked aggregate, the region A is the striated central overlap zone, and its margins are marked by the arrows. B is the region containing unsupported molecular shanlw. l’ha region C, which contains the molecular heads, illustrates the degree of overcrowding of the lat,ter. Bar .= I O(10 .4.

I'LATE VI. J~isodxocl segments cuncentration of 0.53 nr-ammonium uranyl oxalato. The arrows show thv nbscnrc: of st,riatiorra pqxdicular

of rnyoain prepared by dialysis wit,h gradual dilution I o n find acetate with 5.3 mlcI each of Ca2+ and Mgz’-, stained with the rather ragged margins of the cent,ral ovwlrtp zones. Not,<* to the molecular tails in t,hc central moues. Fiar 1000 .\.

PIATE VII. Crossed segment, si,~~r~r,turesof myosin. Bar : 1000 .k. !a) .A specimen in an early growt#h stage. It reveals 13alher clearly the constituent disordered segment structures of which it is composed (pr~pa~~l IIV dialysis with gradual dilution to a final conr.r;l,trat.ion of O-48 M-ammonium acetate. 0.048 nr-M&i, ; s&neci with m-any1 osalatc containing 13rij-35). Icads tu a -- filled-111 SCIII~LI I,” Ih) :\ more typical specimen, showing how the simultaneous growth uf t,ha cclnstituont segment structures ultimately

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The transverse banding pattern in the overlap zone is another distinguishing: feature of this aggregate. The banding pattern is most clearly revealed in specimens that have been fixed with glutaraldehyde and stained with uranyl acetate. It is less distinct in specimens stained with uranyl oxalate or ammonium molybdate. In the most clearly defined images of specimens fixed and stained with uranyl acetate, the pattern appears to be bilaterally symmetrical and reveals I6 bands that form eight opposing pairs. The bands follow no exact periodicity, and vary in intensity of staining. Table 2 gives the locations and estimated intensities of the observed bands, TABLE

2

Banding pattern of the aligned segments of myosin Band

number

Distance midline 24 71 140 201 261 307 347 398

from (A)

Intensity

Weak Weak Medium strong Strong Weak Weak Weak

These results are means from measurements on the images of two different measurements on each individual object deviated from the listed means by 5 A, The mean spacing between bands was 53 A. Other noteworthy features of the were a pronounced white interband between bands 5 and 6, and a somewhat white interband between bands 4 and 5.

specimens. The on the average. banding pattern less pronounced.

The mean spacing between bands is 53 8. Thus, aligned myosin segments show a banding pattern that does not exactly match those of either phase F or L of LMM-C, but it resembles them closely in its mean spacing between bands. Presumably, these banding patterns do not represent true periodicities in the structures. Rather, they are analogous to the banding patterns of the segment structures formed by collagen, and they reveal the regions in the aggregates of aligned molecules that are richest in groups capable of binding the stain, or which contain the largest voids. (For discussion of banding patterns of collagen and references to earlier work, see Hodge, Petruska & Bailey (1965) ; for a comparable interpretation of banding patterns of paramyosin, see Cohen, Szent-Gyorgyi & Kendrick-Jones (19’71) and Szent-Gyorgyi, Cohen & Kendrick-Jones (1971).) In some of the specimens, the outer 150 A on either side of the overlap zone appeared to be more ragged than the central 530 A region, and gaps could then be seen into which the stain had penetrated to some extent. Since this feature did not appear in all the specimens, we think that it is an artifact that depends on the fixative and stain that have been applied. Presumably, it reflects mainly the varying tendency of different stains to penetrate between adjacent molecules in regions where they have been cross-linked at relatively infrequent intervals. Indeed, we have found in applying the negative-staining method to aggregates of myosin and its fragments that these objects must be carefully cross-linked to prevent their penetration and destruction by the stain. For example, phosphotungstate stains invariably destroy these aggregates

548

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unlessthey have been fixed, e.g. with glutaraldehyde. In contrast, the uranyl stains can usually be applied directly without prior fixation. Nevertheless, their fixative action depends on the existence of appropriate patterns of uranyl-binding (carboxyl) groups in the surfaces that must be held together in order to preserve continuity of the structures. Similarly, glutaralclehyde fixation succeedsonly when the aggregates contain suitable pairs of amino groups. Thus, we believe that the tendency of the edgesof the overlap zones to fray on occasion arisesprimarily from a lack of effective cross-linking in this region. This feature in no way implies that the molecular tails in this region are not closely packed in the native structures prior to fixation and staining. Others have also remarked on this type of ambiguity in the interpretation of observed “inner” and “outer” fringes in analogous structures as being possible a,rtifacts (Cohen et al., 1970). (ii) Disordered segmentstructures If one tries to prepare myosin segmentsat alkaline-earth concentrations lower than those usedin the preparation of the aligned segments,e.g. by method (2)(b) at calcium or magnesium concentrations from 0.02 to 0.1 M, one generally obtains structures that appear to be a disordered variant. Like the aligned segments,they are objects having a visible overlap zone where tails of myosin molecules seemto form an antiparallel, flat array, while the bulky myosin heads protrude on either side to form irregular masses.Their contrasting feature is that the overlap zones are quite ragged in appearance, and show little or no banding structure upon application of uranyl stains. Plate VI showsan example of this; the overlap zones are the bands acrossthe central shanks of the objects. They are demarcated on both sidesby quite irregular edges. The range of widths of the overlap zones in these aggregates was from 720 to 1190 8, with a corresponding variation in the total (head-to-head) span of from 2000 to 2900 A. Harrison et al. (1971) have observed that both rabbit and chicken myosins give rise to segment structures under conditions comparable to those that we have used in preparing disordered segments.Their micrographs of the preparation from rabbit muscle are indistinguishable from our disordered segments, and they show clearly the feature of a ragged center strip that lacks transverse striations. In their preparation from chicken myosin, the center strip was lessragged, but nevertheless lacked striations. In contrast to the aligned segments, our measurements on disordered segments aid not yield a constant value of (L + a)/2 (where L is the span, and a is the overlap length). Rather, the observed values ranged from 1658 to 19408, i.e. they were equal to or greater than the estimated molecular length of 1654A. All that this means, we think, is that the observed spans of t,heseobjects depend on the myosin molecules inserted least far into the aggregate, while the estimated overlap lengths depend more on those inserted deeper. At even lower alkaline-earth concentrations, 0.01 M or less,the aggregatesobtained began to resemble twigs - the small structures described by Kaminer & Bell (1966). Among the bulk of twig-like aggregates,however, we could generally seea few objects that had grown laterally to some extent, and they closely resembled the disordered segmentstructures seenat higher alkaline-earth concentrations. Theselatter structures could even be seen rarely in specimensgrown in the presence of EDTA (method (2)(c)). Along with these wider structures, the specimensgrown at low alkaline-earth

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concentrations also showed a minority of aggregates longer than the typical twigs (which had lengths about 2200 A, as found by Kaminer & Bell). However, at myosin concentrations of 6 mg/ml. and lower, at which most of our preparations were carried out, these aggregates hardly ever grew into normal thick filaments of any great length. Nevertheless, when myosin solutions at a concentration of 16 mg/ml. or greater were subjected to the same treatment, they readily furnished typical long specimens of artificial thick filaments. The results with chromatographically purified myosin were generally indistinguishable from those obtained with an ordinary myosin preparation at the same protein concentration. An experiment in which magnesium ions were added, together with an amount of EDTA insufficient to complex all the magnesium, gave a pattern typical of the amount of residual magnesium. Since this treatment would be expected to complex thoroughly any traces of transition-metal cations as well as those of calcium, this behavior suggests that the magnesium alone was capable of inducing the growth of normal disordered segments. (iii) Crossed segments A type of structure that almost universally accompanied the above-described myosin segment structures was a larger aggregate consisting of two segment structures, one lying on top of the other, with their overlap zones in contact. Usually, the direction of the molecular tails in one of the constituent segments was normal to that in the other, or nearly so. We have termed these associated pairs of segment structures crossed segments, and two specimens are shown in Plate VII. While they were usually a minor component in preparations that yielded aligned segments, they were mo,st often the major component under conditions yielding disordered segments. When grown under such conditions, they shared with the single segments the characteristic disorder and raggedness of the overlap zones. The widths of the overlap zones ranged from 750 to 1440 A, and the total (head-to-head) spans ranged from 2200 to 36OOA. Often one can see the more or less ragged demarcation lines of the overlap zones Iof both components of a crossed segment, although such a demarcation line often lies near the edge of the shank region of the other constituent segment, and is thus masked by the latter. In some experiments, we included KSCN in the growth medium in concentrations from 0.01 to 0.05 M, or 0.01 M-KI (see also Cohen et al., 1970). The general effect of these additives was to favor growth of single segments over crossed segments; KSCN had a weak effect, and KI more pronounced. However, the effect of KI was complicated by head-to-head adhesion of the segment structures and ultimate gelation of the preparation.

4. Discussion Two rather striking features stand out among the various details that the segment structures have manifested. One is the general prevalence among these phases (of overlap lengths in the range 830 to 890 A. These numbers are very close to the width of the M-line in muscle (estimated by Knappeis & Carlsen (1968) to be about 750 A and by Pepe (1967) to be about 860 A, although Hanson et al. (1971) found a value of 495 A). Second, and equally striking, is the variety of overlap lengths, some of which lie outside this range. Probably, the basis of the disorder in disordered myosin seg-

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ments lies in the fact that myosin molecules, in binding to a growing aggregate, can attach to their antiparallel neighbors in a variety of ways with different overlap lengths. Thus, several modes of packing can coexist within one and the same aggregate. All of this suggests that the factors which govern the packing arrangement involve kinetic factors as well as a considerable sensitivity to slight changes in the external conditions. The commonly observed overlap lengths in the range close to the width of the M-line are of special morphogenetic interest since this is the region in the thick filament in which contacts between tails of myosin molecules in antiparallel orientations must occur. During morphogenesis of the thick filament, the nucleation step in formation of the aggregate probably occurs here. Yet, the thick filaments in. vivo are joined together in this region by another protein, the M-line protein (Pepe, 1966; for studies on M-line structure, see also Knappeis 8.zCarlsen, 1968). Thus the question arises whether a protein other than myosin has a role in the formation of the segment structures of myosin. The appearance of the micrographs does not permit one to decide. We can only infer that this is not the case from the fact that LMM-C forms similar segment structures, though it lacks such components as M-line protein or the light chains of myosin (King et al., 1970). Thus, we infer that no auxiliary protein is essential in providing the necessary information to permit overlaps with the right range of lengths, and suggest that this information resides in myosin itself. A further question in interpreting the segment structures is whether they are formed (with the exception of phase N of LMM-C) by overlap of the distal regions of myosin tails lying in alternating antiparallel orientations with the proximal regions prot,ruding into the surrounding medium unsupported by neighboring molecules. Only for myosin

(a)

(b)

FIG. 2. Schematic illustrations of the segment structures of myosin (not to scale). (a) An aligned segment structure. This model illustrates the precise alignment of the molecular tails and the overcrowding of the molecular heads, a feature which is considered to limit the lateral growth of the structure (of. Plate V). (b) A disordered segment structure. This model reveals how a random stagger in the alignment of the molecular tails partially relieves the overcrowding of the heads, thus permitting an energetically more favorable packing (of. Plate VI).

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itself is this clearly the case, since here we can see the heads in situ. Figure 2(a) shows an interpretation of the aligned segments of myosin on this basis. However, existence of the disordered segment structures indicates that the myosin molecules can adhere to one another with a variety of overlap distances (see Fig. 2(b) for a model of these structures). Although this suggests that the distal region (approximately the cauded 850 A of the myosin molecule) contributes most to the process of molecular aggregation as the ionic strength is lowered, a clear demarcation between the distal and proximal regions is not possible. Figure 3 shows an interpretation of the crossed segments in terms of growth of one

FIG. 3. A schematic illustration of a crossed segment structure of myosin. Eaoh component is a disordered segment structure of the type shown in Fig. 2(b). The diagram shows how the growth of each component is restricted both by its own overcrowding of heads and by presence of its partner. Owing to this, the ultimate growth stage of these objects is the “filled-in-square” depicted (cf. Plate VII(b)).

segment structure on top of the overlap zone of another structure having a perpendicular orientation. Such structures often grow until the width of the shank region of each of the constituent segments is about equal to the overlap length of its partner. Growth then ceases, giving a characteristic “filled-in square” appearance to the aggregate, although often one or both of the constituent segments will remain narrower than this. The exposed surface of the overlap zone of a growing segment may provide a favorable site for nucleation of another segment having a perpendicular orientation. In such an event, both partners may continue to grow, until overcrowding of the heads and the limited widths of the overlap zones limit growth. The behavior of myosin again strikingly diverges from that of its fragments in the ease with which it forms crossed-segment structures, whereas no counterpart is known among the segment structures of the fragments. It would seem that the factors that favor disorder in the alignment of myosin molecules also favor growth of crossed segments. This is suggested by the observation that the crossed segments are often the most abundant species under conditions in which myosin forms disordered segments, but are at most a very minor species in preparations yielding aligned segments. Further, none of the fragments has yielded disordered segment structures. Perhaps the surface of one of these structures of the disordered type serves as a good

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nucleation site for growth of a perpendicular partner segment, but an aligned structure is ineffective. What role do the alkaline-earth cations have in the morphogenesis of the segment structures, and indeed, in that of the thick filament itself? There is quite a lot of evidence from this work, as well as that of Cohen et al. (1970), that calcium and magnesium ions exert marked effects on these systems. However, the situation is confused by the differing behavior of myosin itself and of its fragments. The fragment molecules (LMZM-C, tryptic LMM, and myosin rods) agree in behavior, in that they form typical, highly ordered segment structures in the presenceof small amounts ofalkaline earths. On the other hand, removal of calcium by EDTA suppressesthe formation of these aggregates, at least in the caseof LMM-C. Myosin, however, doesnot follow this pattern at all. One can get a small yield of segmentseven in the presenceof EDTA, but the presenceof alkaline earths favors these structures greatly. Perhaps the growth of either a segment or filament type of aggregate of myosin starts with a common, twig-type nucleus that contains only a few myosin molecules. Then these nuclei have the potential to grow either into disordered segment,sby further lateral aggregation of myosin molecules, or into filaments by longitudinal addition of more molecules along the fiber axis. Since both these types of aggregates differ from the aligned segments in having staggered arrangements of myosin tails, such staggered patterns are probably an intrinsic property of the smallest nuclei, say, those containing as few as three myosin molecules. Then, further lateral growth is apparently favored by the presenceof appreciable amounts of alkaline earths, or by low myosin concentration, while filament growth is favored by high myosin concentration or by lower concentrations of alkaline earths. In sharp contrast to the twig and disordered-segment types of aggregates, the aligned segments of myosin require high alkaline-earth concentrations for growth. It would seemthat the mechanism of growth for this type of aggregate requires that all possibilitiesof addition of myosin moleculesin staggeredarrays must be suppressed. Perhaps, then, the necessary high alkaline-earth concentration acts by saturating a set of very labile binding sites, whereasaddition to a growing aggregate of new myosin moleculesin disregister is otherwise kinetically favored, and may always be possible unlessthese loose binding sites are saturated. Why does myosin behave so differently from its fragments ? Probably, the overcrowding of the heads in myosin interferes with exact alignment in the growth of segment structures, and it possibly has an adverse effect on the kinetics of growth of even small nuclei. Apparently, antiparallel aggregation of myosin tails occurs even in the absenceof alkaline earths. Then, in the myosin fragments, only minimal amounts of alkaline earths suffice to suppresslongitudinal growth of the aggregates, thus favoring growth of segments over that of tactoids. However, with myosin, there is a wide range of alkaline-earth concentrations between the levels required to favor segmentsover filaments and those required to suppressthe disorder that is favored by overcrowding of heads. A further factor to consider, however, is the fact that the fragments of myosin may have suffered damage in addition to the cleavage of the rod at the selectedpoint. Both tryptic LMM and myosin rods contain internal peptide-bond cleavages (SzentGyiirgyi $ Borbiro, 1956; Mihalyi $ Harrington, 1959; Lowey et al., 1969). While LMM-C has no internal peptide-bond interruptions, it may have local lossesof perfect two-stranded coiled-coil structure, presumably too circumscribed to affect the optical

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properties (King et al., 1970). Various forms of local damage are probably not responsiblefor the pronounced differencesbetween the behavior of myosin and of its fragments, but they may have a role in determining the less marked differences in behavior between the different types of fragment molecules, or between different batches of LMM-c. A further point to settle is the number of layers of myosin tails in the various segment structures. The features of phase L of LMM-C that we have interpreted as moire fringes would suggest that this phase at least is a bilayer. There is, thus far, little evidence to favor monolayers, bilayers, or thicker models for the rest of the segment structures. We can, at least, construct plausible models of monolayers and bilayers to fit the features of the micrographs. The most plausible model for a monolayer segment structure is relatively unambiguous (Fig. 4). It shows a flat array of

Fmge

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Fringe

FIG. 4. Schematic illuatr&ion of a monolayer segment struoture (e.g. as postulated for phase F of LMM-C). The molecules alternately protrude in two opposing directions, and presumably have opposite chain directions to account for the bilateral symmetry of the structures (cf. Plate I).

rod-like moleculesalternately oriented in two opposite directions. However, a bilayex structure presents more possibilities of internal structure that are consistent with the observed bilateral symmetry. One model (Fig. 5(a)) is made of layers, each of which consistsof moleculeswith their endsin register (such an array need not have bilateral symmetry). The other layer is oppositely oriented and makes contact with the first layer only in the overlap zone. Another possiblemodel (Fig. 5(b)) has two layers with structures analogous to Figure 4, each being a staggered linear array. The fact that the fringes in phase L appear to consist of free, unsupported molecular ends, rather than forming sheetsthat are continuous but thinner than the overlap zones, would favor the model of Figure 5(b). Observations that one fringe of a segment is more deeply embedded in stain than its partner have been too rare and sporadic to permit adducing this observation as evidence favoring the first model. Indeed, their rarity might argue the converse. Do the segment structures throw any light on the morphogenesis of the thick filament ? It would seemthat the major analogous feature of segmentsand Laments is the existence of a multitude of stable overlap patterns that can be formed by myosin tails lying antiparallel to one another. Probably the main factor operative in causing

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FIG. 5. Two alternative models for a bilayer structure (e.g. as postulated for phase cf. Plate II) : (a) A composite of two unstaggered layers of molecules, each displaced with respect to create a structure having an overlap zone and fringes. (b) A composite of two staggered layers having the structure of Fig. 4.

L of LMM-C: to its partner

either myosin or its fragments to form segments instead of filaments under appropriate conditions lies in selective suppression or enhancement of the various modes of attachment of myosin tails to one another. Presumably, all of the information to determine these modes of aggregation is already inherent in the myosin molecule, but factors such as the alkaline earths serve to favor certain modes. We are grateful to Drs Jerome Gross and Romaine R. Bruns for the extended use of their electron microscope and to Mm Muriel H. Blanchard and Mr James Vaccarino for their excellent technical assistance. This work was supported by The National Institutes of Health research grant AM-09404, by a grant from The John A. Hartford Foundation, Inc., by grant no. 70870 from the American Heart Association, and by N.I.H. Career Development Award no. 5 K03-AM18565 to one of us (M. Y.). A preliminary account of this work was presented at a meeting of the American Crystallographic Association, Ottawa, Canada, 16 to 22 August, 1970.

REFERENCES American Chemical Society (1968). Reagent Chemicals. American Chemical Society tiorrs, 4th edn. Washington : American Chemical Society Publications. Cohen, C., Lowey, S., Harrison, R. G., Kendrick-Jones, J. & Szent-Gyorgyi, A. J. Mol. Biol. 47, 605. Cohen, C., Szent-Gyorgyi, A. G. & Kendrick-Jones, J. (1971). J. Mol. Biol. Hanson, J., O’Brien, E. J. & Bennett, P. M. (1971). J. Mol. Biol. 58, 865. Harrison, R. G., Lowey, S. & Cohen, C. (1971). J. Mol. Biol. 59, 531. Hodge, A. J., Petruska, J. A. & Bailey, A. J. (1965). In Structure and E’unctiorb nective and Skeletal Tissue, ed. by S. Fitton-Jackson, R. D. Harkness, S. M. L G. R. Tristram, London: Butterworths, p. 31. Holland, L. (1961). In Vacuum Depotition of Thin Films, New York: Wiley. p.

SpecijiicaG. (1970). 56,

223.

oj’ ConPartridge 74.

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Huxley, H. E. (1963). J. Mol. Biol. 7, 281. Huxley, H. E. & Brown, W. (1967). J. Mol. Biol. 30, 383. Huxley, H. E. & Zubay, G. (1960). J. Mol. BioZ. 2, 10. Kaminer, B. & Bell, A. L. (1966). J. Mol. BioZ. 20, 391. Kielley, W. W. & Bradley, L. B. (1956). J. BioZ. Chem. 218, 653. King, M. V., O’Hara, D. S., Molberg, P. J. & Young, M. (1970). Fed. Proc. 29, 893Abs. King, M. V. & Young, M. (1970). J. Mol. BioZ. 50, 491. Knappeis, G. G. & Carlsen, F. (1968). J. Cell BioZ. 38, 202. Lowey, S., Slayter, H. S., Weeds, A. G. & Baker, H. (1969). J. Mol. BioZ. 42, 1. Mihalyi, E. & Harrington, W. F. (1959). Biochim. biophyls. Actu, 36, 447. Mordoh, J., Leloir, L. F. & Krisman, E. R. (1965). Proc. Nut. Accd Sci., Wash. 53, 86. Pepe, F. A. (1966). J. CeZZ BioZ. 28, 505. Pepe, F. A. (1967). J. Mol. BioZ. 27, 203. Reissig, M. & Orrell, S. A. (1970). J. Ultrastructure Res. 32, 107. Rice, R. V. (1961). Biochim. biophys. Acta, 52, 602. Richards, E. G., Chung, C. S., Menzel, D. B. & Olcott, H. S. (1967). Biochemistry, 6, 528. Szent-Gyorgyi, A. G. & Borbiro, M. (1956). Arch. Biochem. Biophys. 60, 180. Szent-Gyorgyi, A. G., Cohen, C. & Kendrick-Jones, J. (1971). J. Mol. BioZ. 56, 239. Young, M., Blanchard, M. H. & Brown, D. (1968). Proc. Nat. Acad. Sci.. Wash. 61, 1087. Zobel, C. R. & Carlson, F. D. (1963). J. Mol. BioZ. 7, 78.