The myosin filament

The myosin filament

J. 21102. Biol. (1967) !37,203-225 The Myosin Filament I. Structural Organization from Antibody Staining observed in Electron Microscopy FRUK Univers...

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J. 21102. Biol. (1967) !37,203-225

The Myosin Filament I. Structural Organization from Antibody Staining observed in Electron Microscopy FRUK University

A. PEPE

Department of Anatomy of Pennsylvania, PhiludeZphia, U.S.A.

(Received 31 December1966, and in revisedform 4 April 1967) A model for the myosin filament is presented. The model is based on: (1) the staining pattern observed with anti-myosin in electron microscopy and its relation to the M-line and pseudo-H-zone; (2) the presence of six radially distributed bridges between the filaments in the M-line; (3) the triangular profiles seen in cross-sections of the thick thament through the pseudo-H-zone. The most significant features of the model are that (1) the packing of the myosin molecules along the filament is altered by the tapered ends and the overlap of molecules in the pseudo-H-zone, (2) the myosin filaments are ordered in the A-band in positions restricted by the ability to form M-bridges between the filaments and (3) the approximately 430-A repeat period generally seen in the A-band can be accounted for solely on the basis of superposition of the cross-bridges. The model of the myosin Clament has the following important structural characteristics: (1) myosin molecules are aggregsted in parallel rows; (2) in the pseudo-H-zone the myosin molecules are aggregated tail (L-meromyosin) to tail and everywhere else head to tail; (3) in the M-line region there is tail to tail abutment of myosin molecules in the same row; (4) between the pseudo-H-zone and the tapered ends, there is an overlap of one period (approx. 430 A) in the myosin molecules along each row; (6) there are 12 rows in a cross-section through the M-line region and 18 rows in a cross-section between the pseudo-Hzone and the tapered ends; (6) the myosin molecules are packed so that a triangular profile is obtained in a cross-section through the pseudo-H-zone. Nine rows are on the surface and three centrally located; (7) these 12 rows make up three sets of four rows. In each set there are two pairs. There is a stagger of one-third of a period between the rows of a pair and a stagger of one period between pairs.

1. Introduction Antibody staining techniques have been used to study the structure of striated muscle. The results have been complicated by (1) the presence of antibody to contaminating antigens and (2) blooking of antigenic sites due to interactions between the muscle proteins. Recently these difhculties have been resolved using fluorescein-labeled antibody and absorption techniques in fluorescent microscopy (Pepe, 1966aJ967). Speci6c antibody localization in eleotron microscopy, in general has been consistent with the results of fluorescent mioroscopy (Pepe, 1963,1966o; Pepe & Huxley, 1963; Pepe, Finck & Holtzer, 1961). Using separated 6laments and the negative-staining technique, anti-myosin stained the thick filaments from the A-band and anti-a&in stained the thin filaments from the I-band. Anti-actin staining of the thin filaments 203

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was also observed in sectioned material, but staining with anti-myosin could not be detected in sectioned myofibrils (Pepe & Huxley, 1963). The purpose of the present work is to present observations, made with the electron microscope, of anti-myosin staining of the A-band in sectioned myofibrils. The characteristics of the staining pattern in the A-band reflect the organization of the myosin molecules within the filaments. From these observations and relative measurements of other characteristics of the A-band, a model can be constructed for the arrangement of myosin molecules in the thick filament. This model has the following characteristics. (1) The myosin molecules of the thick flament are not precisely aligned along its entire length. Each half of the A-band is divided into three regions corresponding to small differences in organization of the myosin molecules in the thick filament. (2) The position of each thick filament with respect to its neighbors in the A-band is determined by the M-line material. The filaments must be placed so that the M-line material adhering to one filament can bridge across to a neighboring Lament. (3) The two ends of the filament are not identical. The Slaments must be stacked in the A-band in anti-parallel directions. (4) The approximately 430 A period seen in the A-band can be accounted for solely on the basis of superposition of cross-bridges. The model is useful in explaining the direction of the cross-bridges (chevrons) seen in rigor muscle (Reedy, Holmes & Tregear, 1965) in electron microscopy (Pepe, 1967) as well as the results of fluorescent antibody staining (Pepe, 1966,1967). A possible length-determining mechanism for the filament is suggested by the model.

2. Materials and Methods (a) Preparation of jibem Glycetiated chicken breast muscle was used. The muscle wae excised in long strips parallel to the fiber axis, tied to plastic rods and placed iu 60% glycerol at 2 to 3°C for 2 days. The glycerol had previously been passed over Amberlite MB1 ion exchange resin. The glycerol was then changed and the muscle stored at -24% for at least 3 weeks before use. A small piece of glycerinated muscle was cut off the rod and placed in 26% glycerol containing 7.6 x 10sa rvr-KC1, 7.6 x lo-” M-MgCl,, 7.6 x 10e3 M-phosphate buffer at pH 7-O (buffered glycerol) and allowed to stand & hr at 2 to 3°C. It was then shredded by drawing a needle through it parallel to the fiber axis and allowed to stand an additional 4 hr. It was then homogenized lightly in a Servall omnimixer until a suspension of short rod segments of the muscle fibers was obtained. The container was immersed in ice during homogenization. The fibers were checked for size in the light microscope at low power. The suspension was centrifuged at top speed (approx. 1600 g) in a clinical International bentrifuge at 2 to 3°C for 10 min. The fibers were resuspended in fresh buffered glycerol, recentrifuged and finally suspended in 9 vol. of buffered glycerol. (b) Antibody staining and enabedding for electron rnicroecopy Anti-myosin was prepared as previously described (Pepe, 1966). A solution containing approximately 20 mg protein/ml. in buffered glycerol was used for staining. To 2 drops of the fiber suspension, 12 drops of the anti-myosin was added. Staining was allowed to occur overnight at 2 to 3°C. The fiber segments were then washed twice with buffered glycerol. The washed fiber segments were suspended for 16 min at 0°C in 2 ml. of a 6% glutaraldehyde solution containing 7.6 x lOTa a6-KCl, 7.6 x lo-* na-MgCl,, 7.6 x 10-s Mphosphate buffer at pH 7-O (buffered 6% glutaraldehyde). Following centrifugation, the supernatant fraction was removed and the fibers resuspended for 16 min at room temperature in 2 ml. of Palade fixative (pH 7.4) containing 1% Os04 (Palade, 1962). They were then dehydrated by suspending in 70% ethanol end finally in 100% ethanol. With ethanol they were carried into polyethylene capsules (Beem capsules, L. K. B.

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In&ument

Co., Rockville, Md.). The capsules were capped and centrifuged. The ethanol was drained off and Araldite mixture (Finck, 1960) without accelerator was added. A small air space was left in the capsules before capping. They were placed on a rotator and rotated overnight. They were then centrifuged, and the Araldite mixture was replaced with Araldite mixture containing accelerator. The capsules were rotated for 3 hr at room temperature and for 3 hr in an oven at 40°C. They were finally centrifuged to bring the fibers to the bottom and placed in an oven at 60°C for 2 days for polymerization.

(c) Staining of sections microtome and a diamond knife. They were Sections were made with a Porter-Blum stained in two steps. The grids containing sections were immersed in a 4% solution of uranyl acetate in 70% ethanol for + hr. They were washed thoroughly in 70% ethanol and dried. Subsequent staining was obtained by floating the grids on Reynolds (1963) lead citrate stain for & hr followed by a vigorous wash in water. (d) Electron microscopy Electron micrographs were obtained with a Siemens Elmiskop voltage of 80 kv and a 60 p objective aperture.

I using an accelerating

(e) X-ray absorption p&urea Short segments of plastic straws were fllled with a mixture of plaster of paris and barium sulfate to make them opaque to the X-ray beam. The pattern of the cross-bridges lying in a particular plane perpendicular to the axis of the iilament were drawn on a stiff board aa predicted by the model. The radio-opaque segments were placed in these orientations. The different planes were separated by a radiotranslucent material (plastic foam). X-ray photographs were obtained with the beam perpendicular to the direction corresponding to the axis of the filaments. The model was equivalent to a square rod cut out of the Aband and containing 39 thick fLlaments (see Fig. 9). The cross-section of this rod would be a square approximately 1800 A on a side. Photographs were taken every 5” rotating the model through 180’.

3. Results (a) Normal nzuscle Unstained muscle and muscle treated with normal y-globulin were examined for comparison to the antibody-stained muscle. No change was found as a result of the normal y-globulin treatment. In Plate I(a) is a longitudinal section through an unstained myofibril showing the characteristic banding pattern with a faint approximately 430 B periodicity in the A-band. In Plate I(b), cross-sections are shown through (1) the pseudo-H-zone (area of the A-band where no myosin cross-bridges come off the thick filaments; see Fig. 1 and Huxley, 1965, p. 15), and (2) the M-line (area of the pseudo-H-zone where there are M-bridges between the thick &laments; see Fig. 1). The triangular proties andcross-bridges inthe M-region are evident (FranziniArmstrong & Porter, 1964; Spiro, 1962; Huxley, 1957). The approximately 430 A periodicity of the A-band can be seen in the contracted sarcomere in l’lnte I(c). In addition in some areas two lines can be seen between the 430 .t!~repeat. This is a minor repeat of 140 to 14.51(1.The values for these periodicities are very rough. A precise value would have no special significance in this work. Only relative dimensions are needed for the derivation of the model presented here. (b) Anti-myosin stained muscle Following exposure to anti-myosin, two bands were visible each in the middle portion of one-half of the A-band. Each of these bands consisted of seven equally spaced lines (Plate II(a) ). The separation between the stained bands, and the spacing

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of the lines was constant throughout contraction (Plates II(b) and III(a)). The periodicity was destroyed only when the sarcomere length decreased to the point where the Z-line approached the area of the periodicity. In this case the periodicity was destroyed progressively as the sarcomere shortened further (Plate III(a) and (b)). In Plates II and III, the sarcomere length is such that thin filaments are present in the area of the A-band giving the periodicity. In stretched fib&, where the thin filaments do not overlap this area of the A-band, the periodicity is still seen after anti-myosin staining. In these highly stretched fibrils, there seems to be some slippage of the thick filaments with respect to each other as seen from the irregular A-I junction. The period lines are not as well aligned but they are still limited to approximately the middle portion of each half of the A-band (Plate IV(a)). It was not uncommon to observe one or two extra dark lines immediately adjacent to the pseudo-H-zone as can be seen in the right half of the A-band in Plate II(a). However they were not consistently observed. In Plate IV(b), there are two extra lines immediately adjacent to the normally occurring seven lines in each half of the A-band. These are added on the side closest to the pseudo-H-zone. This was seen only once out of over 100 different stained fiber segments which were studied. The periodicity observed with anti-myosin staining actually consists of a major repeat of two periods. This can be seen in Plate 11(a) by holding the Plate at eye level with the fiber axis from left to right. Looking obliquely along the period lines perpendicular to the fiber axis, the first, third, fifth and seventh lines are slightly wider and denser than those in between. (I am grateful to Dr M. Moody for pointing this out to me.) (c) Measurements In the anti-myosin stained sarcomere, the A-band can be divided into six bands. In the diagram in Fig. 1 these are numbered 1 to 6 from left to right. Bands 1 to 3 form one-half of the A-band and 4 to 6 the other half. The boundary between bands 3 and 4 is the middle of the M-line. Bands 2 and 5 represent the areas stained with antimyosin. These stained areas consist of seven equally spaced lines, the first and seventh line in each area marking the edges of that band. Therefore each stained band is divided into six equal spaces. Relative measurements were made as shown in the dia-

Pseudo -H M-line

FIU. 1. Relative indicated

metwurements made on the A-band measurements are tabulated in Table 1.

of anti-myosin

stained srucomeres. The

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1

Relative measurements made on the A-band of anti-myosin stained sarcomeres

Banda

Length (cm) (measured from

micrographf3) C-A 2 A - (B + B’) 2

1, 6

1.6

6

3, 4

1.5

6

1.5

6

B + B’ 2 A - (D + D’) t Pseudo-H

Units of length (relative)

0.6

M

t Measured

2 4

z2M in unstained

muscle.

gram in Fig. 1. The measurements are summa rized in Table 1. Relative measurements of both the staining pattern and the normal characteristics of the A-band should reflect the organization of the myosin molecules. Bands 2 to 5 are equal in width. Bands 1 and 6 are slightly larger. This is probably due to the adherence of non-oriented antibody to the edges of the A-band (see Discussion). We will therefore consider the six bands as equal in width. Measurements of both the M-line and the pseudo-H-zone were also attempted on the stained fibrils, but the bands were extremely fuzzy. A value of O-6 cm (Table 1) was obtained for the width of the M-line measured from the micrographs as shown in Fig. 1. Consider the periodicity observed in bands 2 and 5. There are six spaces for a width of l-5 cm. Each space is therefore O-25 cm. The width of the M-line measured from micrographs of antibody-stained fib& is therefore approximately two periods. The pseudo-H-zone width was impossible to measure on stained fibrils. Measurements on unstained fibrib showed that the M-line width is approximately one-half the width of the pseudo-H-zone. We can therefore take four periods as the width of the pseudo-H-zone. In this way we have related the periodicity observed with anti-myosin to the other structural features of the A-band. (d) Observations of M-line Preliminary observations were made of the M-line structure as seen in longitudinal sections. These were obtained in collaboration with Dr R. Armstrong. In Plate V, two examples are shown of the cross-bridge patterns observed. Variations in the patterns result from differences in the plane of section and the section thickness. This type of pattern was observed in both the anti-myosin stained and unstained muscle. (e) X-ray absorption pictures As already described, models were made of the arrangement of the myosin crossbridges predicted by the model in a plane perpendicular to the axis of the Claments (see Discussion for details). For parallel stacking of the filaments, the arrangement

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in Fig. 9(a) is obtained. This pattern is rotated by 60” for each successive plane in which cross-bridges occur along the filament. This is every one-third period. That is, there are three planes per period (approximately 430 A). For anti-parallel stacking the three different aross-bridge patterns shown in Fig. 9(b), (c) and (d) are obtained. These repeat every period along the filaments. X-ray absorption pictures were obtained for both the parallel and anti-parallel stacking arrangements as already described. In both cases the full period repeat showed up strongly for certain positions when the model was rotated through 180” in 5” intervals. An evaluation of the periodicity was made rating +3 as strong, +2 as fair, +l as poor and 0 as very poor. As can be seen from Fig. 10, the parallel arrangement of filaments gave a fair to strong periodicity over a span of approximately 15” at three angles, O”, 60” and 120”. Everywhere else a fair or strong periodicity was never seen to span more than 5”. In the case of anti-parallel stacking of the filaments, again there was an approximately 15’ span showing fair to strong periodicity at 0”, 60” and 120”. In addition however, a span of approximately 20 to 25” showing fair to strong periodicity occurred at 25”, 90” and 150”. Therefore the chances of seeing a clear periodicity in a section through the A-band are considerably greater for the anti-parallel stacking of the filaments than for the parallel stacking.

4. Discussion Anti-myosin staining of the myosin components of the striated myofibril has given the following results. (1) Fluorescent anti-myosin localizes in four bands in the A-band of rest-length saroomeres. Two medial bands represent staining of available H-meromysin antigenic sites and two lateral bands represent staining of available L-meromysin antigenic sites (Pepe, 1966a, 1967). (2) With electron microscopy anti-myosin has been observed to stain separated thick Laments all along their length except for the smooth region midway along the length of the filament (Pepe & Huxley, 1963). (3) Anti-myosin staining has not previously been observed in the intact myofibril in electron microscopy (Pepe, Finck & Holtzer, 1961; Pepe & Huxley, 1963). However, in the present investigation, two bands were formed by the antibody, one in the center of each half of the A-band. Each stained band consisted of seven lines equally spaced (Pepe, 1966b). The reason for previous failure is probably that homogenization to fibrils resulted in some damage to the organization of the A-band. In this study damage was prevented by stopping the homogenization at the point where short fiber segments were obtained. The problem now becomes how to relate these several observations to the organization of the myosin molecules in the A-band. Interpretation of these results depends upon understanding the factors which affect visibility of antibody in fluorescent microscopy and in electron microscopy. Comparison of fluorescent antibody staining of the fibril and antibody staining of the separated filament shows that corresponding regions which stain uniformly on the separated filament are not uniformly stained in the intact fibril (Pepe, 1966a). In both of these cases visibility of staining is proportional to the amount of antibody introduced. Therefore, the non-uniformity of fluorescent antibody staining in the intact fibril must result from the interactions between the thick and thin filaments. In fact, this interaction results in the availability of different antigenic sites in different parts of the A-band (Pepe, 1966a, 1967). Also the areas of the myofibril stainedin fluorescent microscopy are not always clearly stained in sections of the myofibril in electron microscopy. In electron microscopy of sectioned material, the visibility of the anti-

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bodyis dependent on (1) the amount of antibody introduced into a region, (2) the amount of background noise in the region, and (3) the orientation of the antibody molecules, If there is considerable background material, very large amounts of antibody may be required to show up as an increase in protein density. However, if the antibody molecules are aligned precisely in register, visibility will be considerably enhanced over the background noise. Therefore, although heavy staining occurs with fluorescent anti-myosin laterally in the A-band, it is more difficult to see it in electron microscopy (Pepe, 1967) due to random alignment of the antibody molecules. The presence of this non-oriented antibody, however, tends to make the edges of the Aband and pseudo-H-zone fuzzy, leading to large values for measurements involving these edges (Fig. 1). The periodicity seen in electron microscopy results from staining of precisely oriented available antigenic sites. This periodicity is clearly seen in the same area of the A-band, whether there is overlap of the thin and thick filaments in this area or not (Plates II, III and IV(a) ). Therefore, the periodicity must be primarily a result of the organization of the myosin molecules within the thick Claments, and not a result of the interaction of the myosin filaments with the surrounding actin filaments. A detailed analysis of the specific antigenic sites involved in the appearance of the periodicity when overlap of thin and thick filaments occurs as well as when it does not occur is given in the accompanying paper. The occurrence of precisely organized myosin molecules only in the middle of each half of the A-band becomes reasonable if we consider what is known about the structure of the thick filament. By growing artificial filaments from solution, Huxley (1963) showed that in the middle portion of the filament, corresponding to the pseudo-H-zone, the L-meromyosin parts of the myosin molecules aggregate and the H-meromyosin parts stick out at each end. This will be referred to as tail to tail aggregation. Further aggregation of the mole&es occurs with the L-meromyosin portions forming the core of the filament and the H-meromyosin available at the surface and giving a lllament the length of the A-band. This further aggregation will be referred to as head to tail aggregation. At the tapered ends of the lilament the number of molecules per orosssection diminishes. It seems reasonable that the tail to tail aggregation in the center and the taper at the ends of the filament would affect the packing of the myosin molecules so that only between these areas can precise head to tail packing occur. The precise alignment of antigenic sites on this portion of the 6laments leads to the periodicity seen with anti-myosin staining in electron microscopy. The seven distinct lines in the middle portion of each half of the A-band are always seen after staining with anti-myosin. Occasionally additional lines are observed (Plates II(a) and IV(b)). These are always located in the central region of the A-band between the two stained bands. Since they are not consistently observed, they probably represent alterations in the packing of the molecules in this region as a result of handling. For instance, if the tail to tail overlap in the center of the A-band is partially disturbed, its disrupting influence on the further head to tail packing would be diminished. This would allow some reorganization to occur in the central region of the A-band leading to more precise head to tail packing and thus to additional lines in this region. The tapered ends would not be affected. (a) Thick jikmwnt model Using the relative measurements in Pig. 1 and the following assumptions, a model for the arrangement of the myosin molecules in the thiok filament can be oonstructed.

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It is assumed that: (1) The L-meromyosinportions of the myosin molecules in the thick filament are all aggregated in parallel. (The triangular profile of the thick 6laments in cross-section through the pseudo-H-zone would favor such an arrangement.) In the pseudo-H-zone, overlap of L-meromyosin from opposite sides of the A-band occurs. (2) The M-line material adheres specifically to the point at which the L-meromyosin ends of the myosin molecules from opposite sides abut tail to tail in the pseudo-H-zone. Therefore, the width of the M-line determines the maximum tail to tail overlap of the myosin molecules in this region. The extra thickness of the filament at the M-line is due entirely to the loosely adhering M-line material (Pepe, 1966a). (3) The repeat period in the anti-myosin stained bsnds represents the repeat period of myosin molecules along the entire filament. The lack of periodicity in some parts of the Aband is due to small displacement of antigenic sites resulting primarily from differences in the packing of the myosin molecules in different parts of the thick 6lament. (4) For each repeat period there will be six cross-bridges distributed to the six surrounding thin filaments (Huxley, 1957). Each cross-bridge will represent a structural unit consisting of n molecules. Therefore in one period there will be 6n myosin molecules. For convenience we will henceforth refer to the structural unit as a molecule (n = 1). From Fig. 1 we see that the M-line width is approximately twice the myosin period stained in bands 2 and 5. The maximum tail to tail overlap in the M-line (Fig. 2 top) is therefore approximately two periods. Since there are six molecules in every period, the filament will have 12 molecules in a cross-section through the Mregion (Fig. 2, bottom). The edges of the pseudo-H-zone are formed by cross-bridges from myosin molecules with maximum tail to tail overlap (Fig. 2, top). The distance from one edge of the pseudo-H-zone to the opposite edge of the M-line represents the length of the myosin molecule exclusive of the cross-bridge. From the measurements shown in Fig. 1 this length is three times the anti-myosin stained period. In order to have a pseudo-H-zone and an M-line of these dimensions, and, in addition, to have six cross-bridges for every period all along the filament, there must be a head to tail overlap of one period along the axis of the filament in each parallel row of molecules.

,

Fro. 2. Representation of the longitudinal arrangement of molecules pseudo-H.-moue, and the triaugubr profiles for the thick filaments.

IOOOS, ,

leading

to the M-line,

, ,rr,,r,,c, ,,

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L’lattb 1 I. Iallgit

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u~uwIts sttlitletl

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Fixation of tissue and staining of sections wits t,he smnc as for t,he tissue ill Plate I. (a) A relaxed smcomere. Not,e two stained bands, one in each half of t)he A-bated. Ewh stained band consists of seven equally spaced lines. (b) .4 contracted sarcomere. The t,hin filarnenk from opposite sides have overlapped slightly in the cw~t~rrofthrt .A-1m1d. The allti-tnyoxill stttillillg pattenl irs urwhttoged. x 67,500.

Gx (It,” a eve I-l& is sl ;ill res1 11t SllO wi

(0)

(b)

M

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This is shown diagrammatically in Fig. 3 where the 12 molecules have been projected onto a plane. As a result of this overlap, outside of the pseudo-H-region there will be 18 molecules in a cross-section of the filament everywhere except at the tapered ends. The molecules in each parallel row in Fig. 3 have been staggered one-sixth of a period with respect to the next row for clarity. However, as will be shown, the results presented here considered with the other structural characteristics of the A-band and of the thick tllaments, impose a stagger of one-third period between rows.

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p q r _______.-__-: y-2 --I ----’ - ______.-___:I- z?____r- ----• ..a --- ____-_. --- r~‘.- .____-__ ..A---- ---* . _-__ ___-_____r:::.- 2. -__. ..r---‘ -* . - -_- _ ______ r::::: ! _-_-_ ----_ J -- _____ i -___ _--___.I _----i ._-_--__ -~-‘-~--~ ---__ - _____r- ------• ____________- ______ -__,__ .-. - ___________,-. ____.____.- .-- ---’ --_ _________;--- ---a . ---__. I::: z-2 ---- z ------- .8.____ ---_ ‘?-3----_-- .,.____ -B -_____ ------ -__- ---. . -_---_----__-_ >- _- _- _ . ,_-_-__- _____-.----..__ _.________I_________

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Fro. 3. Diagram showing the effect of tail to tail aggregation in the M-line and the effect of the tapered ends of the time&a on the pecking of the myoein molecules. A stegger of one-sixth period ia shown for clarity. The moat likely stegger arrangement is one-third period (see text).

From Fig. 3 the possible influence of the tail to tail interaction and the taper on the precise packing of the molecules can be seen more clearly. The cross-bridges in period c are from myosin molecules involved in tail to tail interactions. The same is true for period d. In period e, the cross-bridges are from molecules which are interacting directly with a molecule involved in tail to tail interaction. Therefore any influence of the tail to tail interaction on the packing may still be felt. In period f the same situation obtains. Consider a cross-section through the f?lament at the last cross-bridge in period f. It will pass through 12 tails and 5 overlap regions of myosin molecules all of which are not interacting directly with a molecule involved in tail to tail interaction. Consider the first molecule with a cross-bridge in period g (labeled 1 in Fig. 3). A cross-section through the filament at this cross-bridge will run through 12 tails and 6 overlap regions of myosm molecules all of which are not interacting directly with a molecule involved in tail to tail interaction. It is here that the first period line is observed with anti-myosin staining. The same situation exists for all cross-bridges in periods g through 1. Now consider period r. These molecules have no head to tail interactions. Similarly for the cross-bridge in period q. In period p, the cross-bridges are from molecules interacting directly with end molecules having no head to tail interaction. The same interactions occur for the cross-bridges in period o. A cross-section through the filament at the sixth cross-bridge in period n will pass through the overlap region of one molecule which is not interacting directly with an end molecule. A cross-section through the first cross-bridge in period n will pass through the overlap region of six such molecules. On reaching the first cross-bridge in period m, a cross-section will pass through the overlap region of six molecules and the tails of six molecules all of which are not interacting directly with the end molecule. It is at this point that the last anti-myosin stained period line is seen as one proceeds 16

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from the M-line to the tapered ends of the filament. Therefore, precise alignment of antigenic sites occurs only when all six overlap regions and at least six tails in a crosssection are from molecules not interacting directly with: (1) a molecule involved in tail to tail interaction, or (2) an end molecule. The packing of the myosin molecules in a cross-section through the thick filament must conform to the cross-sectional profile as seen in electron microscopy. Crosssections through the M-line show very clear triangular profiles (Plate II(b) ). Such triangular profiles have been observed in fish (Franzini-Armstrong & Porter, 1964), and vertebrate heart muscle (Spiro, 1962) and have been pointed out in rabbit muscle (Huxley, 1957). A closely packed parallel arrangement of twelve rods as shown in Fig. 2 (bottom) will give a triangular profile in cross-section. In order for head to tail overlap to occur and still maintain a parallel aggregation of the L-meromyosin portions of the myosin molecules, there must be a flexible region approximately two periods from the L-meromyosin end. If one period is taken to be approximately 430 A, the flexible region would be approximately 860 A from the L-meromyosin end. This corresponds to the trypsin-sensitive region of the myosin molecule (Young, Himmelfarb & Harrington, 1965). The overlap region would account for another 430 h of the molecule giving a total of 1290 A, and the rest of the molecule contributes to the cross-bridge. If the repeat period is greater than 430 A, the contribution to the cross-bridge will beless. Precisemeasurements of the periodicity were not made. From the diagram in Fig. 2 (bottom) it can be seen that nine rowa of molecules are at the surface. The overlapping portion of the myosin molecules from one row can fit between that row and one of the rows on either side of it. The tlexible region of the three central molecules must be flexible enough for the overlap region with its cross-bridge to reach the surface by protruding between two rows of molecules. The arrangement of the cross-bridges along the filament will depend on both the stagger of the molecules and the position of the overlap region with respect to the rows of the packed L-meromyosin portions. The three models in Fig. 4 represent three possibilities for staggering the molecules. In model A, the molecules are helically arranged and staggered one-sixth of a period. There will be one cross-bridge at each of six levels along one period. Since there are 12 rows of molecules in the backbone of the filament (this excludes the rows of overlap), we must consider the bridges from 12 molecules or in two periods along the filament. The numbers 1 to 12 in Fig. 4(A) represent the levels at which each of the 12 molecules is positioned with respect to the others along the filament in the two periods. The overlap from molecules 6 and 12 can both occupy positions between the rows 6 and 12. However, the overlap from molecules 6 and 7 cannot both occupy positions between rows 6 and 7 since the overlap for each molecule is one period and they are not staggered a full period with respect to each other. In models B and C the molecules are again helically arranged and this time staggered one-third of a period. In this case two bridges must come off at one level if we are to have six cross-bridges per period. This means that in two periods along the filament the 12 molecules will contribute two cross-bridges at each of six different positions along the filament. The numbers 1 to 6 in Fig. 4(B) and (C) represent these six positions. The molecules have been positioned so that the two cross-bridges at one level come off as nearly opposite as possible. The reason for this will become evident below. For models A and C, a minimum of six radial directions for the cross-bridges can be obtained, whereas for model B a minimum of eight are obtainable. The repeat period in all of these is every six cross-bridges. However, for

MYOSIN

A

FILAMENT

STRUCTURE

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STAINING

213

B

FIO. 4. Representation of the positioning of the overlap regions between the rows of myosin molecules in the filament. The numbew refer to relative positions of the 12 molecules in two perioda along the filament. In model A with a stagger of one-sixth period there ia one molecule in each of 12 positions (1 to 12). In models B and C with a stagger of one-third period there are two molecules ineaoh of sixpoRitions (1 to 6). These arrengements will determine the positioning of the overlap regions and thus will determine the radial dire&ions of the oross-bridges aa shown by the arrowa. Model A: For a atagger of one-&&h period a minimum of six radial directions can be obtained for the crose-bridges. Model B: For e &agger of one-third period with the arrangement shown, E minimum of eight radial direotiona can be obtained for the cross-bridges. Model C: For e atagger of one-third period with the arrangement shown, 8 minimum of sir radial direatione caa be obtained for the cross-bridges.

three rows of cross-bridges along the filament there will be a major repeat of two periods. Along each of these rows of cross-bridges, every other cross-bridge will. be from one of the three centrally located molecules. In model A, these cross-bridges are from overlaps from molecules in rows 1 and 7,3 and 9, end 5 and 11. In model B the cross-bridges are overlaps from molecules in one of the rows 1 and overlaps from molecules in one of the pair of rows 3 and 6, and one of the pair of rows 2 and 5. In model C they are from overlaps from molecules in one of the pair of rows 1 and 4, 3 and 6 and 5 and 2. Such a major repeat of two periods can be seen in the electron micrographs of the anti-myosin stained fibril (Plate II(a)) by looking obliquely along the period lines as described in the Results section. Another aspect of the filament that comes from these models is that it is polarized. In model A of Fig. 4, one end of the filament will taper with the inner set of molecules 1 to 6 and the other end will taper with the outer set of molecules 7 to 12. Model B in Fig. 4 tapers at one end with the inner set of six molecules 1, 1,2,2,3 end 3 and at the other end with the outer set of six molecules 4, 4, 5, 5, 6 and 6. Model C in Fig. 4 is the most nearly symmetrical since both the inner set and the outer set of molecules

214

F. A. PEPE

are each a set of 1 to 6. However, the necessity for a triangular profile in cross-section still imposes asymmetry. This asymmetry can be seen more easily in the diagrammatic representation of the filament in Fig. 6(d) to be described more fully below. In order to get symmetry, a stagger of one-half period is necessary. This would mean t,hat three cross-bridges would come off every half period along the filament. I would expect that, if present, a stagger of one-half period would be fairly easily distinguishable in electron microscopy as a sub-period one-half the normal period. Either a stagger of one-sixth or one-third of a period seems more likely to be the case. The evidence from X-ray diffraction is consistent with a stagger of one-third of a period but not with a stagger of one-half of a period (Dr H. E. Huxley at the February 1966 meeting of the Biophysical Society in Boston, Mass; Elliott, Lowy & Millman, 1965). Also in the micrograph in Plate I(c) there is a strong indication of a sub-period one-third of the normal period. The M-line material can bridge between two neighboring filaments only when tail to tail abutments are opposite one another in the two filaments. This will fix the orientation of the myosin filaments with respect to each other, Cross-sections of the M-line observed by Franzini-Armstrong & Porter (1964) in fish muscle, by Spiro (1962) in cardiac muscle, and by Page (1965) in frog muscle all clearly show six radially distributed connecting links between the filaments in the M-line. Consider model A in Fig. 4. In this case the stagger is one-sixth period. Along the M-line region going from one end to the other, abutments occur at the surface in rows 2, 4 and 6 spaced at intervals of one-third period. These are followed by abutments in rows 7, 8, 9, 10, 11 and 12 spaced at intervals of one-sixth period along the M-line region. For this model, it is impossible to stack the filaments in such a way that abutments on one filament will be directly opposite abutments on the surrounding 6 filaments, and so that 6 cross-bridges can be formed in this way between all filaments. This is true even if the filaments are stacked anti-parallel. In model B in Fig. 4 the same situation occurs. Abutments 1, 2,3 occur along one-half of the M-line and 4,4,5,5, 6 and 6 along the other half. However, in the case of model C of Fig. 4 abutments occur in rows 1, 2, 2, 3,4, 4, 5, 6 and 6. Filaments stacked in paralle1 in this case can form M-bridges between the 2, 4 and 6 abutments of the neighboring filaments and vice versa to give six M-bridges attached to each filament. The abutments 1,3 and 5 would not be involved. If the filaments are stacked in anti-parallel fashion then 1, 3 and 5 on one filament will be opposite 2, 4, 6 of a filament in the opposite direction and once again six cross-bridges can be formed. These arrangements are shown in Fig. 5. Therefore, of the three models in Fig. 4, model C with a stagger of one-third period is the only one that satisfies the requirements for M-bridge formation. A more detailed diagram of the arrangement of the myosin molecules in model C (Fig. 4) is shown in Fig 6(a). Six molecules are shown as they would appear viewed from one side of the filament. Molecules 1 and 3 (shaded) are centrally located and 2, 4, 5 and 6 are on the surface. For each molecule there is another molecule at the same level. The position of the other molecule can be determined by comparison of Fig. 6(a) and (b). In Fig. 6(c) an entire one-half filament is shown. In this case all six molecules corresponding to those in Fig. 6(a) are alternately shaded and unshaded to make the group more distinguishable. The cross-bridges in both Fig. 6(a) and (c) come off at the top end of the overlap region. The top in Fig. 6(c) is the tapered end of the filament and the bottom is in the pseudo-H-zone. The lines along the right side indicate intervals of one period. The overlaps from molecules in rows 3 and 6 lie in

MYOSIN

FILAMENT

I

STRUCTURE

AND

ANTIBODY t

I

D

representation

216

I I I

(b)

(a) FIQ. 5. Diagrammatic

STAINING

of the M-bridges

formed between the filaments

in the M-line.

This is for the most likely model C as seen in a cross-section which includes the M-line. Bridges oan only occur between tail to tail abutments occurring at the same level in neighboring filaments. (a) Possibilities for M-bridge formation in a parallel arrangement of filaments in the A-band. (b) Possibilities for M-bridge formation in an anti-parallel arrangement of timents in the A-band.

the same row. However, the L-meromyosin portions of molecules in row 3 are centrally located and those of molecules in row 6 are along the surface. The overlaps from molecules in rows 2 and 6 are both in the same row between the L-meromyosin portions in rows 2 and 5. The overlaps from molecules in rows 1 and 4 are in the same row. However the L-meromyosin portions of molecules in row 1 are centrally located and those of molecules in row 4 are along the surface. In Fig. 6(c), because of the perspective of the diagram, the L-meromyosin portions of the molecules in row 4 are behind the L-meromyosin portions of molecules in row 5 and the overlaps from molecules in rows 1 and 4. This becomes evident on comparison of Fig. 6(a) and (c). From the diagram in Fig. 6(c) and (d) the asymmetry of the filament can be seen. Note that the top or the tapered end of the filament ends with the two molecules in rows 6. When the other half of the Slament is constructed starting with tail to tail abutments in the pseudo-H-zone (bottom of the diagram in 6(c)) the other tapered end will end with the two molecules in rows 1. The two rows 6 are centrally located in this view and the two rows 1 are slightly to the left, thus giving asymmetry. (b) Thick-$lament alignment in the A-band Let us consider the appearance of the M-bridges in longitudinal sections through the myofibril. If the iilaments are stacked in parallel, a section through the myofibril in the plane marked A in Fig. 5(a) would give the M-line pattern shown in Fig. 7(a). The filaments go from left to right and the M-bridges are between them. Bridges

216

F. A. PEPE

(a)

(4 Fm. 6. Diegmmm&ia representation of the packing of the myosm molecules in the filament. (e) Representation of six molecules viewed from the surface of the 6lement. Molecules 1 and 3 are centrally loceted. The cross-bridges come off at the top of the overlap region of the molecules. (b) A cross-section of the Slament for reference. (c) An entire one-half of the filament. The group of six molecules shown in (a) have been alternately shaded for clarity. The top is the tapered end of the Clement. The bottom is in the pseudoH-zone. Cross-bridges come off at the top of the overlep regions. (d) Representation of the two tapered ends of the Shxnent showing the esymmetry.

MYOSIN

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STRUCTURE

Plane

AND

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217

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FIQ. 7. Diegremm8tic representation of the M-bridge6 formed between the fllsments in the M-line. This ia for the moat likely model C, 8a seen in 8 longitudinal section through the arcomere. (8) For 8 p8r8llel8rmngement of elements in the A-band, the cross-bridge patterns that would be seen in the planes indicated in Fig. 6 8re shown. (b) For an anti-per8llel8rmngement of filaments in the A-band, the cross-bridge patterns thet would be seen in the planes indicated in Fig. 6 8re shown.

between rows 2 on opposite filaments will be oompletely in the seotion whereas those between rows 6 and 4 will extend out of the plane of the section. In the plane marked B only bridges between rows 4 will be entirely in the section and for the plane C only bridges between rows 6 will be entirely in the section. As seen from Fig. 7(s), in all three planes there will be three rows of bridges spaced two-thirds of a period, in longitudinal sections through the M-line. If the filaments are stacked anti-parallel we will get the patterns shown in Fig. 7(b). In this case rows 6 and 1 in anti-parallel neighboring 6llaments (noted as + and - ) will have abutments at the same level and must therefore be opposite. The same occurs for 5 and 2, 4 and 3, etc. In a section through the plane A all filaments are oriented in parallel and cross-bridges between rows 4 on opposite filaments will be entirely within the section. Cross-bridges from rows 1, 2, 3 and 6 will extend out of the plane of the section. As seen from Fig. 7(b), this will give five rows of bridges. Four rows will be spaced one-third of a period and the last, two-thirds of a period from the rest. Similarly a section through plane B will show five rows. However, the two-thirds period gap will occur with three rows of bridges on one side and two on the other side of it. A section through the plane marked C will show a different arrangement. In this plane the filaments are entiparallel. The srrangement shown in Fig. 7(b) for plane C will show up as four bridges

218

F. A. PEPE

between any two filaments in longitudinal sections. The spacing will alternate between two pairs of bridges separated by two-thirds period and four bridges all separated by one-third period. The same occurs for a section through plane D. Therefore an analysis of the patterns obtained in longitudinal sections through the M-line should distinguish between parallel and anti-parallel arrangements of the filaments. Preliminary observations of longitudinal sections through the M-line were made in collaboration with Dr R. Armstrong. Examples of some of the patterns obtained are shown in Plate V. The similarity of these patterns to those in Fig. 7(b) is consistent with anti-parallel stacking of the proposed filament model and excludes completely parallel stacking. The triangular cross-sections of the filaments in the M-line are depicted in Fig. 6 as all oriented in the same direction. If a single triangle in Fig. 5(a) is rotated by 180”, it will still be possible to have six M-bridges. However, some bridges in this case will be between two corners or two sides of the triangles instead of always being from a corner to a side. It will also be possible to form two bridges between two corners, instead of only one. The M-line pattern in longitudinal sections will still be essentially the same as that in Fig. 7(a). The predominant feature will be three lines. Likewise in Fig. 6(b), a single triangle may be rotated 180’. However, in this case, in order to get at least six radially distributed M-bridges it will be necessary to rotate more than one triangle by 180’. In this case the M-line pattern in longitudinal sections will still be essentially the same as that in Fig. 7(b) for anti-parallel stacking. From preliminary evidence (Plate I(b)), it seems that in chicken muscle the triangular profiles of cross-sections of the filaments through the M-line are not all oriented in the same direction. Changing the pattern of orientation will not change any of the arguments presented in this work, although slight changes will be observed in the details of the M-line patterns in longitudinal sections. The exact arrangement of filaments stacked anti-parallel may also be different than that shown in Fig. ‘7(b) in that the + and - may not be neatly arranged in rows. The exact patterns of orientations remain to be determined. (c) Va%Ank3 of the model A closer look at the model in Fig. 6 reveals that it is made up of three sets of four rows of molecules (see Fig. 8(a)). In each set there are two pairs of rows. The rows of a pair are staggered by one-third period (solid lines) and the two pairs are staggered by one period (dotted lines). In the central triangle there are six rows from 1 to 6 in a olockwise direction. Another six rows surround these, also in a clockwise direction. If all the rows in the central triangle are shifted by one position counterclockwise, the model is not changed. It is now equivalent to the model (a) turned anti-parallel. If the rows in the central triangle of Fig. 8(a) are all shifted by two positions counterclockwise, the stagger relationships in Fig. 8(a) are destroyed and it becomes impossible to form the required M-bridges for anti-parallel stacking of the filaments. In this variation, six M-bridges can be formed between filaments in parallel stacking. The M-line pattern seen in Fig. 7(a) would be obtained at all thicknesses. This has been shown not to be the case. Additional variations made by shifting the rows in the central triangle by 3, 4 and 5 positions make it impossible to obtain the required Mbridge formation for either the parallel or the anti-parallel stacking arrangement of the filaments. In Fig. 8(b), the outer six rows are shifted by one position clockwise relative to the positions in Fig. 8(a). The stagger relationships are maintained and it

MYOSIN

FILAMENT

STRUCTURE

FIG. 8. Variationa

AND

ANTIBODY

STAINING

219

of the model for the myosin fflament.

Solid heavy lines denote position of M-bridges. Arrows represent direotion of row6 of overlap regions. Dots represent position of thin filements with respect to the myosin filament. All variationa have three sets of four row8 of molecules. Each set has two pairs of rows. In each pair the stagger is one-third period (solid limes). The pairs are stccggered one period (dotted lines).

is possible to form the required M-bridges between filaments stacked either in parallel or anti-parallel. If all the rows in the central triangle of the model in Fig. 8(b) are shifted one position clockwise the model is not changed. It is now equivalent to that in (b) turned anti-parallel. A shift of two positions clockwise gives a variation in which only the parallel stacking of the filaments permits six M-bridges to be formed between filaments. Also shifts of 3, 4 and 6 positions do not allow the required Mbridge arrangements to be formed. In the two variations shown in Fig. 8(a) and (b), both the central and the peripheral six rows are arranged in a clockwise direction. Another two variations can be obtained where the six rows are arranged in a counterclockwise direction (Figs. 8(c) and (d)). It becomes obvious from these models that in order to get the required M-bridge arrangement, the two molecules at one level must be approximately on opposite sides of the filament. Also, note that the three centrally located rows forming a triangle within the oentral triangle are all 1, 3 and

220

F. A. PEPE

6. For the anti-parallel equivalents they would be 2,4 and 6. Any other combination such as in Fig. 4(B), makes it impossible to get the required M-bridge arrangement. All the variations in Fig. 8 have three sets of four rows of molecules where the stagger relationships, between the four rows in each set, are the same. Is it possible to eliminate all but one of the variations in Fig. 81 The M-line diagrams such as those in Fig. 7 differ only slightly for the variations of the model. In order to distinguish between them, it will first be necessary to determine the exact pattern of arrangement of the triangular cross-sections of the filaments in the M-line region. Then it will be necessary to determine the directions in which the M-bridges project out of the plane of longitudinal section through the M-line and to compare these with the predictions from the M-line diagrams obtained from the models. If this is done, it will be possible to eliminate all but one of the four variations in Fig. 8. (d) Origin of the approximately 430 L! period in the A-band The thick filaments are fixed in position in the A-band by the M-bridges. This determines the relationship between the thin and thick filaments as shown in Fig. 8 and therefore the position of the cross-bridges between them. Rotation of the filament on its axis through 180” will not change the position of the cross-bridges relative to the surrounding thin filaments. As can be seen, the rows of overlaps and therefore the cross-bridges are not directly in line with the positions of the thin 6laments. In order for a cross-bridge to interact with a thin filament, it must rotate either olockwise or counterclockwise. If the cross-bridges interact with the closest thin filament there will be counterclockwise rotation for the cross-bridges in Fig. 8(a) and (c) and clockwise rotation for the cross-bridges in Fig. 8(b) and (d). When a filament is turned anti-parallel, the counterclockwise rotation becomes clockwise, and vice versa. All the cross-bridges from one row of overlaps will be oriented in the same direction and will occur at one-period intervals along the filament. Therefore bridges 1 and 4 are equivalent in orientation, as are 2 and 5, and 3 and 6. Therefore, the three planes through bridges 1, 2 and 3 will be equivalent to the three planes through bridges 4,5 and 6, respectively. If the filaments in any of the variations in Fig. 8 are stacked in parallel, the orientation of cross-bridges shown in Fig. 9(a) is obtained. For each successive plane this pattern will be rotated by 60”. If the filaments in any of the variations in Fig. 8 are stacked anti-parallel, three different patterns are obtained, one for each of the three planes (Fig. 9(b), (c) and (d)). Radio-opaque models of these cross-bridges patterns were made. X-ray pictures were taken perpendicular to the axis of the filament, rotating the models in 5” intervals through 180”. This represents 36 different directions. For each direction the appearance of a one-period repeat (approximately 430 A) was rated from 0 to + 3 where 0 is very poor, + 1 is poor, + 2 is fair and + 3 is a strong period. As can be seen from Table 2, with parallel stacking of filaments a maximum number of directions gave a poor 430 A period, while with anti-parallel stacking a maximum number of directions gave a strong 430 A period. In Fig. 10 the occurrence of the 430 A repeat is shown as a function of the direction of the X-ray beam. With parallel stacking of the filaments (Fig. 10(b)) the 43OA period was strong over a span of approximately 15” in three directions (O’, 60”, 120”). As can be seen from Fig. 9, this corresponds to directions midway between the directions of the cross-bridges. Au angle of 150” gives a direction directly along the cross-bridges in one plane, A strong period shows up at 150”, but a -+ 5” change in angle results in a poor period. For the case of anti-parallel stacking of the filaments (Fig. 10(a)) again

MYOSIN

FILAMENT

STRUCTURE

AND

ANTIBODY

STAINING

221

(b)

Fm. 9. Orientation

of crone-bridges

in planes perpendicular

to the axis of the filaments.

(a) For parallel etwking of the tie&e this pattern is obtained. The pattern is rotated 90° for each successive plane in which cross-bridges are present. (b), (c) and (d) These three different patterna are obtained for anti-parallel stacking of the filaments. They are repeati every period along the Lament. The angles marked on the di~grntna eerve ae reference point8 for the direction of the X-ray beam wed to obtain the dete in Fig. 10.

a strong period occurs at O”, 60” and 120” for a span of approximately 15”. However, a strong periodicity &o occurs at 25”, 90” and 150” for a span of 20 to 25”. As can be seen from Fig. 9, these directions correspond to the directions of the cross-bridges. It is therefore alear that the approximately 430 A period can be accounted for solely on the basis of the orientation of the oross-bridges and that the visibility of the 430 A period is considerably increased by anti-parallel stacking of the filaments.

222

F. A. PEPE TAESLE 2

Occurrence of the approximately 430 d repeat period. Parallel stacking of the filaments No. of directions

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FIU. 10. Visibility of the approximately 430 A period as a function of direction perpendicular to the longitudinal axis of the filaments. Rating of the period: +3, strong; +2, fair; +l, poor; 0, very poor. (a) Anti-parallel stacking of the filaments. Note span of 16” with fair to strong periodicity at 0, 60 and 120”. Also span of approximately 25” with fair to strong periodicity at 26, 90 and 150’. (b) Parallel stacking of the f%ments. Note span of 16’ with fair to strong periodicity at 0, 60 and 120’.

(e) Length of theJiZument An important consideration, with respect to the thick filament, is the length-determining mechanism. This must be built into the myosin molecules in some way. Consider Fig. 11 in which the relative stagger of neighboring molecules in the variations of the model (Fig. 8) is shown. The solid lines join neighboring molecules staggered by one-third period. The broken lines join neighboring molecules staggered by one full period. We see that the rows are arranged in three sets of four. Each set is made up of two pairs. There is a stagger of one-third period in a pair and a stagger of one period between the two pairs of a set. Since the 6lament grows from the M-line out to either end (Huxley, 1963), the stagger is determined by the tail to tail overlap in this region. A possible length-dete rmining mechanism might be as follows. For simplicity let us say that interactions between the rows of a pair result in no distortion, but that

MYOSIN

FILAMENT

STRUCTURE

AND

ANTIBODY

STAINING

223

interaction between pairs does. Along one side of the pair reactive sites, A may be present and along the other side sites B. Let us say there are four interaction sites of A and four of B per period. If eight A sites are required to interact with nine B sites, a distortion of either the myosin molecules or the reactive sites must occur along the rows of a pair (Fig. 11). For a stagger of one period between pairs the distortion will be least at the middle of one half of the A-filament or in the region where periodicity occurred in electron microscopy and will be maximal at the tapered end. At this end, presumably, the required amount of distortion is too great to permit interaction and the filament stops growing. To verify such a mechanism, detailed information concerning the specific sites involved in the interactions between myosin molecules would be required. Taper

M .line t %.

t

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for the thick filament.

Sites A and B interact between pairs (dotted lines). The dagger between pairs is one period (determined by the tail to tail overlap in the M-line). There are eight A sites and eight B sites every two periods along the rowa of molecules. Interaction of nine A sites with eight B siti leada to minimum distortion at the mid-point of one-half of the f%lament and maximum distortion at the tapered end.

(f) Diameter of the jilament The diameters of natural and synthetic myosin filaments observed by negative staining measure 100 to 150 A (Huxley, 1963). Recent estimates of the diameter of the myosin molecule from electron microscopy have been 15 to 20 A for the rod or L-meromyosin part of the molecule and 40 A for the globular region (Huxley, 1963; Rice, 1961a,b, 1963; Zobel & Carlson, 1963). Let us consider the case of 1 myosin molecule per cross-bridge, (n = 1). Using 15 to 20 A for the diameter of the myosin molecule exclusive of the cross-bridge, the diameter of the Lament deduced from this model (including overlap) would be 90 to 120 A. This is in good agreement with the measured value. In the center of the Glament corresponding to the M-line region where no overlap of the head region of myosin molecules along each row occurs, one would expect to see a smaller diameter for the 6lament. However, this is di&ult to observe unequivocally because M-line material tends to cling to this central zone even in separated thick Glaments (Pepe, 1963). From the model, there are 18 periods in each half of the A-band. One-half of the pseudo-H-zone has no myosin cross-bridges, therefore there are 16 sets of six cross-bridges in each half of the thick filament. Huxley (1960) arrived at about 18 sets of bridges in each half of the filament by direct count from electron micrographs. This 6gure may have included some Mbridges. From determinations of the actual amount of myosin in muscle, Huxley (1960) calculated that there are approximately two myosin molecules isolated for every cross-bridge. If each cross-bridge in the proposed model represents two myosin molecules (n = 2), the cross-sectional profile shown in Fig. 12(a) might obtain, where the previously obtained relationships for the model (Fig. 8) are maintained.

224

F. A. PEPE

The diameter of the Lament in this case would be 120 to 160 A, which is still in good agreement with the measured values. In insect fibrillar muscle there seem to be three myosin molecules per cross-bridge (Chaplain & Tregear, 1966). Three molecules per cross-bridge (n = 3) in vertebrate muscle might be arranged as shown in Fig. 11(b) and would give a diameter of 150 to 200 A.

(b)

(a)

FIG. 12. Possible arrangemente for the rowa of molecules in model C if there are (a) two myosiu molecules per cross-bridge and (b) three myosiu mole&m per cross-bridge. The relationahipe shown in Fig. 8 have been maintained. I am indebted to the members of the Myo Bio Group of the University of Pennsylvania and especially to Dr R. E. Davies for many valuable discussions. I also express my appreciation for the substantial improvement in tbis work resulting from the constructive criticisms and helpful suggestions of Dr H. E. Huxley. I am thankful to Dr Mark M&kin and Miss Monville in the Radiology Department of the Hospital of the University of Pennsylvania for their generous help with the X-ray absorption pictures. It is a pleasure to acknowledge the excellent technical assistance of Mm Barbara Drucker and Miss Oksana Korzenioweki throughout this work. This investigation was supported by U.S. Public Health Service grant ROl-AM-4806. REFERENCES Chaplain, R. A. & Tregear, R. T. (1966). J. Mol. Bid. 21, 276. Elliott, G. E., Lowy, J. & Milhnan B. M. (1965). Nature, 206, 1367. Finck, H. (1960). J. Biophys. B&&em. Cytol. 7, 27. Franzini-Armstrong, C. & Porter, K. R. (1964). J. Cell BioZ. 22, 676. Huxley, H. E. (1967). J. Biophy.9. B&hem. Cytd 3, 631. Huxley, H. E. (1960). In The CeU, vol. 4, ed. by J. Brachet t A. E. Mirsky, p. 366. New York: Aoademio Press, Ino. Huxley, H. E. (1963). J. Mol. Bid. 7, 281. Huxley, H. E. (1966). In Muscle, ed. by W. M. Paul, E. E. Daniel, C. M. Kay & G. Monckton, p. 3. Oxford: Pergamon Press. Page, 5. G. (1966). J. Ceil Bid. 26, 477. Palade, G. E. (1962). J. Exp. Med. 96, 286. Pepe, F. A. (1963). In Tech&qua in Endocrine Reaemch. ed. by P. Echstein & F. Knowles, p. 43. London: Academic Press, Inc. Pepe, F. A. (1966rz). J. Cell Biol. 28, 606. Pepe, F. A. (1966a). In E&s&ova Mieroecopy, vol. 2, ed. by R. Uyeda, p. 63. Tokyo: Maruzen Co., Ltd. Pepe, F. A. (1967). J. Mol. Bid. 27, 227. Pepe, F. A., Finck, H. & Holtzer, H. (1961). J. Biophye. Biochem. CytoE. 11, 633. Pepe, F. A. & Huxley, H. E. (1963). In Biochemietry of Mu&e Contraction, ed. by J. Gergely, p. 320. Boston: Little, Brown & Company. Reedy, M. C., Holmes, K. C. & Tregear, R. T. (1965). Nccture, 207, 1276. Reynolds, E. S. (1963). J. Ceh?Bid. 17, 208. Rice, R. V. (1961cr). Bid&m biaphye. AC&, 52, 602.

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Rice, R. V. (1961b). Biochh. Zkphys. Actu, 53, 29. Rice, R. V. (1963). In Biochemistry of Mu-de Contraction, ed. by J. Gergely, p. 41. Boston: Little, Brown & Company. Spiro, D. (1962). Trans. N. Y. Acd Sci., series 2, 24, 879. Young, D. M., Himmelfarb, S. & Harrington, W. F. (1965). J. Biol. Chem. 240, 2428. Zobel, C. R. & Carlson, F. D. (1963). J. Mol. Biol. 7, 78.