J. Mol. Biol. (1976) 100, 379-386

"Hybrid" Myosin Filaments from Smooth and Striated Muscle BENJAI~IIN KAMINER, ESZTER SZONYI A N D CHARLESD. BELCHER

Department of Physiology Boston University School of Medicine Boston, Mass. and Marine Biological Laboratory, Woods Hole, Mass., U.S.A. (Received 30 July 1975)

Reconstituted myosin filaments from vertebrate smooth muscle are shorter than those from striated muscle (Hanson & Lowy, 1964; Kaminer, 1969). In this investigation filaments were made from mixtures of the two types of soluble myosin in varying proportions. Mixtures were made of chicken gizzard myosin and either rabbit skeletal or chicken breast myosin. The filaments were made by rapid dilution of the KC1 concentration to 0.1 ~ at pH 6.5. Negative staining was used for electron microscopy. Histograms showed no bimodal distribution of lengths. Thus the two types of myosin co-assemble to form "hybrid" filaments presumably through the association of complementary specific bonds in the two types of myosin. These "hybrid" filaments showed a progressive decrease of length with increasing proportions of smooth muscle myosin. Hence the smooth muscle myosin in some ways limits the length of the "hybrid" filaments. This limitation is observed when smooth muscle myosin constitutes as little as 5% of the mixture. Possible factors are discussed on the limitation of length of filaments including the possibility that smooth muscle myosin may have less specific bonds available for assembly.

1. I n t r o d u c t i o n Since the first description of the mode of assembly of striated muscle myosin into filaments (Huxley, 1963) various factors influencing in vitro filamentogenesis have been studied. The p H and ionic concentration of the medium (Kaminer & Bell, 1966a,b; Josephs & Harrington, 1966; Sanger, 1971) determined, for example, the length of the filaments which are comparable to the natural ones when made under pH and ionic conditions approximating the intracellular milieu (Huxley, 1963; Kaminer & Bell, 1966b). Under similar conditions shorter filaments are formed from vertebrate smooth muscle (Hanson & Lowy, 1964; Kaminer, 1969). An immediate question arose whether the two types of myosin would co-assemble, and if so, whether the one type of myosin would determine the length of the "hybrid" filaments. We present evidence on the co-assembly into " h y b r i d " filaments, the length of which is determined by the smooth muscle myosin, t These results were reported at a meeting of the American Society of Cell Biology (Kaminer et al., 1974). Similar observations were made independently and reported by Pollard (1974) at the s a m e meeting.

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2. Materials and Methods (a) Preparation of myosin solutions Striated muscle myosin was extracted from r a b b i t a n d chicken breast muscle dissolved in 0.6 M-KCI (Szent-Gyorgyi, 1947) a n d cleared of actomyosin by dilution to 0.3 M a n d centrifugation (Portzell et al., 1950). The procedure for removing actomyosin was repeated. Smooth muscle myosin was extracted from chicken gizzard. Some preparations were extracted for 30 m i n with K I (Szent-Gyorgyi, 1951) which was washed out and replaced b y KC1 according to B a r a n y et al. (1966). Nucleic acid was removed in a few preparations b y means of a DEAE-celhilose column using conditions described b y Harris & Suelter (1967). This was done to determine whether the c o n t a m i n a n t affected the aggregation into filaments. Since the nucleic acids did n o t influence the length of the filaments this procedure was omitted in subsequent preparations. Other preparations were extracted in 0.6 ~-KC1 according to Cohen et al. (1961). The actomyosin was precipitated at 0.05 ~-KCI a n d redissolved twice. Myosin was then separated out b y ultracentrifugation in the presence of A T P (8 • 10 -4 ~) (Weber, 1956). The myosin solutions were well dialyzed to remove excess nueleotides. The preparations often contained aetin a n d tropomyosin a n d these were cleared b y prolonged ultracentrifugation (100,000 g for 12 h ; Lehman & SzentGyorgyi, 1972) in 0-6 M-KC1, 1 m~-MgCl2, 0.025 ~-Tris-maleate, p H 6"5, a n d the supern a t a n t was reprecipitated and dissolved. The myosin solution (in 0.6 M-KC1, 0.025 M-Trismaleate, p H 6.5) was dialyzed against the same solvent to remove a n y traces of free MgC12 since divalent cations (calcium a n d magnesium) influence the length of filaments from striated muscle myosin (Kaminer et al., 1973). All the myosin stock solutions were stored in 50~o glycerol at -- 20~ Fig. 1 depicts sodium dodeeyl sulphate gel electrophoretic bands of the purified preparations.

A

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FIe. 1. Sodium dodeeyl sulphate-polyacrylamide gel eleetrophoresis of smooth muscle myosin from gizzard (Sm M) and actin (A) and striated muscle myosin from chicken breast (St M) and actin (A); the 2 series were run at different times. The myosin preparations are free of aetin and tropomyosin. The Sm M has 2 light chains and the St M has 3 light chains. The electrophoresis was done using 10% acrylamide according to Weber & Osborn (1969).

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(b) Myosin filaments Samples of myosin from the glycinerated stmcks were precipitated by dilution of the KC1 to 0.05 ~ and the precipitate was washed in 0.05 ~t-KC1. The pellets wore then dissolved in the solution containing 0.6 M-KC1and 0.025 ~-Tris-maleate buffer at pH 6-5. The protein concentrations (Lowry etal., 1951) varied around 3 mg/ml. Filaments wore formed from striated, smooth and varying mixtures of the 2 types of myosin by rapid dilution of KC1 to 0-1 ~ at pH 6.5. Separated single filaments are uncommon from smooth muscle myosin containing actin and tropomyosin; the vast majority are stuck to one another end to end commonly forming clumps and irregular meshworks with the stuck ends having bulbous projections. By contrast pure smooth muscle myosin contains few of these "sticky" filaments and single, easily measurable filaments predominate. Wachsberger & Pope (1974) reduced the number of meshworks from pure smooth muscle myosin at 0.1 M-KC1 by means of 10 mMMgC12. Since dialysis produces longer filaments (Huxley, 1963) it was realized that the rate of dilution might influence the length of filaments (Kaminor & Bell, 1966b) and hence the dilution was done at a constant rate over a fixed time period of precisely 2 min using a Sago Syringe pump under constant magnetic stirring. A detailed study on the rate of dilution influencing filament length has been done by Katsura & Nods (1971,1973). (c) Negative staining This was done using the method of Huxley (1963) using 2~/o uranyl acetate. (d) Electron microscopy A Siemens Elmiskop I was used with an accelerating voltage of 80 kV, a 200 ~m aperture in condenser I I and a 35 ~m silver objective aperture.

3. Results Filaments were formed at 0.1 ~t-KCI and p H 6.5 because it was shown previously (Kaminer & Bell, 1966b) t h a t optimum length was obtained with these conditions. Katsura & N o d s (1973) obtained optimum length at p H 7-0 b y dialysis. Rapid dilution of the KC1 produces a more uniform and shorter population of filaments than is formed b y dialysis. Better uniformity of different preparations is also attained by dilution at a constant controlled rate. The length and characteristics of the striated muscle myosin filaments were essentially similar to previous findings (Huxley, 1963; Kaminer & Bell, 1966b) as were also the smooth muscle ones (Kaminer, 1969) which are about half the length of striated muscle filaments. Figure 2 depicts the length of filaments from rabbit striated and gizzard smooth myosin and from combinations of the two types of myosin in varying proportions. Figure 3 shows similar results of filaments formed from mixtures of breast and gizzard myosin. I n both series of experiments there is a progressive decrease in length of the filaments with increasing proportions of smooth muscle myosin and the histograms show no bimodal distribution. Hence the two types of myosin co-assemble and the smooth muscle myosin in some w a y limits the length of "hybrid" filaments. This limitation is observed when smooth muscle myosin constitutes as little as 5~/o of the mixture (Fig. 3). The shorter the filaments the greater the incidence of a bare central zone with irregular projections (HMM) at either end in accordance with the model of assembly proposed by Huxley (1963). I n long filaments the presence of a bare zone is relatively rare as was previously reported and accounted for, on the basis of errors in assembly as more and more building units are stacked (Kaminer & Bell, 1966b).

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F i g u r e 4 i l l u s t r a t e s r e p r e s e n t a t i v e e x a m p l e s o f filaments from b r e a s t a n d g i z z a r d muscle. A t higher magnification, filaments f o r m e d f r o m g i z z a r d show b a r e zones w i t h p r o j e c t i o n s a t e i t h e r e n d (Fig. 4(e)). I t s h o u l d be n o t e d t h a t m o s t o f t h e s m o o t h muscle filaments u n d e r these conditions, like s t r i a t e d m u s c l e ones, are t a p e r e d . T h e p r o j e c t i o n s are i r r e g u l a r in s h a p e a n d d i s t r i b u t i o n a n d m a y b e a s y m m e t r i c a l . T h e lower f i l a m e n t shown in F i g u r e 4(c), as a n e x a m p l e , shows p r o j e c t i o n s on b o t h sides of t h e left end, whereas t h e r i g h t e n d has p r o j e c t i o n s m a i n l y on t h e one side l e a v i n g t h e o p p o s i t e m a r g i n r e l a t i v e l y s m o o t h . This could be d u e to a b b e r a t i o n in s t a c k i n g . A m o r e defined p a t t e r n of a s y m m e t r i c a l d i s t r i b u t i o n of cross-bridges has b e e n

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Length (/J.m ) Fig. 3. Histograms showing the frequency distribution of filament lengths of smooth muscle myosin (gizzard) a n d striated muscle (chicken breast) a n d mixtures of the two in varying proportions. 300 to 400 filaments were measured from each preparation. There is no bimodal distrib u t i o n of lengths of the mixed filaments a n d hence the 2 types of myosin co-assembled. There is a progressive decrease of length with increasing proportions of smooth muscle myosin. The average lengths a n d s t a n d a r d deviation bars are depicted in the insert. The following are the averages a n d (standard errors). Chicken breast muscle (St) 100%, gizzard (Sm) 0 % 1.035 /~m (0"007); St 95~o Sm 5 % 0.823 /zm (0'005); St 90% Sm 10% 0.714/~m (0.004); St 75% Sm 25% 0.674/~m (0"003}; St 5 0 % Sm 50% 0-621 /zm (0-003); St 25% Sm 75% 0.545 /~m (0-003); St 0 % Sm 100% 0-506 /zm (0-004).

described by Sobieszek & Small (1972) and they consider it to be due possibly to the stacking of bipolar aggregates of myosin. 4. Discussion

Little is understood on the assembly of myosin filaments in vitro and on their length-limiting factors. From the electron microscopic findings and proposed model of Huxley (1963) one could assume binding between sites at the tail end of the myosin molecule with complementary sites along the light meromyosin (LMM) to

384

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SZONYI

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FIG. 4. Electron micrographs of myosin filaments : (a) from gizzard myosin a n d (b) breast muscle myosin (magnification 30,000). (c) Gizzard myosin (magnification 90,000) showing t h e following details. The filaments are tapered, there is a central bax:e zone, the projections are irregular usually pointing away at an angle from the center a n d they are asymmetrically distributed.

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MYOSIN

FILAMENTS

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allow for partial overlap and antiparallel stacking thus producing filaments with a bare central zone and projecting heavy meromyosin (HMM) at either end. As the filament grows in length, appropriately positioned complementary sites must presumably be available for parallel stacking with partial overlapping of LMI~ in the core of the filament on either side of the central bare zone. As more molecules stack there is a greater likelihood for errors and imperfections occurring as already pointed out, thus resulting in obliteration of the bare central zone and in other aberrations. The building unit for assembly appears to be a dimer and not a monomer, as was originally assumed, since the myosin monomer in high salt concentration is in rapid reversible equilibrium with a dimer species (Godfrey & Harrington, 1970). Subsequently Burke & Harrington (1972) and Harrington et al. (1972) concluded that the dimers are formed from parallel association of monomers displaced 400 to 500 A ~dth respect to their neighbors. Herbert & Carlson (1971) using laser light scattering came to essentially similar conclusions. Whatever the building unit, it seems clear from the present findings that smooth and striated muscle myosin must have complementary assembly sites for the formation of "hybrid" filaments. What limits the length of filaments in general and particularly the smooth muscle and "hybrid" ones, under given ionic and pH conditions, remains enigmatic. Katsura & Noda (1971,1973) on discussing the dependence of length on rate of salt dilution, considered the possible role of HM~ and also the relationship of nucleation to the growth process. HMM might, through steric hindrance, limit the length, since LMM paracrystals, in contrast to filaments from the intact molecule, are extremely long. The question relevant for this study is whether the organization of HMM in smooth muscle myosin differs from striated muscle. We of course know that the smooth muscle myosin ATPase is low (Needham & Williams, 1959; Barany et al., 1966) and that it has two light chains (Kendrick-Jones, 1973). Nucleation and subsequent growth, a sequence of processes which are known to determine the size of crystals and of some protein aggregates (Waugh, 1957; Wood, 1960) could not however, be quantitated into a simple model to account for the dependency of filament length on rate of ionic dilution (Katsura & Noda, 1971,1973). A major difficulty in such a model is the lack of dependence of filament length upon protein concentration. The authors gave consideration to the possibility that length could be dependent on a width limiting mechanism and that dimers might be the building units. The possibility that nucleation of smooth muscle myosin occurs more rapidly resulting in short filaments must, however, still be considered. Caspar (1966) on discussing general principles of self-assembly points out the self-limitation of assembly of identical units; as each building unit stacks it finds itself in a different and more restricted environment. An important consideration is the number of specific assembly sites and it was previously suggested (Kaminer, 1969) that vertebrate smooth muscle myosin might have fewer such sites than striated muscle myosin to account for the relatively short filaments from smooth muscle myosin. Similar reasoning could also be used to account for the relatively short filaments from non-muscle myosin (see Pollard & Weihing, 1974 for review and references) including brain (Kuczmanski & Rosenbaum, 1974). The restriction of length imposed on the "hybrid" filaments by the incorporated smooth muscle myosin may be similarly explained.

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This work was supported b y grants from the National I n s t i t u t e s of ~Iealth and the National Science Foundation. One of the authors (C. ]). B.) was the recipient of a George B. Wislocki fellowship from H a r v a r d . REFERENCES Barany, M., Barany, K., Gaetjens, E. & Bailin, G. (1966). Arch. Bioehem. Biophys. 113, 205-221. Burke, M. & Harrington, W. F. (1972). Biochemistry, 11, 1456-1462. Caspar, D. L. I). (1966). I n Molecular Architecture in Cell Physiology (Hayashi, T. & SzentGyorgyi, A. G., eds), pp. 191-207, Prentice Hall, London. Cohen, C., Lowey, S. & Kucera, J. (1961). J. Biol. Chem. 236, PC23-PC24. Godfrey, J. E. & Harrington, W. F. (1970). Biochemistry, 9, 894-908. Hanson, J. & Lowy, J. (1964). Proc. Roy. Soc. London, ser. B, 160, 523-524. Harrington, W. F., Burke, M. & Barton, J. S. (1972). Cold Spring Harbor Syrup. Quant. Biol. 37, 77-85. Harris, M. & Suelter, C. H. (1967). Biochim. Biophy8. Acta, 133, 393-398. Herbert, T. J. & Carlson, F. I). (1971). Biopolymers, 10, 2231-2252. Huxley, H. E. (1963). J. Mol. Biol. 7, 281-308. Josephs, R. & Harrington, W. F. (1966). Biochemistry, 5, 3474-3487. Kaminer, B. (1969). J. Mol. Biol. 39, 257-264. Kaminer, B. & Bell, A. L. (1966a). Science, 151,323-324. Kaminer, B. & Bell, A. L. (1966b). J. Mol. Biol. 20, 391-401. Kaminer, B., Szonyi, E. & Belcher, C. D. (1973). J. Gen. Physiol. 62, 657. Kaminer, B., Szonyi, E. & Belcher, C. D. (1974). J. Cell Biol. 63, 160a. K a t s u r a , I. & Noda, H. (1971). J. Biochem. 69, 219-229. K a t s u r a , I. & Noda, H. (1973). J. Biochem. 73, 245-256. Kendriek-Jones, J. (1973). Phil. Trans. Roy. Soc. sec. B, 265, 183-189. Kuczmanski, E. & Rosenbaum, J. L. (1974). J. Cell Biol. 63, 178a. Lehman, W. & Szent-Gyorgyi, A. G. (1972). J. Gen. Physiol. 59, 375-387. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265-275. Needham, D. M. & Williams, J. M. (1959). Biochem. J. 73, 171-181. Pollard, T. D. (1974). J. Cell Biol. 63, 273a. Pollard, T. D. & Weihing, R. R. (1974). C.R.C. Critical Rev. of Biochem. 2, 1-65. Portzell, I-I., Schramm, G. & Weber, H. H. (1950). Z. Naturforsch. B, 5, 61-74. Sanger, J. W. (1971). Cytobiol. 4, 450-466. Sobieszek, A. & Small, J. V. (1972). ColdSpring HarborSymp. Quant. Biol. 37, 109-111. Szent-Gyorgyi, A. (1947). Chemistry of Muscular Contraction, Academic Press, New York. Szent-Gyorgyi, A. G. (1951). J. Biol. Chem. 192, 361-369. Wachsberger, P. R. & Pepc, F. A. (1974). J. Mol. Biol. 88, 385-391. Waugh, D. F. (1957). J. Cell & Comp. Physiol. 49, Sup. 1, 145-164. Weber, A. (1956). Biochim. Biophys. Acta, 19, 345-351. Weber, K. & Osborn, M. (1969). J. Biol. Chem. 224, 4406-4412. Wood, G. C. (1960). Biochem. J. 75, 598-605.