.I. Mol. Bid.
(1979) 131, 133-136
LETTER TO THE EDITOR
Electron Microscope Studies of Thick Filaments from Vertebrate Skeletal Muscle Scparatcd tllick filamerrt)s llavr been prepared for electroll microscopy by H method involving freeze-drying and shadowing. In tile resulting filaments tllca individual Ileads of myosin molecules can he seen surrounding the filament, shaft, \vhich appears relatively smooth. Pairs of Ileads can frequently be seen to bra rlnanatillg from a common origitl. Myosin bea,ds art’ follrrd at distances alp to A00 .r\ from tJle edge of the shaft,. The basic structure of thick filaments from vertebrate skeletal muscle was deduced b,v Huxlt:y (1963) from studies of negatively stained native and synthetic filaments. A major inference drawn from this work was that myosin heads are arranged on thta outside of the thick filaments enabling them t,o form cross-bridges in the intact musclt,. Although this idea, has since gained wide acceptance, direct confirmation of it by electron microscopy has not hitherto been possible. since myosin heads survive negative sta,ining or embedding procedures poorly or not at all. Data obtained more recently using X-rays have shown that cross-bridges are likely to be helically arrayed : however. because of the difficulty in preservation it has been difficult to test directl? thra various helical a’lternatives proposed (Huxley & Brown, 1967: Pepe, 1967a: Squire. 1972). Transverse stripes were seen in some thin longitudinal sections (Huxley. 1966; Sjiist,riim & Squire. 1977) and in A-segments (Hanson et al.. 1971: Craig, 1977) indicat,inp the presence of regular structure but in neither case were the heads of myosin molecules seen clearly. Cross-bridge detail has been observed in thin sections of rigor insect flight muscle (Reedy, 1968), however. deductions from the micrographs concerning t’he structure of thick filaments are hindered by the fact that in rigor muscle the cross-bridge lattice reflects the symmet’ry of the thin rather than the thick filament’ (Huxley & Brown. 1967) and bridges which are not at,tached are probably not visible (Squire, 1972). By contrast. electron microscopy of myosin molecules, the main constituents of t.hick filaments. has been relatively rewarding and the shape of the molecule has been defined in some detail. These data have been obtained from shadowed preparations of myosin. either air-dried (Rice, 1961: Huxley, 1963; Slayter & Lowey, 1967; Lowe? pt al.. 1969). or more recently, dried at low temperature iz vacua (Elliott & Offer, 1978). In t’his letter we describe the application of freeze-drying and shadowing to t’hink tilaments: t,he method used is similar to that described by Nanninga (1968) (see also Nermut. 1977). Separat’ed thick and thin filaments were prepared from fresh rabbit psoas muscle a,> described by Trinick (1973) and Craig (1977). Strips of rabbit psoas muscle were tied t,o perspex rods and incubat,ed overnight at 0°C in 0.1 ~-Nacl. 2 mM-KCl, 2 mMIfgc’12. I InM-EGTAt. 6 mlvr-potassium phosphate, 0.1 y;, glucose (pH 74). The strips
I 34
.I.
‘I’RISl(‘K
ANI)
A.
ELLIc)TT
were then cut into small pieces and homogenized in (I.1 M- tiCi. 10 m~-~MgCl,. 1 tn~EGTA. 5 mM-ATP. 0.5 mM-dit)liothreit,oI. 6 mi\l-potassiuttt phosphate (pH 7.0). UndissociaDed fragment’s of muscle U’CIY removed I)), centrifugation at, lO.(@() g fOJ ten minutes. Soluble proteins were removed using the lo\\- ionic st,rength prccipitatiott procedure for the purification of ntolluscan t)hick tilamenta described 1)~ BzetltGyiirggi rf al. (1971). A drop of filament suspension was applied to a cerbon-coated grid which had heen ma’de hydrophilic by exposurr to ultra.violet radiation. AfW briefly washing wiU1 cold distilled water (previousl>r boiled t,o raise, its pH t,o about 64) the grid was drained and plunged into liquid nitrogen. It, wxs t(hert freeze-dried for three hours at -70°C and rotary shadowed with pla~tinutn (shadow- angle 1 in 5). Flat, carbon filtnh were made hp stripping carbon from mica and ntountittg it) on grids wit,h t#he side that had heen in contact with the mica. uppermost : suc41 tilm. < give a, mow uniform hackground in the final imapc. Figure 1 shows a montage of thick tiletrtc~nts which for t’titi first, time cbxhibit, project,ions recognizahlc as the heads of myosin moltwtles. Tht~ filaments ;trc bipolar and about, 14 pm long, hut, unlike negatively stIaitwtl t’hick filaments. t,hcy appea,r as a rela,tivel\T smooth central shaft’ surrout~~cd by R fringe of globular projections. The fact, tha,t half-way a,long the shaft t,he fringe is alwent ovt’r a dis;tancc of about 1500 x supports t’he view t,hat’ itI is composed of m,voxitt II(w~s. since myosin heads are known to be absent, in this the “l)arr zone” \vttich is 1490 4 long (Craig. 1977). The remains of the M-line can I)e seen as a bhickcning at, t,lrc middle of the 1)at.r’ zone. Tlt~~ dimensions of the projections in t.lre fringe. ahout 190 .A ton p and i0 :\ \vid(l. md also their shapeh are similar to bhose of the heads of myositt tnolwnlw which wn ocw~sionally tw seen in t,he hackground (SW inset on Fig. 1 ). Mort~ovt~r. pairs of projections itt tltc ftingtl frequently appear to emanate from a common poittt (arrow~d). We thrrefow cottcludt~ that, most, of the projections are individual tnyositr heads. though somf~ of tlw lazger ones ma!; contain two or more heads. OlJjccts \\.lriclr migttt~ Iw the suhfragment 2 portion of tnyosin catr i)r found. tIhough only rawI>.. It \\,ould not tw oxpcctctd t’hat all t,htx tnyositt heads ott a filatnent would htt distinguished. for t,how lying undwnenth the shaft, would Iw ohscured, and those tJ.ittg 011 top may tw invisible against Chrb to rough background. The ttumlw of projt~ctiotw swn is ~~ht)~t 300. corrrspotiding 150 (or more) tnyositt molocult~s. ‘L%c tot;11 nrtmtwt~ of tnolccul~3 iii a filwtucmt is probably 300 t,o 400 (Trcgear 8-zSquirch. 1973: 3loritrto~o bt Hitt~t~itlgtott. 1976). The impression gained from t,ltc: trtictwpt~a~ptrs ih that ttto hrwlh have folded do\vn off the shaft on to t,hr su1Arat.e. Kistlcr & l shaft,, suggesting that, in solutiott tn vosin Itt~ads tlavcb )I grt:atet~ affinit>- for hgdrophilic carhon than for untreated cnthon. -LOOand NO r\. dentottstraling that. The ohsrrvrd fringe-width varies hatwwn at least, under the conditions used tttw. m>-ositt Itcwl~ are ahlc to swing out from tht filament hackloom,. 8uctl behaviour has been propw~d 1)~ Huxley (1969) to account for the abilit,y of cross-bridges to abtach to thin iilatnt~nts at, different interfilament spacings and is c.onsis:tetit wit11 tttt, proposal tttat t~ttt~rc~is 3 flexible Iting:1%region itt t,lttb myositt tail (f’rpc~. 1967b: Huxltby bz. RIYJU II. 1967). Blliott. & Offer (1978) havtb recetttly ot~servrd such a Hexit)ltt region in tttyositl. tttr distattuc, from ttrc: tip Of tllcL
146
-1. TRINICK
AN I) .A. ELLTOT’I
heads being 620 d. This figure is the maximum width which the fringe would have if the cross-bridges were at 90” to the shaft. The smaller value for the maximum fringe-width observed is compatible with an inclination of less than 90”. However, the figure of 500 A may be an underestimate since the width of the central shaft is some 290 A which is about twice the accepted value (Huxley, 1969), indicating some distortion of the backbone, possibly due to washing with distilled water prior to freezing. Although these filaments exhibit projections recognisable as myosin heads, attempts to detect order, either in the array of heads or in the backbone, have been largely unsuccessful. However, it-may in the future be possible to cross-link the heads to the backbone and directly obierve the cross-bridge symmetry. We thank Dr Gerald Offer for much helpful discussion, Mr Arnold Fasoli for constructiorl of apparatus and Mr Zoltan Gabor for help with the photography. We are indebted to t,hr Medical Research Council for financial support. Department of Biophysics King’s College 26-29 Drury Lane London WC2B 5RL England Received
29 January
t Present address: BS18 7DY, England.
,J.
TRINICKt
A.
ELLIOTT
1979 hgricult,ural
Research
Council.
&at
Research
Institute,
Langford.
Bristol
REFERENCES Craig, R. (1977). J. Mol. &ioZ. 109, 69-81. Elliott, A. & Offer, G. (1978). J. Mol. Viol. 123, 505- 519. Hanson, J., O’Brien, E. J. & Bennett, I?. M. (1971). ,I. Mol. Biol. 58, 865~-871. Huxley, H. E. (1963). J. Mol. Biol. 7, 281-308. Huxley, H. E. (1966). Harvey Lectures, 1964-65, 60, 85--l 18. Huxley, H. E. (1969). Science, 164, 1356-1366. Huxley, H. E. & Brown, W. (1967). J. MoZ. Biol. 30, 383-434. Kistler, J. & Kellenberger, E. (1977). J. Ultrastruct. Res. 59, 70 -75. Lowey, S., Slayter, H. S., Weeds, A. G. & Baker, H. (1969). .I. Mol. Hid. 42, 1-~29. Morimoto, K. & Harrington, W. F. (1974). J. Mol. BioZ. 83, 83-97. Nanninga, N. (1968). Proc. Nat. Acad.&i., U.S.A. 61, 614--620. Nermut, M. V. (1977). In Principles and Techniques of Electron Microscopy (Hayat,, M. A.. cd.), vol. 7, pp. 79-117, Van Nostrand Reinhold. NPW York. Pepe, F. A. (1967a). J. Mol. Biol. 27, 203-225. Pepe, F. A. (1967b). J. Mol. Biol. 27, 227-236. Reedy, M. K. (1968). J. Mol. BioZ. 31, 155-176. Rice, R. V. (1961). Biochim. Biophys. Acta, 52, 602%6Ol. Sjiistram, M. & Squire, J. M. (1977). J. Mol. Biol. 109, 4%68. Slayter, H. S. & Lowey, S. (1967). Proc. Nat. Acad. Sci.. C.S.A. 58, 1611.1618. Squire, 5. M. (1972). J. Mol. Biol. 72, 125-138. J. (1971). J. Al,1oZ. Biol. 56. 239-258. Szent-Gyargyi, A. G., Cohen, C. & Kendrick-Jones, Tregear, R. T. & Squire, J. M. (1973). J. Mol. Biol. 77, 279-290. Trinick, J. A. (1973). Ph.D. thesis, University of Leicester.
(1979) 131, 133-136
LETTER TO THE EDITOR
Electron Microscope Studies of Thick Filaments from Vertebrate Skeletal Muscle Scparatcd tllick filamerrt)s llavr been prepared for electroll microscopy by H method involving freeze-drying and shadowing. In tile resulting filaments tllca individual Ileads of myosin molecules can he seen surrounding the filament, shaft, \vhich appears relatively smooth. Pairs of Ileads can frequently be seen to bra rlnanatillg from a common origitl. Myosin bea,ds art’ follrrd at distances alp to A00 .r\ from tJle edge of the shaft,. The basic structure of thick filaments from vertebrate skeletal muscle was deduced b,v Huxlt:y (1963) from studies of negatively stained native and synthetic filaments. A major inference drawn from this work was that myosin heads are arranged on thta outside of the thick filaments enabling them t,o form cross-bridges in the intact musclt,. Although this idea, has since gained wide acceptance, direct confirmation of it by electron microscopy has not hitherto been possible. since myosin heads survive negative sta,ining or embedding procedures poorly or not at all. Data obtained more recently using X-rays have shown that cross-bridges are likely to be helically arrayed : however. because of the difficulty in preservation it has been difficult to test directl? thra various helical a’lternatives proposed (Huxley & Brown, 1967: Pepe, 1967a: Squire. 1972). Transverse stripes were seen in some thin longitudinal sections (Huxley. 1966; Sjiist,riim & Squire. 1977) and in A-segments (Hanson et al.. 1971: Craig, 1977) indicat,inp the presence of regular structure but in neither case were the heads of myosin molecules seen clearly. Cross-bridge detail has been observed in thin sections of rigor insect flight muscle (Reedy, 1968), however. deductions from the micrographs concerning t’he structure of thick filaments are hindered by the fact that in rigor muscle the cross-bridge lattice reflects the symmet’ry of the thin rather than the thick filament’ (Huxley & Brown. 1967) and bridges which are not at,tached are probably not visible (Squire, 1972). By contrast. electron microscopy of myosin molecules, the main constituents of t.hick filaments. has been relatively rewarding and the shape of the molecule has been defined in some detail. These data have been obtained from shadowed preparations of myosin. either air-dried (Rice, 1961: Huxley, 1963; Slayter & Lowey, 1967; Lowe? pt al.. 1969). or more recently, dried at low temperature iz vacua (Elliott & Offer, 1978). In t’his letter we describe the application of freeze-drying and shadowing to t’hink tilaments: t,he method used is similar to that described by Nanninga (1968) (see also Nermut. 1977). Separat’ed thick and thin filaments were prepared from fresh rabbit psoas muscle a,> described by Trinick (1973) and Craig (1977). Strips of rabbit psoas muscle were tied t,o perspex rods and incubat,ed overnight at 0°C in 0.1 ~-Nacl. 2 mM-KCl, 2 mMIfgc’12. I InM-EGTAt. 6 mlvr-potassium phosphate, 0.1 y;, glucose (pH 74). The strips
I 34
.I.
‘I’RISl(‘K
ANI)
A.
ELLIc)TT
were then cut into small pieces and homogenized in (I.1 M- tiCi. 10 m~-~MgCl,. 1 tn~EGTA. 5 mM-ATP. 0.5 mM-dit)liothreit,oI. 6 mi\l-potassiuttt phosphate (pH 7.0). UndissociaDed fragment’s of muscle U’CIY removed I)), centrifugation at, lO.(@() g fOJ ten minutes. Soluble proteins were removed using the lo\\- ionic st,rength prccipitatiott procedure for the purification of ntolluscan t)hick tilamenta described 1)~ BzetltGyiirggi rf al. (1971). A drop of filament suspension was applied to a cerbon-coated grid which had heen ma’de hydrophilic by exposurr to ultra.violet radiation. AfW briefly washing wiU1 cold distilled water (previousl>r boiled t,o raise, its pH t,o about 64) the grid was drained and plunged into liquid nitrogen. It, wxs t(hert freeze-dried for three hours at -70°C and rotary shadowed with pla~tinutn (shadow- angle 1 in 5). Flat, carbon filtnh were made hp stripping carbon from mica and ntountittg it) on grids wit,h t#he side that had heen in contact with the mica. uppermost : suc41 tilm. < give a, mow uniform hackground in the final imapc. Figure 1 shows a montage of thick tiletrtc~nts which for t’titi first, time cbxhibit, project,ions recognizahlc as the heads of myosin moltwtles. Tht~ filaments ;trc bipolar and about, 14 pm long, hut, unlike negatively stIaitwtl t’hick filaments. t,hcy appea,r as a rela,tivel\T smooth central shaft’ surrout~~cd by R fringe of globular projections. The fact, tha,t half-way a,long the shaft t,he fringe is alwent ovt’r a dis;tancc of about 1500 x supports t’he view t,hat’ itI is composed of m,voxitt II(w~s. since myosin heads are known to be absent, in this the “l)arr zone” \vttich is 1490 4 long (Craig. 1977). The remains of the M-line can I)e seen as a bhickcning at, t,lrc middle of the 1)at.r’ zone. Tlt~~ dimensions of the projections in t.lre fringe. ahout 190 .A ton p and i0 :\ \vid(l. md also their shapeh are similar to bhose of the heads of myositt tnolwnlw which wn ocw~sionally tw seen in t,he hackground (SW inset on Fig. 1 ). Mort~ovt~r. pairs of projections itt tltc ftingtl frequently appear to emanate from a common poittt (arrow~d). We thrrefow cottcludt~ that, most, of the projections are individual tnyositr heads. though somf~ of tlw lazger ones ma!; contain two or more heads. OlJjccts \\.lriclr migttt~ Iw the suhfragment 2 portion of tnyosin catr i)r found. tIhough only rawI>.. It \\,ould not tw oxpcctctd t’hat all t,htx tnyositt heads ott a filatnent would htt distinguished. for t,how lying undwnenth the shaft, would Iw ohscured, and those tJ.ittg 011 top may tw invisible against Chrb to rough background. The ttumlw of projt~ctiotw swn is ~~ht)~t 300. corrrspotiding 150 (or more) tnyositt molocult~s. ‘L%c tot;11 nrtmtwt~ of tnolccul~3 iii a filwtucmt is probably 300 t,o 400 (Trcgear 8-zSquirch. 1973: 3loritrto~o bt Hitt~t~itlgtott. 1976). The impression gained from t,ltc: trtictwpt~a~ptrs ih that ttto hrwlh have folded do\vn off the shaft on to t,hr su1Arat.e. Kistlcr & l shaft,, suggesting that, in solutiott tn vosin Itt~ads tlavcb )I grt:atet~ affinit>- for hgdrophilic carhon than for untreated cnthon. -LOOand NO r\. dentottstraling that. The ohsrrvrd fringe-width varies hatwwn at least, under the conditions used tttw. m>-ositt Itcwl~ are ahlc to swing out from tht filament hackloom,. 8uctl behaviour has been propw~d 1)~ Huxley (1969) to account for the abilit,y of cross-bridges to abtach to thin iilatnt~nts at, different interfilament spacings and is c.onsis:tetit wit11 tttt, proposal tttat t~ttt~rc~is 3 flexible Iting:1%region itt t,lttb myositt tail (f’rpc~. 1967b: Huxltby bz. RIYJU II. 1967). Blliott. & Offer (1978) havtb recetttly ot~servrd such a Hexit)ltt region in tttyositl. tttr distattuc, from ttrc: tip Of tllcL
146
-1. TRINICK
AN I) .A. ELLTOT’I
heads being 620 d. This figure is the maximum width which the fringe would have if the cross-bridges were at 90” to the shaft. The smaller value for the maximum fringe-width observed is compatible with an inclination of less than 90”. However, the figure of 500 A may be an underestimate since the width of the central shaft is some 290 A which is about twice the accepted value (Huxley, 1969), indicating some distortion of the backbone, possibly due to washing with distilled water prior to freezing. Although these filaments exhibit projections recognisable as myosin heads, attempts to detect order, either in the array of heads or in the backbone, have been largely unsuccessful. However, it-may in the future be possible to cross-link the heads to the backbone and directly obierve the cross-bridge symmetry. We thank Dr Gerald Offer for much helpful discussion, Mr Arnold Fasoli for constructiorl of apparatus and Mr Zoltan Gabor for help with the photography. We are indebted to t,hr Medical Research Council for financial support. Department of Biophysics King’s College 26-29 Drury Lane London WC2B 5RL England Received
29 January
t Present address: BS18 7DY, England.
,J.
TRINICKt
A.
ELLIOTT
1979 hgricult,ural
Research
Council.
&at
Research
Institute,
Langford.
Bristol
REFERENCES Craig, R. (1977). J. Mol. &ioZ. 109, 69-81. Elliott, A. & Offer, G. (1978). J. Mol. Viol. 123, 505- 519. Hanson, J., O’Brien, E. J. & Bennett, I?. M. (1971). ,I. Mol. Biol. 58, 865~-871. Huxley, H. E. (1963). J. Mol. Biol. 7, 281-308. Huxley, H. E. (1966). Harvey Lectures, 1964-65, 60, 85--l 18. Huxley, H. E. (1969). Science, 164, 1356-1366. Huxley, H. E. & Brown, W. (1967). J. MoZ. Biol. 30, 383-434. Kistler, J. & Kellenberger, E. (1977). J. Ultrastruct. Res. 59, 70 -75. Lowey, S., Slayter, H. S., Weeds, A. G. & Baker, H. (1969). .I. Mol. Hid. 42, 1-~29. Morimoto, K. & Harrington, W. F. (1974). J. Mol. BioZ. 83, 83-97. Nanninga, N. (1968). Proc. Nat. Acad.&i., U.S.A. 61, 614--620. Nermut, M. V. (1977). In Principles and Techniques of Electron Microscopy (Hayat,, M. A.. cd.), vol. 7, pp. 79-117, Van Nostrand Reinhold. NPW York. Pepe, F. A. (1967a). J. Mol. Biol. 27, 203-225. Pepe, F. A. (1967b). J. Mol. Biol. 27, 227-236. Reedy, M. K. (1968). J. Mol. BioZ. 31, 155-176. Rice, R. V. (1961). Biochim. Biophys. Acta, 52, 602%6Ol. Sjiistram, M. & Squire, J. M. (1977). J. Mol. Biol. 109, 4%68. Slayter, H. S. & Lowey, S. (1967). Proc. Nat. Acad. Sci.. C.S.A. 58, 1611.1618. Squire, 5. M. (1972). J. Mol. Biol. 72, 125-138. J. (1971). J. Al,1oZ. Biol. 56. 239-258. Szent-Gyargyi, A. G., Cohen, C. & Kendrick-Jones, Tregear, R. T. & Squire, J. M. (1973). J. Mol. Biol. 77, 279-290. Trinick, J. A. (1973). Ph.D. thesis, University of Leicester.
