During the last few years the basicfeatures of the sliding-filament modelof contraction in striated muscle havegained general *acceptance and it hasbeen possible to concentrate attentionon the detailed mechanism by whichthe relative sliding force between theactin and myosin filaments is devel-oped. A number of observations haveindicated in general outline how cross-bridges between the filaments may beinvolved in the generation of this forcebut have also revealed some apparentlyparadoxical properties of the system.The most recent findings show a pos-sible way in which these paradoxes canbe resolved. Furthermore, there is nowa real possibility of solving the prob-lem in complete detail, provided a waycan be found to crystallize a recentlypurified globular subfragment of themyosin molecule. In this article Idiscuss these new findings and their im-plications.

According to the interdigitating fila-ment model of striated muscle (1), thecontractile material consists of longseries of partially overlapping arrays ofactin and myosin filaments (see Fig. 1)which form the myofibrils. These over-lapping arrays give rise to the charac-teristic band pattern visible in the lightmicroscope. In vertebrate striated mus-cle the myosin-containing filaments arespaced out in a hexagonal lattice 400to 450 angstroms apart, with the actin-containing filaments in between themat the trigonal positions of the lattice.The space between the filaments is oc-cupied by sarcoplasm (a dilute aqueoussolution of salts and of other proteins).When the muscle changes length,

The author is a member of the staff of theMedical Research Council Laboratory of Molecu-lar Biology, Hills Road, Cambridge, England.

1356

either actively during contraction orpassively during stretch or during re-lease from an extended length, thesliding filament model (2) supposes thatthe length of the filaments themselvesremains essentially constant but that theoverlapping arrays of filaments slidepast each other, the actin being drawnfurther into the array of myosin fila-ments (which form the A-bands) asthe muscle shortens, or withdrawnagain as the muscle is stretched. Theevidence for this model has been re-viewed in a number of papers (3-5)and is mentioned only incidentally inthis article.

While the overall changes in the ar-rangement of the filaments can be de-duced from light-microscopic observa-tions (once the origin of the band pat-tern is known), the underlying mecha-nism which produces movement of thefilaments past each other is obviouslynot accessible to direct visual observa-tion, and so we have to build up a pic-ture of it in a less direct way, usingwhatever technique seems likely to giveus useful clues.

Early Ideas about Cross-Bridges

The first and most crucial clue camefrom early electron-microscope obser-vations of sections of muscle (4, 6)which showed that cross-bridges linkedthe actin and myosin filaments togetheracross the gap of about 130 angstromswhich exists between their surfaces. Thebridges could still be seen as projectionson the myosin filaments in places whereno actin filament lay alongside them(for example, on the outside of fibrilsand in the H-zone of stretched sarco-meres), whereas they were not visible

The Mechanism ofMuscular Contraction

Recent structural studies suggest a revealing modelfor cross-bridge action at variable filament spacing.

H. E. Huxley

closely linked to contraction isknown (8), it can be estimated that thesplitting of an amount of adenosine tri-

SCIENCE, VOL. 164

on the actin filaments in the I-bands.It was clear, therefore, that they formeda permanent part of the myosin fila-ment structure. As they were the onlyvisible mechanical agents by which aforce could be developed between theactin and myosin-filaments, it was sug-gested (4) that this indeed was theirfunction, and that they very probablyrepresented the heavy-meromyosin sub-unit of the myosin molecule. It wasalready known that the actin-combiningability and adenosine triphosphataseactivity were associated with this partof the molecule (7), and it seemed rea-sonable to suppose that the sites re-sponsible for these properties would bebuilt into the overall structure of themuscle in such a way that they could in-teract directly with the actin filaments.

During the contraction of a muscle,even during a single twitch, the struc-ture may shorten by 30 percent of itsoriginal length or more, and the actinand myosin filaments must thereforeslide past each other (in a frog musclestarting at a resting sarcomere length of2.5 microns) by 0.375 microns (that is,3750 angstroms) in each half-sarcomere.Some variation in orientation of thecross-bridges can be seen in electronmicrographs, but the distal ends neverseem to be displaced by more thanabout 100 angstroms from the positionthey would occupy if the bridges wereaccurately perpendicular to the thickfilaments. It is clear therefore that, inorder to produce the much larger over-all sliding movement, some type of re-petitive interaction of the cross-bridgeswith the actin filaments is necessary.One possibility might be that the cross-bridges move to and fro in a cyclicalmanner, attaching to the actin fila-ments and pulling them toward thecenter of the A-band on one part oftheir stroke, and detaching again priorto their return stroke. Alternatively,the cross-bridges might remain rigidlyfixed in position while repetitive internalchanges in the actin filaments enabledthem to crawl along the series of fixedpoints so provided. But whatever thedetails, the basic idea was that thecross-bridges were in direct contactwith the actin filaments when force wasdeveloped, and that they were the me-chanical agents through which theforce was transmitted.

Since the probable free energy ofthe chemical reaction apparently most

phosphate equivalent to one moleculefor each myosin cross-bridge through-out the muscle would provide sufficientenergy for the actin and myosin fila-ments in each half-sarcomere to movepast each other by 50 to 100 angstromswhen the muscle was shortening againsta maximal load. This is consistent with(but of course, does not prove) a modelin which one molecule of adenosinetriphosphate is split at a cross-bridgeduring one cycle of its action and inwhich each cross-bridge can go throughits tension-generating cycle only onceduring each 50- to 100-angstrom rela-tive movement of the filaments. Theserequirements have the obvious corol-lary that the probability of splitting islow or zero when this cycle is not com-pleted. In this way chemical-energyrelease in the muscle can be controlled(i) by the tension developed (propor-tional to the number of bridges whichhad time to attach at any given short-ening velocity) and (ii) by the distanceshortened (proportional to the numberof cycles of attached bridges). In sucha system, energy release could be effi-ciently matched to the work done, asis known to be the case in muscle (9).Furthermore, a considerable numberof other properties of striated musclecould be explained n this generalbasis (3, 5); it was de4able, therefore,to investigate the natte and behaviorof the cross-bridges in as much detailas possible.

More Detailed Electron-MicroscopeObservations

Although the cross-bridges were firstseen in sectioned muscle in the electronmicroscope, their appearance underthese conditions is unsatisfactory when'more detailed information is required.Their arrangement on the thick fila-ments does not appear very regular,and little internal detail is visible. [Re-cent x-ray diffraction observation (10)has shown that, although the regularstructure of the muscle filaments andof the cross-bridges on them is re-markably well preserved by glutaralde-hyde fixation (now the method ofchoice), a very great deal of the reg-ular order is lost during the subsequentdehydration of the specimens prior toembedding.] However, studies of sep-arated muscle filaments, in which weused the negative staining technique,revealed a number of new structuralfeatures. These were described in de-tail several years ago (11), but it willbe useful to recall briefly some featuresthat are particularly relevant to thepresent discussion.The most straightforward evidence

that the cross-bridges represent theheavy-meromyosin end of the myosinmolecule came from a comparison ofthe filamentous aggregates of lightmeromyosin (formed at physiologicalionic strength) with aggregates formedunder similar conditions by intact myo-

sin molecules (see Fig. 2). The formerwere seen to consist of needle-shapedstructures many microns long and ofvarious widths, up to several thousandangstrom units. The surfaces of theselight meromyosin filaments were per-fectly smooth. On the other hand, thefilaments formed by the aggregation ofwhole myosin molecules had largenumbers of projections on their sur-faces over most of their length. Thesefilaments varied in thickness but wereusually less than about 200 angstromsin diameter, and they were usuallyshorter than the light-meromyosin fila-ments. Moreover, these synthetic myo-sin filaments were very similar inappearance to the "natural" thick fila-ments prepared directly from mechani-cally disrupted muscle. It was appar-ent, therefore, that the projections re-vealed by the negative staining methodwere equivalent to the cross-bridgesin the sectioned material, and that,since they were absent from the light-meromyosin filaments, they must beassociated with the heavy-meromyosinpart of the molecule. Furthermore,since the cross-bridges seen in sectionswere of the order of 40 to 50 ang-stroms wide by 120 angstroms long(and it was known that lateral shrink-age during processing had probablyreduced the longer dimension of thecross-bridge by about 20 percent) andsince isolated heavy-meromyosin mole-cules examined by the shadow-casting

I AH.

I-

I

-.. * * * *...-* *- **~~~ * ..0.0 0 0

....0 ...--.*.. . *. .*. ...-00 0

* 0000X.* *.* . *0X0X* 0 *0 0 0

* 00 0 0

Fig. 1. Diagrammatic representation of the structure of striated muscle, showing overlapping arrays of actin- and myosin-containingfilaments, the latter with projecting cross-bridges on them. For convenience of representation, the structure is drawn with consid-erable longitudinal foreshortening; with filament diameters and side-spacings as shown, the filament lengths should be- about fivetimes the lengths shown.20 JUNE 1969 1357

I Ir.. a a.. -

technique (11, 12) showed a globularregion of about 40 to 50 by 200 ang-stroms with a short tail about 400 ang-stroms long, it was concluded that thecross-bridges represented the globularregion, and that the tail must lie ap-proximately parallel to the backboneof the filament. Continuing this lineof argument, it seemed reasonable tosuppose that the adenosine triphos-phatase and actin binding sites wouldbe located in the globular region ofthe heavy-meromyosin molecule.

Further arguments which need notbe repeated here indicated that themyosin molecules were arranged inthe thick filaments with a definite struc-tural polarity, so that the heads of themolecules were always directed awayfrom the midpoint of the filaments (seeFig. 3); thus all the cross-bridges inone half of an A-band have the samepolarity, and this polarity is reversedin the opposite half of the A-band. Ifthe sets of actin filaments in each half-

sarcomere are to be drawn toward thecenter of the A-band, they must beacted on by sliding forces directed inopposite senses in either half of theA-band. It seems a reasonable arrange-ment that this directional specificityshould be established by the structureof the filaments and be embodied inthe orientation of the active sites. Itseems likely that these sites would in-teract in a stereospecific manner withthe actin filaments, so that reversingthe orientation of the cross-bridgeswould reverse the direction of theforce developed. A corresponding re-versal of polarity in the actin fila-ments would be expected on either sideof the Z lines, and this also was found.

These observations therefore rein-forced the view that the sliding forcewas developed as a consequence ofdirect physical contact between theheavy-meromyosin cross-bridges of thethick filaments and the actin units inthe thin filaments.

Fig. 2. Electron micrographs of negatively stained preparations of (left) natural thick-filaments, prepared directly from homogenized muscle; (middle) synthetic filamentformed by aggregation of purified myosin in O.AM KCI; and (right) synthetic filamentformed by aggregation of light meromyosin in 0. lM KCI. Projecting cross-bridgescan be seen on the natural and synthetic filaments in which whole myosin is present-that is, in which both the heavy- and light-meromyosin parts of the myosin moleculeare represented. The filaments containing light meromyosin alone have no projections(about X 162.000).

1358

Biochemical Evidence about

Actin-Myosin Inteiaction

An intimate interaction betweenthese molecules during contraction isalso indicated by other important linesof evidence, the first of which is con-cerned with the enzymatic behavior ofmyosin. Purified myosin, in the pres-ence of concentrations of magnesiumand calcium ions similar to those ex-pected in muscle during activity, hasrelatively low adenosine triphosphataseactivity (13). However, at the samemagnesium and calcium ion concen-trations, but in the presence of actin,under conditions where combinationbetween actin and myosin is knownto take place in the absence of adeno-sine triphosphate (that is, at low ionicstrength), the adenosine triphosphataseactivity is greatly enhanced (about 20-fold or more) (14) and approaches thatrequired to account for the knownrate of energy release in a muscle (15).There is therefore a very strong pre-sumption that the activating influenceof actin in the presence of adenosinetriphosphate is exerted by a directphysical combination with myosin,even if only a transitory one, for somepart of the cycle in which adenosinetriphosphate is split. Moreover, a force-generating link between the actin andinyosin filaments is required for con-traction, and this link has to providesome form of two-way coupling be-tween the performance of mechanicalwork and the splitting of adenosinetriphosphate, so that not only is theenergy from the reaction transformedinto mechanical work but, iunless themechanical work can be performed, thereaction is inhibited. It is very difficultto believe that this link is not providedby actual combination of actin withthe heavy-meromyosin cross-bridge.

Recent work by Ebashi and his co-workers (16) and by others (17, 18)has added further force to this argu-ment. The work of Annemarie Weber.of Hasselbach, and of Ebashi hadshown earlier (19, 20) that the adeno-sine triphosphatase activity of unpuri-fied actomyosin and of myofibrils can

be regulated in vitro by the concentra-tion of calcium ions, a change in con-

centration from 10-7A -to 10- M beingadequate to increase the activity 20-fold or more, from that characteristicof myosin alone to the full activity ofactin-activated myosin. There is goodevidence (21) that an analogous processoccurs in vivo and that release of cal-

SCIENCE. VOL. 164

cium from the sarcoplasmic reticulumand its subsequent rebinding therewhen the muscle relaxes permits con-traction to be controlled by externalelectrical signals conducted inwardfrom the sarcolemma along the so-called T-system of the reticulum. How-ever, this left unspecified the exact siteof action of the calcium.

It was noticed some years ago (17)that the adenosine triphosphatase ac-tivity of extensively purified actomyo-sin was insensitive to the absence ofcalcium; unlike the activity in unpuri-fied systems, it continued high whencalcium was withdrawn. The signifi-cance of this was not clear at first,for the effect might have been due tosome slight change in the propertiesof the myosin molecule itself. How-ever, the situation was dramaticallyclarified when Ebashi (16) showed thatcalcium sensitivity could be restored tosuch systems by adding back a certainprotein fraction. This fraction wasshown subsequently to contain twoprincipal protein components-tropo-myosin B (22) and a new protein, trop-onin (23). Ebashi and his co-workershave shown (24) in a very ingeniousway that the calcium seems to act onthe troponin moiety rather than directlyon the actomyosin. Moreover, althoughpurified actomyosin can bind about 1mole of calcium per mole of myosin,this calcium is not in itself adequate tocause activation of the adenosine tri-phosphatase in the presence of thetroponin-tropomyosin system, and addi-tional calcium has to be provided (20),presumably to combine with the tropo-nin.

There has been a good deal of evi-dence for several years (5, 25) thattropomyosin is present in the thin fila-ments, as well as actin. This has beenconfirmed by fluorescent antibody stud-ies by Pepe (26) and by Endo andothers (27), who have also demon-strated the presence of troponin in thesame part of the sarcomere-that is,the region occupied by the thin fila-ments. Ebashi and his co-workers havealso shown biochemically that tropo-nin combines with the tropomyosin-actin complex (23) but not with myo-sin (28).

Thus, there is very compelling evi-dence (i) that troponin functions as asafety catch, preventing activation ofmyosin adenosine triphosphatase byactin when calcium is absent, but al-lowing the activation to occur as soonas calcium can be bound by the tropo-20 JUNE 1969

nin (in other words, troponin appearsto act as an allosteric regulatory sub-unit), and (ii) that troponin is struc-turally part of the thin filaments. Onceagain, it is very difficult to believe thatthis highly effective and sophisticatedcontrol system does not depend on adirect physical interaction between theactin filaments and the myosin cross-bridge, and on some form of interfer-ence by troponin with this interactionunless calcium is present.

Fig. 3. Diagrammatic representation of themode of aggregation of myosin moleculesto form filaments whose structural polarityreverses at the midpoint. The light-meromyosin parts of the myosin mole-cules form the backbone of the filaments,and the globular ends of the heavy-meromyosin- components form the project-ing cross-bridges. Since these will be ori-ented in opposite senses in the two halvesof the A-bands, they could generate slidingforces which are always directed towardthe center of the bands.

Problem of VariableFilament Separation

Further information about the waythe cross-bridges are involved in con-traction is given by the relationshipbetween isometric tension and sarco-mere length (29), and by the correla-tion of these observations with thelengths of the actin and myosin fila-ments (30) and the way in which thecross-bridges are distributed (11). Theactive tension generated by a muscleat different lengths (greater than restlength) is very accurately linearly pro-portional to the number of cross-bridges overlapped by the actin, de-creasing to zero when the muscle isstretched to the point where overlapjust ceases. This strongly suggests thateach cross-bridge develops a givenamount of tension whatever the extentof overlap between the filaments, andthat the number of bridges attached atany one time at a given muscle lengthis proportional to the number ofbridges overlapped by the actin fila-ments. It seems much less likely thatthe linear form would arise accident-ally, from a coincidental variation ofthe tension per cross-bridge, and of theprobability of attachment, with sarco-mere length, which happened to givea constant product at all sarcomerelengths.

This behavior may at first seem verystraightforward, and easily accountedfor by supposing that a cross-bridgeundergoes some unique set of structuralchanges when it interacts with actinand splits adenosine triphosphate andthat these changes enable it to developa fixed amount of tension. However,the first signs of a real difficulty withthis simple mechanical picture appearwhen we take into consideration mea-surements of the side spacing betweenthe actin and myosin filaments at dif-ferent muscle lengths. It was foundsome years ago (31), and later con-firmed (32), that the filament latticein a live muscle exhibits the same con-stant-volume behavior as the wholemuscle itself; the filaments move closertogether as the muscle is stretched and-move further apart when it is allowedto return toward rest length, their sep-aration varying inversely as the squareroot of the muscle length. Thus, be-tween sarcomere lengths of 2.8 and2.0 microns (equilibrium length), theside spacing will increase by about 18percent of its original value. If the cen-ter-to-center separation of the actin

1359

and myosin filaments has a value ofabout 210 angstroms at a sarcomerelength of 2.8 microns, the increase indistance will be about 40 angstroms.This is a very large distance indeedwhen one is thinking in terms of thekinds of interaction between proteinmolecules or protein subunits neces-sary to produce the highly specific con-formational changes associated withthe regulation of enzyme activity,which is one of the outstanding fea-tures of the system we are considering.If close contacts are to be preservedbetween the cross-bridge and the actinfilanment, then the cross-bridges must inson-ie way be able to adapt themselvesto these changes in spacing between thefilaments and yet must still functionin precisely the same way.Assumed changes in orientation of

the globular region which would enableit to adopt a more perpendicular orien-tation and hridge the larger gap at theshorter sarcomere length do not reallyprovide a satisfactory solution to thisproblem, since the change in anglewould be so great; a cross-bridge 160angstroms long would have to alter itsinitial tilt by about 41 degrees. If a spe-

cific set of structural changes occur atthe cross-bridges during adenosine tri-phosphate splitting and tension develop-ment, it is difficult to imagine how thecross-bridges could develop the samelongitudinal component of tension oversuch a wide range of orientations.Nevertheless, such constancy in be-havior is strongly indicated by thelinear form of the length-tension dia-granm.At one time a conceivable way out

of this difficulty was to suppose thatthe interfilament spacing, although vari-able in resting muscle, always adjusteditself to a constant value during con-traction. However, this has been shownnot to be the case by Elliott, Lowy, andMillman (33), and almost the samerange of interfilamentous spacings isexhibited by an actively contractingmuscle as by a muscle at rest. Indeedone can begin to see why such varia-tions in spacing should be inherent inthe system. A fast and efficient muscleshould always operate with a low co-efficient of internal friction, whetherit is actively shortening or being pas-sively stretched. It appears that this isachieved in nature by sliding of the

filaments past each other on a cushionof long-range electrostatic forces of thekind envisaged by Rome (34) and byElliott (35). In such a system, changesin the extent of overlap of the filamentsseem bound to alter this force balance(35) and hence to change the equilib-rium separation of the filaments. Thus,satisfactory operational characteristicsfor a muscle may not be compatiblewith a fixed side spacing between fila-ments.

Nevertheless, a considerable body ofstrong evidence does indicate, as wehave seen, that physical contact be-tween the cross-bridges and the actinfilaments must take place during con-traction; thus a very real and interestingdifficulty does exist here, and I will nowdiscuss some recent structural evidencewhich may provide clues as to how thisparadox can be resolved.

Subunit Order and Negative Staining

Electron-microscope observations onseparated muscle filaments, made bymeans of the negative staining tech-nique, show significant differences in

--

Fig. 4 (left). Electron micrograph of negatively stained actin filament, showing the double-helical arrangement of two chains ofglobular subunits twisted around each other. The subunit repeat in each chain is about 55 angstroms, and the cross-over pointsof the two chains are 360 to 370 angstroms apart. Fig. 5 (center). Electron micrograph of negatively stained actin filament"decorated" with heavy meromyosin. The polarity of the structure is shown by the "arrowhead" appearance, and it is evident thata quite regularly ordered arrangement of the heavy-meromyosin units is preserved. (Compare the arrangement of cross-bridgesin Fig. 2, left and middle) (about X 155,000). Fig. 6 (right). Low-angle x-ray diffraction pattern from living frog sartoriusmuscle (fiber axis vertical). The reflections form a system of horizontal layer lines (with a repeat of -429 angstroms) whicharise from the helical arrangement of cross-bridges on the thick filaments.

1360SCIENCE, VOL. 164

the regularity of the visible subunit re-peats (11). The actin filaments showbetter structural preservation in this re-spect than the myosin filaments do. Inthe former, the double-helical arrange-ment of G-actin units can be seen quitewell (Fig. 4), whereas virtually no traceof an ordered helical structure can beseen in the arrangement of the projec-tions on the myosin filaments (Fig. 2).Since it was believed on generalgrounds [and was demonstrated byx-ray diffraction (36)] that the cross-bridges are arranged in a regular fash-ion in the intact muscle, it was appar-ent that this regular arrangement musthave been greatly disturbed during thenegative staining process. In view of theknown lability of myosin, this was notaltogether surprising, but it was some-what unexpected to find that myosin orheavy meromyosin complexed to actingave a compound filament showing aconsiderable amount of structural reg-ularity in the arrangement of the sub-units bound to the outside of the actinfilament (Fig. 5). Though the signifi-cance of the differing amounts of orderin the two situations (that is, cross-bridges on the outside of myosin fila-ments and isolated cross-bridges at-tached to actin filaments) is not easy toassess, it is apparent that the attach-ment to actin must in some sense be amuch more rigid one than that to thebackbone of the myosin filaments.

New X-ray Diffraction Results

Further evidence about the structureand the character of -the filaments hascome from detailed studies on the low-angle x-ray diffraction patterns frommuscle under differing conditions (37).These observations have been describedat length elsewhere; here I will mentionbriefly those findings which bear closelyon the present problem. The resultsshow that in live striated muscle ofvertebrates the projections on the thickfilaments are arranged on a 6/2 helix.At a given level, two bridges projectout directly opposite each other oneither side of the backbone of the thickfilament. The next two bridges occur143 angstroms further along the fila-ments and are rotated relative to thefirst pair by 120 degrees. This arrange-ment continues, so that the structureas a whole repeats at intervals of 3 X143, or 429 angstroms (see Figs. 6 and7).The distribution of x-ray intensity

along the layer lines can be accounted20 JUNE 1969

143A --

CD.

C =D--b-

Fig. 7. Diagram of cross-bridge arrange-ment on thick myosin-containing filamentsof frog sartorius muscle, which would ac-count for the observed x-ray pattern.

for very satisfactorily on the basis ofa model in which projections are at-tached to the backbone of the thick fila-ments at a radius of about 60 angstromsfrom the center of the thick filamentand extend outward to a total radiusof about 130 angstroms. Now the dis-tance to the surface of the actin fila-ments from the center of the myosinfilaments is about 190 angstroms in amuscle at rest length, and bridges ex-tending out to this distance would givea predicted x-ray intensity appreciablydifferent from the observed one. Ofcourse, the result might merely indicatethat the effect of disorder on the cross-bridges was more marked at largerradii, but the simpler explanationwould be that the cross-bridges in aresting muscle do not extend all theway out to the actin filaments.A most significant feature of the

cross-bridge pattern is the fact that,although the pattern is strongly de-veloped at low angles (at spacingsgreater than about 50 angstroms), thereflections, especially the off-meridionalones, fade out very quickly at higherangles. This shows that on any giventhick filament there must be a consid-erable amount of disorder in the helicalarrangement of the bridges. The regu-larity of the arrangement can be con-trasted, for example, with the highly

ordered arrangement of protein sub-units in the rods of tobacco mosaicvirus, oriented gels of which will givedetailed x-ray patterns out to spacingsof only a few angstrom units. It isclear that the bridges in a resting mus-cle are not at all precisely fixed inposition on the thick filaments. Again,this is a surprising result, for one mighthave expected that structures involvedin the precise and intricate mechano-chemical interactions of contractionwould need to be positioned in a veryprecise and rigid fashion.The x-ray reflections from the actin

filaments show that the G-actin unitsare arranged on a nonintegral helixwith subunits repeating at intervals of54.6 angstroms along either of twochains which are staggered relative toeach other by half a subunit period(27.3 angstroms), and which twistaround each other with cross-overpoints 360 to 370 angstroms apart, sothat the pitch of the helix formed byeither of the two chains is 720 to 740angstroms (see Fig. 8). Again, the actinreflections show something of the samedisorder that characterizes the reflec-tions from the myosin filaments; in thiscase, prominent meridional reflectionsout to 6 angstroms or less show goodordering in a purely axial sense, butoff-meridional reflections at higherangles are extremely weak, indicatingrelatively poor helical ordering of thesubunits. This suggests that the helicesmay be able to twist and untwist tosome extent, but that the axial repeatof the subunits remains rather constant.When a muscle loses adenosine tri-

phosphate and goes into rigor, it be-comes rigid and inextensible, a phe-nomenon which has been interpreted interms of the attachment of a largenumber, if not all, of the cross-bridgesto the actin filaments. Since neitherthe subunit repeats nor the helical re-peats of the myosin and actin filamentsare the same-indeed, a near matchof one cross-bridge with an actinmonomer oriented in the right direc-tion occurs only once every severalthousand angstrom units along the fila-ments-such an attachment can takeplace only if some part of the structurealters its configuration from that in theresting state. When muscles in rigorwere examined, it was found that thelow-angle x-ray pattern from the cross-bridges changes very considerably,while the part of the pattern arisingfrom the actin filaments remains almostconstant. The entire system of myosinlayer lines based on the 429 angstrom

1361

A

helical period disappears, showing thata high proportion of the whole mass ofeach of the cross-bridges moves awayfrom the position it occupied in theresting muscle. The layer-line patternis replaced by a somewhat more dis-ordered but nevertheless quite strongsystem of layer lines now based on ahelix of pitch about 720 angstroms-approximately the pitch of the chainsof monomers in the actin filaments.This indicates that a high proportionof the cross-bridges have attached ina systematic way to the subunits in theactin filaments.The most surprising feature of the

new pattern, however, is the continuedpresence of a strong meridional reflec-tion at a spacing almost unchanged invalue from that in resting muscle at143 angstroms. This reflection arisesfrom the meridional repeat of thecross-bridges, and its continued pres-ence shows that, although the helicalfeatures of the arrangement of bridgeshas changed so as to enable them tomatch up more easily with actin mon-omers-that is, although they havemoved in an azimuthal (and possibly aradial) direction, so as to lie on a helixof different pitch-this has been ac-complished with very little movementin an axial direction. We can see thatfive subunit periods with an unchangedrepeat of 143 angstroms would fit quiteclosely with the new pitch of about 720angstroms and that, with this repeat,a considerable number of near matchescould be made with subunits on theactin filaments (37). Thus, it seems thatthe cross-bridges are able to swing bodi-ly around the thick filaments, keepingtheir axial positions approximately con-stant but changing their azimuth, andprobably their radius too.A similar type of behavior is ob-

served in actively contracting muscles,in which the off-meridional layer-linepattern becomes very much weaker,although here the "resting" pattern isnot replaced by a new one-a factwhich indicates that the bridges do notsettle down to some new regularly or-dered arrangement. Once again, thestrong 143-angstrom meridional reflec-tion decreases in intensity to a muchsmaller extent than the off-meridionalreflection and changes its spacing byonly about 1 percent from the valuecharacteristic of resting muscle. Again,the actin pattern appears to be un-changed in the actively contractingmuscle.The constancy of meridional repeat-

1362

ing distances fitted in very well withthe earlier observations of constancyof filament length during contraction,but the behavior of the cross-bridges,as in the case of rigor, was at first veryunexpected and puzzling. How couldthe bridges move to a new helical ar-rangement yet leave undisturbed thebonding pattern which gave rise totheir meridional repeat?

Possible Solution to the Paradox

This puzzle began to resolve itselfas the concept of cross-bridges attachedat their bases to the backbone of thefilaments but able to tilt was abandonedin favor of a model having a ratherdifferent construction (37). This wasgiven support by Lowey's timelycharacterization (38) of a helical sub-fragment, derived from heavy mero-myosin and soluble at physiologicalionic strength; this subfragment does

- --

A M

Fig. 8. Diagram of the arrangement ofG-actin monomers in actin (A) filaments,derived from x-ray diffraction and elec-tron-microscope observations. Both thepitch of the helix and the subunit repeatdiffer from those of the myosin (M)filaments, indicated schematically along-side. Thus, cross-bridges between filamentswould act asynchronously, and a sequenceof them would develop a fairly steadyforce as the filaments moved.

not aggregate under these conditionseither with itself or with light mero-myosin. Now, heavy meromyosin itselfis soluble at physiological ionic strengthand is known to possess a globularhead, and a short linear tail similarin appearance to the rest of the myosinmolecule-that is, to the light-mero-myosin subunit, which forms aggregatesat physiological ionic strength. Previous-ly it had seemed possible that the linearportion of heavy meromyosin might ag-gregate too, if it were separated fromthe head part of the molecule. How-ever, this turned out not to be the case,and it therefore became apparent (37,39) that the globular part of myosin-the part forming the visible cross-bridge-could be attached to the back-bone of the thick filaments by a linearregion of the molecule (about 400 ang-stroms long), which would not bebonded along its length to the surfaceof the thick filaments but would beattached only at one end, at the junc-tion to the light-meromyosin part of themolecule.The possibility thus began to emerge

that this link might provide the mov-able attachment which was being calledfor by the results of the x-ray experi-ments. Since the junction betweenheavy and light meromyosin is suscep-tible to tryptic' digestion, it might rep-resent a less perfectly a-helical regionof the molecule and hence a region ofgreater flexibility; similarly, the junctionbetween the linear part of heavy mero-myosin and the globular part of themolecule is also susceptible to enzy-matic digestion (by papain, or by moreprolonged tryptic action), and so it toomight provide a flexible coupling. Thus,with two flexible joints, the orientationof the "head" of the molecule could bemaintained when the link swung fur-ther out from the backbone of the fila-ments (Fig. 9).

The great advantage of this modelis that it permits direct myosin-actininteraction to take place over a widerange of interfilament spacing, as il-lustrated in Fig. 10. It may be seenthat the cross-bridges can be attac'hedat the same orientation to the actinsubunits over a considerable range offilament spacings. Thus all the dif-ficulties discussed above are circum-vented.

Models having some analogies tothis one but not concerned with thetwo major structural components ofheavy meromyosin have been suggestedby Pepe on the basis of his antibody-

SCIENCE, VOL. 164

-3651

staining experiments (40), but I havereservations about some of the argu-ments involved. Hanson (41) has alsorecently reviewed the possibilities.

This model of course makes it pos-sible to account for the meridionalx-ray data, which show, essentially,that the globular part of the heavy-meromyosin molecules can move cir-cumferentially around the long axis ofthe thick filaments to some new posi-tion yet still be held with approximatelythe same axial repeat. The axial re-peat of the bridges is fixed by the pack-ing of the light-meromyosin part of themolecules in the backbone of the thickfilaments, which remains constant. Ifthe end of the cross-bridge is con-strained to move to a new position byattachment to actin, then, instead ofrequiring a major change in orientationof the globular region, this model willallow the whole globular region tomove circumferentially to a new posi-tion, keeping its orientation approxi-mately constant, so as to match upwith a site at the appropriate level onone of the actin filaments nearby. Thespacing of the 143-angstrom meridionalreflection could be maintained and itsintensity would still be quite strong,yet virtually all traces of the original429-angstrom helical reflections wouldbe lost.

Furthermore, the flexibility of the at-tachment of the cross-bridges to thebackbone also offers a possible expla-nation for the disordered appearanceof the x-ray reflections at higher anglesand for the difficulty of preserving anordered arrangement in material proc-essed for examination in the electronmicroscope. Additionally, the modelhas the advantage of providing a plausi-ble role for the various structural partsof the myosin molecule, especially the"soluble" linear portion of heavy mero-myosin.

Additional X-ray Evidence

It seemed worth while, therefore, toconsider this model seriously and tolook for additional evidence concerningits validity. For this reason, a furtherstudy was made of the equatorial x-rayreflections from resting muscles andfrom muscles in rigor. These reflectionswould be expected to show up anychanges in the average radial densitydistribution in a direction at rightangles to the long axis of the filamentswhich might take place when the20 JUNE 1969

HMM S2 HMM S1I(

LMM Backbone of Myosin filament

HMM Si-+

HMM S2

LMM Myosin

muscle went into rigor, and they couldtherefore make it possible to detectchanges in the radial positions of thecross-bridges. These changes wouldalso show up in the axial reflections,and indeed strong indications that theywere present had already been detected

A

Fig. 9. Suggested behavior of myosinmolecules in the thick filaments. The light-meromyosin (LMM) part of the moleculeis bonded into the backbone of the fila-ment, while the linear portion of theheavy-meromyosin (HMM) componentcan tilt further out from the filament (bybending at the HMM-LMM junction),allowing the globular part of HMM (thatis, the St fragment) to attach to actin overa range of different side-spacings, whilemaintaining the same orientation.

(37). However, the effects are more dif-ficult to interpret in this case becauseof the disorder present in the helicalstructure.

It may be recalled that striking dif-ferences had already been noticedsome years earlier (31) in the relativeintensities of the equatorial reflectionsfrom muscle, when patterns from liverelaxed specimens were compared withpatterns from muscle in rigor or

I Actin I

I__ _

I !

I1. Actin Il-

I

B

IMyosin I

C

Actiti :-I

I I

,,i

I II~MyosinI

Fig. 10. Diagram showing relative positions of filaments and cross-bridges at twodifferent interfilament spacings [(A) 250 angstroms and (B) 200 angstroms] corre-sponding, in frog sartorius muscle, to sarcomere lengths of -..2.0 and .3.1 microns.The x-ray diagram (not shown) suggests that in a relaxed muscle the cross-bridgesdo not project very far toward the actin filaments. During contraction, or in rigor,the cross-bridges could attach to the actin filaments by bending at two flexiblejunctions, as shown in (C).

1363

after glycerol extraction. Subsequently,doubts were expressed (32) about thevalidity of these findings, but whenthis question was reinvestigated recently(42) it was demonstrated that thereare indeed large differences in the rela-tive intensities of the 10 and 11 reflec-tions when patterns from live muscleand from muscle in rigor are comparedat the same sarcomere length (Fig. 11).Quantitatively, the changes indicatethat an amount of material equal toabout 30 percent of the total originalmass of the thick filaments is trans-ferred to the vicinity of the thin fila-ments, at the trigonal positions, when amuscle passes into rigor. This transfercould be accounted for very well if theglobular ends of the heavy-meromyosinmolecules, originally extending onlypartway out from the backbone of thethick filaments, reached farther outwhen the muscle was in rigor, and at-tached to the surface of the actin fila-ments. Further support for this inter-pretation is given by electron-micro-scope observations of cross sections ofmuscle. Not only is the change in therelative amount of material associatedwith the thick and thin filaments veryevident in such cross sections when themuscle passes into rigor but, further-more, a readily visible reversal of thechange can be produced by treating(before fixation) a muscle in rigor witha "relaxing" solution containing adeno-sine triphosphate and ethylenedinitrilo-tetraacetic acid, a procedure whichwould be expected to detach the cross-bridges again from the thin filaments.Thus a number of different structural

1364

Fig. 11. Low-angle equatorial x-ray pat-terns from rabbit psoas muscle: (top)live; (bottom) in rigor. The patterns showthe 10 and 11 reflections from the hexa-gonal lattice of myosin and actin fila-ments. The reversal of the relative inten-sities of the reflections is believed to becaused by the cross-bridges reachingfarther out from the myosin filaments inrigor and attaching to the actin filamentsat the trigonal positions.

observations all strongly support theidea that the active part of the myosinmolecule-namely, the globular-headregion containing the adenosine tri-phosphatase and actin binding sites-is attached to the backbone of the thickfilament by means of two separate flex-ible couplings with a linear region inbetween them, so that the "heads" onthe thick filaments can attach them-selves to the actin filament over a con-siderable range of different actin-myo-sin spacings, and yet always preserveexactly the same orientation relative tothe actin. Let us examine some of theconsequences of this possible model.

Site of the Structural Change

Since it is one of the postulates ofthe model that the junction betweenlight and heavy meromyosin is flexible,and since we are supposing that thelinear part of heavy meromyosin liesapproximately parallel to the axis ofthe filaments, it is clear that activebending of this particular junction isnot a likely source of the longitudinalcontractile force. The linear portion ofheavy meromyosin does not seem likelyto be the seat of the contractile ma-chinery either, since the actin-activatedadenosine triphosphatase activity of thehead region can function normallyafter it has been removed (43), andsince it is not easy to imagine thatrelatively distant changes in the headregion could cause this two-chain a-helical structure to fold up to a shorterlength. The junction between the linearpart of heavy meromyosin and theglobular region also seems an unfavor-able position for the force-generatingmechanism, for two reasons. First, ifthe globular part of heavy meromyosinattaches at a constant angle to actin,then the angle formed between thelinear and the globular parts will varysomewhat with filament spacing (andindeed it is for this very reason thatwe have supposed the junction to be

a flexible one); thus the mechanismwould have to be capable of actingover a variable range of configurations.Second, such a mechanism would re-quire that the linear part of heavymeromyosin be a completely rigid rod,since it would have to sustain the samecouple that was being developed at thejunctional region.

Thus, it seems unlikely that any ofthe structural elements through whichthe globular parts of cross-bridges areattached to the backbone of the thickfilaments could provide the structuralrequirements necessary for the develop-ment of a longitudinal sliding force,though these attachments could per-fectly well sustain a force developedelsewhere. Indeed, the orientation ofthe myosin molecules in the filamentsis such that the linear part of heavymeromyosin would always be undertension during contraction, a form ofstress which this type of structureseems well adapted to sustain.

Clearly, the most likely seat of theforce-developing mechanism is theglobular part of heavy meromyosinand its attachment to the actin fila-ments. We have already seen, fromthe ordered structures visible in nega-tively stained preparations of actin"decorated" with heavy meromyosin,that this attachment seems to be arather rigid one. It is therefore per-haps more profitable to reverse one'susual picture of the structure and tothink of the cross-bridges (for part oftheir cycle, anyway) as being based onthe actin filaments, and as being at-tached to the myosin filament by a linkwhich could be as flexible as a pieceof thread, provided it was inextensible.Changes in orientation of the cross-bridge relative to the actin filament towhich it is attached will then give riseto a relative sliding force between thefilaments in the manner required.

Such a change of orientation could bebrought about in several ways. The twoglobular units which make up the headof heavy meromyosin could function es-sentially independently, and each couldundergo either a change in shape or achange in the angle at which it attachedto the actin filaments (Fig. 12A). Al-ternatively, the mechanism might dependspecifically on the dual structure in thehead region. Perhaps one subunit couldattach with the head perpendicular tothe actin filament, while the secondsubunit would attach only with thehead bent at an angle to the actin fila-

SCIENCE, VOL. 164

ment. Another possibility is that, in theintact molecule, interactions occur be-tween the two head subunits, and thatduring the splitting of adenosine triphos-phate the relative positions of the sub-units change. This could alter the profileof the surface which is applied to theactin filament and hence could alter theangle of attachment (Fig. 12B).No doubt other schemes could be

devised, all having in common the basicfeature that they depend on an activechange in the effective angle of attach-ment of the globular part of the cross-bridge to the actin filament during theactive stroke, rather than on a change inorientation of the cross-bridge whichcould, in principle, fully manifest itselfin the absence of actin. (Given such ascheme, it would not be surprising tofind that the configurational changes thatoccur in purified myosin during thesplitting of adenosine triphosphate arerelatively minor ones). And while thisview has arisen from consideration ofthe x-ray diffraction observations on ver-tebrate striated muscle, it derives addi-tional support for some of its features(especially the tilt direction) from theelegant observations of Reedy, Holmes,and Tregear (44) on the angling of thecross-bridges when they attach, in rigor,to the actin filaments in insect ffightmuscle. Both Reedy and his associatesand Pringle (45) have suggested that

such "angling" also occurs during theoscillatory contraction of this muscle.

Thus, interest is focusing now on themode of attachment of the cross-bridgeto the actin filament, and there are ob-vious ways of exploring this in greaterdetail. However, it is becoming increas-ingly clear that a full understanding ofthis or indeed of any other molecularmechanism is likely to require that wesolve the full three-dimensional struc-ture of the molecules concerned downto atomic resolution by crystallographictechniques. It has always seemed veryunlikely that intact myosin itself couldbe assembled into crystalline arrays ofthe requisite degree of regularity, butthe isolation in a fairly pure form (46)of a globular subunit still possessingmany of the relevant properties of myo-sin has at last brought a real possibilityof solving the problem in detail, pro-vided this protein subunit can be crys-tallized.

Summary

To summarize, then: the contrac-tion of striated muscle is brought aboutby some mechanism which generates arelative sliding force between the partlyoverlapping arrays of actin and myosinfilaments. There is very strong evidencethat cross-bridges projecting out from

the myosin filaments, and carrying theadenosine triphosphatase and actin bind-ing sites, are involved in the genera-tion of this force in some cyclical proc-ess. However, it appears that the mech-anism must satisfy two conflicting re-quirements: (i) that the force beproduced as a result of a precisely de-termined set of structural changes ina protein complex consisting of actin,myosin, and other components, and beassociated with the splitting of a mole-cule of adenosine triphosphate; (ii) thatthe force-generating mechanism canwork equally well over a considerablerange of side spacings between the actinand myosin filaments. Recent evidencesuggests that these requirements may besatisfied in the following way: the actualforce-generating structure is attached tothe backbone of the myosin filaments bya linkage, 400 angstroms long, whichhas flexible couplings at either end; theforce-generating structure can thereforeattach itself to the actin filament, in aconstant configuration, and undergo ex-actly the same structural changes andproduce the same longitudinal forceover a wide range of interfilament sepa-rations. The muscle structure is arrangedso that the linkage is under tension, notcompression, when a contractile force isbeing generated, and so the force can betransmitted without difficulty. It is sug-gested that the characteristic feature of

I Actin I

I °2

I I

I I

Actin I

- , Myosin,AFig. 12. Diagram illustrating possible mechanisms for producing relativesliding movement by tilting of cross-bridges. (A) If separation of filamentsis maintained by electrostatic force-balance, tilting must give rise to move-ment of filaments past each other. (B) A small relative movement betweentwo subunits of myosin could give rise to a large change in tilt, by themechanism shown.20 JUNE 1969

!Actin I*

F

-I

I I

:Myosin I

B

I II ----Ia

1365

- a

the contraction mechanism may be arigid attachment of the globular headof the myosin molecule to the actinfilament and an active change in theangle of attachment associated with thesplitting of adenosine triphosphate. Theavailability of purified preparations of"head" subunits now opens up the prob-lem to detailed attack.

References and Notes

1. J. Hanson and H. E. Huxley, Nature 172,530 (1953).

2. H. E. Huxley and J. Hanson, ibid. 173, 973(1954); A. F. Huxley and R. Niedergerke,ibid., p. 971.

3. J. Hanson and H. E. Huxley, Symp. Soc.Exp. Biol. 9, 228 (1955); A. F. Huxley,Progr. Bfophys. Biophys. Chem. 7(1956),255 (1957); H. E. Huxley, in ProceedingsAlberta Muscle Symposium (Pergamon, Ox-ford, 1965), p. 3; , Harvey LecturesSer. 60(1964-65), 85 (1966).

4. H. E. Huxley, J. Biophys. Biochem. Cytol. 3,631 (1957).

5. , in The Cell, J. Brachet and A. E.Mirsky, Eds. (Academic Press, New York,1960), vol. 4, p. 365.

6. , Biochim. Biophys. Acta 12, 387(1953).

7. A. G. Szent-Gyorgyi, Arch. Biochem. Bio-phys. 42, 305 (1953).

8. D. F. Cain, A. A. Infante, R. E. Davies,Nature 196, 214 (1962).

9. W. 0. Fenn, J. Physiol. London 58, 175(1923); ibid., p. 373.

10. H. E. Huxley, unpublished manuscript.11. , J. Mol. Biol. 7, 281 (1963).12. R. V. Rice, Biochim. Biophys. Acta 52, 602

(1961).

13. W. F. H. M. Mommaerts and I. Green, J.Bfol. Chem. 208, 833 (1954).

14. W. Hasselbach, Z. Naturforsch. 7b, 163(1952).

15. S. V. Perry, Physlol. Rev. 36, 1 (1956).16. S. Ebashi, Nature 200, 1010 (1963); -

and F. Ebashi, J. Bfochem. Tokyo 53, 604(1964).

17. S. V. Perry and T. C. Grey, Biochem. J. 64,SP (1956).

18. N. Azuma and S. Watanabe, J. Biol. Chem.240, 3847 (1965); , ibid., p. 3852; H.Mueller, Biochem. Z. 345, 300 (1966).

19. A. Weber, J. Biol. Chem. 234, 2764 (1959);and S. Winicur, ibid. 236, 3198 (1961);

A. Weber, R. Herz, I. Reiss, J. Gen. Physiol.46, 676 (1963); , Federation Proc. 23,896 (1964); , Proc. Roy. Soc. LondonSer. B 160, 489 (1964); T. Nagai, M. Maki-nose, W. Hasselbach, Biochim. Biophys. Acta43, 223 (1960); W. Hasselbach and M. Maki-nose, Biochem. Z. 333, 518 (1961); -Ibid. 339, 94 (1963); W. Hasselbach, Proc.Roy. Soc. London Ser. B 160, 501 (1964);S. Ebashi, J. Biochem. Tokyo 48, 150(1960); , ibid. 50, 236 (1961);and F. Ebashi, Nature 194, 378 (1962).

20. A. Weber and R. Herz, J. Biol. Chem. 238,599 (1963).

21. R. Niedergerke, J. Physlol. London 128, 12P(1955); K. R. Porter and G. E. Palade,J. Blophys. Biochem. Cytol. 3, 269 (1957);A. F. Huxley and R. E. Taylor, J. Physiol.London 144, 426 (1958); P. C. Caldwell andG. Walster, ibid. 169, 353 (1963); H. E.Huxley, Nature 202, 1067 (1964); R. J. Podol-sky and L. L. Constantin, Federation Proc.23, 933 (1964); S. Winegrad, J. Gen. Physiol.48, 997 (1965).

22. K. Bailey, Blochem. J. 43, 271 (1948).23. S. Ebashi and A. Kodama, J. Biochem.

Tokyo 58, 107 (1965); ibid. 59, 425 (1966).24. S. Ebashi, F. Ebashi, A. Kodama, ibid. 62,

137 (1967).25. H. E. Huxley and J. Hanson, Biochim. Bio-

phys. Acta 23, 229 (1957); S. V. Perry and

A. Corsi, Blochem. 1. 68, 5 (1958); 3. Han-son and J. Lowy, 3. Mol. Blol. 6, 46 (1963).

26. F. A. Pepe, J. CeU RIol. 28, 505 (1966).27. M. Endo, Y. Nonomura, T. Masakd, I.

Ohtsuki, S. Ebashi, J. Blochem. Tokyo 60,605 (1966).

28. S. Ebashl, In Proc. Intern. Congr. Physlol.23rd, Tokyo (965), p. 405; D. R. Kominzand K. Maruyama, 3. Bfochem. Tokyo 61,269 (1967).

29. A. M. Gordon, A. F. Huxley, P. 3. Julian,J. Physiol. London 184, 170 (1966).

30. S. Page and H. E. Huxley, 3. CeU Biol. 19,369 (1963).

31. H. E. Huxley, thesis, University of Cam-bridge (1952).

32. G. F. Elliott, J. Lowy, C. R. Worthington,J. Mol. Blol. 6, 295 (1963).

33. G. F. Elliott, J. Lowy, B. M. Millman, Ibid.25, 31 (1967).

34. E. Rome, ibid. 27, 591 (1967); ibid. 37, 331(1968).

35. G. F. Elliott, J. Theoret. Blol. 21, 71 (1968).36. - , Proc. Roy. Soc. London Ser. B 160,

467 (1964).37. H. E. Huxley and W. Brown, 3. Mol. Biol.

30, 383 (1967).38. S. Lowey, L. Goldstein, S. Luck, Biochem.

Z. 345, 248 (1966); S. Lowey, L. Goldstein,C. Cohen, S. Luck, J. Mol. Biol. 23, 287(1967).

39. S. Lowey, in Symposium on Fibrous Proteins,Australia, 1967 (Butterworths, London, 1968),p. 124.

40. F. A. Pepe, J. Mol. Biol. 27, 203 (1967).41. J. Hanson, Quart. Rev. Blophys. 1, 53 (1968)42. H. E. Huxley, J. Mol. Biol. 37, 507 (1968).43. E. Eisenberg, C. R. Zobel, C. Moos ko-

chemistry 7, 3186 (1968).44. M. K. Reedy, K. C. Holmes, R. T. Tregear,

Nature 207, 1276 (1965).45. J. W. S. Pringle, Progr. Biophys. 17,1 (1967).46. D. R. Kominz, E. R. Mitchell, T. Nihei, C.

M. Kay, Biochemistry 4, 2373 (1965); S.Lowey, H. S. Slayter, A. Weeds, H. Baker,J. Mol. Biol., in press.

-Elephants, which are among themost popular and decorative of ani-mals, stand as a witness of prehistory,having been a part of the environmentof our ancestors. The dinosaur was notcontemporary with early man, as manyfilms and stories insist, but the mam-moth was. Although prehistoric or ex-

The author is professor in the Departmentof Paleontology of the Universidad de Madrid,Spain, and dean of the Colegio Mayor NuestraSefiora de Africa, Madrid, Spain.

1366

tinct elephants are frequently referredto as mammoths, such a designationis not always correct. The true mam-moth is but one of many species ofextinct elephants; furthermore, it be-longs to one of a few genera, whichinclude four or five species that haveaffinities with the woolly elephant.These different genera and species aregrouped by zoologists into a family,Elephantidae. Because this family orig-inated by the beginning of the Pleisto-

cene period, elephants can be consid-ered contemporary with man.

Anthropologists and prehistorianshave often attempted to establish achronology of sites of fossil manthrough correlations based upon thespecies of elephant associated withthem (1), but the systematics of theElephantidae is quite confused. Thedocumented monograph of Osborn (2)established 10 genera and some 59species of elephants; to these Garutt(3) added two more genera. However,many taxonomists have recognizedonly one genus and no more than fiveor six valid species. In the museumcollections from most major sites thereare many samples with dubious iden-tifications and many intermediateforms labeled either with two namesor with a composite or new name. Ithas been assumed that many differentspecies have lived contemporaneouslyin a single area, as was the case forthe sample excavated in the railwaytrench of San Paolo, Italy, in the firstyears of this century. Explanations ofthe phylogeny of elephants have hadone feature in common: the patternsfor the phyletic trees have agreed withthe fashionable evolutionary theories

SCIENCE, VOL. 164

Evolutionary History of theElephant

A tentative phylogeny of Elephantidae based on

morphological and quantitative analysis is given.

Emiliano Aguirre