Biophysical Journal Volume 107 October 2014 1493–1501 1493

Biophysical Review

Hugh E. Huxley: The Compleat Biophysicist

Sarah E. Hitchcock-DeGregori1,* and Thomas C. Irving21Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey; and 2CSRRI andDept. BCHS, Illinois Institute of Technology, Chicago, Illinois

ABSTRACT The sliding filament model of muscle contraction, put forward by Hugh Huxley and Jean Hanson in 1954, is60 years old in 2014. Formulation of the model and subsequent proof was driven by the pioneering work of Hugh Huxley(1924–2013). We celebrate Huxley’s integrative approach to the study of muscle contraction; how he persevered throughouthis career, to the end of his life at 89 years, to understand at the molecular level how muscle contracts and developsforce. Here we show how his life and work, with its focus on a single scientific problem, had impact far beyond the field ofmuscle contraction to the benefit of multiple fields of cellular and structural biology. Huxley introduced the use of x-raydiffraction to study the contraction in living striated muscle, taking advantage of the paracrystalline lattice that wouldultimately allow understanding contraction in terms of single molecules. Progress required design of instrumentation withever-increasing spatial and temporal resolution, providing the impetus for the development of synchrotron facilities used formost protein crystallography and muscle studies today. From the time of his early work, Huxley combined electron microscopyand biochemistry to understand and interpret the changes in x-ray patterns. He developed improved electron-microscopytechniques, thin sections and negative staining, that enabled answering major questions relating to the structure and organi-zation of thick and thin filaments in muscle and the interaction of myosin with actin and its regulation. Huxley establishedthat the ATPase domain of myosin forms the crossbridges of thick filaments that bind actin, and introduced the idea thatmyosin makes discrete steps on actin. These concepts form the underpinning of cellular motility, in particular the study ofhow myosin, kinesin, and dynein motors move on their actin and tubulin tracks, making Huxley a founder of the field of cellularmotility.

INTRODUCTION

The year 2014 is the 60th birthday of the sliding filamentmodel of muscle contraction, first put forward in 1954by Hugh Huxley and Jean Hanson. The formulation ofthe model, and its subsequent proof, was driven in alarge part by the pioneering work of Hugh Huxley(1924–2013). In this memorial review, we celebrate hisintegrative approach to learning how muscles contract.We illustrate how his ideas and approach to science ledto seminal new concepts and developments in technologythat continue to have wide-ranging impact on biophysics,cellular biology, and structural biology. Although thesemethods have gone on to enable biophysicists and cellbiologists to study numerous processes at the molecularlevel in living cells and tissues, we recognize that Huxley’sfocus and passion were always to understand the contrac-tile process at the molecular level in living muscle in realtime. Several fine tributes to Hugh Huxley have been pub-lished elsewhere (1–5). Our aim here is to give a flavor ofhow Huxley thought about and did science, informed byhis ingenuity and inventiveness, his consummate hands-on approach, focus and drive. It is, therefore, not intendedas a comprehensive, critical review of the field, or even of

Submitted July 1, 2014, and accepted for publication July 25, 2014.

*Correspondence: hitchcoc@rwjms.rutgers.edu

Editor: David Warshaw.

� 2014 by the Biophysical Society

0006-3495/14/10/1493/9 $2.00

just the work of Huxley and his colleagues. We apologizein advance to those whose work we short-shrifted in thisprocess.

WHY MUSCLE?

Skeletal muscle was likely the best-studied tissue whenHuxley began his graduate work with John Kendrew in1949 at the newly-formed Medical Research Council(MRC) Unit for Work on the Molecular Structure of Biolog-ical Systems at the Cavendish Lab in Cambridge, England.For an historical review of early research on muscle contrac-tion, see Needham (6) and Szent-Gyorgyi (7). For a century,the striations of the sarcomere (Fig. 1) had been known fromlight microscopy studies to change during contraction. Actinand myosin were understood to be separate proteins thatform filaments and interact to produce ATP-dependentcontraction. Cambridge was for decades a center of muscleresearch with luminaries such as Kenneth Bailey with S. V.Perry, A. V. Hill, and Joseph and Dorothy Needham study-ing the biochemistry, energetics, and mechanics of skeletalmuscle. Although theories of muscle contraction abounded,there was no consensus. Huxley’s reviews and memoirs (8–10) provide personal accounts of his early career, and howhe chose to work on muscle contraction within this intellec-tual milieu.

http://dx.doi.org/10.1016/j.bpj.2014.07.069

FIGURE 1 (A) Sarcomere pattern of striated muscle. Longitudinal sec-

tion of frog muscle and diagram of corresponding overlapping thick,

myosin-containing filaments and thin, actin-containing filaments (from

Huxley (9), with permission). (B) First published scheme showing sliding

filaments, related to changes in the sarcomere pattern at different lengths

(from Hanson and Huxley (29), with permission).

1494 Hitchcock-DeGregori and Irving

X-RAY DIFFRACTION: A TOOL TO STUDYPHYSIOLOGY

Huxley has recounted how, after an early (and somewhatpainful, it seems) brush with macromolecular crystallog-raphy, he preferred to work on a more biological systemthan that of individual proteins (8,9). Then as now,x-ray diffraction remains the only technique to detectnanometer-scale structural changes in real physiologicaltime in living muscle. However, this was far from obviouswhen Huxley started his work. The Astbury group inLeeds, England pioneered x-ray fiber diffraction of muscleproteins (11), showing that these proteins had the diffrac-tion features expected from a-helices but that these fea-tures did not change after stretch of the muscle or aftercontraction. The first small angle x-ray diffraction studiesof muscle from the F. O. Schmitt lab at MIT (12) andearly electron microscopy (EM) images showed filaments(13,14), indicating some degree of order (as recounted inHuxley (8)). Based on these meager clues, Huxley real-

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ized he would need an exceptionally bright source ofx-rays coupled to a specialized instrument to resolve the~40-nm basic repeats in muscle in hydrated specimens(9,10).

True to a pattern that would characterize the rest of hiscareer, whenever Huxley needed a new instrument or tech-nique, he looked around for possible solutions and thenapplied his ingenuity to adapt them to his use. In thiscase, he designed and built a compact small-angle x-raycamera with a fine-focus x-ray tube developed by Ehrenbergin Bernal’s lab (15) and a high-voltage supply made of ex-war-surplus parts that could deliver relatively high-intensityx-rays through very narrow slits to live frog muscle toresolve the weak x-ray reflections. With this then-innovativesetup, Huxley was able to make some profound observationsand some prescient predictions (16–18), reviewed in Huxley(10). He was able to resolve multiple reflections on the equa-tor in x-ray patterns from resting muscle indexing on the~40-nm periodicity, corresponding to the inter-thick fila-ment spacing in the sarcomere (Fig. 2 A). He also obtainedx-ray patterns from muscle in rigor (Fig. 2 B) where henoticed that although the interfilament spacings were thesame, the relative intensities of the first two reflectionswere reversed compared to resting living muscle. Longbefore crossbridges were seen in the EM, Huxley postulatedthat the filaments at the hexagonal lattice position weremade of myosin and those at the trigonal position weremade of actin, and that the changes in equatorial intensitieswere due to the formation of cross-links between them. Atthat time, there was no other direct evidence for thisarrangement. Axial x-ray patterns showed periodicitiesthat he measured to be orders of 41.5 nm (now known tobe 43 nm) that did not change with stretch. Perhaps incompensation for his lucky guess about the equatorialpattern, he mistakenly ascribed these periodicities to actin(reviewed in Huxley (10)), later correctly assigned tomyosin by Worthington (19) and Elliott and Worthington(20). These x-ray observations set the stage and preparedHuxley’s mind for sliding filaments, as would be revealedin the light and electron microscopy studies that followed.

SLIDING FILAMENTS: ELECTRON MICROSCOPY,THINNER AND THINNER SECTIONS

Upon completing his Ph.D. in 1952, aware that EM had thepotential to reveal muscle structures in a way he could relateto his x-ray studies, Huxley moved to the MassachusettsInstitute of Technology in Cambridge, Massachusetts, tolearn EM with the biophysics pioneer, F. O. Schmitt. Ithappened that Jean Hanson, who had been studying thechanges in the sarcomere pattern of myofibrils duringcontraction using light microscopy at King’s College,London, England (21), soon joined the lab, also to learnEM. True to form, from the start Huxley was constantlyimproving the existing EM methods for application to

FIGURE 2 Representative x-ray diffraction patterns from muscle

collected at various stages of Huxley’s career. (A and B) Equatorial patterns

from resting and rigor muscle taken with his first x-ray camera and reported

in his Ph.D. thesis (86). Note the reversal of relative intensities of the 1,0

and 1,1 equatorial reflections when going from relaxed to rigor (adapted

from Huxley (9), with permission). (C) Resting pattern from frog muscle

taken with a rotating anode generator and a Huxley-Holmes mirror mono-

chromator camera (adapted from Huxley and Brown (47), with permission).

(D) Integrated intensity of the 14.5-nm myosin-based meridional reflection

during a quick release experiment collected at DESY (adapted from Huxley

et al. (55) with permission). (E) Comparison of the ~2.7-nm reflection from

actin (A) and the ~2.8-nm reflection from myosin (M) under isometric con-

tracting conditions (C) as compared to relaxed (R) conditions. The inward

movement of the reflections reflects the stretch of the filaments under con-

tracting conditions (adapted from Huxley et al. (74) with permission). (F)

The fine structure in the 14.5-nm meridional reflection (M3) in a diffraction

pattern from contracting frog muscle due to the interference of myosin

heads on either side of the M-line in a thick filament (H. E. Huxley and

T. C. Irving, unpublished).

Hugh E. Huxley 1495

muscle. The x-ray studies told him that the thick-to-thickfilament center-to-center distance was ~40 nm. Isolatingsingle layers of thick filaments would require sectionsmuch thinner than this. He partnered with A. J. Hodge and

D. Spiro in developing a microtome that could make thinnersections that would allow visualization of the double arrayof filaments predicted by his x-ray studies (22,23). Andthere they were. Hanson and Huxley wrote, ‘‘thin filamentsof actin extend from the Z-line through the I-band andthrough one half of the A-band’’ (24), noting that therewere two sets of filaments in the A-band. When the myosinwas selectively extracted in the presence of ATP, the A-banddisappeared, leaving thin filaments. These too disappearedwhen the actin was selectively extracted.

It was not until the landmark 1954 paper (25) thatHuxley and Hanson proposed the sliding filament model,after quantifying parameters of glycerinated myofibrilsduring stretch and shortening, and relating the results totheir EM studies. They noted that as the I-band shortens,the H-zone disappears whereas there is no change in theA-band at different sarcomere lengths (Fig. 1 B). A. F.Huxley (no relation) and Niedergerke (26) saw the sameband pattern changes at the light microscope level in mus-cle fibers using an interference microscope. The classiclength-tension relationship that related function to thesliding filament model was years away (27), although itwas known from A. V. Hill’s studies (and even earlier inwork by Schwann) that during isometric contraction, ten-sion is maximal at the rest length of muscle (reviewed inNeedham (6)). Reflecting the caution of the investigatorsthat was also typical of the times, the models of the slidingfilaments were not published until 1957 (Fig. 1 B) (28),after additional quantitative measurements using interfer-ence microscopy, biochemistry, and electron microscopy(28–30).

SKEPTICISM: EXACTING EXPERIMENTS ANDEVEN THINNER SECTIONS SUPPORT THESLIDING FILAMENT MODEL

Huxley returned to Cambridge, England in 1954, and at theend of 1955 took a position at University College, London tocontinue his EM studies. In 1962, he became a foundingmember of the MRC Laboratory of Molecular Biology(LMB) in Cambridge, England. The micrographs publishedin the 1953–1954 papers showed filaments, but to see indi-vidual filaments would require even thinner sections as wellas precise orientation of the section in relation to the myofi-bril axis. The idea of contraction based on sliding filamentswas still avant-garde, because at the time the prevailing viewwas that the protein filaments themselves contract (7). Hisremarkable images, such as those in Fig. 3, A and B (30),clearly showed the overlap between thick and thin filaments,the crossbridges between the filaments, the relationship be-tween the size of the H-zone and sarcomere length, and theconstant distance between the Z-line and the edge of theH-zone. He recognized that the bridges between the thickand thin filaments were sites of actin-myosin interaction,and introduced the idea that they attach and detach with

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FIGURE 3 (A) Thin section of skeletal muscle showing interdigitating thick and thin filaments, from a circa-1957 image. In this image, and others like it,

Huxley noted that the thin filaments end at the H-zone, the crossbridges between the thick and thin filaments, the thickening of the thick filaments at the

M-band, the dimensions of the thick and thin filaments, and the interfilament spacing (from a photo in Huxley’s collection). (B) Cross-section through

the overlap region of skeletal muscle showing the double hexagonal array of thick and thin filaments. One of the fundamental insights driving all Huxley’s

work was that the paracrystalline lattice of filaments offered an ensemble approach to understand the behavior of single molecules (from a circa-1957 photo

in Huxley’s collection). (C) Negatively-stained native thick filaments showing crossbridges except for bare central region and tapered ends (adapted from

Huxley (37), with permission). (D) Negatively-stained synthetic myosin filaments showing bare central region free of crossbridges, leading to postulation of

the bipolar nature of the thick filaments (adapted from Huxley (37), with permission). (E) Actin filaments decorated with myosin S1 (showing the polar

arrowheads) with the same polarity along the entire filament (from Huxley’s collection, date unknown).

1496 Hitchcock-DeGregori and Irving

a minimum step distance of 5.4 nm—the size of an actinmonomer—during shortening, well within the range of laterexperimental determinations of the crossbridge workingstroke (31). Huxley suggested the crossbridges were partof myosin, although they ‘‘do not reveal the basic mecha-nism involved in contraction’’ (30).

Looking back, it is difficult to appreciate the skepticismthat greeted the sliding filament model, and that themodel was not widely accepted until the mid-to-late1960s. Sliding mechanisms as a mode of mechanochemicaltransduction are now the paradigm for dynein-based ciliaryand flagellar motility, and motility of filamentous andnonfilamentous myosins and kinesins in a wide varietyof cytoskeletal motile processes. Beyond the technicaladvances made in the course of their work, Hanson andHuxley were among the first to use electron microscopyfor a physiological experiment accompanied by rigorousquantitative analyses at a time when EM was predominantlyused to describe the ultrastructure of cells and tissues. Wenote in passing that Huxley was also the first to show thatthe T-tubules of skeletal muscle are invaginations of theplasma membrane, continuous with the extracellular space(32), a critical aspect of the excitation-contraction couplingmechanism.

Hanson and Huxley (24), followed up by other groups(33,34), noted fine S-filaments that run from Z-line toZ-line, but they did not incorporate a third filament systeminto the sliding filament model or their thinking, engen-

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dering years of controversy (reviewed in dos Remediosand Gilmour (35)). We now know the elastic protein, firstcalled connectin, now titin, forms a third set of filamentsin the sarcomere that have multiple, critical roles in themechanics and intracellular regulation of muscle even ifthey do not appear to have a direct role in the centralactin-myosin force generating mechanism (36).

ELECTRON MICROSCOPY TO STUDYSTRUCTURE AND FUNCTION OF ISOLATEDPROTEINS

In the 1950s, the structures of actin and myosin moleculesor filaments and origin of the crossbridges in thin sectionswere unknown. By comparing negatively-stained EM im-ages of myosin filaments assembled from purified myosinwith thick filaments isolated from muscle (37), Huxleysaw for the first time the bipolar nature of myosin filamentswith crossbridges extending from both ends, leaving a barezone in the middle (Fig. 3, C and D). The structure ofnative thin filaments was similar to that Hanson andLowy had seen in filaments made from purified actin(38). By 1963, the proteolytic fragments of myosin,HMM and LMM, were well known (39). One can imagineHuxley’s excitement at seeing arrowheads on actin fila-ments when he added HMM (Fig. 3 E). The polarity in-ferred in the sliding filament model was realized in thebipolar structure of myosin combined with the opposite

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polarity of actin filaments extending from Z-lines. In the1963 paper, Huxley proposed that myosin formed the cross-bridges and that sliding of thick and thin filaments pasteach other depended on crossbridges interacting with actinin an ATP-dependent manner (37). In years to come,myosin HMM, and later myosin S1 decoration of actin,became diagnostic tools to identify filaments as actinand to define their polarity in cells. The terms ‘‘barbed’’and ‘‘pointed’’ have become generic.

The classic images in Huxley’s 1963 article (37) againdepended on improved methodology. Huxley and Zubay(40) modified an earlier negative-staining method (41) byusing uranyl acetate as the stain, and perforated grids where‘‘one gently breathed on the [drying collodion] so that thesurface appeared slightly cloudy’’ (40) to create holes inthe collodion film. On the EM grids, after dissolving thecollodion and staining, the specimen over the holes wasthin, embedded only in a layer of stain, allowing visualiza-tion of structural details.

Further development of EM methods to study macromo-lecular structure remained a focus within the StructuralStudies Division at the MRC-LMB, where Huxley servedas joint head from 1975 to 1987 (and Deputy Director ofthe LMB from 1978 to 1987) until he moved to BrandeisUniversity in Waltham, Massachusetts. DeRosier and Klug(42) developed a method for three-dimensional reconstruc-tion of EM images. The first application in muscle researchwas to actin filaments. Because the actin filament is helical,the image of a single filament contains all the structuralviews, without needing to tilt the specimen (43). Althoughlow-resolution by present standards, the structures thatMoore et al. (44) obtained of pure actin and native thinfilaments (that contain tropomyosin-troponin), with andwithout myosin-S1, were astounding (Fig. 4, A and B).The structures revealed the tilted and slewed appearanceof the myosin head that became the basis of future studiesand modeling of x-ray diffraction data. In addition, theymodeled the position of tropomyosin along the helical actinfilament, reinforcing Hanson and Lowy’s proposal thattropomyosin lies in the grooves of the actin helix (38),

setting the stage for the steric blocking model (see below).The Structural Studies Division at the LMB continued tobe a frontier for EM methodology and structure determina-tion, a focus that continues to this day. Electron microscopyis now a mainstream technique for structure determinationof macromolecular assemblies at near-atomic resolution.

FIRST X-RAY DIFFRACTION MEASUREMENTSFROM CONTRACTING MUSCLE

After 1963, Huxley’s major focus returned to x-ray diffrac-tion. This was a period of competition between Huxley andcolleagues (including Ken Holmes, John Haselgrove, BillLongley, and Wilf Brown) at Cambridge and the King’sCollege, London group (that included Roy Worthington,Gerald Elliott, Jack Lowy, and Barry Millman). This rivalrywas a kind of technological arms-race whereby considerableingenuity in both groups was devoted to extractingmaximum performance out of their x-ray generators tocollect diffraction data from contracting muscle. TheCambridge group held a technological edge primarily inthe development of the Huxley-Holmes mirror-monochro-mator diffraction camera, and rotating anode x-ray genera-tors of ever-increasing power (see Fig. 3 C). Huxley givesa lively account of these developments (45). This competi-tion culminated in two monumental papers by Elliott et al.(46) and Huxley and Brown (47), which compared resting,isometrically contracting, and rigor muscle. In addition tothe rich harvest of structural details for the myofilaments re-sulting from these studies, one of the key conclusions wasthat the major axial periodicities from the thick and thin fil-aments are constant, within experimental error, between restand isometric contractions, confirming the sliding filamenttheory of contraction. In either contraction or rigor, the ma-jority of the structural changes could be attributed primarilyto changes in the thick filament, presumably due to move-ment of myosin crossbridges, not to large-scale changesin the thin filament. This work ushered in a time ofsynthesis in the muscle field culminating in Huxley’s 1969paper in Science (48) proposing the well-known swinging

FIGURE 4 Three-dimensional reconstruction of

thin filaments. (A) End-on view of a model of actin-

myosin S1. The S1 heads are tilted and skewed rela-

tive to the actin filament axis (from a circa-1969

photo in Huxley’s collection). (B) Superposition of

a reconstruction of a native thin filament (actin with

tropomyosin-troponin, red) with actin-myosin S1

(blue) to illustrate the extra material (tropomyosin)

in the thin filament (adapted from Moore et al. (44),

with permission). (C) A composite end-on view of

actin-tropomyosin-myosin S1 based on EM and

x-ray data where tropomyosin (TM) is shown in

the active state (solid contours) and relaxed state

(dotted contours), where it was postulated that it

could block cross-bridge attachment (steric-blocking

model) (adapted fromHuxley (64), with permission).

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1498 Hitchcock-DeGregori and Irving

crossbridge mechanism for muscle contraction. We notethat Huxley put forward a cycling crossbridge model ina 1958 Scientific American article (49), showing he hadbeen thinking in those terms for a long time.

Although there was a widespread sense that the so-calledproblem of muscle was solved at the time of the 1972 ColdSpring Harbor Symposium on muscle contraction, it was nodoubt frustrating for Huxley that the work he and others haddone did not provide direct evidence that individual myosincrossbridges actually move axially during contractionin synchrony with the force-producing events as suggestedby the swinging cross-bridge model. For such evidence,he had to wait for the next major technological advance.And it was a while in coming.

DEVELOPMENT OF SYNCHROTRON RADIATIONAS A TOOL FOR STRUCTURAL BIOLOGY

The now widespread availability of synchrotron radiationfor structural biology is one of the most outstanding suc-cess stories in science. How the use of synchrotron radia-tion began has been recounted in the literature (45,50,51).In the mid-1960s, Ken Holmes realized that electron syn-chrotrons, large machines initially developed for high-energy physics experiments, emitted hard x-rays that mightbe useful for structural biology. In 1971, Rosenbaum,Holmes and Witz (52), using the Deutsches Elektronen-Synchrotron (DESY), in Hamburg, Germany, publishedan x-ray pattern from insect flight muscle, the first diffrac-tion pattern taken from anything using synchrotron radia-tion, using a beam that was then ~100-fold brighter thanthat available from the best rotating anodes. In 1969, Hux-ley chaired a committee of the planned European Labo-ratory of Molecular Biology (EMBL) that proposed anorganization to foster technology development that wasbeyond the scope of any one university or laboratory. Thefuture promise of synchrotron radiation for structure deter-mination was quickly appreciated, and the opportunity pre-sented by the synchrotron radiation facilities at Hamburg,Germany helped tip the balance toward creation of theEMBL in 1974. The EMBL, headquartered in Heidelberg,Germany, now has outstations in Hamburg and Grenoble,France that are critical components of the world’s scientificinfrastructure.

Whereas development of beamline technology continuedfor some years on DESY, the first production x-ray beam-lines (X11 and X13) had to wait until the construction ofthe DORIS storage ring and the EMBL outstation atDESY in 1975. The flux delivered by these beamlines, alongwith fast electronic detectors, allowed ~1000� faster dataacquisition than with the best rotating anodes of the time(53). The goal of the experiments was to try to detectevidence that myosin crossbridges moved axially duringrapid mechanical transients, designed to synchronize cross-bridges, of the type pioneered by Huxley and Simmons (54).

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Huxley was then able, for the first time, to obtain x-raydiffraction evidence (Fig. 3 D) that there were structuralchanges in the myosin heads, as reported by changes inthe intensity of the myosin meridional x-ray reflection at14.5 nm. The changes did indeed appear to be synchronouswith the force-producing events (55–57), as predicted byReedy et al. (58), based on studies comparing relaxed andrigor glycerinated insect flight muscle. The X11 and X13beamlines at the DESY storage ring at Hamburg went onto have a long and productive life with a significant impacton many fields of structural biology.

REGULATION OF CONTRACTION: STERICBLOCKING MODEL

As the sliding filament mechanism became accepted, atten-tion turned to how muscle contraction is regulated, whereEM and x-ray studies from Huxley’s group were again crit-ical. By the 1960s, it was established that Ca2þ is the regula-tory ion, and tropomyosin-troponin on the actin filament is thetarget (59–61). The pieces of the puzzle were in place to pro-pose the steric blockingmodel. The focus became the second-order actin layer line attributed to tropomyosin, consistentwith its position along the actin filament (62). Haselgrove(63), Huxley (64), Lowy and Vibert (65), Vibert et al. (66),and Parry and Squire (67) reported changes in the actin layerlines during contraction in x-ray studies. The patterns weremodeled and interpreted to show an azimuthal shift in the po-sition of tropomyosin away from the outside of the filament,wheremyosin binds toward amore central position upon acti-vation, or in rigor when myosin binds to actin (Fig. 4 C). Thechange does not depend on myosin, because it takes placewhen muscles are stretched to lengths where the thick andthin filaments do not overlap (63,64,66,68).

Subsequent improvements in sensitivity and time resolu-tion at DESY (see above), enabled Huxley working withKress et al. (69) to show that the changes in the second actinlayer line occur after activation but before the movement ofthe myosin crossbridges toward the actin filaments. The re-sults pointed to the change in the position of tropomyosin onthe actin filament as the first observable structural step incontraction, in response to Ca2þ binding to troponin C onthe thin filament—a prerequisite for myosin binding andfull force development. Definitive proof for the structuralchanges in the thin filament upon Ca2þ binding came later(70). It is now accepted that in vertebrate skeletal muscle,both Ca2þ binding to troponin and myosin binding to actinare required for full activation (71,72); however, the detailsof this process remain unresolved.

STRETCHY MYOFILAMENTS

When Huxley retired from the LMB, he moved to BrandeisUniversity in Waltham, Massachusetts where he wasProfessor of Biology (1987–1997), Professor Emeritus

FIGURE 5 Huxley’s team at the Advanced Photon Source, Argonne

National Laboratory, July 2004. (Left to right) Massimo Reconditi, Tom

Irving, and Hugh Huxley. Huxley, ever the hands-on experimentalist,

preferred to do as much as possible himself, from dissecting the muscles

and setting up the electronics, to running the experiments themselves—

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(1997–2013), and Director of the Rosenstiel Basic MedicalSciences Research Center at Brandeis from 1988–1994.While continuing with EM studies (73), his main interestwas to use the more powerful beam and the then-newimaging-plate detector technology at the Cornell HighEnergy Synchrotron Source in Ithaca, New York. To hissurprise, the experiments revealed unanticipated smallspacing changes, ~0.2–0.3% in the myofilament lengths,in the higher-order meridional reflections at ~2.7 nm (actin)and ~2.8 nm (myosin) (Fig. 2 E). These studies (74) wereparalleled by those conducted by K. Wakabayashi et al.(75) in Japan, and the two papers appeared back-to-backin Biophysical Journal in 1994. These changes in filamentlength were near the limit of detectability, circa. 0.3%, butnonetheless required radical reformulation of the maincrossbridge theories (e.g., A.F. Huxley and Simmons(54)), which assumed that all the compliance was in thecrossbridges and none in the myofilament backbones (seeA.F. Huxley and Tideswell (76)).

showing remarkable stamina for synchrotron x-ray experimentation long

after the retirement ages of most scientists (photo from T. C. Irving).

SEEING CROSSBRIDGES MOVE: LATE X-RAYDIFFRACTION EXPERIMENTS

Huxley was a major motivator, through his collaborationwith Tom Irving, behind the development in the late1990s of the BioCAT x-ray beamline 18ID at the AdvancedPhoton Source (APS), Argonne National Laboratory inLemont, Illinois, which continues to this day as a facilityfor diffraction studies of noncrystalline biological materials,including muscle. Huxley was well aware that despite yearsof research including in vitro motility assays and crystallo-graphic studies of myosin S1 in various nucleotide states,direct evidence for axial motions of crossbridges in intactmuscle as envisaged in the Huxley 1969 swinging cross-bridge model (48) remained elusive. A possible wayto achieve such evidence emerged when it was realizedthat the fine structure in the ~14.5-nm meridional reflection(Fig. 2 F), first reported by Bordas et al. (77), arose frominterference between the diffracted rays from the arrays ofcrossbridges in the two halves of each thick filament, as firstreported by Linari et al. (78). The relative intensities of theinterference peaks would be sensitive to small axial motionsof crossbridges (78). A productive period followed wherethe Lombardi group in Florence worked on interferencestudies on single muscle fibers, e.g., Piazzesi et al. (31),Linari et al. (77), and Reconditi et al. (79,80), mainly atthe European Synchrotron Radiation Facility in Grenoble,France, but also at the APS, in friendly competition withHuxley on whole muscle at the APS with Massimo Recon-diti working with both groups at different times (Fig. 5).Huxley’s data allowed him to determine crossbridge anglesduring synchronized isometric contraction after a quickrelease or during isotonic shortening, and how these angleschange with adjustments in load and estimates of the degreeof dispersion of crossbridge angles about these averages

(81,82). Although this interpretation is not without its critics(83), this led Huxley to feel satisfied that he had finallyachieved his goal of seeing axial motions in crossbridgesin contracting muscle (9).

HUXLEY’S LEGACY

The sliding filament mechanism was a paradigm shift in ourview of muscle contraction. However, proving the idea ofsliding filaments to the skeptics was just the beginning. Asatisfactory explanation of the mechanism behind how mus-cle contracts and develops force, and how it is regulated atthe molecular level, required advances in 1), both x-raydiffraction and electron microscopy, 2), x-ray crystallog-raphy beyond what is described herein, 3), development ofin vitro motility and single molecule assays, and 4), applica-tion of a lot more contractile protein biochemistry. In theend, it took another 50 years until Huxley convinced himselfthat he had proven the swinging crossbridge mechanism ofmuscle contraction. Ever a perfectionist, Huxley would onlypublish when he was ‘‘good and ready,’’ leading to a smallnumber of primary publications by modern standards. Buthis influence has been disproportionately far-reaching.

We have seen how Huxley’s single-minded devotion toone important problem—the molecular mechanism of mus-cle contraction—led to an impressive number of technicaladvances that he either produced himself or motivated inothers, which profoundly influenced multiple fields in cellbiology, including cellular motility. Although he was keenlyinterested in cellular motility (84,85), he left the area toothers at the LMB, such as Linda Amos, John Kendrick-Jones, Murray Stewart, and Alan Weeds, and the many post-doctoral fellows and visiting scientists who worked there.

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1500 Hitchcock-DeGregori and Irving

Honored as a founder of the field of cellular motility in itsbroadest sense (3), the American Society for Cell Biologyrecognized Huxley with the E. B. Wilson Award, the highesthonor of the Society, in 1983. As the field moves forward,the goal of deciphering mechanisms of muscle contractionand cellular motility at the single molecule level and eventhe atomic level are within reach. More realistic moleculardynamics simulations and other improved modeling ap-proaches offer the best hope to relate the x-ray patterns ofliving muscles to molecular structures. The ability to visu-alize single molecules in living cells coupled with methodsto transition between superresolution light microscopy ofliving cells and cryo-EM may fulfill a dream Huxley surelyhad, that we carry on.

We thank Maggie McNeely, archivist at Brandeis University, for access to

Hugh Huxley’s papers. We also thank the reviewers and numerous other

colleagues who provided helpful suggestions and information to us as we

tried to sort out details and their essence, reflecting their admiration and

respect for Hugh Huxley.

Use of the Advanced Photon Source, an Office of Science User Facility

operated for the U.S. Department of Energy Office of Science by Argonne

National Laboratory, was supported by the U.S. Department of Energy

under Contract No. DE-AC02-06CH11357. This project was supported

by grant No. 9-P41-GM103622 from the National Institute of General

Medical Sciences, National Institutes of Health (to T.C.I.), and National

Institutes of Health RO1 grant No. GM-093065 (to S.E.H.-D.).

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