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Despite having all the evidence needed to come to the right conclusions in the middle of the 1800s, it was not until the 1950s that it was realised by two unrelated Huxleys and their collaborators that striated muscle sarcomeres contain overlapping sets of filaments which do not change much in length and which slide past each other when the muscle sarcomere shortens. It then took quite a while to convince others that this was the case, but now the idea of sliding filaments is fundamental to our understanding of how any muscle works. Here a brief overview of the history of the discovery of sliding filaments and the factors that were missed in the 1800s is followed by an analysis of the more recent experiments which have added to the conviction that all muscles operate on the same guiding principles; two sets of sliding filaments, independent force generators and a mechanism of protein rowing that makes the filaments slide.
Look at any school biology textbook and the muscle chapter will show a muscle sarcomere, the building block of striated muscles, containing overlapping arrays of myosin and actin filaments ( Figures 1 and 2(f) ). The idea of muscle filament sliding is now a fundamental concept in biology, but it was not always so. In the 1800s, quite impressive light microscopy of striated muscles showed the sarcomeres to have substructure; a central region (the A-band; although the terminology then was different), which often appeared dark, flanked by two lighter regions (the I-bands) which ended at the Z-discs (or Z-bands or Z-lines). We now know that the A-bands contain filaments of the protein myosin, and the I-bands have filaments of actin, which start at the Z-band, pass through the I-band and overlap the ends of the myosin filaments in the A-band. The part of the A-band not overlapped by actin filaments is called the H-zone ( Figures 1 and and2 2 ).
Schematic diagrams showing the hierarchy of structures in vertebrate skeletal muscles, going from an anatomical muscle (top right), to a group of muscle fibres, to a single muscle fibre showing cross-striations and then a single myofibril with sarcomeres, A-bands, I-bands, H-zones, Z-discs (Z-bands, Z-lines) and M-lines (M-bands).
Vertebrate striated muscle sarcomeres are often around 2.2 to 2.3 µm long at rest length. Myofibrils may be 1 to 3 µm in diameter and very long, and individual fibres might often be 30 to 100 µm in diameter. Redrawn from Bloom & Fawcett (1975)[t].
(a) Actin filament composed of actin molecules, A, two tropomyosin strands, TM, and troponin molecule complexes, TN.
(b) Bridge region of myosin filament composed of myosin molecules shown in (c) with the rod of the myosin molecules forming the backbone of the filament and the myosin heads arranged on the surface of the filament backbone. (d) The bipolar packing of the myosin molecules showing the anti-parallel arrangement giving rise to a heads-free bare zone region at the centre of the filament. This is also illustrated in (e). (f) Sarcomere structure extending between two successive Z-bands, M: Myosin, A: Actin. (g-j) Cross-sectional views through different parts of the sarcomere, showing (g) the square lattice of actin filaments in the I-band, (h) the hexagonal lattice between overlapping arrays of actin and myosin filaments in the A-band, (i,j) the hexagonal lattice of myosin filaments in the M-band (i) and bare-zone (j) regions, with the extra M- protein density linking the myosin filaments at the M-region in the centre of the sarcomere (i). (From Squire et al., 2005, with permission).
Some of the key observations in the studies which outlined the sliding filament model of contraction (Huxley & Hanson, 1954[alp]; Huxley & Niedergerke, 1954[alp]) were actually rather simple (but technically innovative) observations. Firstly, it was found that, as the sarcomere length changed, for example by stretching a relaxed muscle, the length of the A-band remained virtually constant. At the same time the edges of the H-zone appeared to move with the Z-band so that the distance from the Z-band to the H-zone edge remained constant. These two observations alone are almost enough to postulate the presence of sliding filaments, so why were these simple observations missed in the 1800s? [For detailed overviews of the history of muscle research and the conclusions reached see, for example, Needham (1971)[t]; Huxley (1980)[t]; Squire[author] (1981[year]; 1986[year]); McMahon (1984)[t]; Squire & Parry (2005)[t]; Huxley (2008)[t]; Rall (2014)[t].]
Andrew Huxley, in his fascinating book ‘Reflections on Muscle’ (1980)[t], discusses many ways in which early observations or knowledge of muscle from work in the 1800s was ignored or forgotten by the early 1900s. For example, Boeck (1839)[t] showed that muscle is birefringent, with the slow direction being along the fibre axis. Bowman (1840)[t] knew about fibres, myofibrils, the sarcolemma (muscle fibre membrane) and the presence of striations within each myofibril. Dobie (1849)[t] showed that most of the length change in sarcomeres occurred in the I-band. Brücke (1858)[t] showed that the birefringence is confined to the A-band. He also showed that this birefringence was not increased by stretching the muscle, so it must be due to rodlets which are not themselves stretched when the sarcomere length changes. Kühne (1864)[t] characterised myosin. Krause (1869)[t] showed that the A-band length stays virtually constant when a muscle is stretched and that the high refractive index and birefringence of the A-band were due to birefringent rodlets which extend the whole length of the A-band. He also described how solvents known to remove myosin only extracted material from the A-band, an observation later confirmed by Schipiloff & Danilevsky (1881)[t], so the A-band contains myosin rodlets. When a muscle shortened substantially, dense contraction bands were observed to appear at the Z-band (Engelmann, 1873[p]).
With hindsight we can see that there was probably enough information available by the late 1800s to postulate a sliding filament mechanism for sarcomere shortening, the only really vital piece of missing evidence perhaps being that the I-band (despite being non- birefringent) also contained rodlets. So how did people think muscles contracted in those days? There were a variety of views; Krause believed that the rodlets in consecutive A- bands attracted each other; Engelmann thought that the A-band swelled on muscle activation, mostly in a lateral direction, and that fluid was then drawn into the A-bands. Then, after the observation of transverse and longitudinal elements through muscle fibres (what we now call t-tubules and the sarcoplasmic reticulum) after the introduction of gold chloride staining and in a retrograde step, some authors thought that these structures were rather like the protoplasm of other cells and they tried to suggest that, since all contractile behaviour probably had a common origin, it was the transverse and longitudinal elements which were associated with movement, not the myofibrils. However, Kölliker (1888)[t] argued convincingly that myofibrils are the structures that shorten actively during contraction.
To quote Andrew Huxley from his book ‘Reflections on Muscle’:
“But whatever the rights and wrongs of arguments based on the assumption that all ‘contractility’ is essentially similar, I think there can be no doubt that they helped to reduce the interest that physiologists took in the striation pattern and its changes during contraction”.
So what happened early in the 1900s? One of the first observations was superbly carried out, but rather unfortunate. A new microscope had been developed with the help of the Zeiss works in Jena, Germany. This was an ultraviolet microscope which, with its short wavelength, greatly improved the available resolution. Meigs (1908)[t] used this microscope to study the myofibrils of the ‘asynchronous’ muscles of a fly. The resulting images were beautifully clear and they showed sarcomeres with Z-bands between which there was relatively little substructure. No A-bands or I-bands were apparent, so Miegs concluded that these must be artefacts of the limited resolution of previous microscopes.
What he did not know was that in these particular insect muscles the sarcomere length changes involved in normal contraction are tiny, that the myosin filaments almost fill the whole length of the sarcomere and that the I-bands are very short and not easily seen. Then there was a study by Hürthle (1909)[t] who used cinematography of the leg muscles of the water beetle (Hydrophilus), which sometimes showed spontaneous contractions. He followed waves of contraction down these muscles using polarized light and showed that most of the shortening appeared to be in the A-bands; the reverse of the results obtained in the 1800s.
Because he was using photography of living tissue his results were believed and became generally accepted. Other studies in the next few decades appeared to support his conclusions. In addition it was generally believed that myosin was present right through the sarcomere and that the darker appearance of the A-band was due to some other material. How had the field come to such opposite and erroneous views compared with what was known earlier? We should take this as an object lesson in being careful about what we believe.
The 1950s were an astonishing time in biology. Following the ravages of the second world war, many scientists, including many refugees from Europe, focused their attention on studies beneficial to mankind, namely on the nature of biological molecules and assemblies. They also had at their fingertips new emerging techniques such as X-ray diffraction and electron microscopy, so the time was ripe for some major discoveries, such as the α-helix structure of protein chains proposed by Pauling et al. (1951)[t].
This was soon followed by the discovery of the DNA double helix by Watson & Crick (1953)[t]. At that time all seemed set fair for significant advances to be made in understanding muscle. But there was an immediate setback. Pauling & Corey (1951)[t], who recognised that the α-helix could be converted to a β-sheet by stretch, thought that this might apply to muscle as well. In this 1951 paper “The structure of hair, muscle and related proteins” they argued that muscles contain continuous filaments through the whole sarcomere and that they can convert from α to β and back as part of the contractile mechanism. However, this conclusion was quickly refuted by Huxley & Perutz (1951)[t]. Perutz had already confirmed the existence of the α-helix by recording the 1.5 Å meridional X-ray reflection, which comes from the axial separation of successive amino acids along a protein chain in an α-helix, using a synthetic polypeptide Poly-γ-benzyl-L-glutamate (Perutz, 1951; see also Squire & Elliott, 1972[p]). In the next paper of the same issue of the journal Nature, Perutz and Huxley found that the 1.5 Å peak showed up in X-ray patterns from both stretched and shortened muscle. The α-helices in muscle did not appear to convert to β-structures on stretch. They concluded that: “Our results are incompatible with the mechanism of muscle contraction proposed by Pauling & Corey (1951)[t]. ”
So we come to the definitive studies by HE Huxley and Hanson, and AF Huxley and Niedergerke in 1954. Hugh Huxley (1924–2013: Figure 3(b) ) studied at Cambridge, United Kingdom, served in the RAF and then started research at the Medical Research Council Unit linked to the Cavendish Laboratory in Cambridge. His early work used X-ray diffraction to study muscle, and also the work with Perutz on the 1.5Å reflection, but his main work used a different sort of X-ray diffraction camera.
(a) Jean Hanson, (b) Hugh Huxley, (c) Andrew Huxley, (d) Rolf Niedergerke.
Some of the important axial repeats in myosin and actin filaments are of the order of 350 to 450 Å. The diffraction angles involved in X-ray diffraction are given by Bragg’s Law: nλ = 2d sin θ. Here d is the spacing involved in the structure doing the diffracting, n is any integer, λ is the wavelength of the X-rays being diffracted (usually about 1.0 to 1.5 Å in most muscle studies), and the angle of diffraction is 2θ (see for example Squire & Knupp, 2005[p]). For a d- spacing as in the α-helix at 1.5Å, sin θ for n = 1 is 1.5/2 × 1.5 = 0.5 for a wavelength of 1.5 Å. So 2θ is 60°. If the d spacing involved is 400 Å, then 2θ is only about 0.2° and special X- ray cameras need to be used to study diffraction patterns at such small angles. We’ll discuss later some of Huxley’s results with his low-angle X-ray cameras.
Jean Hanson (1919-1973; Figure 3(a) ) was a trained zoologist who in 1948 had joined the Biophysics Research Unit in J.T. Randall’s Department of Physics at King’s College London [See Squire, 2013 for descriptions of this laboratory and of Jean Hanson’s role with Gerald Elliott, who will be mentioned later]. Schick & Hass (1950)[t] and Perry (1951)[t] had shown that preparations of isolated myofibrils could be obtained which showed the normal striation pattern and the normal ATPase activity. Jean Hanson studied these preparations by phase contrast microscopy to see how the striation pattern changed with sarcomere length. Then both she and Hugh Huxley wanted to extend their studies to electron microscopy, which was being successfully employed in Francis Schmitt’s Laboratory at the Massachusetts Institute of Technology (MIT), so they both arrived there independently, Huxley in 1952 and Hanson in 1953. Very soon in 1953 they were working together.
Andrew Huxley (1917-2012; Figure 3(c) : later Professor Sir Andrew Huxley PRS, OM, Nobel Laureate; not related to Hugh Huxley) graduated from Trinity College, Cambridge, UK and then in 1939 joined Alan L Hodgkin at the Marine Biology Association at Plymouth and at Cambridge. They worked on and successfully recorded the transmembrane resting and action potentials of the squid giant axon. After the war, Hodgkin and Huxley eventually published their squid axon work in 1945.
Following this, Huxley was joined in Hodgkin’s laboratory at Cambridge by Robert Stämpfli, with whom he published several papers on nerve conduction in frogs. Hodgkin and Huxley then carried out pioneering and definitive experiments on squid giant axons controlled by voltage clamping. This led to five classic papers (Hodgkin et al., 1952; Hodgkin & Huxley, 1952a; 1952b; 1952c; 1952d) and the eventual award of the Nobel Prize in Physiology or Medicine (1963; jointly awarded with JC Eccles).
After his membrane work, and inspired by the work of another giant of the muscle field A.V. Hill, Andrew Huxley started to think about muscle contraction and this was the main focus of the remainder of his long career. Although unrelated to Hugh Huxley, Andrew was part of the famous Huxley family. His grandfather was Thomas Henry Huxley, well-known in the nineteenth century as a supporter of Charles Darwin. Andrew’s half -brothers were the writer Aldous Huxley and the famous biologist Julian Huxley (see Clark, 1968[p]). Interestingly, despite his meteoric career, Andrew Huxley never carried out a PhD; it was not necessary in those days, but he was the only one of our four heroes who was not a doctor. There are interesting comments on PhDs and the British class system from that era in Maruyama (1995)[t] whom we will hear of later in another context.
Andrew had been a near contemporary of David Hill, son of A. V. Hill, at Trinity College Cambridge. They knew each other quite well at Trinity and then, when the second world war intervened, Huxley and David Hill worked together on the application of radar to anti-aircraft gunnery. Interestingly, in the 1914-18 war, A.V. Hill had actually been a pioneer of anti-aircraft gunnery and around 1924 was the main author of the Text Book of Anti-Aircraft Gunnery. To quote AF Huxley (1977):
‘This comprehensive two-volume work, issued for H.M Stationery Office in 1924–1925 for the War Office, was still a valuable reference book in the second world war. It was ‘for official use only’ and is not easily found in general libraries …The list of contributors contains at least seven who were, or subsequently became, Fellow of the Royal Society’.
It is also notable that J.T. Randall, who brought Jean Hanson to Kings College (and was also the author’s first boss), was very much involved in developing radar in the second world war. He greatly improved the cavity magnetron, an essential component of centimeter-wavelength radar, which was one of the keys to the Allied victory in the second world war. It is also the key component of microwave ovens.
Rolf Niedergerke (1921-2011; Figure 3(d) ), born in Mülheim-Ruhr, West Germany, joined Andrew Huxley’s muscle laboratory in Cambridge in 1952. He had worked on isolated nerve fibres in the Berne Institute of Alexander von Muralt, and was a demonstrator in physiology in Göttingen. He was recommended to Andrew Huxley by Robert Stämpfli as someone who could dissect single intact skeletal muscle fibres, and he also introduced Huxley to many aspects of the available light microscopy techniques.
By 1953 both teams were working on muscle using light microscopy, with Hugh Huxley and Hanson using electron microscopy as well. What did they find and what was different from before?
In that same year Hugh Huxley reported on the X-ray diffraction work that he had done in Cambridge (Huxley, 1953[p]). The first sentence of that paper sets the scene:
“The present day picture of muscle is as follows: muscle is a machine for converting chemical energy into mechanical work; the ‘moving parts’ of this machine are built up of two proteins myosin and actin; the known energy producing reaction most closely linked to the contractile process is the dephosphorylation of adenosine triphosphate (ATP)”. He used low-angle X-ray diffraction, especially of the equator of the diffraction pattern (diffraction at right angles to the fibre axis; Figure 4 ) to conclude: “the transverse X-ray pattern from living muscle reveals the presence of very long molecules, arranged in a hexagonal array, parallel to the fibre axis and 450 Å apart”.
The muscle axis was vertical and the diffraction is at right angles to the fibre axis and shows some of the equatorial reflections, labelled 10 and 11. (a) is from resting muscle and (b) from rigor muscle. In (a) the 10 reflection is stronger than the 11; in (b) the 11 reflection is stronger. These observed intensities can be used to generate electron density maps as in (c) and (d), where the myosin filament (M) and actin filament (A) positions can be seen on a hexagonal lattice. In (d) there is much more material at the actin positions than there is in (c), suggesting movement of material (crossbridges, side-pieces) from the myosin filaments towards the actin filaments. Adapted from Huxley (1968)[t].
He goes on to say that when ATP is removed from the muscle, the diffraction pattern changes, but the lateral spacings remain at around 450Å. The axial pattern was also studied and right at the end of that paper Hugh Huxley said: “If the ATP-containing muscle is stretched by up to 40% then the axial pattern remains unchanged. This is rather a surprising result, and it may be an important one. However, there is not time now to discuss its possible implications”.
The first paper produced by Hanson and Hugh Huxley working together at MIT (Hanson & Huxley, 1953[p]) had the ambitious title: “Structural basis of the cross-striations in muscle”. It went quite a long way towards what was needed. Jean’s isolated myofibril preparations were treated with solutions known to extract myosin, and they confirmed that the A-bands in the myofibrils virtually disappeared, leaving only the Z-bands which appeared intact. There was also some ground substance.
The myofibrils were no longer birefringent and would not contract. If the myofibrils were then treated with an actin-extracting solution on the microscope slide, the myofibrils, which in solution virtually collapsed, could be observed to remain structurally intact, but with no A-bands or ground substance. Hanson and Huxley also reported on electron microscopy observations in which they found two sets of filaments in A-band cross-sections, with the second set of filaments also in the I-band and very much thinner than the A-band filaments. The thinner filaments formed a hexagonal ring around a thicker A-band filament, except in the H-zone where the thinner filaments were absent.
Despite this enormous progress, it is clear that they had still not quite grasped what was going on. Their summary was that: “In its simplest form our picture of muscle is as follows: thin filaments of actin extend from the Z-line through the I-band and through one half of the A-band, until they join up with the H-band filaments, the composition of which is unknown. Myosin is located primarily in the A-band, in the form of filaments about 100 Å in diameter, which extend from the A-I junction up to the H-band, where they too join up with the H-band filaments.” So what are these unknown H-band filaments?
The two classic Nature papers of 1954 started with one by AF Huxley and Rolf Niedergerke. Andrew Huxley was one of those brilliant scientists who could almost do anything, as required. One of his multiplicity of talents was to be able to manufacture his own equipment. As a child he had learnt how to use a lathe, and later in life he used such skills to help with his experiments. He also didn’t waste his time. He is reputed to have thought long and hard about each experiment that he carried out; was it the best way to achieve his aims, would it be reliable, would it answer the right questions? It is said that he spent 90% of his time thinking about the right experiments to do, designing the equipment and so on and then 10% of his time actually doing them. It often then took several years and a great deal of analysis before the results were published, and he had a great analytical mind.
In the case of his early muscle studies, Andrew Huxley was influenced by Niedergerke in his knowledge of microscopy and also his familiarity with some of the papers of the nineteenth century, such as those by Krause (1869)[t] mentioned earlier. Huxley wanted to study intact muscle fibres, which Niedergerke could dissect, but he realised they would be too thick (perhaps 50 to 100 µm) to provide reliable measurements of the sarcomere and A-band lengths and other sarcomere features in the 2–3 µm range using a conventional light or phase contrast microscope. He also realised that an interference microscope could provide what he needed.
Here, the light beam through the microscope is split into two spatially separated beams, one of which goes through the specimen and the other through a background region to serve as a reference beam. The two beams are then recombined and contrast is generated by interference. Such a system can provide an optical section of the specimen. Andrew Huxley made the carcase of his microscope with the optical components being made by Messrs R and J Beck.
[On a personal note, the author’s PhD supervisor, Dr. Arthur Elliott, was another superb scientist who made some of his own equipment, including the toroid X-ray camera which focused X-ray beams using the inside of a hollow toroidal-shaped (i.e. barrel–shaped) mirror 60 to 100 mm long, but only about 3 mm in diameter which Elliott manufactured himself (Elliott, 1965[p]). It was Arthur Elliott who gave Perutz the sample of poly-γ-benzyl-L-glutamate with which Perutz demonstrated the existence of the 1.5 Å reflection from the α-helix (Squire & Vibert, 1987[p]).]
Huxley & Niedergerke (1954)[t] described the results from their interference microscopy of single frog muscle fibres. The contrast in their images could be changed from positive to negative by altering the path difference between the two beams and they found that measurements of the A-band length, for example, were not changed by this procedure. Figures 5 and and6 6 show some of their results. Figure 5 shows the effects of passive stretch on the fibres, viewed in positive contrast with the A-bands dark. They noted that almost all the change in length within sarcomeres of different length was in the I-band, except at very short sarcomere lengths. They also studied fibres undergoing isometric (constant length) twitches and isotonic (constant load) shortening ( Figure 6 ). In all cases the A-band length was more or less constant except at extreme shortening.
Interference microscopy results of Huxley & Niedergerke (1954)[t] showing what happens when a fibre is passively stretched.
Sarcomere lengths are shown on the left hand side. The A-bands (dark) remain almost constant in length as the sarcomere length changes, whereas most of the shortening is in the I-bands (light). Reproduced with permission.
