I began the work that ultimately led to the
sliding filament theory of muscle contraction in 1950.
I was a research student in a small MRC unit with John Kendrew as my supervisor
and Max Perutz being the head of the unit and that was it.
It was just three people.
I'd been a student in physics, and my main interest had been in experimental particle physics.
But, after graduation I decided to change fields completely, joined this unit,
and was in fact in complete freedom to choose my own particular subject of research.
Now during the first couple of years I spent doing some work on hemoglobin
and learning a bit of biology of which I was totally ignorant originally.
And then I chose muscle as a possible topic because
I'd found that although an awful lot was known about the
mechanics and energetics and biochemistry of muscle,
virtually nothing was known about its submicroscopic structure
nor about what actually happened during contraction.
Now, one way of getting that information might be
that if muscle was composed of regular, repeating or semi-crystalline units of these
two structural proteins then one might get evidence about what was happening from X-ray diffraction.
However, people had looked at wide-angle X-ray diffraction,
which showed you any changes in the polypeptide chain configuration,
and essentially it didn't show anything.
And they'd also looked at dried muscle by low angle diffraction, which showed you
structural regularities and the size of protein molecules, but that hadn't been informative either.
However, I'd learnt that one had to keep protein crystals fully hydrated
if you were going to get anything useful and an X-ray diagram from them.
So I thought, "Well, maybe that would be true of muscle and
maybe muscle really did contain lots of regular structures if only one could attack it the right way."
And so for that purpose I built a miniaturized X-ray camera,
which would optimize the sensitivity of images collected on film.
And I was also very fortunate that my supervisor John Kendrew knew
a man called Bernal in London in whose X-ray group two people were
actually building a very fine-focus, high-intensity X-ray generator (X-ray tube) for another purpose,
and they were kind enough to give me an early prototype of this,
which formed an essential part of the X-ray set-up that I built.
I was very thrilled and relieved to see that I did in fact get quite a well defined X-ray diagram.
Not very detailed, but it consisted basically of two reflections.
One here and then another one a bit further out.
And, the spacing of these told one they came from a hexagonal arrangement of objects,
and I concluded that this must be a hexagonal arrangement of myosin filaments.
But, what was particularly interesting was that in a muscle in rigor,
the pattern had the same spacing, approximately,
but the relative intensity of the two spots had changed,
and this told one that there was some alteration in the character of the lattice.
And that in fact you can get a projection of the electron density from those diagrams.
This is a myosin filament here on my picture, and then this is another myosin filament there
and another one in the lattice over here.
In between the myosin filaments is what I took to be actin filaments,
which appeared at low density in the relaxed muscle, but in rigor muscle,
their density was much increased.
Now I knew that actin and myosin tended to combine with others in the absence of ATP, that's in a rigor muscle.
So I concluded, and it turned out I was correct, that
was in fact what I was seeing: a double array of actin and myosin filaments interpenetrating with each other.
I could also see some reflections, axial reflections, which give you information
about the longitudinal regularities along the length of the muscle.
This is a spacing of about 145 angstroms, and there are another series of reflections not shown here.
The interesting...so it showed that muscle was a sort of semi-crystalline structure at this scale.
And these reflections didn't change in spacing when the muscle was passively stretched.
Now, I made a mistake at that point.
I thought that these reflections probably all came from the actin filaments.
So I concluded the actin filaments didn't change in length,
but I still had this picture that perhaps the myosin filaments somehow shortened down.
Anyway, I finished my PhD and went to MIT to Schmitt's lab,
which was a new center for doing electron microscope work,
because I wanted to make sure that this double array of filaments really existed.
So by fixing, embedding and sectioning a muscle,
I was able to look at cross sections in the electron microscope,
and lo and behold one could see the double array of filaments very, very clearly.
This was a rabbit muscle in rigor.
This is a myosin filament, and that's another myosin filament.
And then the actin filaments are in this little ring of six of them around each myosin center.
So that was very encouraging and then shortly after this, early in 1953,
Jean Hanson came to the same lab to learn electron microscopy too
because she'd been studying isolated myofibrils as seen in the light microscope.
She was zoologist by training.
And we decided, we were both doing the same thing essentially,
we decided to combine our different approaches, and this worked out very successfully.
One of the first experiments we did
was to look at myofibrils in the light microscope on a slide with a coverslip over them
in the phase contrast microscope, and you get very nice images of the A and I bands. I'll show you one in a moment.
And when we irrigated these with solutions known to extract myosin from muscle,
we found that the dense material in the A band here was rapidly removed,
leaving a sort of ghost fibril which consisted of the original Z lines,
and a segment of the filament which we concluded were probably actin.
And we later found that applying actin extraction procedures
would remove this second set of filaments as well.
These are just showing the density plots before and after myosin extraction.
So, this was a sort of, quite a revolutionary finding for us.
And after a few hours we sort of reoriented our way of thinking about things.
And we realized that we were looking at two partially interpenetrating but overlapping arrays
of myosin filaments and actin filaments.
And that the cross-sections I had seen had been through the overlap region here.
So this is what I showed before-the overlap region.
In the H zone, which is where the actin filaments have stopped, you just see the thick filaments.
And in the I bands you can see the cross-section of the thin filaments.
So we then had a quite new model of the structure of the muscle.
And in between the two sets of filaments were these cross-bridges
that I had postulated must be there to enable
the two sets of the actin and myosin filaments to interact with each other.
So we published a short paper on this in Nature, but at Schmitt's request
we omitted any speculation about how this system would actually work.
But what we knew was that there were these two axial series of X-ray reflections
which didn't change during passive stretch.
And so we thought, "Well maybe they didn't change during contraction either."
And that would mean that the two sets of filaments would themselves remain constant in length
and that contraction would be achieved by
the actin filaments sliding further into the array of myosin filaments.
And when we...after a lot of hard work we were able to get sufficient images
of muscle at different stages of contraction to prove that point.
And you can see, this is a slightly stretched myofibril.
Then, we give it a little bit of ATP, and it shortens down a little.
The I bands, the clearer regions, become shorter, but the A bands stay constant in length.
And as it shortens further still, the I bands get even shorter, and ultimately they almost disappear.
We can also extract myosin from a stretched fibril,
and we can see the large gap between the ends of the actin filaments.
But when, on of course a different fibril, which is contracted, we can see
this gap between the actin shortening down and then disappearing.
So we can measure the lengths of the I filaments and that was constant during contraction.
So, this was written up for Nature and published in late May in 1954,
along with another paper by Andrew Huxley, who's actually no relation of mine,
and Niedergerke who had used a special interference microscope to look at intact fibers.
And they'd found the same thing in so far as the A band stayed constant in length,
but their optics weren't such as to be able to see to measure the length of the actin filaments.
So that we thought was pretty strong evidence for this type of model.
But, it still remained quite controversial for a number of years
particularly because I think some of the other workers in the field
hadn't sort of got used to this combination of X-rays and EM and phase microscopy.
But I think a few more people were probably convinced by some electron micrographs
I was able to get two or three years later,
which showed the longitudinal sections through the myofibril, through the muscle fiber,
in which you can see the thick filaments, very clearly I think,
stopping at the end of the A band
whereas the thin filaments continue on into the I bands out here.
And you can also, at higher magnification, you can see these cross-bridges
between the actin and myosin filaments
that I'd supposed must be able to produce the relative sliding force that produces contraction.
If there's any general conclusions about how you go about a problem you can draw from this,
well, I'm not sure that there are.
I think I was very lucky because I had a clearly defined problem,
and I was able to find some new techniques which provided a way into it.
So, if you can repeat that process, you might get lucky like I did.
Thank you.