How Muscle Contracts
Transcript of Part 1: How Muscle Contracts
00:00:17;09 I began the work that ultimately led to the 00:00:19;28 sliding filament theory of muscle contraction in 1950. 00:00:25;01 I was a research student in a small MRC unit with John Kendrew as my supervisor 00:00:36;01 and Max Perutz being the head of the unit and that was it. 00:00:40;26 It was just three people. 00:00:43;14 I'd been a student in physics, and my main interest had been in experimental particle physics. 00:00:53;07 But, after graduation I decided to change fields completely, joined this unit, 00:01:00;09 and was in fact in complete freedom to choose my own particular subject of research. 00:01:07;06 Now during the first couple of years I spent doing some work on hemoglobin 00:01:12;09 and learning a bit of biology of which I was totally ignorant originally. 00:01:17;15 And then I chose muscle as a possible topic because 00:01:24;23 I'd found that although an awful lot was known about the 00:01:28;23 mechanics and energetics and biochemistry of muscle, 00:01:33;10 virtually nothing was known about its submicroscopic structure 00:01:37;26 nor about what actually happened during contraction. 00:01:41;23 Now, one way of getting that information might be 00:01:46;28 that if muscle was composed of regular, repeating or semi-crystalline units of these 00:01:55;20 two structural proteins then one might get evidence about what was happening from X-ray diffraction. 00:02:04;12 However, people had looked at wide-angle X-ray diffraction, 00:02:09;03 which showed you any changes in the polypeptide chain configuration, 00:02:14;13 and essentially it didn't show anything. 00:02:17;02 And they'd also looked at dried muscle by low angle diffraction, which showed you 00:02:23;20 structural regularities and the size of protein molecules, but that hadn't been informative either. 00:02:31;04 However, I'd learnt that one had to keep protein crystals fully hydrated 00:02:37;18 if you were going to get anything useful and an X-ray diagram from them. 00:02:41;27 So I thought, "Well, maybe that would be true of muscle and 00:02:44;09 maybe muscle really did contain lots of regular structures if only one could attack it the right way." 00:02:51;21 And so for that purpose I built a miniaturized X-ray camera, 00:02:58;00 which would optimize the sensitivity of images collected on film. 00:03:08;12 And I was also very fortunate that my supervisor John Kendrew knew 00:03:14;05 a man called Bernal in London in whose X-ray group two people were 00:03:20;03 actually building a very fine-focus, high-intensity X-ray generator (X-ray tube) for another purpose, 00:03:30;09 and they were kind enough to give me an early prototype of this, 00:03:34;02 which formed an essential part of the X-ray set-up that I built. 00:03:39;08 I was very thrilled and relieved to see that I did in fact get quite a well defined X-ray diagram. 00:03:50;13 Not very detailed, but it consisted basically of two reflections. 00:03:56;29 One here and then another one a bit further out. 00:04:03;01 And, the spacing of these told one they came from a hexagonal arrangement of objects, 00:04:11;05 and I concluded that this must be a hexagonal arrangement of myosin filaments. 00:04:17;21 But, what was particularly interesting was that in a muscle in rigor, 00:04:22;08 the pattern had the same spacing, approximately, 00:04:26;07 but the relative intensity of the two spots had changed, 00:04:31;11 and this told one that there was some alteration in the character of the lattice. 00:04:39;15 And that in fact you can get a projection of the electron density from those diagrams. 00:04:47;16 This is a myosin filament here on my picture, and then this is another myosin filament there 00:04:54;03 and another one in the lattice over here. 00:04:56;27 In between the myosin filaments is what I took to be actin filaments, 00:05:03;28 which appeared at low density in the relaxed muscle, but in rigor muscle, 00:05:10;08 their density was much increased. 00:05:13;13 Now I knew that actin and myosin tended to combine with others in the absence of ATP, that's in a rigor muscle. 00:05:21;14 So I concluded, and it turned out I was correct, that 00:05:25;25 was in fact what I was seeing: a double array of actin and myosin filaments interpenetrating with each other. 00:05:36;19 I could also see some reflections, axial reflections, which give you information 00:05:43;03 about the longitudinal regularities along the length of the muscle. 00:05:48;00 This is a spacing of about 145 angstroms, and there are another series of reflections not shown here. 00:05:56;25 The interesting...so it showed that muscle was a sort of semi-crystalline structure at this scale. 00:06:06;26 And these reflections didn't change in spacing when the muscle was passively stretched. 00:06:14;06 Now, I made a mistake at that point. 00:06:17;20 I thought that these reflections probably all came from the actin filaments. 00:06:25;10 So I concluded the actin filaments didn't change in length, 00:06:28;22 but I still had this picture that perhaps the myosin filaments somehow shortened down. 00:06:34;25 Anyway, I finished my PhD and went to MIT to Schmitt's lab, 00:06:41;07 which was a new center for doing electron microscope work, 00:06:47;03 because I wanted to make sure that this double array of filaments really existed. 00:06:53;13 So by fixing, embedding and sectioning a muscle, 00:06:58;14 I was able to look at cross sections in the electron microscope, 00:07:03;15 and lo and behold one could see the double array of filaments very, very clearly. 00:07:09;27 This was a rabbit muscle in rigor. 00:07:12;28 This is a myosin filament, and that's another myosin filament. 00:07:17;16 And then the actin filaments are in this little ring of six of them around each myosin center. 00:07:26;00 So that was very encouraging and then shortly after this, early in 1953, 00:07:34;28 Jean Hanson came to the same lab to learn electron microscopy too 00:07:40;10 because she'd been studying isolated myofibrils as seen in the light microscope. 00:07:47;18 She was zoologist by training. 00:07:50;11 And we decided, we were both doing the same thing essentially, 00:07:55;05 we decided to combine our different approaches, and this worked out very successfully. 00:08:01;25 One of the first experiments we did 00:08:05;00 was to look at myofibrils in the light microscope on a slide with a coverslip over them 00:08:12;00 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. 00:08:19;15 And when we irrigated these with solutions known to extract myosin from muscle, 00:08:28;00 we found that the dense material in the A band here was rapidly removed, 00:08:38;24 leaving a sort of ghost fibril which consisted of the original Z lines, 00:08:44;26 and a segment of the filament which we concluded were probably actin. 00:08:53;06 And we later found that applying actin extraction procedures 00:08:59;24 would remove this second set of filaments as well. 00:09:05;26 These are just showing the density plots before and after myosin extraction. 00:09:11;21 So, this was a sort of, quite a revolutionary finding for us. 00:09:17;09 And after a few hours we sort of reoriented our way of thinking about things. 00:09:23;21 And we realized that we were looking at two partially interpenetrating but overlapping arrays 00:09:32;00 of myosin filaments and actin filaments. 00:09:35;27 And that the cross-sections I had seen had been through the overlap region here. 00:09:43;23 So this is what I showed before-the overlap region. 00:09:49;23 In the H zone, which is where the actin filaments have stopped, you just see the thick filaments. 00:09:59;07 And in the I bands you can see the cross-section of the thin filaments. 00:10:03;14 So we then had a quite new model of the structure of the muscle. 00:10:10;04 And in between the two sets of filaments were these cross-bridges 00:10:15;11 that I had postulated must be there to enable 00:10:19;10 the two sets of the actin and myosin filaments to interact with each other. 00:10:24;11 So we published a short paper on this in Nature, but at Schmitt's request 00:10:32;04 we omitted any speculation about how this system would actually work. 00:10:37;20 But what we knew was that there were these two axial series of X-ray reflections 00:10:47;08 which didn't change during passive stretch. 00:10:50;23 And so we thought, "Well maybe they didn't change during contraction either." 00:10:55;22 And that would mean that the two sets of filaments would themselves remain constant in length 00:11:02;00 and that contraction would be achieved by 00:11:05;10 the actin filaments sliding further into the array of myosin filaments. 00:11:10;24 And when we...after a lot of hard work we were able to get sufficient images 00:11:17;10 of muscle at different stages of contraction to prove that point. 00:11:23;07 And you can see, this is a slightly stretched myofibril. 00:11:28;14 Then, we give it a little bit of ATP, and it shortens down a little. 00:11:35;12 The I bands, the clearer regions, become shorter, but the A bands stay constant in length. 00:11:41;03 And as it shortens further still, the I bands get even shorter, and ultimately they almost disappear. 00:11:50;11 We can also extract myosin from a stretched fibril, 00:11:58;01 and we can see the large gap between the ends of the actin filaments. 00:12:02;03 But when, on of course a different fibril, which is contracted, we can see 00:12:08;15 this gap between the actin shortening down and then disappearing. 00:12:12;17 So we can measure the lengths of the I filaments and that was constant during contraction. 00:12:18;03 So, this was written up for Nature and published in late May in 1954, 00:12:26;23 along with another paper by Andrew Huxley, who's actually no relation of mine, 00:12:33;20 and Niedergerke who had used a special interference microscope to look at intact fibers. 00:12:41;11 And they'd found the same thing in so far as the A band stayed constant in length, 00:12:46;24 but their optics weren't such as to be able to see to measure the length of the actin filaments. 00:12:56;23 So that we thought was pretty strong evidence for this type of model. 00:13:05;07 But, it still remained quite controversial for a number of years 00:13:12;15 particularly because I think some of the other workers in the field 00:13:18;03 hadn't sort of got used to this combination of X-rays and EM and phase microscopy. 00:13:23;26 But I think a few more people were probably convinced by some electron micrographs 00:13:31;13 I was able to get two or three years later, 00:13:34;12 which showed the longitudinal sections through the myofibril, through the muscle fiber, 00:13:43;11 in which you can see the thick filaments, very clearly I think, 00:13:49;19 stopping at the end of the A band 00:13:52;18 whereas the thin filaments continue on into the I bands out here. 00:13:58;19 And you can also, at higher magnification, you can see these cross-bridges 00:14:03;29 between the actin and myosin filaments 00:14:07;21 that I'd supposed must be able to produce the relative sliding force that produces contraction. 00:14:16;15 If there's any general conclusions about how you go about a problem you can draw from this, 00:14:24;21 well, I'm not sure that there are. 00:14:27;00 I think I was very lucky because I had a clearly defined problem, 00:14:31;01 and I was able to find some new techniques which provided a way into it. 00:14:37;28 So, if you can repeat that process, you might get lucky like I did. 00:14:43;22 Thank you.