Discovery Talk Questions and Answers: How Muscle Contracts

Hugh Huxley
Assessments created by Dr. Rachna Sharma

Questions

What was the specific research problem pursued by Dr. Huxley?
1. The energetics of muscle.
2. The biochemistry of muscle.
3. The mechanics of muscle.
4. The submicroscopic structure of muscle.
5. None of the above
What was Dr. Huxley able to show using X-Ray technology and a projection of the electron density from the diagrams he obtained?
1. That actin filaments appeared as a high density image in relaxed muscles
2. That actin and myosin combined with each other
3. That actin density varied with ATP levels.
4. None of the above
In his talk, Dr. Huxley refers to muscle structures called “crossbridges”. What is the role of these crossbridges?
1. To interact with actin filaments.
2. To interact with myosin filaments
3. To enable myosin and actin to interact with each other.
4. None of the above
What type of muscle preparation is useful for obtaining successful X-ray images?
1. Hydrated muscle preparations
2. Dry muscle preparations
3. None of the above
What was the procedure used to confirm the existence of the two proteins actin and myosin in the muscle submicroscopic structure? Indicate the corresponding experiment in the research paper.
Which experimental evidence helped the authors conclude that the A-band of muscle contains myosin?
In his talk, Dr. Huxley describes the first experiment that he did after he reached Schmitt’s MIT laboratory.
What was the goal of this experiment, and which procedure did he use?
In his talk, Dr. Huxley refers to the experimental study that helped him decipher the mechanism of muscle contraction that also remained controversial for a long time and is now referred to as “the sliding filament theory of muscle contraction.”
Discuss this mechanism.
At the end of his talk, Dr. Huxley gives a very important message to people pursuing scientific research about having a clear research question, and finding ways to resolve it.
Discuss a real research problem that you would like to address. Which tools would help you address this problem?

Answers

d, submicroscopic structure of muscle. Talk 1:2423 to 1:33:10.
Video (01:17): “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.”
bVideo (03:39): “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.”
c.Video (10:03): “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.”
hydrated muscle preparation.Protein crystals need to be kept hydrated to get precise structural information.
Video (02:31): “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.”
Phase contrast microscopy before and after myosin extractionVideo (08:01): “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.”
Also refer to Fig. 1 on p.531

p.531: “Methods have become available during recent years for extracting exclusively myosin and actin from muscle, with very little mutual contamination; minced fresh muscle in the form of broken fibers is used as the starting materials. We have examined this material microscopically (in phase contrast illumination, in polarized light and in the electron microscope) at various stages of extraction.”
During the structural studies of the muscle, ATP and pyrophosphate along with solutions that cause exclusive myosin extraction, the A-band is lost and results in a ghost-like myofibril.Video (08:01): “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.”
Also, refer to the discussion p. 532, (a) and (b).
The goal was to confirm the X-ray data that indicated that myosin and actin interacted and showed up as double array of filaments. To do this, Dr. Huxley used histology and microscopy techniques at MIT.Video (6:34): “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.”
Video (10:37): “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 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.”
Ask students to come up with a research problem which they might want to address and the tools which will help them to do so. The problems can be categorized into structural (to find the microscopic structure) using various kinds of histology and microscopy techniques and functional like (gene function using molecular biology tools).