|Philosophical overview: method to the madness|
n case you haven't noticed, our basic approach to understanding myosin is to take a perfectly good (actually, an absolutely astounding) machine and kick it into submission. What can we possibly hope to learn about how the machine works by breaking it?
irst, it's important to note how very difficult it is to learn from a tiny, fast, efficient little machine such as the myosin motor. This thing has spent a billion years being refined into an awesome device for powering almost everything you do (anything that involves muscle involves some form of myosin, and many things that don't involve muscle, such as the division of cells, also require myosin!) What the machine isn't designed to do is to be scientifically analyzed. One goal of a mutation-driven approach is to beat up the motor somewhat such that its properties begin to fall closer to our abilities to watch. Much like watching a slow-motion video of a bird flying or something.
ut there's a much more subtle and important aspect to this approach. We hope to gain insights into the machine by 'taking it apart', just as you might do with an (old) car engine.
ne useful analogy for explaining why making damaging mutations helps us to understand how protein machines works comes from my primitive understanding of my car. Having bottomed it out (thus inducing damage, similar to our approach with mutagenesis, except we do that on purpose), I was suddenly in a position to learn something about how it works. Starting up the car the next day, I was greeted by a roar, and I noticed steam emerging from the middle of the car rather than the tail end where it usually exited. Upon close inspection, I found that the muffler was emitting the steam, and achieved much enlightenment. First, I learned that there was such a thing as a muffler--from driving a functional car, I had been blissfully unaware of such things. Second, I learned from the smoke and site of damage where the muffler was--toward the middle of the car on the underside. Finally, I learned what the functional roles of the muffler were--to dampen sound and to conduct the exhaust to the rear of the car! An amazing series of findings, all achieved by running my car over a rock.
hus it also is with our mutant-making strategy. By taking a perfectly good machine and damaging it (and then searching carefully for machines showing partially impaired function--I would've learned little about mufflers if I had totaled the car or ripped out the engine!) we seek to learn what the function of parts is, where they are, and hope to gain insights into how they perform that function. It is important to note that an analogy to a macroscopic mechanical device, such as an auto engine, is useful, but there are also key differences. In looking at an engine, we observe discrete parts--pistons, wires, gas lines, valves.... In looking at a protein, it's all kind of "one thing"--sure there are 20 discrete building blocks that have been strung together to make the machine, but they're all wadded up into one blob. The key here is that we are only beginning to have some grasp of what the "parts" of a protein machine are! Also, a protein machine is much more homogeneous than a mechanical device--if you take out almost any chunk of it, there's a good chance the whole machine will "melt" into a shape that bears essentially no resemblance to the original! So we need to "dissect" a protein machine in subtle ways that preserve the general shape and workings of the device, but nonetheless perturb it in ways we can detect and try to understand.
hat's the problem with the story so far--I learned what goes wrong when the muffler is damaged, but I have gained little insight into how the muffler carries out its chores. Here comes the second tier of our approach: to further damage the motor such that its function is improved! This is a pretty counter intuitive concept, and works much better in the protein world than it does with cars. It has a lot to do with the differences between protein machines and mechanical devices, particularly the way the elements of each are put together and the kinds of forces that hold them together. Given these differences, I hope I will be forgiven for the following rather contrived and implausible example.
uppose I continued to drive my roaring vehicle about, since I had no idea how to go about restoring function to my defunct muffler. And suppose that some idiot rear-ended my innocent self with pretty good force. Thus my poor car has been damaged again. But let's imagine that out of the millions of possible outcomes of the damage, this time the tail pipe was accordioned and rammed through the defective muffler. As I zoom away from the scene of the crime, I notice that my car is now strangely quiet! What's more, when I get out to look, I see the steam is exiting from my foreshortened vehicle in more or less the right place! Now I am in a position to learn some very interesting facts about the exhaust system! If I am clever, I notice that my previously straight tail pipe is now greatly kinked--and somehow this provides "muffler function" (by baffling the sound waves and dissipating the force of the exhaust). Further, I learn that the tail pipe is another part of the machinery that conveys the exhaust to the rear of the car. Note that the muffler is in no better shape than it was before--I have learned all this by compensating for loss of muffler function, not by fixing the muffler itself.
t least that's the general idea! There are several things that are important to note. First, until recently, we hadn't even been able to see the motor "up close"! After many years of mighty struggle, Ivan Rayment's group at Madison Wisconsin has managed to get some "close-ups" of the motor locked in a couple of different states. These form the basis of all the images of myosin you see throughout this document.
qually important is the power the Dictyostelium system gives us for mutant hunting (for the nuts and bolts of this phase of the operation, see the Page of Mutant Hunting). Briefly, since we can grow and chemically treat "billions and billions" of these little guys quite easily, we have the opportunity to generate the rare "informative" mutations that damage the motor in ways that we can learn from, as well as the extremely rare second mutations that allow "broken" motors to function again even in the presence of damaging mutations. This is one place where the car analogy breaks down badly (no pun intended)--I am not allowed to go out and wreck my car 100 million times in order to uncover the "special" damage described above. But in Dictyostelium, we routinely sift through millions or billions of mutagenized cells to find those rare few that have benefited from the treatment!
f course, the key measure of whether all this stuff is worth doing or not is whether it works! As you will see throughout this document, we have had good success in making mutations that damage but do not destroy the motor. And we're managing to learn some important things about what regions of the motor perform critical functions. But the most ambitious and (to me) exciting part is that we're having tremendous good fortune in generating further mutations ('suppressors') that restore function to the damaged motors ! The challenge before us now is to figure out exactly why the motor was functioning poorly following the damage, and by what means the suppressor "fixes" it! This is a mighty challenge, as you can well imagine that even with the crumpled tail pipe, I could have looked at my "collision-improved" car and concluded that bursting open the trunk or shearing off the license plate had restored the muffling and steam-directing functions!
o that is the challenge before us: to figure out, using some powerful but nonetheless blunt tools, how a string of amino acids performs the intricate and precise dance that results in movement of actin filaments. The clues we have generated are to be found in these pages, as well as our current interpretation of what they're trying to tell us. And since the data is largely of the "who fixes whom, and what's wrong with who and whom by themselves" type, I truly hope that anybody who wants to take a crack at making sense out of the emerging picture can and will do so. Your thoughts are welcome at %firstname.lastname@example.org.