Mutant Hunter!

Mutant Hunter Intro

How we hunt myosin mutants in Dicty

"Come & see the violence inherent in the system!" --Monty Python

Background: that Dicty lifestyle


ll the work we do is carried out in an organism called Dictyostelium discoideum. We choose to use this organism because it offers us a variety of ways to assess and manipulate the function of the myosin motor. This is because many of the things a Dictyostelium does in its day-to-day life require myosin function. Following is a brief list of what goes on in a normal ('wildtype') Dictyostelium cell, and what goes wrong if the cell's myosin is not up to its tasks. First, a little information about the lifestyle of a Dictyostelium out living in the wild. Dicty are little amoeboid cells that ooze around the forest floor eating bacteria. As with the rest of us, the more they eat, the bigger they get. Whenever a Dicty cell gets big enough, it will pinch itself in half (a process known as cytokinesis, which it shares with your cells), creating two daughter cells. However, they are also capable of a much more social lifestyle. Should a traveling Dicty run out of food and begin to starve, it sends out chemical signals alerting its neighbors to its distress. Any nearby Dicty sensing these signals drop what they're doing and rush to their companions side. Then all the Dicty that have gathered begin acting as a single organism rather than a bunch of individuals. They form a structure rather like a slug, in which each cell retains its identity but acts as part of the whole. The slug travels, "looking" for a sunlit spot. Upon finding one, the organism alters itself to form a fruiting structure, consisting of a mass of toughened cells, or spores, atop a "trunk" of dead cells called the stalk. From this point, the spores "wait" for rain to disperse and awaken them, and the cycle begins again.


Consequences of myosin deficiency: Genetic "handles" on the motor



o our great good fortune, a failure of the myosin motor in Dicty cells disrupts a number of conspicuous events in the cycle. Perhaps the most striking is the requirement of myosin function to perform cell division, or cytokinesis. Dicty cells without myosin grow in size, but cannot divide. Instead of increasing in number, individual cells get bigger and bigger and bigger....and finally burst. That would seem to be the end of the road, except for a critical unexpected finding. If we grow the myosin-less Dicty on a plastic surface, it turns out they can pull themselves apart "gracefully", i.e. each of the two parts is intact and can continue life as a reasonably normal Dicty cell. This allows us to propagate Dicty cells without any myosin at all, despite its critical role in the process of normal cell division.


nother point in the cycle where myosin facilitates a key process is in consumption of bacteria. In the laboratory, we grow Dicty on of bacterial marshes or "lawns". These are prepared by inoculating nutrient agar on petri dishes with a bacterial strain that Dicty likes to eat. If we place a Dictyostelium cell or population on this marsh, the Dicty will begin munching their way into the lawn, 'clearing' a circular patch.
The size of the cleared patch will grow as the Dicty continue to invade the lawn and eat it. As it turns out, a Dicty population with functional myosin performs this process MUCH better than one without myosin. So if we were to put down equal numbers of wild type and myosin-less Dicty on a bacterial lawn, after several days' time, we would notice that the circle made by the wildtype Dicty was much larger than that made by Dicty lacking myosin.


he final critical behavior of Dicty that involves myosin function is the maintenance of cell shape and size. Throughout the periphery of a Dicty cell are two myosin and its sidekick, actin, form an interlinked meshwork. The myosin motors keep this meshwork taut by tugging on the actin filaments. However, there is another system, consisting of actin filaments only, that combats the "sphere-forming" tendencies of the actomyosin meshwork and allows the cell to maintain a flat shape, which is better for attaching to surfaces, like that of a petri dish. However, if we chemically treat these cells in the appropriate way, we can "turn off" the flattening pathway, and the now unrestrained sphericalizing force ('cortical tension') will cause the cell to spring into a rounder shape and lose its ability to grip the surface. In other words, if a cell has myosin and is chemically treated appropriately, it will let go of the surface it is growing on. This contrasts with the behavior of a cell with defective myosin, which will remain flat and stuck to the surface since its cortical tension pathway is not operative. Notice that in this instance, the behavior of cells without myosin is actually advantageous, in that these cells remain stuck to a plate while their myosin-functional brethren are lost to the surrounding medium. In other words, we can actually go "fishing" for cells that have inferior myosin function!


In the beginning: the quest for mutant motors

Scheme for selection of cs myosin mutants using alternating cold and warm cycles of azide treatment


ur first goal was to identify "broken" motors of a very special type: ones that were "stuck" at a particular point in the motor cycle. This would be as opposed to motors that never got assembled, or ones that were unstable or ones that bore such a poor resemblance to motors that their actions were not informative about how the real motor worked. In other words, we wanted motors that "froze up" under a certain set of conditions, but that under better circumstances could pretty much do what they were supposed to. Technically speaking, we went after motors that were 'conditional' or cold-sensitive--ones that were able to do their jobs in the Dicty cells at a warm temperature, but that seized up or otherwise failed in the cold.
To achieve this we took advantage of one of the myosin-dependent behaviors mentioned above: the difference between wildtype cells that pull themselves off of a plastic surface when exposed to azide and myosin-deficient cells that remain firmly stuck to the surface under these conditions. Briefly, after damaging the DNA of billions of cells, we "sorted through" them to find the very, very few that showed the behavior of interest to us: sticking to plates in the cold, but releasing from them in the warm. In the diagram on the left, the cells whose behavior is of interest (and which were therefore retained after the azide treatment) are shown in green; those doomed to be discarded are depicted in red. After performing these procedures over and over and over again (due to the extreme rarity of the desired mutants and the fact that each round of selection was only 95-99% efficient), we were left with only those cells with this behavior and their descendants. Partly due to our good fortune, virtually all of these cells behaved the way they did because of a defect in their myosin motor. We extracted the myosin gene from these cells and determined its DNA sequence. By comparing this sequence to that of "normal" myosin, we deduced the specific amino acid substitution that rendered the motor bearing the change sensitive to cold temperature. The following is a list of these cold-sensitive ('cs') mutations:
V192F G240C G240V E476Q Y494K W501L E531Q P536R R562H R562L E586K G624D G680V G691C G740D
The problems plaguing these motors are dealt with in the DataDungeon section. Suffice it to say, several of them have characteristics that merit further study--such as how to rebuild them--make them faster, stronger...


Bring 'em back alive: finding motors whose function has been restored


n order to really understand how the motor works, we want to learn not only how to break it, but also how to fix it. If we can truly understand how to "put Humpty Dumpty back together again", the we have a meaningful picture of what makes the motor tick. The Dicty system makes it very easy for us to identify rare Dicty cells whose myosin motors have been "fixed" by the process of mutation. The procedure we use relies on the bacterium-eating facet of the Dicty lifestyle. As mentioned above, when Dicty cells are grown on lawns of bacteria, the rate at which a colony expands into and eats the bacteria is dependent on the degree of functioning of their myosin motors. Thus if we deposit some Dicty cells whose only myosin motor is a broken one, the cells and their descendants will "clear" or expand into and eat the bacterial lawn very slowly. Visually, we observe this as a slowly growing circular clearing of the cloudy bacterial lawn. When the cells have cleared an area about a centimeter in diameter, we abuse them with UV light. This treatment has the same effect on Dicty cells as it does on you and I--the DNA is heavily damaged, and some of the damage is repaired incorrectly--mutating the DNA sequence so that it codes something other than is normal. In a population of millions of cells, such as we have in each cleared circle on the bacterial lawn, many cells will receive damage to their myosin gene (other genes will be damaged as well, but we don't care about those). The process is random, so it is likely that each cell that takes damage in its myosin gene undergoes a different mutation. Most of these mutations will render the myosin motors even more useless than they were originally. But rarely, the new change will actually have a beneficial effect on the defective motor, in some cases actually restoring its ability to function!
When the repair event occurs, it happens in a single cell--one of millions or billions on a plate. In the animation, the yellow dot represents a cell that contains a beneficial mutation in its defective myosin gene. Repaired myosin gives faster growing cleared section How can we find this cell? Fortunately, it identifies itself for us! Since this cell and all its progeny expand into the bacterial lawn more rapidly than its neighbors, the smoothly expanding circle develops a "blister", "bubble", or "petal", depending on who is describing it. In the animation, this is represented by the pinkish region. Of course, in the actual experiment, we rely only on shape; there are no color differences! Regardless, after 4-5 days, we now have a pure culture of cells expressing "repaired" myosin (the animation represents time lapse of 1 day/image). We can harvest these, recover their myosin gene, and determine the change that has "brought it back to life."


For the aficionado: some other facets of the Dicty system that make it work


or now, just a brief listing: Dicty cells as used in most labs are heavily derived from those living in the North Carolina forests; the following characteristics are for the lab versions: haploid; can be transformed by electroporation; perform reasonably efficient homologous recombination; can be grown in liquid media (in suspension or on plastic surfaces) or on bacterial lawns; can be stored as frozen stocks; extrachromosomal plasmids are routinely used; myosin is not essential for viability under some conditions; are a rich source of myosin protein; can be used to produce virtually any variant of the myosin protein imaginable in good quantity and in the absence of the normal myosin.

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Philosophy

Why mutants
can solve motors
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The Origins
of Myosin Truths

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2 Wrong
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Fixing mutants
the hard way
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Bruce Patterson
http://research.biology.arizona.edu/myosin