An Experiment on Flagellar Regeneration

A true scientist is interested in explaining natural phenomena regardless of what we can do with the information. Curiosity drives his or her research. A successful scientist pursues such interests and can make a strong argument for funding the studies. Foundations, the guv'mint, and other organizations that fund biomedical research are interested in (1) addressing issues related to human-focused problems, and (2) acquiring new knowledge of basic biological mechanisms that can facilitate addressing such issues. Typically, the more basic the research the more valuable are the findings. For example, consider the impact of the research leading to the Watson and Crick paper identifying the DNA double helix as the carrier of genetic information. Can any research focusing on this or that particular disease have had anywhere near the impact as that single publication in the journal Nature?

If we develop a thorough understanding of how a universally important organelle functions and how its structure and function are regulated, we can exploit that knowledge in many ways. For example, a great breakthrough in biological science was accomplished with the discovery of the "fluid mosaic" nature of cell membranes (another area of research leading to a Nobel Prize). Another type of structure, the microtubule, is critical to the function of all eurkaryotic organelles and therefore is vital to their survival. We can accomplish a great many things with knowledge of how cells regulate the assembly, disassembly, and function of microtubules.

Acting upon the axiom that "different organisms solve similar problems in similar ways," we might begin a study of microtubule regulation with a relatively simple biological model. Flagella of the protist Chlamydomonas are typical of eukaroytic cilia and flagella, in that they are extensions of the cell with microtubule-based structures at their core. Chlamydomonas cultures consist of independent, vegetative cells that are essentially all alike. We can raise large numbers of them, divide cultures into experimental groups, and vary just one thing at a time without concern that we will initiate a cascade of events that will obscure the cause of any observations that we make. That is, we can use these cultures to conduct well-controlled experiments. The outcomes of such experiments gives us a "leg up" toward learning how more complex organisms regulate microtubule structure and function.

Hypothesis

Ultimately, we are interested in everything we can find out about the structure, function, and regulation of microtubules. However, to design a meaningful experiment we have to focus our ambitions. Science advances just one step at a time. A study might focus on regulation of the assembly of microtubules, specifically on the assembly of microtubules to form flagella. It might focus on regulation of synthesis of microtubules, or on the roles of different types (isoforms) of subunits. One might ask how cellular mechanisms control when microtubules will be assembled or disassembled. What determines the final length of a microtubule? These questions are very broad, and would be very difficult (if not impossible) to address in a single experiment.

One way to pursue a scientific investigation is to conduct experiments. A common way to begin to design a good experiment is to pose a question in the form of a testable hypothesis. Not all questions or statements can be tested experimentally. For example, what experiment could you design to answer the question "why is the sky blue?" On the other hand, with a bit of thought you can probably come up with an experiment to test the statement "the sky is blue because the air contains particulate matter."

It has been well establish that if one amputates flagella from cells of Chlamydomonas without damaging the cells themselves, the membranes re-seal and the flagella regenerate back to normal length. They do not grow any longer, or grow back "stunted." We would like to know why they regenerate and why a pre-set length is maintained. We cannot expect to answer those questions with one experiment, but we can make progress by posing a well-chosen hypothesis. We will test the hypothesis that protein synthesis must take place in order for flagella to regrow in Chlamydomonas from which flagella have been amputated. Because flagella are composed in large part of microtubules, the outcome of our experiment also addresses the question, "must new protein be synthesized for microtubules to assemble? To appreciate the rationale behind this question you will need some background information on microtubules in general.

One reasonable question is whether or not all of the tubulin has to be synthesized "fresh." A hypothesis is a statement to be tested. The hypothesis for our experiment will be that protein synthesis must take place in order for flagella to regrow in Chlamydomonas from which flagella have been amputated. It makes sense to start here because it is easy to test and protein synthesis is central to the expression of genes and hence to the regulation of intracellular processes.

Experiment

Cycloheximide inhibits protein synthesis by preventing the translocation step after the first two amino acids are linked by the ribosome. The messenger RNA is unable to index, the next transfer RNA cannot be attached, the next amino acid cannot be lined up, and protein synthesis stops. Cycloheximide will be added to a freshly deflagellated culture to a final concentration of 10 micrograms/ml , and periodic measurements will be taken to determine the effect of inhibition of protein synthesis on assembly of flagella.

Controls

An experimental control is a standard to which to compare an experimental group. For this experiment we need a positive control to ensure that the deflagellated Chlamydomonas can grow back in the first place. If flagella grow back in an untreated culture and if they do not grow in the treated culture then you can draw the conclusion that the lack of growth is due to lack of protein synthesis. Without the untreated culture we have no way of knowing if some other factor, such as the amputation procedure itself, prevented growth.

Colchicine is obtained commercially, of course. The natural source is the plant Cochicum autumnale, known as the autumn crocus. It is highly poisonous, however the substance has traditionally been used in the treatment of gout, and in fact is still the drug of choice for that ailment. Extreme care is used in its administration, of course. Colchicine binds the soluble tubulin heterodimers. After binding with colchicine a heterodimer is still capable of assembing to the growing end of a microtubule. However, the colchicine molecule 'caps' the end, so that no additional heterodimers can be added. All microtubule growth, including flagellar growth, stops, however tubulin is still free to detach from assembled microtubules. A culture in a medium that includes 3 mg/ml colchicine serves as an effective negative control group.

Controls for stability of intact flagella

For all we know, the length of flagella of our model species may vary as part of some circadian (daily) rhythm. Either of the poisons used in our experiment could have an effect on assembled flagella. This would complicate matters, of course. To control for possible effect of poisons on intact flagella and to control for possible variation in assembled flagella length, samples should also be taken of treated and untreated cultures that have not had flagella removed. It is not necessary to determine a time course, but non-deflagellated cells should be sampled at least at the beginning and end of the experiment.

Sample collection and data recording

Sampling at timed intervals will enable us to record the progress of regrowth of flagella. Cultures can be maintained under identical conditions and small representative samples removed for measurement, leaving the rest of the culture to continue. Samples must be fixed and stained to preserve the structures as they were at the exact moment of sampling, and to enable visualization of flagella.

A complete data set of data should include measurements taken at approximately ten minute invervals at the beginning of the experiment, perhaps with wider intervals (e.g., 15 minutes) toward the end if rates of change appear to be slowing. For non-deflagellated samples it should be sufficient to record average flagella length at the beginning and again at the end of the experiment.