Derek Wong
Biology 275L-02
Lab 7
Assembling Microtubules of Chlamydomonas in Cycloheximide and Colchicine
Introduction:
Microtubules are long polymers of tubulin subunits that are used for cell motility and mobility. Each microtubule is approximately 25 nanometers that form a circle of 14 vertical rows of protofilaments, made up of alpha-beta dimers. Besides intracellular movements, microtubules may be used for cell mobility such as in flagella and cilia.
In this lab we will be examining the microtubule assembly of the unicellular photosynthetic organism Chlamydomonas in different medias to examine the affect that certain chemicals have. The chemicals to be used in this lab are colchicine which is an antimitotic drug which inhibits microtubule assembly by binding tightly to the alpha-tubulin of each free tubulin preventing further polymerization, and cycloheximide which inhibits protein synthesis by preventing the movement of ribosomes on the mRNA blocking the translation of proteins.
Results:
See next three pages for raw data tables and graph.
Discussion:
In this lab the affects of cycloheximide and Colchicine on microtubule assembly of Chlamydomonas were examined. The alga was deflagellated and then exposed to either cycloheximde, colchicines, or only media. The results were then compared to the lengths of the non-delagellated Clamydomonas.
After the 90-minute trail, the length of the non-deflagellated was 12.58 um compared to a flagella length of 10.6um for the media only, 8.43 um for the colchicines, and the 4.8um for the cycloheximide. It can be concluded that the flagella in all three medias may not have grown to complete length. The cycloheximide regenerated the least of all three medias, and the Medium I grew the most.
The results were as not as expected. Since the media only (Medium I) had no inhibitors preventing regeneration, there would be flagella regrowth within the given time period. The cycloheximide works to inhibit flagella regrowth by preventing the movement of ribosomes on mRNA, thus the production of proteins used for flagella synthesis does not occur. However, cycloheximide has no effect on the tubulin molecules itself and therefore the free tubulin remaining in the solution can still bind to the cell and flagella regrowth may occur. Flagella regrowth will only continue until all the free tubulin is used up. In contrast, the colchinicine binds to the free-floating alpha-beta tubulin dimmers in the media and prevents further polymerization, therefore no polymerization or minimal should take place. The results seen, however, were reversed. The colchicine had more regrowth then the cycloheximide. It is possible that other groups mistakenly took into their calculations the zeros and/or did not measure correctly. If there was any human error in the measurement or in the calculations, then the class average data would then be incorrect. It is possible also that several of the samples contained Chlamydomonas no longer capable of regeneration (i.e. samples were dead) causing the numbers for the class to be distorted due to the original pH shock that the Chlamydomonas underwent in order to become deflagellated.
In Figure 1, the length of the flagellas was graphically expressed for each time interval for each type of media. Although the flagella in Medium I did not it regrow entirely (at least compared to the non-deflagellated) Medium I had the highest regeneration. Cycloheximide should have had a higher regeneration curve than the colchincine because in the cycloheximide solution there was still free tubulin dimmers that could polymerize.