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The regeneration of Flagella in Chlamydomonas Reinhardtii

Abstract: Chlamydomonas reinhardtii is a unicellular, photosynthetic and a bi-flagellated organism that is found in water and can excise their flagella and then regenerate them. Chlamydomonas has a cell wall and a single, large chloroplast, which occupies about 40% of the cell volume. In order to study the regeneration of their flagella we examined the effects of EGTA, and cyclohexamide on the cells. Using a pH shock to de-flagellate the cells we were able to determine if any of the chemicals influenced the regeneration of their flagella. Colchicine was used as the negative control because it showed to stop flagellar regeneration when added to the cells. We hypothesized that EGTA and cyclohexamide would have an effect on flagellar regeneration. Our first hypothesis that EGTA would have an effect on flagellar regeneration was accepted and the null hypothesis that EGTA would have no effects was rejected. Our second hypothesis that cyclohexamide would have an effect on flagellar regeneration was rejected and the null hypothesis that cyclohexamide would have no effects was accepted. Our data supported the rejection of the hypothesis for cyclohexamine and the acceptance of the hypothesis for EGTA. Introduction: Chlamydomonas Reinhardtii is a unicellular green algae found in seawater, freshwater, stagnant water, damp soil, and even snow. It has been widely studied because of its adaptability to different environments and short generation time. These organisms are best used as model organisms for their fast generation time and they are easy to access and can be manipulated. Also, they are best studied for studying various biological processes such as flagellar regeneration, photosynthesis, genetics, flagellar motility, cells movement, response to light, mineral

nutrient

and

regulation

of

ciliary

assembly-disassembly

(Lab

manual).

Chlamydomonas cells can be chemically or mechanically induced to shed their flagella (deflagellation). After amputation, they can regenerate flagella to predeflagellation lengths rapidly within 2 hours. Flagellar assembly and disassembly are precisely controlled throughout cell cycle progression and cell division. During cell division, flagella are disassembled naturally (dutta & Avasthi, 2017). Under optimal conditions, C. reinhardtii grows so rapidly that its numbers can double approximately every 8 hours (Keller, 1984). The fact that it can grow in the dark on acetate-containing medium while retaining a functional photosynthetic apparatus, has allowed even light-sensitive photosynthesis mutants to be isolated (Cheshire 1991). They have a cell wall and an eyespot that helps them see the direction of light and its intensity. They have two anterior flagella that allow movement and these two flagella originate from basal bodies showing the typical 9+2 arrangement of microtubules found in eukaryotic flagella and cilia (lab manual). These microtubules are singlet microtubules that are surrounded by outer doublet microtubules (Lindemann, 2007). These singlet and doublet polymers are formed from heterodimers that are made up -tubulin and -tubulin monomers. Moreover, chlamydomonas are very useful for studying flagellar regeneration because it is very easy to make them lose their flagella through a pH shock. The isolated cells can regenerate their flagella when they are separated and placed in fresh medium. In order to study flagellar regeneration, the effects of Colchicine were analyzed. Colchicine is extracted from the Autumn crocus plant. It is known to prevent microtubule polymerization by binding to tubulin monomers inhibiting the assembly of the heterodimer and disassembling the existing ones which leads to inhibiting flagellation. It does that by binding to alpha tubulin and beta tubulin that forms a tubulin-colchicine complex that prevents flagellar regeneration. The presence of colchicine causes the number of non-motile cells that lost their flagella, to stay constant because

flagella are not expected to regenerate. Using Colchicine, it can be confirmed that microtubules are one of the primary building blocks of the flagella. And since colchicine prevents flagellar regrowth, it is used as the negative control in this experiment. In this experiment, we also studied the effect of calcium on flagellar regeneration. Calcium ions are known to be the universal secondary messengers that have been implicated in the deflagellation process in Chlamydomonas (Quarmby and Harzell, 1994) A Ca 2+ influx stimulated by acidification of Chlamydomonas results in flagellar excision. One pathway that involves calcium ions is the IP 3 pathway that leads to cellular responses such as de-flagellation. Ca2+ entry into the cell following acid treatment may be signaled by the second messenger inositol 1,4,5-triphosphate (IP 3). IP3 is made by a signaling molecule binding to a receptor that activates Phospholipase C. The IP3 binds to the IP3-gated calcium channel and allows an influx of calcium ions (Ca2+) from the endoplasmic reticulum to the cytosol increasing flagellar excision. In order to determine whether calcium is required for the regeneration of flagella, TAP medium made without calcium must be used. And in order to do this, EGTA (Ethylene glycol tetraacetic acid) is added to the TAP media. EGTA is a calcium ion chelating reagent, and has a high affinity to calcium. Therefore, it will form EGTA-Calcium complexes with all the calcium ions in the media yielding calcium free TAP media. The EGTA is expected to have an effect on regeneration of the flagella. Another drug that was used to study the regeration of flagella was cycloheximide, an antibiotic and an eukaryote protein synthesis inhibitor. It interferes with translation process in protein synthesis by blocking the elongation phase of eukaryotic translation. It binds the E-site of the ribosome and inhibits translocation (Gokal, Cavanaugh, and Thompson, 1986). 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. According to a previous experiment by Rosenbaum in 1969 on “flagellar elongation and shortening in Chlamydomonas”, it was found that a concentration of cycloheximide which inhibits protein synthesis permits less than one-third of each flagellum to form when both flagella are amputated. When only one is amputated in cycloheximide, shortening proceeds normally and the degree of elongation in the amputated flagellum is greater than if both were amputated in the presence of cycloheximide. The alternative hypothesis is that removal of calcium ions from the environment using EGTA will affect flagellar regeneration as EGTA is expected to stop flagellar regeneration. The null hypothesis is that the removal of calcium ions from the environment using EGTA will have no effect on flagellar regeneration. And the alternative hypothesis for the cyclohexamide experiment is it will affect flagellaer regeneration. Because cycloheximide inhibits the protein synthesis but it doesn’t completely block flagellar regeneration. Also, protein synthesis is required for the formation of the full-length flagella even though it was not needed in the initial regeneration. So, it will inhibit regeneration of flagella at some point.

Methods: A. week 1 The first week of the experiment consisted of de-flagellating the cells through an acid shock which acted as a positive control. 1mL of well mixed Chlamydomonas culture was obtained using a cut pipette tip and placed it into a 1.5mL microcentrifuge tube. Then 10ul of the culture was loaded onto the hemocytometer and observed under the microscope to ensure they were

moving. In order to de-flagellate the cells, 24ul of sodium acetate (pH 4) was added to the remaining cells in the microcentrifuge tube and gently flipped the tube to properly mix them then incubated the tube for 60 seconds. Then the cells were neutralized immediately after the 60 seconds by adding 48 ul of potassium hydroxide (KOH) and gently flipping the cell to mix it in. This gave the cells an acid shock because sodium acetate has a low pH and KOH has a high pH. After the neutralization the tubes were centrifuged for 10 seconds at maximum speed. Next, the supernatant was discarded and 1mL of normal TAP media was added on top of the cell pellet and the cells were resuspended. Then it was centrifuged again for 10 seconds, discarding of the supernatant, and resuspending of the cells using TAP media, two more times. After the last resuspension 10ul of the cells was loaded onto the hemocytometer for observation, and the rest of the cells was put under a source of light. Once they were placed under the light a timer was started for 10 minutes. Then the number of non-motile cells in the small squares was counted. 10ul sample from the tube was observed and counted every 10 minutes for 60 minutes. B. Week 2 The second week of the experiment involved developing the negative control using colchicine. For this experiment the same procedure for de-flagellation and neutralization and the three resuspensions were followed as week 1. For the third resuspension in this experiment, only 970ul of TAP media was added to the two tube. Also, 30ul of 95% Ethanol was added to tube 1 and resuspended and 30ul of Colchicine was added to tube 2 and resuspended. Then, 10ul from each tube was taken and loaded onto the hemocytometer. And the two tubes with the rest of the cells were put under a light source. The cells were observed under the microscope and the non-motile cells were counted the same way as week 1 for 60 minutes. C. Week 3

For week 3 the experiment involved examining the role of calcium ions in flagellar regeneration. In order to do this four 1.5mL microcentrifuge tubes were obtained and labeled as tuve 1, 2, 3, and 4. 1mL of Chlamydomonas was added into all four tubes. Then tubes 1, 2, and 3 were deflagellated by adding sodium acetate into them and 24ul of distilled water was added to tube 4, followed by a 60 second incubation. Right after the 60 seconds tubes 1, 2, and 3, with 48ul of KOH were neutralized but 48ul of distilled water was added to tube 4. The fourth tube was used to ensure that the concentration of EGTA will not kill the cells. Then the cells were centrifuged for 10 seconds, the supernatant was discarded and the cells were then resuspended. For tubes 1 and 2 1mL of normal TAP media was used and resuspended. For tubes 3 and 4 1mL of calcium free TAP media (EGTA+TAP) was used and resuspended those cells. Then the centrifugation, supernatant and resuspension steps were repeated two more times. In the last resuspension step 970uL of fresh TAP media was added to tube 1 and 2 and to tube 3 and 4, 970uL of calcium free TAP media was added. Then 30ul of 95% ethanol was added to tubes 1, 3, and 4, and 30ul of Colchicine was added to tube 2, all tubes were resuspended. From each tube 10ul of culture were taken and 2ul of 12mM CaCl2 solution was added to each. These 12ul were then loaded onto the hemocytometer and were observed and non-motile cells in the small squares were counted. Every 15 minutes a fresh sample from all four tubes was observed and the CaCl 2 was added before observing the cell every 15 minutes for 75 minutes.

D. Week 4: Week four of the experiment involved seeing the effects of cyclohexamide on the cells. For this experiment four 1.5mL microcentrifuge tubes were used and labeled 1-4. The same deflagellation and neutralization procedure was followed as week 3. The tubes were then

centrifuged and the supernatant was discarded and added. 1mL TAP media was added to tube 1 and 2 and 1mL TAP+ cyclohexamide was added to tube 3 and 4. The pellets were resuspended and centrifuged for 10 seconds. This step was repeated two times. After the final resuspension, 970uL of TAP media was added to tube 1 and 2 and 970uL of TAP+cyclohexamide was added to tube 3 and 4. After that 30ul of 95% ethanol was added to tubes 1, 3, and 4 and 30ul of Colchicine was added to tube 2 and resuspend. Then a fresh 10ul sample from the tubes were observed under the microscope every 15 minutes for 75 minutes. RESULTS:

Figure 1: Shows the percentage of non-motile cells counted in 10-minute intervals for 60 minutes after a pH shock which served a positive control. The percentage of the nonmotile cells after the addition of sodium acetate shows the gradual decrease in the number of cells because of the acid shock. Week 2

Figure 2: Illustrates the percentage of nonmotile cells after the acid shock with a positive control in tube 1, receiving the same treatment as experiment 1 and with colchicine in tube 2, negative control. The graph shows that the percentage of nonmotile cells decreased. Week 3

Figure 3: Illustrates the percentage of nonmotile cells with positive control in tube1 treated with sodium acetate, negative control in tube 2 treated with colchicine, experimental in tube 3 treated with EGTA+TAP, sodium acetate, and KOH and experimental in tube 4 treated with only EGTA+TAP. Tubes 3 and 4 stayed constant the first 30 minute interval then decreased gradually. Week 4

Figure 4: illustrates the percentage of nonmotile cells after adding the cycloheximide to tube 3 and 4 This graps shows positive control in tube 1 and colchicine in tube 2. The experimental in tube 3 was treated with sodium acetate and cycloheximide and the experimental in tube 4 was treated without sodium acetate. Tube 2 stayed constant throughout the 75 minutes interval. Tube 3 and 4 decreased constantly and the number of non-motile cells observed was low. ANOVA

Factor A: Factor B: AxB A x subj: B x subj A x B x subj: Total Line 2 EGTA

Sum of sqrs 41540.6 4571.61 1822.14 701169 8310.95 11060.4 834712

Line 1 positive control

df 1 5 5 9 45 45 59

Mean square 41540.6 914.323 364.429 77907.7 184.688 245.787

F 0.5332 4.951 1.483

p(same) 0.4838 0.001075 0.2144

Figure 5: The class data for the percentage nonmotile cells for the EGTA experiment. Line 1 represents positive and line 2 represents treatment with EGTA.

Factor A: Factor B: AxB A x subj: B x subj: A x B x subj: Total

Sum of sqrs 4330.26 10993.1 3857.2 300879 34479.8 41615.7 237408

Line 2 cycloheximide Line 1 positive control

df 1 5 5 9 45 45 59

Mean square 4330.26 2198.62 771.439 33431 766.218 924.794

F 0.1295 2.869 0.8342

p(same) 0.7272 0.02479 0.5324

Figure 6: shows the class data for the percentage non-motile cells for the cycloheximide experiment. Line 1 represents positive control and line 2 represents treatment with cycloheximide. DISCUSSION: In this study of the regeneration of flagella in Chlamydomonas, the effects various treatments on the cells were measured. Figure 1 shows that the pH is a positive control therefore the number of non-motile cells is decreasing which means the number of motile cells should be increasing since they will re-flagellate after the pH shock. The graph shows a steady decrease all throughout. In figure 2, the effects of colchicine on flagellar regeneration is demonstrated. Colchicine as a negative control was expected to inhibit the regeneration of the flagella which was shown in all the experiments Also, colchicine was used as negative control to compare the effects of other treatments such as EGTA and cycloheximide and how they affect flagellar regeneration. Graph 2 shows that the line for colchicine has a steady line around the same values which is shown in orange. This was expected because colchicine stop tubulin formation therefore there should be no increase in motile cells or decrease in non-motile cells. In graph 3, the effects of EGTA is demonstrated through tubes 3 and 4. However, tube 4 was deflagellated. The

numbers for tube three are hovering around the same values. The line stayed steady which was expected because the cells require calcium to re-flagellate. Tube 4 was also expected to show a steady line but at a lower percentage because the cells weren’t deflagellated therefore small number of non-motile cells would be observed. In figure 4, the effects of cyclohexamide on cells have been seen through tube 3 and 4. Cells treated with cycloheximide were expected to display little to no flagella regeneration. Both tubes 3 and 4 showed decrease with deflagellated cells and cells that have not been deflagellated. Moreover, ANOVA was used to test hypothesis based on their statictical significance. In order to be statistically significant and support the hypothesis the p-value must be less than 0.05, if the p-value is greater 0.05 the null hypothesis is supported. The p-value for the EGTA results is 0.4838 which is less than 0.05 therefore we supportt our hypothesis and reject the null hypothesis. The p-value for the ANOVA cyclohexamide results is 0.7272 which is greater than 0.05 therefore we reject our hypothesis and accept the null hypothesis. For both the experimental groups our statistical significance helped accept the hypotheses the graphs also further supported the hypotheses. For EGTA the results showed it did have an effect on flagellar regeneration therefore our null hypothesis was rejected, and the ANOVA results confirm that. Furthermore, the cycloheximide experiment showed decrease in tube 3 and 4 and AVOVA also shows a decrease which might suggest that regeneration occurred immediately. Therefore, more data would be required to dee the effect of cyclohexamide. Although cyclohexamide inhibits the protein synthesis which could affect the regeneration of flagella and it is expected to have no effect on re-flagellation because it only inhibits one pathway. And although the data was supported by the p value which was greater than 0.5, there are numerous factors that could’ve led to errors during the experiment. For instance, lack of consistency,

possibility of contamination of cycloheximide or of the growth media could affect the data. More importantly, time was important for this experiment due to having a correlation between regrowth of flagella and time under each of the treatments. Although the experiments were conducted effectively, the trials

may have been subject to minor errors due to unprecise

pipetting, or imprecise hemocytometer cell counts. In future experiments, it would be best if these samples ran more times to have better accuracy and understanding of flagellar regeneration in Chlamydomonas and to prevent error in cell counts. In addition, the amount of variability can be reduced by increasing sample sizes with further experimentation and data points.

Citation Gokal, P. K., et al. The effects of cycloheximide upon transcription of rRNA, 5 S RNA, and tRNA

genes, 25 February 1986. https://www.ncbi.nlm.nih.gov/pubmed/2419318 Rosenbaum, et al. Flagellar elongation and shortening in Chlamydomonas. The use of cycloheximide and colchicine to study the synthesis and assembly of flagella proteins. May 1969. https://www.ncbi.nlm.nih.gov/pubmed/5783876 Schneider-Poetsch, et al. “Inhibition of Eukaryotic Translation Elongation by Cycloheximide and Lactimidomycin.”, 30 Jan. 2010. Https://Www.ncbi.nlm.nih.gov/Pmc/Articles/PMC2831214/ Quarmby, L. M., Yueh, Y. G., Cheshire, J. L., Keller L. R., Snell, W. J., and Crain, R. C. (1992). Inositol phospholipid metabolism may Chlamydomonas reinhardtii. The Journal of doi:10.1083/jcb.116.3.737

trigger flagellar excision in Cell Biology, 116(3), 737-744.

Lindemann, C. B., & Lesich, K. A. (2010). Flagellar and ciliary beating: The proven and the possible. Journal of Cell Science, 123(4), 519-528. doi:10.1242/jcs.051326 Dutta, Soumita and Prachee Avasthi. “Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies” Sphere vol. 2,2 e0000317. 8 Mar. 2017, doi:10.1128/mSphere.00003-17 Keller L. R., Schloss J. A., Silflow C. D. and Rosenbaum J. L.(1984). Transcription ...