The Role of Calcium and Phospholypase C Signaling in Flagellum Regeneration in Chlamydomonas
Autor: Mikki • February 12, 2019 • 1,617 Words (7 Pages) • 876 Views
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In experiment 2, the effects of neomycin were measured. We had 4 test tubes in total. The first two test tubes received acid shock treatment, neutralization, and cell washings. Test tube 3 didn’t receive any acid shock treatment, while tube 4 received acid shock treatment but no neutralization. Centrifugation and cell washings were done similarly as in experiment 1. However, test tube 3 and 4 were washed by adding 1mL of neomycin (not 1mL of distilled water). After three washings, the supernatant was removed and each tube received their designated solutions: Tube 1: 1mL distilled water; tube 2: 1mL colchicine mixture; tubes 3 and 4: 1mL TAP and neomycin solution. As before, the tubes were placed under the light and the number of non-motile cells were calculated.
Results
During the 10-minute intervals, data was collected for both experiments. The number of non-motile cells were counted using a hemocytometer; this number was recorded. The data was plotted.
Table 1: Effect of Calcium on Chlamydomonas Reflagellation
Mins
Test tube 1 - Acid Shock treatment
Test tube 2 - Acid shock treatment + colchicine treatment
Test tube 3 – Acid shock treatment + TAP solution with Calcium
Test tube 4 – TAP solution with EGTA
10
20
42
36
11
20
34
25
17
23
30
43
2
27
18
40
42
27
39
11
50
29
37
26
8
60
19
28
23
6
[pic 1]
Graph 1: Visual representation of the effect of calcium on Chlamydomonas reflagellation based on the number of non-motile cells.
Table 2: Effect of Neomycin on Chlamydomonas Reflagellation
Mins
Test tube 1 - Acid Shock treatment
Test tube 2 - Acid shock treatment + colchicine treatment
Test tube 3 – No acid shock treatment + washing with neomycin + TAP solution with neomycin
Test tube 4 – Acid shock treatment + washing with neomycin + TAP solution with neomycin
10
6
12
1
82
20
15
29
1
67
30
7
18
8
49
40
6
28
10
101
50
9
60
9
81
[pic 2]
Graph 2: Visual representation of the effect of neomycin on Chlamydomonas reflagellation based on the number of non-motile cells.
Discussion
The purpose of this lab was to explore this the effects of colchicine, calcium, and neomycin on the reflagellation of Chlamydomonas. Based on the data collected, the effects we should have observed were observed for the most part. It also can be concluded that colchicine inhibits flagella regeneration and Chlamydomonas requires Ca2+ to swim around and regenerate flagella. Overall, colchicine, no calcium ions, and adding neomycin all contribute to inhibiting the regeneration of the flagella. While our data is a little off, general trends can be observed
In the first experiment, it was expected that cultures treated with an acid shock, tubes 1, 2 and 3, should all be non-motile at time zero. EGTA chelated with the calcium ions and reduced the amount of free calcium ions available to promote regeneration. Therefore, those cells remained largely immotile since they could not regenerate their flagella. However, this tube shows a slight decreasing trend, concluding that the flagella regenerated and were able to swim due to the calcium in the TAP medium. In the second experiment, it was expected that cultures treated with an acid shock, tubes 1, 2 and 4, should all be non-motile at time zero. Tube 4, treated with neomycin, which should have inhibited phospholipase activity, would demonstrate a high number of immotile cells. However, the number of non-motile cells decreased, not following the expected results. This could be due to human error since these cells may have not been properly acid-shocked.
References
CCNY Biology 22900 Lab Manual.
Quarmby LM, Yueh YG, Cheshire
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