Question

As a continuation of the experiments in Question 1 you want to study the timing of the APC's activity more distinctly. To do this you treat your cells with MG132 - a proteasome inhibitor. When MG132 is added to cells proteins can still be ubiquitinated, but they are not degraded, because the proteasome is inhibited. In cells that are treated with MG132 you can monitor APC activity on Cyclin B using just the cyclin B a. antibody and a western blot, and monitoring the size of cyclin B. Describe how and why this works. b. You take a group of cells and synchronize them so that they are all transitioning through the cell cycle simultaneously. At 4 different transition times you remove some cells, treat them with MG132 and allow them to (attempt to) undergo the transition. You then run a western blot using your cyclin B antibody. Below is the start of the Western Blot that you would put in your paper. Complete this blot and write a figure legend to accompany the figure. Be sure to add all relevant labels to the figure and write the figure legend you would expect to find in the paper. The cells are allowed to transition into the final stage listed, so G1 S would look like cells in the S phase, unless those cells got stuck somewhere along the way. Remember MG132 inhibits ALL of the proteasome activity. Figure 1: MG13 2 Using your knowledge of the cell cycle, the cyclins and the APC explain the results you drew above. c. f you looked under the microscope at the cells from your final lane (Metaphase->G1 would you expect to see? d MG132) what NLadder G1-S G1S G2- Metaphase Metaphase DE Anaphase Anaphase

Answer 1

a. The cell devision takes place in 4 important stages, G1, S, G2 and M phase. Cyclin levels increase and decrease in each step to facilitate the cell division. The cyclinB is found during G2 to M phase transition. Its levels are high in the beginning of prophase and metaphase. During anaphase its level goes down. The progression to anaphase is carried out by degradation of cyclinB by APC. APC is anaphase promoting factor.
Now, in the presence of MG132 which is an inhibitor of proteosome activity, would degrade APC. If APC is not there, then cyclinB levels would not go down even after metaphase. This can be studied with the help of antibody which binds to cyclinB.
Whever the cylcinB is degraded, the band would not appear on the western blot. In the presence of MG132, the cyclinB is not degraded and antibody can bind to it and band would appear. This way the cyclin B degradation can be studied.

b. The cyclins and CDKs levels fluctuate during cell cycle. The cyclinA is dominant during G1 and S phase. In Prophase and metaphase cyclin B is high, later during anaphase it gets degraded. The degradation is done by the proteosome.
In the iamge, The G1 to S withour MG132, will have no bands on the gel.
No cyclin B in this phase; hence anti cyclin B antibiotic will not bind to anything.
In the second column, the MG132 is present, but no Cyclin B. so no bands.
In third column: The G2 to Metaphse, cyclin B levels rise and hence the band would show up on the band.
in 4th : G2 to metaphase with MG132. Only cyclin B band would show up.
5th: Metaphse to G1, the cell has no cyclin B, so no band.
6th. This has MG1132, which inhibits the proteosome activity, Thus cyclinB is retained. SO band would come up.
7th: Anaphase to G1, the cell has no cyclin B, so no band.
8th. This has MG1132, which inhibits the proteosome activity, Thus cyclinB is retained. SO band would come up.

c. The cell is in Metaphase --> G1 + MG132. This cells is arrested in G1. But also has MG132 which inhibits cyclin degradation. This cell would have completed cell division and looks like normal cell with oval shape. Chromosomes will not be visible and cell wall formation is complete.