Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007A real time RT-PCR experiment will be carried out for the proteins affected by changed ROS levels, andfor actin as a control. RNA will be purified using the RNEasy kit (QIAGEN). The reverse transcription step,the polymerase chain reaction, and detection of the labeled target will be performed using QuantiTect Kits(QIAGEN), as described in Appendix B3.3.2. The amount of cycles necessary for signal detectionrepresents the amount of transcript present.Question 3.3.2: Are the affected proteins regulated at the level of translation?After transcription, second regulatory level is translational control. Even though cyclins A and B have notbeen reported to be regulated in translational level during the normal cell cycle, it should still be examinedto see whether translation of the affected proteins is redox-regulated. 35 S pulse-chase experiments will beconducted which can indirectly quantify how much the mRNA is translated. A comparison will be madebetween normal cells and two groups of experiment cells to see if there is a difference in translation of theprotein.Experiment 3.3.2: 35 S pulse-chase experimentsCells will be synchronized as explained in Appendix B1 and cultured as described in Appendix B1.1. ROSlevels will be manipulated, together with the addition of nocodazole, 2 hours before the start of mitosis asdescribed for experiments 3.1.1 and 3.1.2.To see whether the proteins affected by ROS are regulated at the level of translation, the rate oftranslation will be monitored through a 35 S pulse-chase experiment. This technique uses a radioactiveamino acid that is incorporated into the translated protein. Incorporation of 35 S-cysteine into the protein ofinterest can be observed in SDS-PAGE and can later be quantified using a phosphorimager (AppendixB3.3.3).Question 3.3.3: Are the affected proteins regulated on the level of degradation?In the G1/M checkpoint, the stability of the cyclins was shown to be ROS dependent (Havens et al., 2006).Checking the stability of the cyclins will show whether stability of the proteins changes with altered ROSlevels at G2/M transition. If a change in stability of the protein is observed, activity of APC/C will bechecked to investigate whether altered activity of APC/C is the reason behind altered cyclin levels.Question 3.3.3.1: Are cyclin A and cyclin B less stable in cells with manipulated ROS levels compared tocontrol cells?Experiment 3.3.3.1: Cycloheximide treatmentCells will be synchronized as explained in Appendix B1 and cultured as described in Appendix B1.1. ROSlevels will be manipulated, together with the addition of nocodazole, 2 hours before the start of mitosis asdescribed for experiments 3.1.1 and 3.1.2.Experiment groups and control cells will be treated with cycloheximide after cells are arrested in mitosis.Cycloheximide inhibits translation of all proteins and observation of the cyclin A and cyclin B levels inwestern blot at different intervals will give us information about how quickly proteins get degraded. CyclinA and cyclin B antibodies will be used together with actin antibody as a control (Appendix B3.3.4). Cellextracts will be gathered at three intervals for western blotting. Protein levels at these intervals will give usa clear indication of how stable the protein is. Comparison of protein stability in cells of the experimentcells to control cells will indicate if there is an altered regulation on the level of degradation resulting inaltered cyclin levels.Question 3.3.3.2: Is APC/C activity the reason behind instability of cyclin A and cyclin B?At the G2/M transition, APC/C activity is inhibited by preventing association of Cdh1 to APC/C. Cdc20, theother APC/C co-factor, is not present in this transition and is transcribed only after entry into mitosis andbinds to APC/C starting in metaphase. If APC/C is unexpectedly active in this transition, it is most likelydue to association with Cdh1. In G1/S cell cycle arrest due to altered ROS levels, it is shown that APC/Cdue to changed ROS levels binds to Cdh1 and thus becomes active (Havens et al., 2006) If degradation isshown to be the regulatory step, co-immunoprecipitation analysis (see Appendix A) of APC/C will beperformed in order to confirm that APC/C is the reason behind altered stability of the cyclins.Experiment 3.3.3.2: Co-immunoprecipitationSCI 332 Advanced Molecular Cell Biology Research Proposal 28