Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Cells 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. When halted in prometaphase cells will be fixed as describedin Appendix B3.1.3.Fixed cells will be lysed and incubated with antibodies. Antibodies against cyclin A and cyclin B will beused for the immunoblot analysis. These antibodies are chosen so that all isoforms of cyclin A and cyclinB will be observed in the immunoblot analysis. Actin antibody will be used as a loading control (AppendixB3.2.2a)Question 3.2.2.2: Is phosphorylation/inactivation of cdks in control cells different from cells withmanipulated ROS levels?To see whether the alteration in cdk-cyclin complex activity is caused by different levels of active cdkspresent, a western blot will be applied to see the levels of phosphorylated cdk1 and cdk2 together withtotal amounts of cdk1 and cdk2 present. The difference between the total amount present and the amountof phosphorylated, inactive, cdks will show the extent of cdks inactivation by phosphorylation inexperiment and control cells. A change in levels of active cdks in experiment cells would mean that thechange in activity of the complex is (partly) due to the cdk.Experiment 3.2.2.2: Phospho-specific Western Blot:Cells 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. After cells are halted in prometaphase, they will be fixed asdescribed in Appendix B3.1.3.Fixed cells will be lysed and incubated with phospho-specific cdk1 antibodies and phospho-specific cdk2antibodies. Normal (non-phospho-specific) cdk1 and cdk2 antibodies will be used to see whether there isa change in total cdk levels (Appendix B3.2.2b).Question 3.3: At what level are the redox-dependent processes regulated by ROS?After it has been established whether the cdk or the cyclin is affected by ROS in the G2/M transition, thelevel of regulation should be examined. Redox regulation of proteins can occur at the level of transcription,translation, or post-translation (activation or stability). From previous studies (Havens et al., 2006), it isexpected that the stability of cyclins is affected by redox-modulation of APC. Nonetheless, all options areheld open. All experiments are carried out on all experimental and control groups.Question 3.3.1: Are the affected proteins regulated on the level of transcription?To investigate whether transcription of the affected proteins are redox-regulated, Northern blot and realtime RT-PCR experiments will be carried out. (See Appendix A)Experiment 3.3.1.1: Northern blotCells 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. When halted in prometaphase, cells will be fixed as describedin Appendix B3.1.3.A Northern blot will be carried out for the proteins affected by changed ROS levels, and for actin as acontrol. Primers for the proteins affected by changed ROS levels can be found using the PrimerBankdatabase. Labeled primers will be synthesized by the Massachusets General Hospital DNA Core Facility.RNA will be purified using a QIAGEN RNEasy kit and the Northern blot will be carried out usingNorthernMax (Ambion) (Appendix B3.3.1).Experiment 3.3.1.2: Real time RT-PCRCells 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. When halted in prometaphase, cells will be fixed as describedin Appendix B3.1.3.SCI 332 Advanced Molecular Cell Biology Research Proposal 27