Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Taking into account the full length of the cell cycle of the cell line used (Appendix B1A), incubation withPF-1 probes dissolved in DMSO will be performed every two hour of that cycle, beginning with t=0. Adifferent well will be incubated every two hours. As a control, a cell culture will receive DMSO only.4. Fixation & Measuring fluorescence and cell cycle progressionAfter the incubation with BrdU and with PF-1 dissolved in DMSO, the cells will be washed with PBS and-fixated by incubation by formaldehyde in order to measure cell cycle progression and O 2 levels,respectively. To detect the fluorescence of the PF-1 probe, confocal laser scanning microscopy will beused to pinpoint a single cell and measure that cell’s fluorescence. As a control the experiment will beperformed on five different single cells. Subsequently, the cells will be lysed and BrdU incorporation will bemeasured (Appendix B1.2). Intensity will be semi-quantified using the appropriate software.Experiment 2.1.2 Cytosolic H 2 O 2 levels throughout the cell cycleIn this experiment cyclic fluctuations of cellular hydrogen peroxide levels will be measured. A probe,named HyPer, will be designed, using the protocol of Belousov et al. (2006, see Appendix B2.3). Thisprobe can be directed to any organelle or the cytosol, by adding a targeting sequence. Additionally, theprobe is highly selective for H 2 O 2 , more so than other commercially available probes (Belousov et al.,2006).In this experiment, the HyPer probe will be targeted to the cytosol instead of at a specific organelle.Therefore, no targeting sequence will be used while constructing the probe, so that it will be targetedsolely to the cytosol. The probe oxidation is reversible, which allows for real time measurements byfocusing on the same cell.1. Construction of HyPer expression vectorA HyPer expression factor will be constructed and checked according to protocol as described byBelousov et al. (2006) explained in Appendix B2.3.2. Plating and growing cells on a coverslipThe cells will be grown on a coverslip, as indicated in Appendix B2.2.3. Cell cycle controlAs a control, thirteen cell cultures will be taken from the same synchronized batch as the cells which areplated on a coverslip. These cells will then be plated in wells with appropriate medium and every twohours a different well will be checked for its cell cycle progression by BrdU.4. Transfecting the cell with HyPerThe cells will be transfected with a C1 mammalian expression vector containing HyPer, as explained inAppendix B2.3. To control for the effects of transfection on the cells, a different cell culture will be mocktransfected.5. Measuring fluorescenceAt two-hour intervals, starting with t=0, corresponding with the cell cycle length of the cell line, H 2 O 2 levelmeasurements will be done by real-time confocal laser scanning microscopy. The laser is fixed on a singlecell and at each different time interval the laser will be activated and the emitted fluorescence measured.Intensity will then be semi-quantified using the appropriate software. The entire experiment will be done infive different single cells.Experiment 2.1.3: Mitochondrial O - 2 levels throughout the cell cycle-In this experiment, the levels of mitochondrial O 2 will be measured by using a commercially availableprobe, MitoSOX (Invitrogen), which is specifically targeted to the mitochondrial matrix and is highlyspecific for superoxide.1. Cell synchronization & Plating of cellsIn this experiment, as in the previously mentioned experiments, the cells will be synchronized and platedinto the medium. Again, the number of cell cultures is dependent on the total cell cycle length of thedifferent cell lines.SCI 332 Advanced Molecular Cell Biology Research Proposal 19