Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Research question 2: How do ROS levels fluctuate throughout the cell cycle?The results of the experiments for question 1 will answer the question whether or not there is a metaboliccycle present in mammalian cells. If there are metabolic fluctuations throughout the cell cycle, it isinteresting to see whether ROS level also fluctuate, since ROS production is an important side effect ofmitochondrial activity. However, metabolic fluctuations do not necessarily mean that ROS levels fluctuateas well:1. Mitochondria are not the sole ROS producers in the cell: peroxisomes and NADPH oxidase arealso producers of ROS (Schrader and Fahimi, 2006; Foster et al., 2006).2. There is an antioxidant system that scavenges ROS, so an increase in ROS production does nothave to mean that there are higher ROS levels in the cell.In question 2, ROS levels and antioxidants will be examined. Takahashi and colleagues (2003) haveshown that ROS levels fluctuate throughout, and apparently synchronous with the cell cycle. However, theROS probes used in the study were neither specific nor localized to compartments in the cell, making itimpossible to assess which ROS producer was the cause of the fluctuation. In the experiments conductedto answer question 2, more specific probes will be selectively targeted to different compartments of thecell where ROS is produced.First, cytosolic ROS levels will be examined, in order to replicate the results obtained byTakahashi et al. (2003). Then, it will have to be determined what the relative ROS production bymitochondria is to that of other ROS producers like peroxisomes (Schrader and Fahimi, 2006) or NADPHoxidase(Foster et al., 2006).Question 2.1: How do cellular O 2-and H 2 O 2 levels fluctuate throughout the cell cycle?In researching fluctuations in ROS levels throughout the cell cycle, two specific ROS will be studied:superoxide (O 2 -) and hydrogen peroxide (H 2 O 2 ). These are the ROS produced directly by mitochondria,peroxisomes and NADPH oxidase. Other ROS are formed by reactions of compounds with O 2-and H 2 O 2 .To assess the role of mitochondria in ROS production, mitochondrial levels of O 2-and H 2 O 2 will beexamined. Technical limitations disable the option of measuring superoxide in the peroxisomes, sincecurrently there are no good specific superoxide probes which are targeted to the peroxisomes. Therefore,only H 2 O 2 levels will be measured in peroxisomes throughout the cell cycle. There are no probes formeasuring NADPH oxidase ROS production. Therefore, cytosolic ROS levels will be measured before andafter inhibition of NADPH oxidase, to get a view of the ROS production by this enzyme.Since the laser used to obtain ROS levels (confocal laser scanning microscopy will be used, seeAppendix A) might influence ROS levels, quiescent cell lines will be used as a control. In theseexperiments multiple synchronized cell cultures will be used, after which single cells from these cultureswill be analyzed for experimental data. Furthermore these experiments will be done twice for everydifferent cell line.-Experiment 2.1.1: Cytosolic O 2 levels throughout the cell cycleThe levels of superoxide present in the cytosol will be studied to determine if these levels fluctuate in a-cyclic pattern. The commercially available probes for measuring cellular O 2 levels are not selectiveenough, nor react fast enough to rapid fluctuations. Thus, a PF-1 probe will be designed, as described inthe protocol of Xu et al. (2007). The PF-1 probe is both very specific for O - 2 and has a short incubationtime, which is relevant for a reactive component such as O - 2 (Xu et al., 2007).1. Construction of Pf-1 probeThe PF-1 probe will be constructed and checked according to protocol as described by Xu et al. (2007)(see Appendix B2.2).2. Cell synchronization & plating the cellsCell cultures will be synchronized using mitotic shake-off with nocodazole, as described in question 1, andwill then be plated in wells with their normal medium (Appendix B0; B1.1).3. Incubation with PF-1 probe and BrdUSCI 332 Advanced Molecular Cell Biology Research Proposal 18
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
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