Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Proposed ResearchResearch question 1: Are there cyclic fluctuations in mitochondrial metabolicactivity that are coordinated with the cell cycle?Mitochondrial metabolic activity can be characterized by ATP production, oxygen consumption andmitochondrial membrane potential. Measuring ATP or ADP levels will not provide conclusive evidence onthe state mitochondria are in, since differences in these levels can also be attributed to glycolytic activityand cellular ATP consumption. To investigate whether there are cyclic fluctuations in mitochondrialmetabolic activity, oxygen consumption is a better measure, since mitochondria are the major source ofcellular oxygen consumption and it is directly related to metabolic activity.As a second indication of mitochondrial metabolic activity the mitochondrial membrane potentialwill be measured throughout the cell cycle.Question 1.1: Are there cyclic fluctuations in oxygen consumption by the cell during cell proliferation?The oxygen consumption of a single cell will be measured for the duration of one cell cycle to investigate ifthere are cyclic fluctuations in the amount of oxygen that is used by the cell for its metabolic activity.Although oxygen consumption is fundamental for mitochondrial metabolic activity, it has never beforebeen measured throughout an entire mammalian cell cycle.Oxygen flux into a single cell will be measured with a self-referencing microelectrode throughoutone entire cell cycle in proliferating human fibroblasts and retinal epithelial cells.Experiment 1: Oxygen measurements throughout the cell cycleKnowing exactly at what time the cells are in which phase is important to compare the fluctuations inoxygen consumption with cell cycle progression. Therefore, a large population of cells will besynchronized and it will be determined what phase these cells are in at intervals of two hours for theduration of one entire cell cycle.Human fibroblasts and retinal epithelial cells will be grown in air-saturated medium that containsenough glucose.1. Cell synchronizationCells will be synchronized in mitosis with nocodazole treatment and mitotic shake-off. (see AppendixA) The synchronized cells will be divided over different wells and will be used for either cell phasedetermination or oxygen measurements (see Appendix B1.1 for specific information).2. Cell phase determinationThe cell phase is determined with BrdU pulse-labeling and histone H3 phosphorylation in thesynchronized cells (Appendix A; Appendix B1.2). With BrdU pulse-labeling the onset and offset of theS-phase can be determined while histone H3 phosphorylation shows the onset of M-phase. Thelength of G1 can be calculated as the time between mitotic shake-off and the start of BrdUincorporation. By subtracting the time of the onset of M-phase from the time of the end of BrdUincorporationthe length of G2 can be determined.Measurements will be taken at time intervals of two hours.From this experiment a plot can be made of BrdU-incorporation and histone H3 phosphorylationagainst time and so the lengths of the cell cycle phases can be determined for this culture.To validate the data the experiment (cell synchronization and cell phase determination) will beconducted 5 times in both cell lines.3. Oxygen consumption measurementThe oxygen consumption can be measured real-time in a single cell with a self-referencing electrode.(Appendix A) This is a very accurate amperometric method to measure the analyte flux into a cell in amedium with high background levels of the analyte (Appendix B1.3) (Osbourn et al., 2005).The measurements will be done 5 minutes per hour, throughout one entire cell cycleSCI 332 Advanced Molecular Cell Biology Research Proposal 16
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007In order to correlate the results of oxygen flux at certain time points with cell cycle progression, cellswill be used that are synchronized together with the cells used for cell phase determination. Therefore,oxygen consumption will also be measured in 5 different cells in 5 different wells for 5 cell cycles.Oxygen consumption will also be measured in non-synchronized cells in order to assure thatnocodazole treatment does not influence differences in oxygen consumption. As a second control,oxygen consumption will be measured in quiescent cells (treated as described in Appendix B1). Sucha control is necessary in order to see if the observed fluctuations in mitochondrial metabolic activityare specific for cell cycle progression and do not occur, or occur in a different pattern in quiescentcells.4. Plot dataThe data of oxygen flux into a cell and the cell cycle progression will be plotted against time. This willprovide us with a graph that shows oxygen consumption potential is related to cell cycle progression.Question 1.2: Are there cyclic changes in mitochondrial membrane potential throughout the cell cycle?As a second measure of fluctuations in mitochondrial metabolic activity, the mitochondrial membranepotential will be measured. In previous research the results on how the mitochondrial membrane potentialchanges during a cell cycle are contradictory. Some studies show that the potential is the highest duringG2 and M phase (Martinez-Diez et al., 2006), while other studies show that a high membrane potentialcauses high ROS levels and that these levels are high in G1/ S phase (Lee et al., 2007). Therefore, wewill measure the fluctuations in mitochondrial membrane potential in order to resolve the contradictoryindications from different studies.Experiment 1: JC-1 Dual-emission potential-sensitive probe to measure mitochondrial membrane potentialin single cells1. Cell synchronization and determinationCells will be synchronized as described in Appendix B1.1 and divided over several wells. Persynchronization one well will be used for the membrane potential and the other wells will be used forphase determination (Appendix B1.2).For every cell type synchronization and determination will be done five times.2. Membrane potential measurementThe membrane potential will be measured in the synchronized (but not BrdU-treated) fibroblast andepithelial cells using a JC-1 probe (Appendix B1.4). The JC-1 probe is used because it is a verysensitive measure of membrane potential and is not sensitive to loss of staining, cell density and dyebleaching (Foster et al., 2007). This probe emits green light when it is in the cytosol and red light whenit is in the mitochondria. The higher the membrane potential, the more probes are attracted to themitochondria and so the higher the ratio of red light emitted as compared to green light.The membrane potential will be measured five times in five separate synchronized cultures of bothcell lines.4 Plot DataThe data obtained from the red/green fluorescence can be plotted against cell cycle progression. Thiswill provide us with a graph that shows how mitochondrial membrane potential is related to cell cycleprogression.SCI 332 Advanced Molecular Cell Biology Research Proposal 17
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Tour-de-Force

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