Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Proposed ResearchHypothesis 1: Mfn2 levels fluctuate throughout the cell cycle and this istranscriptionally regulatedAs indicated by previous research fusion is promoted in certain phases of the cell cycle (Margineantu et al.,2002). Since Mfn1 and Mfn2 play an important role in fusion accordingly, either their levels or their activitymust be regulated in coordination to the necessity of performing fusion. In addition, as hypothesized bygroup 1, it seems likely that during the cell cycle OXPHOS activity is constantly being regulated (Tu et al.,2005). In the G1 phase the biosynthetic activities of the cell cycle increase, consequently causing a rise inthe energy demand (Martínez-Diez et al., 2006), whereas a downregulation of OXPHOS would bedesirable from pre-S-phase through S-phase. Since Mfn2 is able to upregulate OXPHOS, it might be thatthis protein is targeted according to the energy demands expressed in each various cell cycle phase.Derived from these findings, it is viable to propose that levels of Mfn2 are regulated throughout the cellcycle, presumably in a coordinated manner. Therefore, this hypothesis will research the fluctuation of theprotein throughout the different phases of the cell cycle. It will aim to establish the regulation of the levelsof Mfn2. For that purpose, expressed mRNA levels, next to those of the protein itself, will be measured.Question 1.1: Are there fluctuations in the levels of Mfn2 corresponding to the various phases of the cellcycle?As prior background knowledge was mainly obtained from research using human aortic smooth musclecells (Chen et al., 2004), unless indicated otherwise, experiments throughout this research will also makeuse of the same cell line; HAoSMC-c (Promocell) (for cell culture medium, see Appendix E1).Firstly, in order to determine the protein levels of Mfn2 throughout the cell cycle, measurementswill be taken at various intervals; in the middle and end of each phase of the cell cycle (i.e. M/G1, mid G1,G1/S, mid S, S/G2, G2/M, mid M). This will be done over the length of two cycles in order to exclude anypossible side effects attributed to re-entry into the cell cycle. Additional intervals may be added to furtherinvestigate the fluctuation of Mfn2 protein levels after these initial results have been obtained. The cellswill be synchronized by the use of mitotic shake off (Appendix A). Not only is this a non-disruptive methodfor synchronizing cells but also, as opposed to other methods, it doesn’t influence OXPHOS neither doesit disturb the energy balance (Davis et al., 2001). This is of crucial importance, since those factors mightinfluence the expression of Mfn2. Followed by this, the protein levels will be quantified using western blotanalysis (Appendix A).Experiment 1.1In order to determine the length of each cell phase, cells will be labeled with bromo-deoxyuridine (BrdU)(Appendix A), using the standardized protocol, provided by the manufacturing company (Invitrogen).Mitotic shake-off will be carried out as discussed in (Muray, 2006).To label the cells, DAPI (Appendix A)will be used, thus enabling the detection of the desired cell phase (Exalpha Biologicals).A Western Blot Kit (Invitrogen) will be used to perform western analysis. This kit contains twosecondary anti-bodies, namely anti-rabbit IgG-HRP (starting dilution 1:5000) and anti-mouse IgG-HRP(starting dilution 1:2000). The procedure will be performed according to the manufacturer’s manual. As aprimary antibody Mfn2 H-68: rabbit polyclonal IgG (starting dilution 1:50) will be used (Santa CruzBiotechnology). As an internal control β-tubilin, a household protein, will be blotted using 3F3-G2, amouse monoclonal IgM (Santa Cruz Biotechnology).Research Question 1.2: Are there fluctuations in the Mfn2 mRNA levels corresponding to the variousphases of the cell cycle?The same procedure discussed in the previous experiment 1.1, will be executed, with the change ofmeasuring mRNA levels rather than those of the protein itself. mRNA levels will be examined using realtime PCR (Appendix A). As this is a highly sensitive method, it will exclude closely related mRNA levelsand yield accurate results of Mfn2 mRNA levels. Since mitotic shake-off will be used to synchronize thecells, the amount of available cells will be limited. Real time PCR is therefore particularly useful, since itSCI 332 Advanced Molecular Cell Biology Research Proposal 77