Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Since we have very little insight in the cellular distribution and difference of the functionality of the Mfn2isoforms, we want to investigate Mfn2 regulation by cyclin D1 and E and their partner cdk’s on two levels:we will research Mfn2 protein levels in addition to the (de)activation of the protein.Another reason to look at both protein levels and (de)activation, is that cyclins have the tendencyto be translocated during the cell cycle; cyclin D1 accumulates in the nucleus during G1 phase and istranslocated to the cytosol during S phase (Alt 2000). This makes it very complex to research the (in)directinteraction between cyclins and Mfn2. Investigating two levels of regulation is therefore imperative.Question 5.1: Is there a correlation between Mfn2 protein levels and cyclin D1 / cyclin E levels?In order to investigate the influence of the active forms of cyclin D1 and E (i.e. together with their partnercdk’s) on Mfn2 protein levels, we propose a quantitative research to start off with. The main reason forperforming a quantitative research rather than a qualitative research is that our knowledge of Mfn2 in theirinteraction with cyclins is extremely limited. The proposed research will indicate whether it is viable toassume that the active forms of cyclin D1 and E are somehow able to regulate Mfn2 protein levels. Fornow, it is then sufficient to measure only cyclin D1 and cyclin E levels, and leave the cdk’s out ofconsideration. A quantitative research in this case thus means that we will look at the correlation betweencyclin D1 / E levels and Mfn2 protein levels in a large set of mammalian cell lines that differ in theironcogenic state and tissue type. We will conduct our research in 14 cell lines that are known (or expectedto) differ in their cyclin D1 or E expression (for a detailed description including motivation and cell culturingmedia, see Appendix E4.1). We chose this many cell lines not merely because it will contribute to thesignificance of our research, but also because it allows us to generalize conclusions to multiple cell lines.We decided to use this method, as apposed to using one cell line in which microinjection or transfectionwith cyclins is executed, in order to prevent unforeseen side effects that may occur with the latter methods.As said before, both cyclins are important in G1/S phase transition (Resnitzky et al., 1994), andtherefore we will conduct our experiments at the beginning, the middle and the end of G1 and S phase.We will thus perform cell synchronization (Appendix A) after which we will execute western analysis(Appendix A) to measure all protein levels. Since very little is known about the two types of Mfn2, we willtake into account the protein levels of both cytosolic and mitochondrial Mfn2. In order to do this, we willperform cell fractionation (Appendix A). Once the results are obtained, we will use SPSS (StatisticalPackage for the Social Sciences) to perform the statistical analysis.Experiment 5.1As in hypothesis 1, cells will be synchronized using mitotic shake-off. Cell phase determination will bedone using BrdU and DAPI.Western analysis will be executed with help of a Western Blot Kit (Invitrogen). The kit contains twosecondary antibodies: anti-mouse IgG-HRP (starting dilution 1:2000) and anti-rabbit IgG-HRP (startingdilution 1:5000). The procedure will be carried out according to the manufacturer’s manual. For cyclin D1detection we will use DCS-6: mouse monoclonal IgG 2a (Santa Cruz Biotechnology). For cyclin E, ourprimary antibody will be E-4: mouse monoclonal IgG 1 (Santa Cruz Biotechnology).In order to measure the levels of mitochondrial and cytosolic Mfn2 separately, two samples haveto be generated. Cell fractionation will be executed in the same way as in hypothesis 3, namely with acytosol / mitochondria cell fractionation kit (BioVision). We will perform a control to see whetherfractionation was successful. The cytosolic sample will be tested through western analysis for Tom40, aprotein that is only present in mitochondria. The western blot kit used in this experiment contains differentsecondary antibodies than the one in hypothesis 3. Therefore, the primary antibody for Tom40 will be H-300: rabbit polyclonal IgG (starting dilution 1:200) (Santa Cruz Biotechnology). The mitochondrial samplewill be tested through western analysis for the presence ribosomal protein L28. The primary antibody forthis protein will be F-137: polyclonal rabbit IgG (starting dilution 1:200) (Santa Cruz Biotechnology).After cell fractionation Mfn2 levels can be detected. We will use H-68 (Santa Cruz Biotechnology)as a primary antibody. This is a rabbit polyclonal IgG (starting dilution 1:200).As an internal control to demonstrate similar protein loading among the samples (all except for themitochondrial samples), ß-tubulin will also be blotted. The primary antibody for ß-tubulin will be 3F3-G2:mouse monoclonal IgM (starting dilution 1:200).Question 5.2: Can cyclins mediate the (de)activation of Mfn2?SCI 332 Advanced Molecular Cell Biology Research Proposal 86