Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007To research the influence of the Mfn2-Stoml2 complex on mitochondrial functioning, a mutant ensuring nodisplay of complex formation will have to be synthesized. The likely mutation site to achieve this would bethe Mfn2 intermembrane space loop. The tryptophan on location 626 is the only amino acid in theintermembrane space loop of Mfn2, causing it to be a relevant mutation target. Proline is commonly foundin turns and does not readily form hydrogen bonds, which makes it suitable for this type of mutation.Experiment 2.1Using custom synthesized oligonucleotides replacing amino acid VGGVVWKAVGW withVGGVVPKAVGW (Integrated DNA Technologies) in site-directed mutagenesis (QuickChange, Stratagene)an Mfn2 W626P mutant will be generated (Appendix A).As indicated earlier, human aortic smooth muscle cell line: HAoSMC-c (Promocell). The cells willbe transfected with a plasmid containing the Mfn2 W626P mutant. To silence wild type (wt) expression ofMfn2, siRNA against Mfn2 (siGenome SMARTpool, Dharmacon) will be used.As control groups, firstly wt Mfn2 cells will be used, thus enabling the complex formation.Secondly, as a negative control, untransfected cells with siRNA against Mfn2, in which no complexformation would be anticipated, will be utilized.Results will be measured using co-immunoprecipitation (Appendix A) using Dynabeads-Protein A(Dynal). The primary antibody for Mfn2 H-68: rabbit polyclonal IgG, starting dilution 1:50 (Santa CruzBiotechnology). Subsequent western blotting will be performed using a Western Blot Kit (Invitrogen). Thiskit containsa secondary antibody,namely mouse anti-rabbit IgG.Question 2.2: How does mitochondrial membrane potential change when Mfn2-Stoml complex formationis inhibited?Inhibition of Stoml2 has been shown to decrease the mitochondrial membrane potential (Hájek et al.,2007). A decrease in Stoml2 will also cause a decrease in Mfn2-Stoml2 complex formation. Thisexperiment will help ascertain whether it is the lack of Mfn2-Stoml2 complex presence rather than theexpression of Stoml2 that causes the decrease in membrane potential.Experiment 2.2In order to research the influence of the Mfn2-Stoml2 complex on mitochondrial membrane potential, wewill use cells transfected with the Mfn2 W626P mutant and stained using JC-1 (Molecular Probes), whichexhibits a fluorescence emission shift when the mitochondrial membrane potential changes. We willexamine whether a downregulation of Mfn2-Stoml2 complexes will lead to a downregulation in membranepotential. We will measure the membrane potentials of cells containing different amounts of Mfn2-Stoml2complexes. These different amounts of complex formation are generated by using different levels ofsiRNA Mfn2 wt and siRNA Mfn2 W626P in the cells.To visualize the change in membrane potential in Mfn2-Stoml2 complex-deficient cells versus themembrane potential in wt Mfn2 cells, we will use different levels of wt Mfn2 and Mfn2 mutant inhibitionthrough siRNA (Table 4.1).The Mfn2 W626P mutant siRNA will be acquired in custom made form(Dharmacon).As a control group, wild type cells will be stained with JC-1 and measured without transfection orsiRNA inhibition. The fluorescence emission will be measured using confocal fluorescence microscopy(Appendix A).SCI 332 Advanced Molecular Cell Biology Research Proposal 79