Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Mfn2 wtMfn2 mutant0 nM siRNA Mfn2 wt Mfn2 W626P100 nM siRNA60 nM siRNA Mfn2 wt Mfn2 W626P40 nM siRNA80 nM siRNA Mfn2 wt Mfn2 W626P20 nM siRNA100 nM siRNA Mfn2 wt Mfn2 W626P0 nM siRNATable 4.1: Amounts of siRNA Mfn2 wt and siRNA Mfn2 W626P to be simultaneously administered to cells.Question 2.3: How does OXPHOS complex I, II, III and V subunit expression change when Mfn2-Stoml2complex formation is inhibited?Mfn2 knockdown results in a decrease in OXPOS complex I, II, III and V subunit expression (Pich et al.2005). In this experiment, we will look at the influence of the Mfn2-Stoml2 complex on OXPHOS complex I,II, III and V subunit expression.Experiment 2.3To assess the influence of the Mfn2-Stoml2 complex on OXPHOS complex subunit expression, we willuse western blots (Appendix A) to measure subunit abundance in the Mfn2 W626P mutant cells comparedto wild type cells, using actin as a control measure. The subunits looked at in particular are the ones thathave shown to be decreased when Mfn2 expression is downregulated (Pich et al., 2005), being subunitp39 from complex I, p70 from complex II, p49 from complex III and the α-subunit of complex V, allencoded by nuclear DNA.The antibodies that will be used are anti-NDUFA9 (Santa Cruz Biotechnology) against complex I,anti-Fp (Molecular Probes, A11142) against complex II, anti-core 2 (Molecular Probes, A11143) againstcomplex III and anti-subunit α from H + -F 1 ATP synthase (Molecular Probes, ) against complex V. As antiactin,CGA7, a smooth muscle actin antibody (Santa Cruz Biotechnology) will be used.Question 2.4: Are Mfn2-Stoml2 complexes still formed in the absence of Mfn2 GTP binding domains?Mfn2 contains three GTP binding domains. Since GTP binding might be necessary for the formation of theMfn2-Stoml2 complex, it is useful to create GTP binding domain-deficient Mfn2 mutant to see if Mfn2-Stoml2 complexes are still being formed.Experimental ProceduresWe will use a custom made mutant of Mfn2 which lacks the three GTP domains 103-110, 199-203 and258-261, Mfn2 GTP- (GenScript). We will transfect cells with this plasmid along with siRNA Mfn2 wt to silencewild type expression. As controls we will use wild type cells, which will form complexes, and untransfectedcells with siRNA Mfn2 wt as a negative control.The results will be measured using co-immunoprecipitation and western blotting as describedabove.Hypothesis 3: Cytosolic Mfn2 weighs 68 kilo Dalton and is formed after cleavageof the heavier type Mfn2Apart from the role of Mfn2 in the mitochondria, multiple studies conclude a role for Mfn2 in the cytoplasm.A recent study illustrated an emerging role of Mfn2 in inducing apoptosis. There are multiple pathwaysinvolving Mfn2 through which apoptosis could be induced (Shen et al., 2007). Firstly, apoptosis can beachieved through a mitochondrial pathway, also known as the intrinsic pathway, through whichmitochondrial Mfn2 can act upon the activation of both Caspases 9 and 3 (Shen et al., 2007). Secondly,binding of cytosolic Mfn2 to inhibit Ras can provoke apoptosis through a mitochondria independentmanner involving Akt (Shen et al., 2007). Moreover, In a study conducted on vascular smooth muscle cells,researchers noted reduced presence of Mfn2 in hyper-proliferative vascular smooth muscle cells (Chen etSCI 332 Advanced Molecular Cell Biology Research Proposal 80