Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Stratagene). siRNA to silence the wild type expression of Mfn2 will be used (Dharmacon). Afterintroduction of both into a cell culture (cell line as described above), western blots (as described above)will again be used to evaluated the occurrence of both Mfn2 variants. If only the 86 kD Mfn2 appears to bepresent after mutation it has been shown that the 68 kD variant is formed after cleavage of the 86 kD, andthe approximate cleavage site will have been identified.Hypothesis 4: Levels of cytosolic Mfn2 fluctuate throughout the cell cycle andthere is a threshold at which Mfn2 inhibition of Ras will lead to cell cycle arrestMfn2 can be present at a minimal of two locations within the cell, being in the outer mitochondrialmembrane and in the cytosol (Pich et al., 2005). Mfn2 located in the mitochondrial membrane has positivefunctions on mitochondrial activity, as has been discussed above (Pich et al., 2005). However, cytosolicMfn2 has the capability of binding to Ras, and subsequently inhibiting its function. This interaction withRas inhibits the MEK dependant signaling pathway. In vascular smooth muscles cells, this will ultimatelylead to cell cycle arrest throughout ERK-2 arrest (Shen et al., 2007). In conclusion, it is conceived thatcytosolic and mitochondrial Mfn2 have distinct functions. Mitochondrial Mfn2 function has been discussedabove. Here, cytosolic Mfn2 will be elaborated upon.Cell hyper-proliferation has long been thought to be one of the main etiologies in the causation of cancerand cardiovascular diseases. Especially vascular proliferative disorders, such as atherosclerosis, are themain common cause of cardiovascular diseases. These diseases are all characterized by uncontrolledproliferation of vascular smooth muscle cells. Proliferation of cells is influenced by a variety of externalfactors. All these factors have one common goal, namely influencing the cell cycle. One central regulatorin this targeting process is in the protein Ras. Ras is required to allow transition from G1 into S phase. Themain downstream pathway of Ras involves factors such as Raf, Mek1/2, ERK1/2 and MAPK. Thispathway ultimately leads to activation of cdk’s that in turn interact with cyclins, and thus allow cell cycleprogression. Mfn2 has been found to interfere with this pathway, and thus with hyper-proliferation. Mfn2has also been found to be capable of leading to cell cycle arrest throughout different pathways, andmoreover, the protein is capable of inducing apoptosis. Cytosolic Mfn2 appears to be a target ofupregulation when interference with cell cycle is needed. Because of this role for Mfn2, the protein hasalso been called hyperplasia suppressor gene. Various death-inducing stimuli, such as oxidative stress,can induce higher (cytosolic) Mfn2 expression (Shen et al., 2007). It is hypothesized that these effects ofMfn2 are due to cytosolic Mfn2 rather than to mitochondrial Mfn2. Therefore, by exploration of cytosolicMfn2, and its regulation, knowledge on diseases related to hyper-proliferation might be obtained, and newdrug targets could be discovered.Question 4.1: How do the levels of cytosolic Mfn2 fluctuate through out the cell cycle?Research has gone to indicate the presence of Mfn2 in the cytosol throughout the cell cycle (Pich et al.,2005). In the cytosol Mfn2 is capable of inducing cell cycle arrest in the course of binding to RAS.However, Mfn2 does not seem to be capable to elucidate a specific function at the cytosolic location at alltimes. Based on the previous working frame presented in the third hypothesis, the following experimentwill be aimed towards testing the levels of cytosolic Mfn2 throughout the various phases of the cell cycle.Experiment 4.1For the conductance of all the experiments to follow under this hypothesis, the human aortic smoothmuscle cell line, HAoSMC-c (Promocell), will be used yet again. Furthermore, the same measuresprovided in hypothesis 1 will be taken for cell synchronization. In order to achieve the goal of thisexperiment, the mitochondria first need to be fractioned from the cytoplasm. For that purpose, cellfractionation (Appendix A) will be carried out, with the aid of mitochondria/cytosol fractionation kit(Biovision). The procedure will be conducted according to the instructions provided by the manufactures'protocol. The same procedure used in hypothesis 3, will be repeated in order to determine the purity of theobtained fractions.The step to follow will entail conducting western blot (Appendix A), for which a BreezeChemiluminescence Kit anti-goat (Invitrogen) will be used. A C-terminal mouse monoclonal anti-humanSCI 332 Advanced Molecular Cell Biology Research Proposal 83