Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007The difference between the two types of Mfn2 is hypothesized to be constituted by the cleavage ofregions that are responsible for mitochondrial targeting. Firstly, a TMD is normally sufficient to target aprotein to the mitochondria (Santel and Fuller, 2001). As cytosolic Mfn2 is not targeted to the organelle,the TMD might partially account for the difference in weight between cytosolic and mitochondrial Mfn2.Secondly, Mfn2 contains coiled-coil domains that are upstream and downstream of the TMD, whichinteraction is also important for mitochondrial targeting. When any of these domains is deleted, part of theMfn2 population remains cytosolic (Rojo et al., 2002). Furthermore, it has been noticed that the C-terminalis also essential for mitochondrial targeting of Mfn2 (Rojo et al., 2002).The region of Mfn2 reaching from the first TMD to the cytoplasmic C-terminal regions contains alldomains that have been identified as mitochondrial-targeting regions. The difference between the light andheavy type Mfn2 accounts for 18 kD. Through a simple calculation one can conclude that the regionsresponsible for mitochondrial targeting must altogether weigh approximately 18 kD. This can be concludedas the region contains 152 amino acids and an average amino acid weighs 0,115 kD. Thus, 0,115 times152 equals 17,48 kD.From the information presented above it is hypothesized that the N-terminal cytosolic region ofmitochondrial Mfn2 is also present in cytosolic Mfn2. After cleavage of both TMD’s, the intermembraneloop and the C-terminal cytosolic region, Mfn2 becomes cytosolic.Experiment 3.2Firstly, the western blots obtained in experiment 1 will be used. Antibodies specific for the N-terminal havealready been added to the western blots, as was described above. Additionally, mouse antibodies 6A8(Starting dilution 1:200) specific for the C-terminal (Abnova) will be added as well. Thereafter, secondarydonkey anti-mouse IgG (starting dilution 1:2000) conjugated with HRP (Santa Cruz Biotechnology) will beadded. A luminal reagent obtained from the same company will be used as HRP substrate. It will bevisualized which Mfn2 populations still contain the C-terminal region.In a new cell culture, from the same cell line as used in previous experiments, immuno-fluorescencemicroscopy (Appendix A) will be used to asses the localization of the N-terminal and C-terminal antibodiesin formalin fixated paraffin-embedded cells. The primary C-terminal and N-terminal specific antibodies thatwill be used are the same as in previous experiments, although as indicated, different dilutions must beused in western analysis and immuno-fluorescence microscopy. It is hypothesized that the N-terminalantibody (starting dilution 1:50) will bind to all present Mfn2, whereas the C-terminal antibody (startingdilution 1:50) will only be seen in the mitochondria. A secondary rabbit anti-goat antibody (starting dilution1:100), labeled with FITC (green) (Santa Cruz Biotechnology), will be used to visualize the N-terminalprimary antibody. A secondary donkey anti-mouse antibody (starting dilution 1:100) (Santa CruzBiotechnology) labeled with biotin (red) will be used to identify the C-terminal specific primary antibody.After having shown the distribution of the two variants of Mfn2 over the previously mentioned fractions,and after having determined whether there is a difference in C-terminal region between the two, it will befurther explored how this difference has come to being. It is hypothesized that the light 68 kD variant isformed after cleavage of the heavier 86 kD Mfn2. However, to proof that the difference is not explainedthrough the occurrence of alternative splicing, a northern analysis (Appendix A) will be performed toexamine mRNA populations, in a cell culture obtained from the cell line stated above. A probe specific forthe N-terminal will be used to see whether one or two bands of Mfn2 mRNA will appear, as it ishypothesized that the two variants share identical N-terminals. Northern analysis will be performed usingthe Northern Max-Gly kit (Ambion). As a probe, a N-terminal specific custom made oligonucleotide will beused (Integrated DNA Technologies). The probe will be labelled using StarFire (Integrated DNATechnologies).Lastly, to show that cleavage is really the mechanism behind the two different forms of Mfn2, the expectedcleavage site will be deleted. At the same time it will be tested whether the hypothesized cleavage site isin fact the cleavage site. It is expected that the region reaching from the first TMD to the C-terminal regionis cleaved off to form cytosolic Mfn2. Therefore, cleavage is expected to take place somewhere betweenamino acids 595 and 604 (Appendix E4.2). Through the deletion of the 30 base pairs that form this region,we will try to disable cleavage. To form this mutant, a custom made oligonucleotide will be used(Integrated DNA Technologies) along with site-directed mutagenesis (Appendix A) (QuickChange,SCI 332 Advanced Molecular Cell Biology Research Proposal 82
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
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