Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007minute intervals using a confocal microscope. We intend to use this information to construct a graph of thechanges in fission and fusion over time.3.2 How does mitochondrial morphology change when the cell cycle is arrested through AMPK activation?Once we know how the mitochondrial network changes throughout a normal cycle of our cell type, wewould like to see what happens during AMPK induced cell cycle arrest.Experiment 3.2We will prepare a cell culture with constitutively active AMPK, as described earlier. A synchronous cellculture will be obtained through mitotic shake-off, and sodium arsenite will be added to the growth mediumimmediately following synchronization. This should induce CA- AMPK and cause the cell cycle to arrestduring late G1, at the metabolic, AMPK-dependent checkpoint. The changes in the mitochondrial networkmorphology will be observed by GFP fluorescence and confocal microscopy, as described in the previousexperiment.3.3 What happens to mitochondrial network morphology when the cell cycle is arrested independently ofAMPK activation?In order to see whether any changes in the mitochondrial network observed in the previous experimentare related particularly to AMPK activated cell cycle arrest, we want to determine whether these changesoccur when the cell cycle is arrested through any other means.Experiment 3.3We will first inhibit AMPK in a cell culture. This will be done through a direct AMPK inhibition withcompound C as described earlier. The cell culture will be synchronized through mitotic shake-off.Following mitotic shake off, we will arrest the cell cycle just prior to the restriction point in G1 (Hullemanand Boonstra, 2001). About one hour before the restriction point (measured in experiment 2.1) we willremove the growth factor rich medium, wash the cells and replace them on a serum free media (CellularTechnology Ltd.). In the course of this experiment we will observe the changes in mitochondrialmorphology as the cells enter G1 following mitosis and pass through part of G1 until they are forced intoG0 in late G1 due to absence of growth factors.3.4 Does AMPK act on any of the fusion or fission proteins to produce changes in the mitochondrialnetwork?If in the previous experiments we see changes in the mitochondrial network morphology as a result ofAMPK activation and cell cycle arrest, we intend to see whether these are the result of an interactionbetween AMPK and one or multiple fission or fusion proteins. If we see increased fission we will test Drp1,should we see increased fusion we will test the mitofusins (Mfn1 and Mfn2). Opa1 is probably not relevantbecause it does not have a cytosolic domain and therefore cannot interact with AMPK.Experiment 3.4.1We will determine whether or not there is an interaction between AMPK and any of these proteins bymeans of an in vitro kinase assay. This assay combines the kinase and possible substrates in vitro, andallows us to analyze whether the kinase phosphorylated any of the substrates. The proteins will be purifiedfrom our cells through immuno-precipitation. We will grow a culture of cells on normal, nutrient richmedium so that AMPK is inactive. A series of these cells will be lysed and targeted antibodies willprecipitate AMPK. A polyclonal rabbit IgG antibody which targets both AMPKα1 and AMPKα2 will be used.Protein G PLUS-Agarose beads, which have a specificity for the IgG antibodies, will be used to precipitatethe protein (Santa Cruz Biotechnology Inc.). We will use the same procedure to isolate any substrateproteins we want to study. Antibodies for Mfn1, Mfn2 and Drp1 are also commercially available from SantaCruz Biotechnology Inc., and will be ordered depending on the results of experiments 3.2 and 3.3. PurifiedAMPK is mixed with a solution of radioactive [32P]-ATP. This solution is incubated with the possiblesubstrates we wish to study (one substrate per well). Since we need the AMPK in the wells to be activated,we will add AICAR to the solution. After 15 minutes the mixtures will be resolved on SDS-Polyacrylamidegels, which are stained with Coomassie Blue and exposed to x-ray film (Zhang and Prives, 2001). Fromthis it is possible to determine whether AMPK phosphorylated any of the substrates.SCI 332 Advanced Molecular Cell Biology Research Proposal 50
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Experiment 3.4.2Since kinase activity in vitro is not necessarily representative of the interactions within a cell, we wouldalso check protein-protein interactions through co-immunoprecipitation of AMPK. A synchronous cellculture will be forced into cell cycle arrest through nutrient deprivation, and subsequent AMPK activation.Cells from this culture will be lysed and AMPK will be immunoprecipitated through the same procedure asin experiment 3.4.1. In this case AMPK will be precipitated along with any proteins it might be bound to.The precipitates will be analyzed on a western blot. The results will probed with fluorescent antibodiesspecific for the fission or fusion proteins that we chose to investigate.Experiment 3.4.3If the kinase assay and the immunopercipitation show that AMPK can bind one of the fusion or fissionproteins, we want to see whether this occures in vivo as well. We intend to use FRET to see whetherAMPK activates the proteins in the specific case of cell cycle arrest at the metabolic checkpoint. FRETinvolves labeling the kinase and substrate with fluorophores, a donor and acceptor pair. When theacceptor fluorophore is in close proximity to the donor, it emits a certain frequency of light. Twosulfoindocyanine dyes, Cy5 and Cy5.5, will be used to label the molecules. Cy5 emits light at 665nm andCy5.5 at 693nm. Cy5.5 will be targeted to AMPK while Cy5 will be tagged onto the substrate. Thesespecific dyes were chosen because they are a pair that can achieve energy transfer at relatively largedistances, achieving a 50% effective energy transfer at a distance of 69 Ǻ (Schobel et al., 1999), whichmost likely makes them especially suitable for large molecules like AMPK. Additionally, the fluorophore willbe tagged to the N-terminal part of the α-subunit of AMPK. This is the subunit which interacts with thesubstrate, and placing the fluorophore there ensures it is close enough to the substrate for fluorescence tooccur (Hardie, 2007). The fluorescence can be observed through confocal microscopy.SCI 332 Advanced Molecular Cell Biology Research Proposal 51
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