Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007accelerated AMPK activation. Now, by measuring protein levels indicative of quiescence (see: 2.2) it isdetermined whether AMPK can induce quiescence at multiple points in G1.In a subsequent experiment, constitutively active AMPK (CA-AMPK) (Appendix C) with aninducible promoter will be used to activate AMPK at the same points in the cell. This alternative method forAMPK activation is used as a control to assure complete AMPK activation.Experiment 2.3.2 How does activating AMPK under normal energy levels affect cell cycle progression?This experiment will be performed as described in 2.3 but now under normal physiological energy levels(25mM) to see whether AMPK activation alone is sufficient to induce cell cycle arrest. Whether the cellsenter quiescence will again be determined by the measurement of the “quiescent markers”.2.4 Is AMPK-induced quiescence followed by apoptosis or reentry into the cell cycle?The aim of the following experiments is to confirm the results of 2.3, and to investigate whether AMPKinducedquiescence at different points in G1 is followed by apoptosis or reentry into the cell cycle. This isinvestigated at three points in G1: immediately after M, before passage through the previously determinedrestriction point R, and after R.Experiment 2.4.1 AMPK activation and quiescence in early G1In order to activate AMPK, AICAR will be added to synchronized cells in mitosis. Furthermore, the nucleiof cells treated with AICAR will be stained with DAPI and examined via immuno-fluorescence microscopy(Appendix C) to assess whether they display similar nuclear morphology as untreated control cells in earlyG1. Subsequently, the “quiescent markers” will be measured during early G1. This will establish whethercells with active AMPK actually leave mitosis and enter an AMPK induced quiescent state early in G1.The procedure will be repeated using glucose deprivation in mitosis instead of AICAR addition, inorder to mimic physiological conditions of AMPK activation.Experiment 2.4.2 AMPK induced apoptosis after quiescence in early G1As mentioned in the background, an early G1 checkpoint depending on PI3-kinase inhibition within 10min.after M has been proposed previously (Hulleman et al., 2004). Cells arrested at this point cannot reenterthe cell cycle and become apoptotic. Furthermore, IRS-1 has been shown to be phosphorylated by AMPKunder conditions of glucose deprivation, inhibiting the PI3-kinase/Akt pathway and thereby inducingapoptosis. Therefore, we hypothesize that AMPK will induce early G1 cell cycle arrest followed byapoptosis through IRS-1 activation in low glucose conditions.To test this hypothesis we will deprive synchronized cells of glucose during mitosis and assesswhether the cells become apoptotic within 72 hours following completion of mitosis. If cells do not becomeapoptotic but re-enter the cell cycle after AMPK induced quiescence, we have established the possibility ofan AMPK energy checkpoint early in G1. If the cells, however, do not re-enter the cell cycle and becomeapoptotic, it will be investigated if IRS-1 and p53 play a role in this process. Apoptosis will be assessed viaa caspase activity assay (Appendix C).In order to investigate whether the proposed AMPK – IRS-1 pathway leads to IP3-kinase inhibitionand apoptosis, phosphorylation of AMPK at Thr-172, of IRS-1 at Ser-794 and of Akt at Thr-308 will bequantified by Western blot analysis and compared to control levels.Next, it will be determined whether cell cycle arrest in early G1 and/or subsequent apoptosis isp53 and/or IRS-1 dependent. For this purpose, the action of IRS-1 on Akt will be inhibited via a pointmutation of IRS-1 on Ser-794. Furthermore, p53 will be completely inhibited via RNA interference(Appendix C). In 3 consecutive experiments, firstly IRS-1 and secondly p53 will be inhibited. Lastly, bothproteins will be inhibited simultaneously. In all cases apoptosis and quiescence will be assessed aspreviously described.2.5 AMPK induced quiescence before and after RIn the final experiments regarding hypothesis 2 it will be established whether AMPK is able to cause cellcycle arrest and/or apoptosis specifically before and after the restriction point R.As mentioned earlier, it is generally thought that cell cycle arrest can only occur before R.However, it has not been previously investigated whether this is true under conditions of glucosedeprivation. Therefore, we will deprive synchronized cells of glucose before and after R and observewhether these cells still continue into S-phase.SCI 332 Advanced Molecular Cell Biology Research Proposal 48
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Experiment 2.5As in the previous experiment we will assess cell cycle arrest and apoptosis as a consequence of AMPKactivation. In addition we will determine whether these processes depend on p53, using RNAi. This willclarify the role of p53 in AMPK induced cell cycle arrest and apoptosis in the later stages of G1.2.6 Does active AMPK cause apoptosis after prolonged energy depletion?Since AMPK is thought to be able to cause apoptosis (Jones et al., 2005), the question is whether it doesthis in result to energy depletion, and whether it can induce apoptosis in G1 phase.Experiment 2.6Synchronized cells will be put on a BrdU/uridine medium containing low glucose. The energy producingmechanisms will be inhibited as discussed in 1.8, but all three are inhibited simultaneously. Progressioninto S-phase will be measured as described in experiment 2.13: Effects of AMPK-induced cell cycle arrest on mitochondrial networkmorphologyHypothesis 3: Activated AMPK can cause morphological changes in mitochondrial network following cellcycle arrest in G1.3.1 How does mitochondrial network morphology change throughout the cell cycle?Several studies have observed the changes in the mitochondrial network throughout the cell cycle(Arakaki et al., 2006 ; Margineantu et al., 2002). However, these studies have produced varying results,most likely because the network morphology varies greatly between cell types (Arakaki et al., 2006). Forthis reason, we will start by observing the mitochondrial network change throughout the cell cycle of ourmouse fibroblast cells.Experiment 3.1.1We will visualize the mitochondria through fluorescent microscopy. The mitochondria will be made visiblethrough transfection with a vector containing mitochondria-targeted Green Fluorescent Protein. We willuse a transfection solution specialized for DNA transfection into fibroblast cells (Altogen Biosystems,Fibroblast Transfection Reagent). The cells will be transfected with “pTurboGFP-mito vector,” amammalian expression vector containing GFP with a mitochondrial targeting sequence (Evrogen). Thefluorescent mitochondria will be observed and images recorded with a confocal microscope.In this experiment we will observe mitochondrial morphology in cell cultures stuck in three major phases.First we will force a culture into quiescence (G0) by nutrient deprivation. Cells will be removed from thegrowth medium, washed and grown on medium containing 0.1 mM glucose (Jones et al., 2005), for 64hrs.We will fix the culture using paraformaldehyde (Margineantu et al., 2002) and observe mitochondrialmorphology. Secondly, we will synchronize another cell culture through mitotic shake-off (Appendix A),and plate them on normal growth medium. When the cells are about half-way through G1 we will fix theculture using paraformaldehyde (Margineantu et al., 2002). The time the cell culture will be allowed toproceed will be decided bases on the length of G1 in our cell type, determined in experiment 0.1. Finally,we will take a new, synchronized cell culture and treat cells with Aphidicolin, a DNA replication inhibitor,for 16hrs, to stop the cell cycle in S-phase (Margineantu et al., 2002). The cells of this culture will also befixed with paraformaldehyde. Observation of these three cultures should give us an overview of thenetwork morphology in the early phases of the cell cycle as well as in G0. We will use these observationsto decide on categories for the different morphologies of the mitochondrial network.Experiment 3.1.2Secondly, we will observe the changes in mitochondrial morphology in real time. We will visualize themitochondria as in experiment 3.1.1. A synchronous cell culture will be obtained through mitotic shake-off.We will observe the changes in morphology through one cell cycle, by taking images of multiple cells at 30SCI 332 Advanced Molecular Cell Biology Research Proposal 49
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