Tour-de-Force

More documents

Recommendations

Info

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Experiment 0.2The cells will be synchronized and a western blot (Appendix A) will be performed every 2 hours throughoutthe cell cycle. The antibodies used are obtained from Abcam (www.Abcam.com):Rb: (phospho S249 + T252) antibodyCyclin A: Cyclin A antibody (rabbit polyclonal, ab7956)Cyclin B: Cyclin B antibody (rabbit polyclonal, ab32053)Cyclin D1: cyclin D1 antobody (rabbit polyclonal 31450)AMPK: AMPK beta 1 antibody (Y367, ab 32382)pAMPK: AMPK beta 1 phospho S181 (rabbit polyclonal, ab55311).Protein levels will be determined and fluctuations throughout the cell cycle will become visible.0.3 What are the physiological concentrations of ATP, Cyclin A, B and D1,phosphorylated/unphosphorylated AMPK, phosphorylated/unphosphorylated Rb throughout G1?The cell cycle phase of our interest is G1, therefore a detailed knowledge of the fluctuations of theseproteins in G1 is required as a comparison for our later research.Experiment 0.3The experiments in 0.2 will be repeated, this time throughout G1, every 30 minutes.0.4 Measurement of AMPK activity.Throughout our experiments it will be useful to check the activity of AMPK, for example after inducingconstitutively active AMPK, glucose deprivation or AICAR activation. We will use this method to verifyAMPK activation in the rest of our experiments.Experiment 0.4We will use synchronized cells in which AMPK is activated. AMPK activity will be measured by a syntheticpeptide, SAM, as described by Zhou et al. (2001).1: AMPK and its effect on glycolysis, oxidative phosphorylation and fatty acidoxidation during G1 PhaseHypothesis 1: AMPK activation mediates energy production to maintain the cell’s energy balance underacute metabolic stress. It will do this by influencing the following Energy generating processes: glycolysis,Oxidative Phosphorylation (Oxphos) and Fatty Acid Oxidation (FAO).1.1 What are the fluctuation of glycolysis, Fatty Acid Oxidation and Oxidative Phosphorylation throughoutthe cell cycle?In order to gain an understanding of periodic changes in glycolysis in relation to AMPK, we initially want toestablish the changes that occur during the normal cell cycle of synchronized, proliferating cells. The rateof glycolysis has been observed to increase and reach a peak in S-phase (Brand et al., 1997). We willcheck the fluctuations throughout the cell cycle in our cell lines.We use a cell culture that is grown on complete medium and that has not been exposed to interveningconditions. Cells will be synchronized and divided into four samples. We will perform:1) Extracellular flux (FX) method, to determine the overall changes in glycolysis and oxidativephosphorylation, with the XF24 Extra Cellular Flux Analyzer as described by Hue et al. (2003).This machine will measure the uptake of oxygen and the production of lactate in real time(Appendix A).2) Metabolomics assay. With the metabolomics assay the intermediates of the energy generatingpathways can be screened for and relative quantities measured. Cells will be lysed and frozenimmediately at -45 degrees Celsius, to prevent changes in the metabolome. The analysis will beperformed by the Metabolomics Centre Utrecht at the UMC, or alternatively as described by vander Werf et al. (2007). An assay will be performed every 2 hours throughout the cell cycle andevery 30 minutes during G1.SCI 332 Advanced Molecular Cell Biology Research Proposal 44
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 20073) ATP levels will be measured using CellTiter-Glo Luminescent Cell Viability Assay as explained byWu et al. (2007). A luminescent signal will be generated that is proportional to the amount of ATPpresent in cells. The advantage of this method is that it yields a rapid, simple and sensitivedetermination of the ATP level in cells (Appendix A).4) During all these experiments small samples of cells will be taken to determine the cell phase andensure that the methods do not have an influence on cell cycle progression. This determinationshould correspond to the in experiment 0.1 established phases (Appendix A).1.2 Does AMPK activation regulate levels of glycolysis, FAO or Oxphos in G1phase of proliferating cells?The goal here is to measure the influence of AMPK on catabolic, energy generating processes. In theliterature it can be found that glycolysis as well as FAO is up-regulated through AMPK, but that oxidativephosphorylation is possibly inhibited in differentiated cells (Dyck and Lopaschuk, 2006). In this experimentAMPK will be activated under normal energy conditions, as determined in experiment 0.3, during the G1phase of the cell cycle.Experiment 1.2We use a synchronized cell culture in which ATP levels correspond to the ones determined in experiment0.3. These cells are transfected with constitutively active AMPK (CA-AMPK) with an inducible promoter.(Appendix C). In G1 AMPK will be allowed to be expressed by adding sodium arsenite. Subsequently,after 2 hours, the experiments as described in 1.1 will be performed. The results will be compared to thoseobtained in 1.1, to show the influence of AMPK on these pathways. If the data are clear enough, themetabolomics assay will also give us an indication of the specific enzymatic conversions in the pathwaysthat are up-regulated.1.3 Does AMPK that is activated by AMP also regulate energy production?This experiment is a control to 1.2. Here we activate AMPK in the “natural way” to check that the effectsinduced by AMPK in 1.2 are similar to “natural conditions”. If the effects are different, there might be adirect effect of the energy levels on energy production. We expect the latter, since AMP by itself is aregulator of the glycolytic enzyme phosphofructokinase (Kühn et al., 1974 & Pinilla and Luque, 1977).Experiment 1.3We will use glucose deprivation to lower ATP levels. Cells not treated with any intervening conditions willbe washed and then provided with medium containing 5mM glucose (this will be the standard amount ofglucose used for glucose deprivation throughout all experiments of hypothesis 1). The experiments in 1.1will be repeated and the results will be compared to 1.2.1.4 Is there a change in glycolysis, Oxphos or FAO under lower than normal energy conditions, withoutactive AMPK?To determine the effects of low energy conditions without the influence of AMPK we will inactivate AMPKand determine the effects on the energy producing mechanisms. The effects here should equal thedifference in results of 1.2 and 1.3. We theorize that these cells with inactive AMPK will most likely not beable to restore energy balance.Experiment 1.4In synchronized cells AMPK will be inhibited by compound C, a specific inhibitor of AMPK, obtained fromMerck. We will add compound C (C-(6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo[1,5-a]pyrimidine) (Zhou et al., 2001) to the medium at a 40µM concentration suspended in Me 2 SO for 3 hourspreceding analysis. Then the experiments described in 1.1 will be repeated and the results compared to1.2 and 1.31.5 When exactly in G1 does AMPK cause up-regulation of glycolysis, Oxphos or FAO?AMPK is thought to be mainly active during the checkpoint in late G1 - however, it also plays a role in anenergy checkpoint in late G2 and possibly at other points in the cell cycle (Hardie et al., 2005). Toelucidate the role AMPK can have throughout G1, AMPK will be up-regulated at several points in thisphase and the effects will be examined.SCI 332 Advanced Molecular Cell Biology Research Proposal 45
Page 1 and 2: Tour-de-ForceInterplay between mito
Page 3 and 4: Tour-de-Force: Interplay between Mi
Page 43: Tour-de-Force: Interplay between Mi
Page 95 and 96:
Tour-de-Force: Interplay between Mi
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109 and 110:
Page 111 and 112:
Page 113 and 114:
Page 115 and 116:
Page 117 and 118:
show all

Tour-de-Force

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?