Tour-de-Force

More documents

Recommendations

Info

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007G2/M transition (Harborth et al., 2001), and because they are regulated by APC/C, which has been shownto be redox-dependent (Havens et al., 2006).The major function of cdk2-cyclin A is in the G1/S transition, and neither the cdk4&6-cyclin Dcomplex is vital for G2/M transition. However, to get a clear overview of all cdk-cyclin complexes presentat the G2/M transition their activity will also be assessed, even though they might not be needed for entryinto mitosis.After having identified which cdk-cyclin complexes are affected by ROS in the G2/M transition, it isnecessary to establish whether a change in activity is due to ROS-dependency of the cyclins or of thecdks. Cyclins A and B are degraded by APC/C and might therefore by under the control of ROS. However,it also possible that altered levels of activated cdks are responsible for this change.Question 3.2.1: Is activity of cdk-cyclin complexes different in cells with altered ROS levels?The activity of the previously mentioned cdk-cyclin complexes in the G2/M transition will be tested. Activityof these complexes will be examined both in control groups and the experimental groups of experiment3.1.1 and 3.1.2 (lower-than-physiological ROS levels and G1 ROS levels) using an immunoprecipitationkinase assay in order to see if manipulation of ROS levels has an effect on the activity of cdk-cyclincomplexes.Experiment 3.2.1: Immunoprecipitation Kinase AssayCells will be synchronized as explained in Appendix B1 and cultured as described in Appendix B1.1. ROSlevels will be manipulated, together with the addition of nocodazole, 2 hours before the start of mitosis asdescribed for experiments 3.1.1 and 3.1.2. When halted in prometaphase, cells will be fixed as describedin Appendix B3.1.3.1. Immunoprecipitation kinase assayFixed cells from all groups will be lysed and incubated with antibodies against cyclin D, cyclin A, and cyclinB (Appendix B3.2.1). The kinase assay will be performed with the Trulight Universal Kinase/PhosphataseAssay Kit using the immunoprecipitated extracts as explained in the manual. Histone H1 will be used as asubstrate for cell extracts isolated by incubation with cyclin A and B, whereas glutathione transferase(GST) C terminus pRb will be used as a substrate for cell extracts isolated by cyclin D incubation. Afterthe reaction is stopped, kinase activity will be measured very accurately by standard fluorescence intensitymeasurements by a fluorometer.2. Interpretation of resultsA change in activity of the cyclin B kinase assay signifies a change in cdk1-cyclin B complex. However, achange in activity of the cyclin A kinase assay could mean a change in activity of cdk1-cyclin A and/orcdk2-cylin A complex. A change can also be observed in kinase assay for cyclin D signifying an alteredactivity in activity of cdk4&6-cyclin D.Question 3.2.2: Are cyclin levels and/or active cdk levels different in cells treated with ROS?Since this research focuses on the redox-dependency of cyclin-cdk complexes regulated by APC/Cactivity, here the focus will lie on the cdk1-cyclin A&B and cdk2-cyclin A complexes. Here, it will beinvestigated whether a change in activity is due to altered cyclin levels, or altered levels of activated cdks.In case cyclin D-cdk4&6 complexes seem to be affected after Experiment 3.2.1, a similar approach can befollowed.All experiments are carried out on all experimental and control groups.Question 3.2.2.1: Are levels of cyclins in control cells different from cells with manipulated ROS levels?The levels of cyclin A and B in control and experiment cells will be tested with a Western blot (seeAppendix A). A change in cyclin levels would indicate that the change in activity of the complex is (partly)due to the cyclin.Cyclin A is degraded by APC/C in the start of metaphase and cyclin B in the start of anaphase(Reed et al., 2003). Because control cells will be arrested in prometaphase, the amount of cyclins will notdiffer from the amount present at the G2/M transition, so that this is a good control point.Experiment 3.2.2.1: Western BlotSCI 332 Advanced Molecular Cell Biology Research Proposal 26
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Cells will be synchronized as explained in Appendix B1 and cultured as described in Appendix B1.1. ROSlevels will be manipulated, together with the addition of nocodazole, 2 hours before the start of mitosis asdescribed for experiments 3.1.1 and 3.1.2. When halted in prometaphase cells will be fixed as describedin Appendix B3.1.3.Fixed cells will be lysed and incubated with antibodies. Antibodies against cyclin A and cyclin B will beused for the immunoblot analysis. These antibodies are chosen so that all isoforms of cyclin A and cyclinB will be observed in the immunoblot analysis. Actin antibody will be used as a loading control (AppendixB3.2.2a)Question 3.2.2.2: Is phosphorylation/inactivation of cdks in control cells different from cells withmanipulated ROS levels?To see whether the alteration in cdk-cyclin complex activity is caused by different levels of active cdkspresent, a western blot will be applied to see the levels of phosphorylated cdk1 and cdk2 together withtotal amounts of cdk1 and cdk2 present. The difference between the total amount present and the amountof phosphorylated, inactive, cdks will show the extent of cdks inactivation by phosphorylation inexperiment and control cells. A change in levels of active cdks in experiment cells would mean that thechange in activity of the complex is (partly) due to the cdk.Experiment 3.2.2.2: Phospho-specific Western Blot:Cells will be synchronized as explained in Appendix B1 and cultured as described in Appendix B1.1. ROSlevels will be manipulated, together with the addition of nocodazole, 2 hours before the start of mitosis asdescribed for experiments 3.1.1 and 3.1.2. After cells are halted in prometaphase, they will be fixed asdescribed in Appendix B3.1.3.Fixed cells will be lysed and incubated with phospho-specific cdk1 antibodies and phospho-specific cdk2antibodies. Normal (non-phospho-specific) cdk1 and cdk2 antibodies will be used to see whether there isa change in total cdk levels (Appendix B3.2.2b).Question 3.3: At what level are the redox-dependent processes regulated by ROS?After it has been established whether the cdk or the cyclin is affected by ROS in the G2/M transition, thelevel of regulation should be examined. Redox regulation of proteins can occur at the level of transcription,translation, or post-translation (activation or stability). From previous studies (Havens et al., 2006), it isexpected that the stability of cyclins is affected by redox-modulation of APC. Nonetheless, all options areheld open. All experiments are carried out on all experimental and control groups.Question 3.3.1: Are the affected proteins regulated on the level of transcription?To investigate whether transcription of the affected proteins are redox-regulated, Northern blot and realtime RT-PCR experiments will be carried out. (See Appendix A)Experiment 3.3.1.1: Northern blotCells will be synchronized as explained in Appendix B1 and cultured as described in Appendix B1.1. ROSlevels will be manipulated, together with the addition of nocodazole, 2 hours before the start of mitosis asdescribed for experiments 3.1.1 and 3.1.2. When halted in prometaphase, cells will be fixed as describedin Appendix B3.1.3.A Northern blot will be carried out for the proteins affected by changed ROS levels, and for actin as acontrol. Primers for the proteins affected by changed ROS levels can be found using the PrimerBankdatabase. Labeled primers will be synthesized by the Massachusets General Hospital DNA Core Facility.RNA will be purified using a QIAGEN RNEasy kit and the Northern blot will be carried out usingNorthernMax (Ambion) (Appendix B3.3.1).Experiment 3.3.1.2: Real time RT-PCRCells will be synchronized as explained in Appendix B1 and cultured as described in Appendix B1.1. ROSlevels will be manipulated, together with the addition of nocodazole, 2 hours before the start of mitosis asdescribed for experiments 3.1.1 and 3.1.2. When halted in prometaphase, cells will be fixed as describedin Appendix B3.1.3.SCI 332 Advanced Molecular Cell Biology Research Proposal 27
Page 1 and 2: Tour-de-ForceInterplay between mito
Page 3 and 4: Tour-de-Force: Interplay between Mi
Page 25: Tour-de-Force: Interplay between Mi
Page 77 and 78:
Tour-de-Force: Interplay between Mi
Page 79 and 80:
Page 81 and 82:
Page 83 and 84:
Page 85 and 86:
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Page 93 and 94:
Page 95 and 96:
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?