Tour-de-Force

More documents

Recommendations

Info

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Since we have very little insight in the cellular distribution and difference of the functionality of the Mfn2isoforms, we want to investigate Mfn2 regulation by cyclin D1 and E and their partner cdk’s on two levels:we will research Mfn2 protein levels in addition to the (de)activation of the protein.Another reason to look at both protein levels and (de)activation, is that cyclins have the tendencyto be translocated during the cell cycle; cyclin D1 accumulates in the nucleus during G1 phase and istranslocated to the cytosol during S phase (Alt 2000). This makes it very complex to research the (in)directinteraction between cyclins and Mfn2. Investigating two levels of regulation is therefore imperative.Question 5.1: Is there a correlation between Mfn2 protein levels and cyclin D1 / cyclin E levels?In order to investigate the influence of the active forms of cyclin D1 and E (i.e. together with their partnercdk’s) on Mfn2 protein levels, we propose a quantitative research to start off with. The main reason forperforming a quantitative research rather than a qualitative research is that our knowledge of Mfn2 in theirinteraction with cyclins is extremely limited. The proposed research will indicate whether it is viable toassume that the active forms of cyclin D1 and E are somehow able to regulate Mfn2 protein levels. Fornow, it is then sufficient to measure only cyclin D1 and cyclin E levels, and leave the cdk’s out ofconsideration. A quantitative research in this case thus means that we will look at the correlation betweencyclin D1 / E levels and Mfn2 protein levels in a large set of mammalian cell lines that differ in theironcogenic state and tissue type. We will conduct our research in 14 cell lines that are known (or expectedto) differ in their cyclin D1 or E expression (for a detailed description including motivation and cell culturingmedia, see Appendix E4.1). We chose this many cell lines not merely because it will contribute to thesignificance of our research, but also because it allows us to generalize conclusions to multiple cell lines.We decided to use this method, as apposed to using one cell line in which microinjection or transfectionwith cyclins is executed, in order to prevent unforeseen side effects that may occur with the latter methods.As said before, both cyclins are important in G1/S phase transition (Resnitzky et al., 1994), andtherefore we will conduct our experiments at the beginning, the middle and the end of G1 and S phase.We will thus perform cell synchronization (Appendix A) after which we will execute western analysis(Appendix A) to measure all protein levels. Since very little is known about the two types of Mfn2, we willtake into account the protein levels of both cytosolic and mitochondrial Mfn2. In order to do this, we willperform cell fractionation (Appendix A). Once the results are obtained, we will use SPSS (StatisticalPackage for the Social Sciences) to perform the statistical analysis.Experiment 5.1As in hypothesis 1, cells will be synchronized using mitotic shake-off. Cell phase determination will bedone using BrdU and DAPI.Western analysis will be executed with help of a Western Blot Kit (Invitrogen). The kit contains twosecondary antibodies: anti-mouse IgG-HRP (starting dilution 1:2000) and anti-rabbit IgG-HRP (startingdilution 1:5000). The procedure will be carried out according to the manufacturer’s manual. For cyclin D1detection we will use DCS-6: mouse monoclonal IgG 2a (Santa Cruz Biotechnology). For cyclin E, ourprimary antibody will be E-4: mouse monoclonal IgG 1 (Santa Cruz Biotechnology).In order to measure the levels of mitochondrial and cytosolic Mfn2 separately, two samples haveto be generated. Cell fractionation will be executed in the same way as in hypothesis 3, namely with acytosol / mitochondria cell fractionation kit (BioVision). We will perform a control to see whetherfractionation was successful. The cytosolic sample will be tested through western analysis for Tom40, aprotein that is only present in mitochondria. The western blot kit used in this experiment contains differentsecondary antibodies than the one in hypothesis 3. Therefore, the primary antibody for Tom40 will be H-300: rabbit polyclonal IgG (starting dilution 1:200) (Santa Cruz Biotechnology). The mitochondrial samplewill be tested through western analysis for the presence ribosomal protein L28. The primary antibody forthis protein will be F-137: polyclonal rabbit IgG (starting dilution 1:200) (Santa Cruz Biotechnology).After cell fractionation Mfn2 levels can be detected. We will use H-68 (Santa Cruz Biotechnology)as a primary antibody. This is a rabbit polyclonal IgG (starting dilution 1:200).As an internal control to demonstrate similar protein loading among the samples (all except for themitochondrial samples), ß-tubulin will also be blotted. The primary antibody for ß-tubulin will be 3F3-G2:mouse monoclonal IgM (starting dilution 1:200).Question 5.2: Can cyclins mediate the (de)activation of Mfn2?SCI 332 Advanced Molecular Cell Biology Research Proposal 86
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007In the previous study we tried to find a correlation between cyclin D1 / E presence and Mfn2protein levels. We hypothesized that the active form of cyclin D1 / E, i.e. together with its partner cdk,either was able to decrease Mfn2 transcription or translation. When this hypothesis would be refuted, itcould be the case that cyclins are able to directly interact with Mfn2. In the counterpart of fusion, thefission machinery this type of interacting between Cdk1/cyclin B and Ser 585 on Drp1 has already beendemonstrated (Taguchi 2007). In 2004 Chen et al. demonstrated that Mfn2 has a similar PKA/PKGphospholylation site at Ser 442 as Drp1. We hypothesize that Mfn2 can be phosphorylated by cyclin D1 /E, in turn inhibiting its activity. In regard of the cell cycle and Ras this will mean that increased levels ofcyclin/Cdk holoenzymes can phosphorylate cytosolic Mfn2, leading to increased Ras activity and at thesame time decrease OXPHOS by mitochondrial Mfn2 deactivation.Experiment 5.2To investigate this we will look at the protein-protein interaction of Mfn2 with cyclin D1 / E by performingan in vitro kinase assay. Verification of the phosphorylation site of Mfn2 will be done by site directedmutagenesis on Ser 442 of the PKA domain in Mfn2. Regarding the Cdk/cyclin complexes, twoconstitutions come to mind:1. Cdk4/Cyclin D12. Cdk2/Cyclin EA third constitution, i.e. Cdk6/Cyclin D1, could be possible, cdk4, however, appears to be their mostprominent partner (Matsushime, 1994). The reason to limit our research to only cyclin D1 and notincluding cyclin D2 and D3 is due to the fact that cyclin D1 appears to be the major player in G1/S phasetransition, which is our main interest. In a later stage we could include additional cyclin/Cdk complexes.The majority of the proteins we will use in this experiment are commercially available. This incombination with the fact that only small quantities of these proteins will be used, makes the purchaseinstead of the manufacture of these items (Table 4.2) evident.Code Size Price RetailerCdk4 + Cyclin D1 protein ab55695 2 x 10 µg €660.00 AbcamCdk2 with GTS tag H00001017-P02 10 µg1 unknown AbnovaCyclin E with GTS tag H00000898-P01 10 µg1 unknown AbnovaMfn2: GST Expression and 554803/ 554763/ Kit/cells/culture2 unknown BD BiosciencesPurification Kit + Sf9 insectcells + TNM-FH Insect CellMedium554760mediumpRb, control cyclin D1/Cdk4 H00005925-Q01 10 µg1 unknown AbnovaNPM1 control Cyclin E/Cdk2 H00004869-P01 10 µg1 unknown AbnovaTable 4.2: Overview of proteins and their retailers.Since Mfn2 protein is not available commercially it will be purified using a GTS-tag expression vector andSf9 insect cells. These cells will be transfected with recombinant plasmid DNA containing our gene(according to BD biosciences handbook). These plasmids will be custom made by GeneScript. To assistin the proper folding of Mfn2 and keep them from precipitating we will make use the solubilization agentGST. This GST Expression and Purification Kit, containing all necessary products for Mfn2 expression andpurification, will be purchased from BD Bioscience’s. We will use HUAoSMC-c cells, purchased for earlierexperiments, to obtain the Mfn2 gene, this way we will use solely mouse proteins, which will preventincompatibility of protein originating from different animals.As a positive control to cyclin D1 / E phophorylation activity we will use pRb, which is known to bephosphorylated by Cdk4/cyclin D1 (Zhang, 2001). NPM1, which is phosphorylated by cyclin E/Cdk21 Estimated price €300,-2 Estimated price €800,-SCI 332 Advanced Molecular Cell Biology Research Proposal 87
Page 1 and 2:
Tour-de-ForceInterplay between mito
Page 3 and 4:
Tour-de-Force: Interplay between Mi
Page 5 and 6:
Page 7 and 8:
Page 9 and 10:
Page 11 and 12:
Page 13 and 14:
Page 15 and 16:
Page 17 and 18:
Page 19 and 20:
Page 21 and 22:
Page 23 and 24:
Page 25 and 26:
Page 27 and 28:
Page 29 and 30:
Page 31 and 32:
Page 33 and 34:
Page 35 and 36: Tour-de-Force: Interplay between Mi
Page 85: Tour-de-Force: Interplay between Mi
show all

Tour-de-Force

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

Delete template?

Save as template?