Name: experimental approaches to study mitochondrial localization and function of a nuclear cell cycle kinase, cdk1
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Description: Watch this Scientific Journal Video about Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1 at JoVE.com

This work was supported by NIH grants CA133402, CA152313 and Department of Energy Office of Science DE-SC0001271. We thank the University of California Davis Flow Cytometry Shared Resource Laboratory with funding from the NCI P30 CA0933730, and NIH NCRR C06-RR12088, S10 RR12964 and S10 RR 026825 grants and with technical assistance from Ms. Bridget McLaughlin and Mr. Jonathan Van Dyke for their help with the flow cytometry experiments.

1. Isolation of Mitochondria from Cultured Cells
<ol>
	<li>Preparation of Isolation Buffer for Cells (IBc) Buffer
	<ol>
		<li>Prepare 0.1 M Tris/MOPS (tris(hydroxymethyl)aminomethane/3-(N-morpholino) propanesulfonic acid): Dissolve 12.1 g of Tris in 800 ml of distilled water, adjust pH to 7.4 using MOPS powder, add distilled water to a total volume of 1 L and store at 4 oC.</li>
		<li>Prepare 0.1 M EGTA (Ethylene glycol bis(2-aminoethyl ether)tetraacetic acid)/Tris: Dissolve 38.1 g ...

<ol>
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E., Sesaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mitochondrial+fusion+and+division.">Regulation of mitochondrial fusion and division.</a> Trends Cell Biol. 17 (11), 563-569 (2007).</li><li>Brandt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+converting+NADH:quinone+oxidoreductase+(complex+I).">Energy converting NADH:quinone oxidoreductase (complex I).</a> Annu Rev Biochem. 75, 69-92 (2006).</li><li>Hofhaus, G., Weiss, H., Leonard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+analysis+of+the+peripheral+and+membrane+parts+of+mitochondrial+NADH+dehydrogenase+(complex+I).">Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I).</a> J Mol Biol. 221 (3), 1027-1043 (1991).</li><li>Weiss, H., Friedrich, T., Hofhaus, G., Preis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory-chain+NADH+dehydrogenase+(complex+I)+of+mitochondria.">The respiratory-chain NADH dehydrogenase (complex I) of mitochondria.</a> Eur J Biochem. 197 (3), 563-576 (1991).</li><li>Janssen, R. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+10,+Preparation+of+proteins+and+peptides+for+mass+spectrometry+analysis+in+a+bottom-up+proteomics+workflow.">Chapter 10, Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow.</a> Curr Protoc Mol Biol. , Unit 10.25 (2009).</li><li>Davies, D., Macey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11,+Cell+Sorting+by+Flow+Cytometry.">Chapter 11, Cell Sorting by Flow Cytometry.</a> Flow Cytometry. , 257-276 (2007).</li><li>van den Heuvel, S., Harlow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+cyclin-dependent+kinases+in+cell+cycle+control.">Distinct roles for cyclin-dependent kinases in cell cycle control.</a> Science. 262 (5142), 2050-2054 (1993).</li><li>Terry, N. H. A., White, R. 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Protcols. 1 (2), 859-869 (2006).</li><li>Becker, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preprotein+translocase+of+the+outer+mitochondrial+membrane:+reconstituted+Tom40+forms+a+characteristic+TOM+pore.">Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore.</a> J Mol Biol. 353 (5), 1011-1020 (2005).</li><li>Karniely, S., Pines, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+translation--dual+destination:+mechanisms+of+dual+protein+targeting+in+eukaryotes.">Single translation--dual destination: mechanisms of dual protein targeting in eukaryotes.</a> EMBO Rep. 6 (5), 420-425 (2005).</li><li>Candas, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CyclinB1/Cdk1+phosphorylates+mitochondrial+antioxidant+MnSOD+in+cell+adaptive+response+to+radiation+stress.">CyclinB1/Cdk1 phosphorylates mitochondrial antioxidant MnSOD in cell adaptive response to radiation stress.</a> J Mol Cell Biol. 5 (3), 166-175 (2013).</li><li>Nantajit, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+B1/Cdk1+phosphorylation+of+mitochondrial+p53+induces+anti-apoptotic+response.">Cyclin B1/Cdk1 phosphorylation of mitochondrial p53 induces anti-apoptotic response.</a> PLoS One. 5 (8), e12341 (2010).</li><li>Cappella, P., Gasparri, F., Pulici, M., Moll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+based+on+click+chemistry,+which+overcomes+limitations+of+cell+cycle+analysis+by+classical+determination+of+BrdU+incorporation,+allowing+multiplex+antibody+staining.">A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining.</a> Cytometry A. 73 (7), 626-636 (2008).</li><li>Niwa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+nature+of+the+light+emitter+of+the+Aequorea+green+fluorescent+protein.">Chemical nature of the light emitter of the Aequorea green fluorescent protein.</a> Proc Natl Acad Sci U S A. 93 (24), 13617-13622 (1996).</li><li>Tsien, R. 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Like the proteins destined for other subcellular organelles, the mitochondrial targeted proteins possess targeting signals within their primary or secondary structure that direct them to the organelle with the assistance of elaborate protein translocating and folding machines21,22. Mitochondria targeting sequences (MTS) obtained from exclusively mitochondrial resident proteins such as COX8 can be added to N-terminus of any gene sequence to target specific proteins into the mitochondria11,23,24. Here...

In mammals, cell cycle progression is dependent upon highly ordered events controlled by cyclins and cyclin-dependent kinases (Cdks)1. Through its cytoplasmic, nuclear, and centrosomal localization, CyclinB1/Cdk1 is able to synchronize different events in mitosis such as nuclear envelope breakdown and centrosome separation2. CyclinB1/Cdk1 protects mitotic cells against apoptosis3 and promotes mitochondrial fission, a critical step for an equal distribution of mitochondria to the newly formed daughter cells4.
In proliferating mammalian cells, mitochondrial ATP is generated via oxidative phosphorylation (OXPHOS) machinery (electron transport chain), which is composed of 5 multi-subunit complexes; complex I - complex V (CI-CV). Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase or complex I (CI) is the largest and least understood of the five complexes5. The complex consists of 45 subunits, 14 of which form the catalytic core. Once assembled, the complex assumes an L-shaped structure with one arm protruding into the matrix and the other arm embedded in the inner membrane6,7. Mutations in CI subunits are the cause of a variety of mitochondrial disorders8. A functionally efficient CI in OXPHOS is required not only for overall mitochondrial respiration9, but also for successful cell cycle progression10. Unravelling the mechanisms underlying the functioning of this membrane-bound enzyme complex in health and disease could enable the development of novel diagnostic procedures and advanced therapeutic strategies. In a recent study, we have found that the CyclinB1/Cdk1 complex translocates into mitochondria in the (Gap 2) G2/(Mitosis) M phase and phosphorylates CI subunits to enhance mitochondrial energy production, potentially to offset increased energy needs of cells during cell cycle11. Here we showcase experimental procedures and strategies that can be used to study mitochondrial translocation of otherwise nuclear/cytoplasmic kinases, their mitochondrial substrates as well as functional consequences of their mitochondrial localization using CyclinB1/Cdk1 as an example.
The finding that the CyclinB1/Cdk1 complex translocates into mitochondria when needed prompted the studies of mitochondria-specific overexpression and knockdown of this complex. To achieve mitochondria-specific expression of proteins, one can add a mitochondria targeting sequence (MTS) in the N-terminus of the protein of interest. Mitochondria targeting sequences allow the sorting of the mitochondrial proteins into the mitochondria where they normally reside12. We have used an 87 base mitochondria targeting sequence derived from the precursor of human cytochrome c oxidase subunit 8A (COX8) and cloned it into Green Fluorescent Protein (GFP)-tagged CyclinB1 or Red Fluorescent Protein (RFP)-tagged Cdk1 containing plasmids in frame. This method allowed us to target CyclinB1 and Cdk1 into the mitochondria, specifically changing the mitochondrial expression of these proteins without affecting their nuclear pool. By fluorescently tagging these proteins, we were able to monitor their localization in real time. Similarly, we have introduced MTS into a plasmid containing RFP-tagged dominant negative Cdk1, which allowed us to specifically knock down the mitochondrial expression and functions of Cdk1. It is essential to distinguish between mitochondrial and nuclear functions of the kinases that have dual localizations like Cdk1. Engineering MTS into the N-terminal of these dual functional kinases offers a great strategy that is easy to be employed and effective.
Since Cdk1 is a cell cycle kinase, it is fundamental to determine the cell cycle progression when Cdk1 is localized into mitochondria. To achieve this, we have utilized a new method to monitor DNA content in cells. Traditional methods include using BrdU (bromodeoxyuridine), a synthetic thymidine analogue, which incorporates into the newly synthesized DNA during the S phase of the cell cycle to substitute thymidine. Then the cells that are actively replicating their DNA can be detected using anti-BrdU antibodies. One disadvantage of this method is that it requires denaturation of DNA to provide access for the BrdU antibody by harsh methods like acid or heat treatment, which may result in inconsistency among results13,14. Alternatively, we utilized a similar approach to monitor the actively dividing cells with a different thymidine analog, EdU. EdU detection does not require harsh DNA denaturation as mild detergent treatment enables the detection reagent to access the EdU in newly synthesized DNA. The EdU method has proven to be more reliable, consistent and with potential for high-throughput analysis15.
Finally, to determine the mitochondrial substrates of Cdk1, we used a proteomics tool called 2D-DIGE, which is an advanced version of classical two-dimensional gel electrophoresis. Two dimensional electrophoresis separates proteins according to their isoelectric point in the first dimension and molecular weight in the second. Since post-translational modifications such as phosphorylation affect the isoelectric point and molecular weight of the proteins, 2D gels can detect the differences between phosphorylation statuses of proteins within different samples. The size (area and intensity) of protein spots changes with the expression level of proteins, allowing quantitative comparison between multiple samples. Using this method, we were able to differentiate the phosphorylated proteins in wild type versus mutant mitochondria-targeted Cdk1 expressing cells. The specific protein spots that showed in the wild type but were missing in the mitochondria-targeted mutant Cdk1 preparation were isolated and identified via mass spectrometry.
In traditional 2D gels, triphenylmethane dyes are used to visualize the proteins on the gel. 2D-DIGE uses fluorescent protein labels with minimal effect on protein electrophoretic mobility. Different protein samples can be labeled with different fluorescent dyes, mixed together and separated by the identical gels, allowing the co-electrophoresis of multiple samples on a single gel16. This minimizes the gel-to-gel variations, which is a critical problem in gel-based proteomics studies.

<table border="0" cellpadding="0" cellspacing="0" width="765">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:273px;">32P ATP&#160;</td>
			<td style="width:207px;">PerkinElmer</td>
			<td style="width:119px;">BLU002001MC&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse secondary antibody</td>
			<td>Invitrogen&#160;</td>
			<td>A-11003</td>
			<td>Alexa-546 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-rabbit secondary antibody</td>
			<td>Invitrogen&#160;</td>
			<td>A11029</td>
			<td>Alexa-488 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATP</td>
			<td>Research Organics</td>
			<td>1166A</td>
			<td>For in vitro kinase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cdk1 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cdk1 kinase buffer</td>
			<td>New England Biolabs</td>
			<td>P6020S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Click-iT EdU Alexa Fluor 488 Imaging Kit</td>
			<td>Life Technologies</td>
			<td>C10337</td>
			<td>For cell cycle analysis with EdU labeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">COX IV antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4844S</td>
			<td>For mitochondrial immunostaining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyclin B1 antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-752</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CyclinB1/Cdk1 enzyme complex</td>
			<td>New England Biolabs</td>
			<td>P6020S</td>
			<td>Avoid freeze/thaw</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CyDye DIGE Fluor Labeling Kit</td>
			<td>GE Healthcare Life Sciences</td>
			<td>25-8009-83</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DIGE Gel and DIGE Buffer Kit</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-9480-26 AA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethylformamide&#160;</td>
			<td>Sigma Aldrich</td>
			<td>319937</td>
			<td>DMF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol</td>
			<td>Bio-Rad</td>
			<td>161-0611</td>
			<td>DTT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP</td>
			<td>EMD Millipore</td>
			<td>71004</td>
			<td>For site-directed mutagenesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dpn I enzyme</td>
			<td>Stratagene</td>
			<td>200519-53</td>
			<td>For site-directed mutagenesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry Strip cover fluid</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-1335-01</td>
			<td>Used as mineral oil</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>J.T. Baker</td>
			<td>4040-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Acros Organics</td>
			<td>409910250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf Vacufuge Concentrator</td>
			<td>Fisher Scientific</td>
			<td>07-748-13</td>
			<td>Used as vacuum centrifuge concentrator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoromount G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Anti-fade mounting solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fortessa Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>649908</td>
			<td>For cell cycle analysis with EdU labeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Histone H1</td>
			<td>Calbiochem</td>
			<td>382150</td>
			<td>For in vitro kinase assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>For purifying DNA fragments from agarose gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Immobiline DryStrip Gels</td>
			<td>GE Healthcare Life Sciences</td>
			<td>18-1016-61</td>
			<td>IEF (isoelectric focusing) strips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immobilized Glutathione</td>
			<td>Thermo Scientific</td>
			<td>15160</td>
			<td>Glutathione-agarose beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodoacetamide</td>
			<td>Sigma Aldrich</td>
			<td>I1149</td>
			<td>IAA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPGphor 3 Isoelectric Focusing Unit</td>
			<td>GE Healthcare Life Sciences</td>
			<td>11-0033-64</td>
			<td>IPGphor strip holders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropyl-b-D-thio-galactopyranoside&#160;</td>
			<td>RPI Corp</td>
			<td>156000-5.0</td>
			<td>IPTG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leupeptin</td>
			<td>Sigma Aldrich</td>
			<td>L9783</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668027</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysine</td>
			<td>Sigma Aldrich</td>
			<td>L5501</td>
			<td>For CyDye labeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysozyme</td>
			<td>EMD Chemicals</td>
			<td>5960</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mitoctracker Red/Green</td>
			<td>Invitrogen&#160;</td>
			<td>M7512/M7514</td>
			<td>Mitochondrial fluorescent dyes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MOPS</td>
			<td>EMD Chemicals</td>
			<td>6310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pEGFP-N1</td>
			<td>Clonetech</td>
			<td>6085-1</td>
			<td>GFP-expressing vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pfu</td>
			<td>Stratagene</td>
			<td>600-255-52</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pGEX-5X-1&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-9545-53</td>
			<td>GST-expressing vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylmethylsulfonyl fluoride</td>
			<td>Shelton Scientific</td>
			<td>IB01090</td>
			<td>PMSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>14040</td>
			<td>PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectra/Por 4 dialysis tubing</td>
			<td>Spectrum Labs</td>
			<td>132700</td>
			<td>as porous membrane tubing for dialysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pro-Q Diamond Phosphoprotein Gel Stain</td>
			<td>Life Technologies</td>
			<td>P-33300</td>
			<td>For staining phosphoproteins on 2D gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase inhibitor cocktail</td>
			<td>Calbiochem</td>
			<td>539134</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QuikChange site-directed mutagenesis kit</td>
			<td>Stratagene</td>
			<td>200519-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>MiniPrep Plasmid Isolation Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RO-3306</td>
			<td>Alexis Biochemicals</td>
			<td>270-463-M001</td>
			<td>Cdk1 inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotenone</td>
			<td>MP Biomedicals</td>
			<td>150154</td>
			<td>Complex I inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium carbonate</td>
			<td>Fisher Scientific</td>
			<td>S93359</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>EMD Chemicals</td>
			<td>SX0420-5</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium orthovanadate</td>
			<td>MP Biomedicals</td>
			<td>159664</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyrophosphate decahydrate</td>
			<td>Alfa Aesar</td>
			<td>33385</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium &#946;-glycerophosphate</td>
			<td>Alfa Aesar</td>
			<td>L03425</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpectraMax M2e&#160;</td>
			<td>Molecular Devices</td>
			<td>M2E</td>
			<td>Microplate reader</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Fisher Scientific</td>
			<td>57-50-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Grinder pestle</td>
			<td>Kimble Chase</td>
			<td>885301-0007</td>
			<td>For mitochondria isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Grinder tube</td>
			<td>Kimble Chase</td>
			<td>885303-0007</td>
			<td>For mitochondria isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichloroacetic acid solution</td>
			<td>Sigma Aldrich</td>
			<td>T0699</td>
			<td>TCA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>MP Biomedicals</td>
			<td>103133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-x-100</td>
			<td>Teknova</td>
			<td>T1105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td>Calbiochem</td>
			<td>650211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Typhoon Imager</td>
			<td style="width:207px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">28-9558-09</td>
			<td>Laser gel scanner fro 2D-DIGE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ubiquinone</td>
			<td>Sigma Aldrich</td>
			<td>C7956</td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental approaches to study mitochondrial localization and function of a nuclear cell cycle kinase, cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Sub-mitochondrial localization of CyclinB1 and Cdk1
Sodium carbonate extraction is used to determine whether a protein is located inside the mitochondria or on the outside surface, namely outer membrane. Once a protein is shown to localize inside the mitochondria, further determination of sub-mitochondrial localization can be made via mitoplasting combined with protease digestion. To specify the sub-mitochondria...

Watch this Scientific Journal Video about Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1 at JoVE.com

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

The overall goal of the following procedure is to determine the submitochondrial localization of a normally nuclear cell cycle kinase and then to analyze how its mitochondrial localization affects the progression of the cell cycle. This method can help answer key questions in the mitochondrial research field, such as how nucleus communicates with the mitochondria during the cell cycle. By tagging proteins with a mitochondria leading sequence, we can take advantage of specifically over expressions in protein in the mitochondria, so we can study their mitochondria-specific functions.Though this method can provide insight into mitochondria targeting of certain proteins, it can be also be applied to the other organelles, such as the nucleus, ER, golgi, and a lysosome. After culturing, homogenizing, and pelleting cells according to the text protocol, transfer the supernatant into a new tube. And centrifuge the sample at 7, 000 g and 4 degrees Celsius for 10 minutes.Then use 200 microliters of ice-cold IBC buffer to resuspend the pellet, and divide the homogenate into two aliquots. Centrifuge the samples again at 7, 000 g and 4 degrees Celsius for 10 minutes. And repeat the wash.After discarding the supernatant, add 30 microliters of cell lysis buffer to one of the pellets, and store the lysate at 80 degrees Celsius for immunoblotting. To carry out sodium carbonate extraction with the second pellet to separate soluble and membrane-bound proteins, add 250 microliters of 0.1 molar sodium carbonate pH 11.0, and incubate on ice for 30 minutes. Centrifuge at 100, 000 g for 20 minutes.Then collect the supernatant and add an equal volume of freshly made, 20%trichloroacetic acid to precipitate the proteins. Keep on ice for 30 minutes. In the meantime, add 30 microliters of cell lysis buffer to the pellet and sonicate according to the text protocol before storing at 80 degrees Celsius for immunoblotting.After the 30-minute TCA incubation, centrifuge the reaction at 15, 000 g for 10 minutes. Discard the supernatant, then use 80 microliters of cell lysis buffer to resuspend the pellet. After isolating mitochondrial fractions from cells as described in the text protocol, separate the mitochondrial fractions into 10 equal portions.Pellet the samples at 7, 000 g and 4 degrees Celsius for 10 minutes. Use 30 microliters of a range of concentrations of hypotonic sucrose buffers with or without trypsin, to dissolve each pellet. And incubate on ice for 30 minutes.Add 3 microliters of 10 millimolar PMSF to the trypsin-containing vials to stop the trypsin digestion. And incubate on ice for 10 minutes. Centrifuge the samples at 14, 000 g and 4 degrees Celsius for 10 minutes.And transfer the supernatant into a new tube. To lyse the pellet, add 30 microliters of cell lysis buffer. Sonicate the samples as described in the text protocol and store at 80 degrees Celsius.To construct mitochondria targeted GFP/RFP-tagged cyclinB-1 Cdk-1 vectors, clone the mitochondria targeting sequence from the precursor of human cytochrome-c oxidase subunit 8A, and frame with the n-terminus of GFP or RFP at the Nhe1 and bAmH1 sites, of pEGFP-N1 or pERFP-N1, using standard molecular cloning techniques. Using the primers outlined in the text protocol, amplify the Cdk1 and cyclinB1 genes following standard techniques. Then use BamH1 to digest the PCR products.Run the reactions on a 1%agarose gel, before using a razor blade to cut out the correct size DNA fragments. Then use a gel extraction kit to purify the DNA. Next, digest one microgram of MTS-pEGFP-N1 and MTS-pERFP-N1 plasmids with 1 microliter of BamH1 at 37 degrees Celsius for 2 hours.Then add 1 microliter of calf intestinal alkaline phosphatase and incubate at 37 degrees Celsius for 30 minutes. After running the digestion products on a 1%agarose gel and purifying the DNA as just described, set up a ligation reaction using the reagents listed in the text protocol. Then incubate the reaction at 4 degrees Celsius overnight.Transform e. coli dH5-alpha competent cells with 10 microliters of the ligation mixture. And grow the bacteria on plates of LB agar plus 10 milligrams per milliliter of kanamycin at 37 degrees Celsius overnight.The following day, use a sterile pipette tip to pick a colony from a plate. And insert the tip into a tube containing 5 milliliters of LB kanamycin. Incubate the culture overnight and the following morning, use a mini prep kit according to the text protocol to isolate the plasmid.To tranfect exponentially growing MCF-10A cells, use Cdk1 or cyclinB-1 plasmids to prepare plasmid transfection reagent at a ratio of 1:2 in 100 microliters of serum and antibiotic-free medium. Transfect the cells and incubate at 37 degrees Celsius for 48 hours. Stain and visualize the mitochondria according to the text protocol.To carry out cell sorting, tranfect 2 times 10 to the 5th cells with the desired vectors in a 6-well plate using a 1:2 ratio of DNA to tranfection reagent prepared in 2.5 milliliters of serum and antibiotic-free medium. After incubating the transfection for 48 hours, use flow cytometry to live-sort the cells stably expressing the GFP-tagged Cdk-1 and RFP-tagged clyclinB1 proteins according to the text protocol. To measure cell cycle length using the EdU labeling flow cytometry assay, seed sorted cells in 6-well plates at a density of 2.5 times 10 to the 5th cells per well.After an overnight incubation, add EdU to the culture medium at a final concentration of 25 micromolar and incubate for an additional hour. Then at two hour intervals, use 1%BSA in 500 microliters of PBS to wash one well of cells. And collect in a 1.5-milliliter tube.Centrifuge the cells at 350 g for 5 minutes. Then discard the supernatant. Dislodge the pellet by adding 100 microliters of fixing solution.Mix well and incubate at room temperature for 15 minutes. Next, use 1 milliliter of 1%BSA in PBS to wash the cells three times. Then use 0.5 milliliters of 70%ethanol to fix the cells at 4 degrees Celsius overnight.When performing EdU labeling with cells transfected with GFP and RFP tagged proteins, it is critical to quench the fluorescent signals from GFP and RFP. To achieve this, we add an extra step of overnight cell fixing using 70%ethanol. The following day, after washing and permeabilizing the cells according to the text protocol, add 0.5 milliliters of reaction cocktail into each tube and mix well.Following the incubation in the dark and another wash, use 50 micrograms per milliliter of propidium iodide or PI in 1%BSA PBS to stain the DNA. Analyze the cells by flow cytometry to follow the EdU-positive population. Present a scattered dot plot of EdU-labeled cells stained for DNA content and EdU.Use the APC channel for Alexa 647 EdU utilizing a 670 30-bandpass filter with all light present, less than 685 nanometers hitting that filter and the phycoerythrin channel for PI with a 581 15-bandpass filter in front of it, with all light present less than 600 nanometers hitting that filter. With a standard gating strategy for acquistion, plot FSC area by SSC area for morphology, followed by PI by Alexa 647 EdU for cell staining. Record data for all tubes one by one, acquiring 10, 000 events per sample.In this figure, mitochondrial matrix protein Hsp60 and intermembrane space protein Timm13 were used as submitochondrial localization markers. Similar with Hsp60, but unlike Timm13, cyclinB1 and Cdk1 were protected from trypsin digestion, indicating that they localize to the mitochondrial matrix. As shown here, by Western blotting of the isolated mitochondrial fractions, using the MTS and GFP-tagged cyclinB1 and Cdk-1 constructs, overexpression of cyclinB1 and/or Cdk-1 was achieved in the mitochondria.Using an EdU pulse chase assay, it was demonstrated that labeled S phase cells progressed through G2M phase and appeared in G1 phase as fast as 4 hours in cells expressing wild-type mitochondrial cyclinB1 Cdk-1. As compared to 6 hours, in cells transfected with a vector control or mutant cyclinB1 Cdk-1, indicating that enhancement of mitochondrial cyclinB1 Cdk-1 accelerates cell cycle progression. Following this procedure, other methods like mitochondrial ATP generation, oxygen consumption, membrane potential, and reactive oxygen species can be measured in order to answer additional questions like how mitochondrial localization of cell cycle kinase Cdk-1 alters mitochondrial respiration and energy output.After watching this video, you should have a good understanding of how to study the mitochondrial localization and the mitochondria-specific functions of a nuclear kinase.

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