Eksperimentelle tilgange til at studere mitokondrie Lokalisering og funktion af en nuklear Cell Cycle kinase, Cdk1
Summary
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.
Abstract
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′-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.
Introduction
I pattedyr cellecyklusprogression er afhængig af stærkt ordnede arrangementer kontrolleres af cycliner og cyclinafhængige kinaser (CDK'er) 1. Gennem sin cytoplasmatiske, nukleare, og centrosomal lokalisering, CyclinB1 / Cdk1 er i stand til at synkronisere forskellige begivenheder i mitosen såsom opdeling nukleare kuvert og centrosom adskillelse 2. CyclinB1 / Cdk1 beskytter mitotiske celler mod apoptose 3 og fremmer mitokondrie fission, et afgørende skridt for en ligelig fordeling af mitokondrier til de nydannede datterceller 4.
I prolifererende pattedyrceller, er mitokondrie ATP genereres via oxidativ phosphorylering (OXPHOS) udstyr (elektrontransportkæden), som er sammensat af 5 multi-subunit-komplekser; kompleks I – kompleks V (CI-CV). Nikotinamidadenindinucleotid (NADH): ubiquinone oxidoreduktase eller kompleks I (CI) er den største og mindste forstået af de fem komplekser 5. Komplekset consists af 45 underenheder, hvoraf 14 danner den katalytiske kerne. Når samlet, komplekset antager en L-formet struktur med den ene arm rager ind i matrixen og den anden arm indlejret i den indre membran 6,7. Mutationer i CI-underenheder er årsag til en række forskellige mitochondriale lidelser 8. Et funktionelt effektiv CI i OXPHOS er ikke kun brug for den overordnede mitokondriel respiration 9, men også for vellykket cellecyklusprogression 10. Opklaringen mekanismerne bag driften af denne membranbundne enzym kompleks i sundhed og sygdom kunne muliggøre udviklingen af nye diagnostiske procedurer og avancerede terapeutiske strategier. I en nylig undersøgelse, har vi fundet, at CyclinB1 / Cdk1 kompleks translokaliserer i mitokondrier i (Gap 2) G2 / (Mitose) M fase og phosphorylerer CI underenheder at øge mitokondrie energiproduktion, potentielt at opveje øgede energibehov af celler under celle cyklus 11. Her sho viwcase eksperimentelle procedurer og strategier, der kan anvendes til at studere mitokondrisk translokation af ellers nukleare / cytoplasmiske kinaser, deres mitokondrielle substrater samt funktionelle konsekvenser af deres mitokondrie lokalisering ved hjælp CyclinB1 / Cdk1 som eksempel.
Den fandt, at CyclinB1 / Cdk1 kompleks translokaliserer i mitokondrier når det er nødvendigt bedt studier af mitokondrier-specifikke overekspression og knockdown af dette kompleks. For at opnå mitokondrier ekspression af proteiner, kan man tilføje en mitokondrier targeting sekvens (MTS) i den N-terminale ende af proteinet af interesse. Mitokondrier rettet sekvenser tillader sortering af mitokondrielle proteiner i mitokondrierne, hvor de normalt bor 12. Vi har anvendt en 87 base-mitokondrier målrettet sekvens afledt fra forstadiet af human cytochrom c-oxidase subunit 8A (COX8) og klonet det i grønt fluorescerende protein (GFP) -mærket CyclinB1 eller rødt fluorescerendeProtein (RFP) -mærket Cdk1 indeholdende plasmider i ramme. Denne metode tillod os at målrette CyclinB1 og Cdk1 i mitokondrierne, specifikt ændrer den mitokondriske ekspression af disse proteiner uden at påvirke deres nukleare pool. Ved fluorescens tagging disse proteiner, var vi i stand til at overvåge deres lokalisering i realtid. Ligeledes har vi introduceret MTS i et plasmid indeholdende RFP-mærkede dominant negativ Cdk1, hvilket tillod os at specifikt vælte det mitokondrielle ekspression og funktion Cdk1. Det er vigtigt at skelne mellem mitokondrielle og de nukleare funktioner af kinaser, der har dobbelt lokaliseringer som Cdk1. Engineering MTS til N-terminalen af disse dobbelte funktionelle kinaser tilbyder en stor strategi, der er let at være ansat og effektiv.
Da Cdk1 er en cellecyklus kinase, er det afgørende at bestemme cellecyklusprogression når Cdk1 er lokaliseret i mitokondrier. For at opnå dette, har vi udnyttet en ny method til at overvåge DNA-indholdet i celler. Traditionelle metoder omfatter anvendelse af BrdU (bromdeoxyuridin), en syntetisk thymidinanalog, som inkorporerer i det nyligt syntetiserede DNA under S-fasen af cellecyklussen at erstatte thymidin. Derefter kan detekteres de celler, der aktivt replikerende deres DNA under anvendelse af anti-BrdU-antistoffer. En ulempe ved denne metode er, at den kræver denaturering af DNA for at give adgang for BrdU antistof ved barske metoder som syre eller varmebehandling, hvilket kan resultere i uoverensstemmelse mellem resultaterne 13,14. Alternativt vi udnyttet en lignende tilgang til at overvåge aktivt delende celler med en anden thymidin analog, Edu. Edu afsløring kræver ikke barske DNA denaturering som mildt rengøringsmiddel behandling gør det muligt for påvisningsreagenset at få adgang til Edu i nyligt syntetiseret DNA. Den edu-metoden har vist sig at være mere pålidelige, konsistente og med potentiale for high-throughput analyse 15.
Endelig to bestemme de mitokondrielle substrater af Cdk1 anvendte vi et proteomics værktøj kaldet 2D-DIGE, som er en avanceret udgave af klassisk todimensional gelelektroforese. Todimensional elektroforese adskiller proteiner på grundlag af deres isoelektriske punkt i den første dimension og molekylvægt i den anden. Da post-translationelle modifikationer, såsom phosphorylering påvirker det isoelektriske punkt og molekylvægten af proteinerne, kan 2D-geler detektere forskellene mellem phosphoryle- status af proteiner i forskellige prøver. Størrelsen (areal og intensitet) protein blev ændringer med ekspressionsniveauet af proteiner, hvilket muliggør kvantitativ sammenligning mellem multiple prøver. Ved hjælp af denne metode, var vi i stand til at skelne de phosphorylerede proteiner i vildtype versus mutant mitokondrier målrettet CDK1 udtrykkende celler. De specifikke proteinpletter der viste i vild type, men manglede i mitokondrier målrettede mutant Cdk1 præparat blev isoleret ogidentificeret via massespektrometri.
I traditionelle 2D-geler, der triphenylmethanfarvestoffer farvestoffer anvendt til at visualisere proteinerne på gelen. 2D-DIGE bruger fluorescerende protein etiketter med minimal effekt på protein elektroforetisk mobilitet. Forskellige protein prøver kan mærkes med forskellige fluorescerende farvestoffer, blandet sammen og adskilt af identiske geler, tillader co-elektroforese af multiple prøver på en enkelt gel 16. Dette minimerer gel-til-gel variationer, hvilket er et kritisk problem i gel-baserede proteomics undersøgelser.
Protocol
Representative Results
Discussion
Ligesom proteinerne bestemt til andre subcellulære organeller, mitokondrier målrettede proteiner besidder målrettet signaler på deres primære eller sekundære struktur, som dirigerer dem til organel med bistand fra omfattende protein translokaliserende og falsning 21,22. Mitokondrier targetingsekvenser (MTS), der udelukkende er mitokondrielle residente proteiner såsom COX8 kan sættes til N-terminalen af enhver gensekvens at målrette specifikke proteiner i mitokondrierne 11,23,24. Her blev C…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 32P ATP | PerkinElmer | BLU002001MC | |
| Anti-mouse secondary antibody | Invitrogen | A-11003 | Alexa-546 conjugated |
| Anti-rabbit secondary antibody | Invitrogen | A11029 | Alexa-488 conjugated |
| ATP | Research Organics | 1166A | For in vitro kinase assay |
| Cdk1 antibody | Cell Signaling Technology | 9112 | |
| Cdk1 kinase buffer | New England Biolabs | P6020S | |
| Click-iT EdU Alexa Fluor 488 Imaging Kit | Life Technologies | C10337 | For cell cycle analysis with EdU labeling |
| COX IV antibody | Cell Signaling Technology | 4844S | For mitochondrial immunostaining |
| Cyclin B1 antibody | Santa Cruz Biotech | sc-752 | |
| CyclinB1/Cdk1 enzyme complex | New England Biolabs | P6020S | Avoid freeze/thaw |
| CyDye DIGE Fluor Labeling Kit | GE Healthcare Life Sciences | 25-8009-83 | |
| DIGE Gel and DIGE Buffer Kit | GE Healthcare Life Sciences | 28-9480-26 AA | |
| Dimethylformamide | Sigma Aldrich | 319937 | DMF |
| Dithiothreitol | Bio-Rad | 161-0611 | DTT |
| dNTP | EMD Millipore | 71004 | For site-directed mutagenesis |
| Dpn I enzyme | Stratagene | 200519-53 | For site-directed mutagenesis |
| Dry Strip cover fluid | GE Healthcare Life Sciences | 17-1335-01 | Used as mineral oil |
| EDTA | J.T. Baker | 4040-03 | |
| EGTA | Acros Organics | 409910250 | |
| Eppendorf Vacufuge Concentrator | Fisher Scientific | 07-748-13 | Used as vacuum centrifuge concentrator |
| Fluoromount G | Southern Biotech | 0100-01 | Anti-fade mounting solution |
| Fortessa Flow Cytometer | BD Biosciences | 649908 | For cell cycle analysis with EdU labeling |
| Histone H1 | Calbiochem | 382150 | For in vitro kinase assay |
| QIAquick Gel Extraction Kit | Qiagen | 28704 | For purifying DNA fragments from agarose gels |
| Immobiline DryStrip Gels | GE Healthcare Life Sciences | 18-1016-61 | IEF (isoelectric focusing) strips |
| Immobilized Glutathione | Thermo Scientific | 15160 | Glutathione-agarose beads |
| Iodoacetamide | Sigma Aldrich | I1149 | IAA |
| IPGphor 3 Isoelectric Focusing Unit | GE Healthcare Life Sciences | 11-0033-64 | IPGphor strip holders |
| Isopropyl-b-D-thio-galactopyranoside | RPI Corp | 156000-5.0 | IPTG |
| Leupeptin | Sigma Aldrich | L9783 | For cell lysis buffer |
| Lipofectamine 2000 | Life Technologies | 11668027 | Transfection reagent |
| Lysine | Sigma Aldrich | L5501 | For CyDye labeling |
| Lysozyme | EMD Chemicals | 5960 | |
| Mitoctracker Red/Green | Invitrogen | M7512/M7514 | Mitochondrial fluorescent dyes |
| MOPS | EMD Chemicals | 6310 | |
| pEGFP-N1 | Clonetech | 6085-1 | GFP-expressing vector |
| Pfu | Stratagene | 600-255-52 | |
| pGEX-5X-1 | GE Healthcare Life Sciences | 28-9545-53 | GST-expressing vector |
| Phenylmethylsulfonyl fluoride | Shelton Scientific | IB01090 | PMSF |
| Phosphate buffered saline | Life Technologies | 14040 | PBS |
| Spectra/Por 4 dialysis tubing | Spectrum Labs | 132700 | as porous membrane tubing for dialysis |
| Pro-Q Diamond Phosphoprotein Gel Stain | Life Technologies | P-33300 | For staining phosphoproteins on 2D gels |
| Proteinase inhibitor cocktail | Calbiochem | 539134 | For cell lysis buffer |
| QuikChange site-directed mutagenesis kit | Stratagene | 200519-5 | |
| QIAprep Spin Miniprep Kit | Qiagen | 27104 | MiniPrep Plasmid Isolation Kit |
| RO-3306 | Alexis Biochemicals | 270-463-M001 | Cdk1 inhibitor |
| Rotenone | MP Biomedicals | 150154 | Complex I inhibitor |
| Sodium carbonate | Fisher Scientific | S93359 | |
| Sodium chloride | EMD Chemicals | SX0420-5 | For cell lysis buffer |
| Sodium orthovanadate | MP Biomedicals | 159664 | For cell lysis buffer |
| Sodium pyrophosphate decahydrate | Alfa Aesar | 33385 | For cell lysis buffer |
| Sodium β-glycerophosphate | Alfa Aesar | L03425 | For cell lysis buffer |
| SpectraMax M2e | Molecular Devices | M2E | Microplate reader |
| Sucrose | Fisher Scientific | 57-50-1 | |
| Tissue Grinder pestle | Kimble Chase | 885301-0007 | For mitochondria isolation |
| Tissue Grinder tube | Kimble Chase | 885303-0007 | For mitochondria isolation |
| Trichloroacetic acid solution | Sigma Aldrich | T0699 | TCA |
| Tris | MP Biomedicals | 103133 | |
| Triton-x-100 | Teknova | T1105 | |
| Trypsin | Calbiochem | 650211 | |
| Typhoon Imager | GE Healthcare Life Sciences | 28-9558-09 | Laser gel scanner fro 2D-DIGE |
| Ubiquinone | Sigma Aldrich | C7956 |
References
- Weinert, T., Hartwell, L. Control of G2 delay by the rad9 gene of Saccharomyces cerevisiae. J Cell Sci Suppl. 12, 145-148 (1989).
- Takizawa, C. G., Morgan, D. O. Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C. Curr Opin Cell Biol. 12 (6), 658-665 (2000).
- Allan, L. A., Clarke, P. R. Phosphorylation of caspase-9 by CDK1/cyclin B1 protects mitotic cells against apoptosis. Mol Cell. 26 (2), 301-310 (2007).
- Cerveny, K. L., Tamura, Y., Zhang, Z., Jensen, R. E., Sesaki, H. Regulation of mitochondrial fusion and division. Trends Cell Biol. 17 (11), 563-569 (2007).
- Brandt, U. Energy converting NADH:quinone oxidoreductase (complex I). Annu Rev Biochem. 75, 69-92 (2006).
- Hofhaus, G., Weiss, H., Leonard, K. Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I). J Mol Biol. 221 (3), 1027-1043 (1991).
- Weiss, H., Friedrich, T., Hofhaus, G., Preis, D. The respiratory-chain NADH dehydrogenase (complex I) of mitochondria. Eur J Biochem. 197 (3), 563-576 (1991).
- Janssen, R. J., Nijtmans, L. G., van den Heuvel, L. P., Smeitink, J. A. Mitochondrial complex I: structure, function and pathology. J Inherit Metab Dis. 29 (4), 499-515 (2006).
- Roessler, M. M., et al. Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron-electron resonance. Proc Natl Acad Sci U S A. 107 (5), 1930-1935 (2010).
- Owusu-Ansah, E., Yavari, A., Mandal, S., Banerjee, U. Distinct mitochondrial retrograde signals control the G1-S cell cycle checkpoint. Nat Genet. 40 (3), 356-361 (2008).
- Wang, Z., et al. Cyclin B1/Cdk1 coordinates mitochondrial respiration for cell-cycle G2/M progression. Dev Cell. 29 (2), 217-232 (2014).
- Omura, T. Mitochondria-targeting sequence, a multi-role sorting sequence recognized at all steps of protein import into mitochondria. J Biochem. 123 (6), 1010-1016 (1998).
- Konishi, T., Takeyasu, A., Natsume, T., Furusawa, Y., Hieda, K. Visualization of heavy ion tracks by labeling 3′-OH termini of induced DNA strand breaks. J Radiat Res. 52 (4), 433-440 (2011).
- Leif, R. C., Stein, J. H., Zucker, R. M. A short history of the initial application of anti-5-BrdU to the detection and measurement of S phase. Cytometry A. 58 (1), 45-52 (2004).
- Li, K., Lee, L. A., Lu, X., Wang, Q. Fluorogenic ‘click’ reaction for labeling and detection of DNA in proliferating cells. Biotechniques. 49 (1), 525-527 (2010).
- Kondo, T., et al. Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool. Proteomics. 3 (9), 1758-1766 (2003).
- Gundry, R. L., et al. Chapter 10, Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr Protoc Mol Biol. , Unit 10.25 (2009).
- Davies, D., Macey, M. G. Chapter 11, Cell Sorting by Flow Cytometry. Flow Cytometry. , 257-276 (2007).
- van den Heuvel, S., Harlow, E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science. 262 (5142), 2050-2054 (1993).
- Terry, N. H. A., White, R. A. Flow cytometry after bromodeoxyuridine labeling to measure S and G2+M phase durations plus doubling times in vitro and in vivo. Nat. Protcols. 1 (2), 859-869 (2006).
- Becker, L., et al. Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore. J Mol Biol. 353 (5), 1011-1020 (2005).
- Karniely, S., Pines, O. Single translation–dual destination: mechanisms of dual protein targeting in eukaryotes. EMBO Rep. 6 (5), 420-425 (2005).
- Candas, D., et al. CyclinB1/Cdk1 phosphorylates mitochondrial antioxidant MnSOD in cell adaptive response to radiation stress. J Mol Cell Biol. 5 (3), 166-175 (2013).
- Nantajit, D., et al. Cyclin B1/Cdk1 phosphorylation of mitochondrial p53 induces anti-apoptotic response. PLoS One. 5 (8), e12341 (2010).
- Cappella, P., Gasparri, F., Pulici, M., Moll, J. A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining. Cytometry A. 73 (7), 626-636 (2008).
- Niwa, H., et al. Chemical nature of the light emitter of the Aequorea green fluorescent protein. Proc Natl Acad Sci U S A. 93 (24), 13617-13622 (1996).
- Tsien, R. Y. The green fluorescent protein. Annu Rev Biochem. 67, 509-544 (1998).
- Marouga, R., David, S., Hawkins, E. The development of the DIGE system: 2D fluorescence difference gel analysis technology. Anal Bioanal Chem. 382 (3), 669-678 (2005).
- Timms, J. F., Cramer, R. Difference gel electrophoresis. Proteomics. 8 (23-24), 4886-4897 (2008).
- Crane, J., Mittar, D., Soni, D., McIntyre, C. Cell Cycle Analysis Using the BD BrdU FITC Assay on the BD FACSVerse System. BD Biosciences Appl Notes. , (2011).
