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.

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The authors declare that they have no competing financial interests.

Wie die Proteine ​​für andere subzelluläre Organellen bestimmt, die mitochondriale Zielproteine ​​besitzen Targeting - Signale in ihrer primären oder sekundären Struktur , die sie dem Organell mit Hilfe von aufwendigen Protein translocating und Falzmaschinen 21,22 lenken. Mitochondrien - Targeting - Sequenzen (MTS) aus ausschließlich mitochondrialen resident Proteine ​​wie COX8 erhalten kann , um N-Terminus von jedem Gen - Sequenz hinzugefügt werden , um spezifische Proteine ​​in die Mit...

Bei Säugetieren ist die Zellzyklusprogression in Abhängigkeit von hoch geordneten Ereignisse gesteuert von Cyclinen und Cyclin-abhängigen Kinasen (Cdks) 1. Durch seine zytoplasmatische, nukleare und zentrosomalen Lokalisierung, ist CyclinB1 / Cdk1 2 verschiedene Ereignisse in der Mitose wie Kernhülle Zusammenbruch und Zentrosom Trennung zu synchronisieren können. CyclinB1 / Cdk1 schützt mitotischen Zellen vor Apoptose 3 und fördert mitochondrialen Spaltung, ein entscheidender Schritt für eine gleichmäßige Verteilung der Mitochondrien an die neu 4 gebildeten Tochterzellen. In proliferierenden Säugetierzellen wird die mitochondriale ATP durch oxidative Phosphorylierung (OXPHOS) Maschinen (Elektronentransportkette) erzeugt, die von 5 Multi-Untereinheiten-Komplexen zusammengesetzt ist; Komplex I - Komplex V (CI-CV). Nicotinamidadenindinucleotid (NADH): Ubichinon Oxidoreduktase oder Komplex I (CI) ist die größte und am wenigsten verstandenen der fünf Komplexe 5. Der Komplex consists von 45 Untereinheiten, von denen 14 bilden den katalytischen Kern. Einmal montiert, nimmt der Komplex eine L-förmige Struktur mit einem Arm in die Matrix hervorstehenden und der andere Arm in der inneren Membran 6,7 eingebettet. Mutationen in CI - Untereinheiten sind die Ursache für eine Vielzahl von mitochondrialen Erkrankungen 8. Eine funktionell effiziente CI in OXPHOS nicht nur 9 zur Gesamt mitochondriale Atmung erforderlich ist , sondern auch für die erfolgreiche Zellzyklusprogression 10. das Funktionieren dieser membrangebundenen Enzymkomplex in Gesundheit und Krankheit, die Mechanismen Unravelling zugrunde liegen könnte die Entwicklung neuer Diagnoseverfahren und erweiterte therapeutische Strategien ermöglichen. In einer aktuellen Studie haben wir herausgefunden, dass die CyclinB1 / Cdk1 Komplex in die Mitochondrien transloziert in den (Gap 2) G2 / (Mitose) M-Phase und phosphoryliert CI Untereinheiten mitochondriale Energieproduktion zu erhöhen, möglicherweise erhöhten Energiebedarf der Zellen während des Zell ausgeglichen Zyklus 11. Hier stellen wir showcase experimentellen Verfahren und Strategien, die mitochondriale Translokation von sonst Kern / Zytoplasma-Kinasen, deren mitochondriale Substrate sowie funktionelle Konsequenzen ihrer mitochondrialen Lokalisation mit CyclinB1 / Cdk1 als Beispiel zu studieren verwendet werden kann. Die Erkenntnis, dass die CyclinB1 / Cdk1 Komplex in die Mitochondrien transloziert, wenn Sie dazu aufgefordert notwendig, um die Studien der Mitochondrien-spezifische Überexpression und Zuschlags dieses Komplexes. In den Mitochondrien-spezifischen Expression von Proteinen zu erreichen, kann man eine Mitochondrien-Targeting-Sequenz (MTS) in den N-Terminus des Proteins von Interesse hinzuzufügen. Mitochondrien - Targeting - Sequenzen ermöglichen die Sortierung der mitochondrialen Proteine ​​in die Mitochondrien , wo sie normalerweise 12 befinden. Wir haben eine 87 Basen Mitochondrien-Targeting-Sequenz, abgeleitet aus dem Vorläufer von humanem Cytochrom c Oxidase Untereinheit 8A (COX8) und kloniert es in Green Fluorescent Protein (GFP) -markierte CyclinB1 oder Red Fluorescent verwendetProtein (RFP) -markierte Cdk1 Plasmide in Rahmen enthält. Diese Methode erlaubt uns CyclinB1 und Cdk1 in die Mitochondrien zu zielen, insbesondere die mitochondriale Expression dieser Proteine ​​zu ändern, ohne ihre Kern Pool zu beeinflussen. Durch fluoreszenz diese Proteine ​​Tagging, waren wir in der Lage, ihre Lokalisierung in Echtzeit zu überwachen. In ähnlicher Weise haben wir MTS in ein Plasmid, das RFP-markierte dominant negative Cdk1, eingeführt, die uns speziell auf die mitochondriale Expression und Funktion von Cdk1 abreißen dürfen. Es ist wichtig, zwischen mitochondrialen und Kernfunktionen der Kinasen, die haben zwei Lokalisierungen wie Cdk1 zu unterscheiden. Ingenieur MTS in die N-terminale dieser Dual funktionelle Kinasen bietet eine große Strategie, die einfach eingesetzt und wirksam werden. Da Cdk1 ein Zellzyklus-Kinase ist, ist es von grundlegender Bedeutung die Zellzyklus-Progression, um zu bestimmen, wenn Cdk1 in Mitochondrien lokalisiert ist. Um dies zu erreichen, haben wir ein neues metho genutztd DNA-Gehalt in Zellen zu überwachen. Traditionelle Methoden umfassen BrdU Verwendung (Bromdesoxyuridin) ein synthetisches Thymidin-Analogon, die während der S-Phase des Zellzyklus in die neu synthetisierte DNA umfasst Thymidin zu substituieren. Dann werden die Zellen, die ihre DNA aktiv replizieren können anti-BrdU Antikörper verwendet werden, detektiert. Ein Nachteil dieses Verfahrens ist , dass es die Denaturierung von DNA erfordert durch harte Methoden wie Säure oder Wärmebehandlung Zugang für den BrdU - Antikörper zu schaffen, die unter Ergebnisse 13,14 in Inkohärenz führen kann. Alternativ dazu verwendeten wir einen ähnlichen Ansatz, um die teilungsaktiven Zellen, die mit einem anderen Thymidin-Analogon, EdU zu überwachen. EdU Erkennung erfordert keine harte DNA-Denaturierung als einem milden Reinigungsmittel Behandlung Reagenz den Nachweis ermöglicht in neu synthetisierte DNA, die die EdU zuzugreifen. Die EDU - Methode hat sich als zuverlässiger zu sein, konsequent und mit Potenzial für die Hochdurchsatzanalyse 15. Schließlich to die mitochondriale Substrate von Cdk1 bestimmen, verwendeten wir eine Proteomik-Tool namens 2D-DIGE, die eine erweiterte Version des klassischen zweidimensionalen Gelelektrophorese ist. Zweidimensionale Elektrophorese trennt Proteine ​​nach ihrem isoelektrischen Punkt in der ersten Dimension und einem Molekulargewicht in der zweiten. Da posttranslationale Modifikationen wie Phosphorylierung den isoelektrischen Punkt und das Molekulargewicht der Proteine ​​beeinflussen, 2D Gele können die Unterschiede zwischen Phosphorylierung Stati von Proteinen in unterschiedlichen Proben nachzuweisen. Die Größe (Fläche und Intensität) von Proteinspots ändert sich mit der Expression von Proteinen, quantitativen Vergleich zwischen mehreren Proben ermöglicht. Mit dieser Methode konnten wir die phosphorylierten Proteine ​​in Wildtyp im Vergleich zu mutierten Mitochondrien-bezogene Cdk1 exprimierenden Zellen zu unterscheiden. Die spezifischen Proteinspots, die im Wildtyp zeigten, wurden aber in den Mitochondrien-Targeting-Mutante Cdk1 Vorbereitung fehlt, wurden isoliert undüber Massenspektrometrie identifiziert. In herkömmlichen 2D-Gelen sind Triphenylmethanfarbstoffe verwendet, um die Proteine ​​auf dem Gel sichtbar zu machen. 2D-DIGE verwendet fluoreszierendes Protein Etiketten mit minimaler Auswirkung auf die Protein elektrophoretische Mobilität. Verschiedene Proteinproben mit unterschiedlichen Fluoreszenzfarbstoffen, miteinander vermischt und durch die identischen Gelen getrennt markiert werden, so dass die Co-Elektrophorese mehrerer Proben auf einem einzigen Gel 16. Dies minimiert die Gel-zu-Gel-Varianten, die ein kritisches Problem in Gelbasis Proteomstudien ist.

<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-mitochondriale Lokalisation von CyclinB1 und Cdk1 Natriumcarbonat Extraktion wird verwendet, um zu bestimmen, ob ein Protein in der Mitochondrien oder an der Außenfläche angeordnet ist, nämlich der äußeren Membran. Sobald ein Protein im Inneren der Mitochondrien, weitere Bestimmung der Unter mitochondriale Lokalisation gezeigt wird zu lokalisieren kann über mitoplasting kombiniert mit Proteaseverdau ...

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.

Experimentelle Ansätze zur Untersuchung Mitochondriale Lokalisation und Funktion eines Kernzellzyklus-Kinase, Cdk1

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

experimental-approaches-to-study-mitochondrial-localization-function

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Unit 2

Unit 1

Cell Proliferation

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11.8K Aufrufe.  University of California, Davis. 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. 

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

Experimental Approaches to Tissue Engineering

Experimentelle Ansätze zur Tissue Engineering

Forschung

Biologie

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualizing RNA Localization in Xenopus Oozyten

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Mit der optokinetischen Reaktion auf visuelle Funktion des Zebrafisch-Studie

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

Überwachung Kinase und Phosphatase-Aktivitäten durch den Zellzyklus durch Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analyse des Zellzyklus Position in Säugerzellen

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Beurteilung der mitochondrialen Funktionen und Zellviabilität in Renal Überexpression Protein Kinase C-Isoenzyme

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Kombiniert Immunfluoreszenz und DNA FISH auf 3D erhaltene Interphase Nuclei, um Änderungen in 3D Nuclear Organization Study

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Verfahren zum Screening und Validierung von Beständig Mutationen Vor-Inhibitoren

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

Ein zellfreien Assay zu Chromatindekondensation Studieren am Ende der Mitose

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

Ein zellfreien Assay unter Verwendung von<em&gt; Xenopus laevis</em&gt; Embryo Extrahiert Mechanismen der Kerngröße Verordnung zum Studium

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...