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.

<ol>
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The authors declare that they have no competing financial interests.

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 ...

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11.8K Views.  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. 

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

Experimental Approaches to Tissue Engineering

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.

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.

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.

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.

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.

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).

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.

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.

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.

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 ...