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.

Como as proteínas destinadas para outros organelos subcelulares, proteínas mitocondriais possuem sinais alvo específicas no interior da sua estrutura primária ou secundária que os dirigem para o organelo, com a assistência de translocação de proteína elaborada e máquinas de dobragem 21,22. As mitocôndrias sequências de direccionamento (MTS) obtidos a partir de proteínas residentes exclusivamente mitocondriais tais como COX8 pode ser adicionado ao terminal-N de qualquer sequência de genes para pr...

Nos mamíferos, a progressão do ciclo celular é dependente de acontecimentos altamente ordenadas controlados por ciclinas e cinases dependentes de ciclina (CDK) 1. Através de sua citoplasmático, nuclear, e localização centrosomal, CyclinB1 / Cdk1 é capaz de sincronizar diferentes eventos na mitose, tais como quebra envelope nuclear e separação centrossoma 2. CyclinB1 / Cdk1 protege as células mitóticas contra a apoptose 3 e promove a fissão mitocondrial, um passo crítico para uma distribuição igual das mitocôndrias das células filhas recém-formados 4. Em proliferação de células de mamíferos, mitocondrial de ATP é gerado através da fosforilação oxidativa (FOX) construção (cadeia de transporte de electrões), o qual é composto por 5 complexos de multi-subunidades; Complexo I - V complexo (CI-CV). Nicotinamida adenina dinucleótido (NADH): oxidorredutase ubiquinona ou complexo I (CI) é a maior e menos compreendido dos cinco complexos de 5. O complexo Consists de 45 subunidades, das quais 14 formam o núcleo catalítico. Uma vez montado, o complexo assume uma estrutura em forma de L com um braço saliente na matriz e o outro braço incorporado na membrana interna 6,7. Mutações em subunidades de IC são a causa de uma variedade de doenças mitocondriais 8. Um IC funcionalmente eficiente em OXPHOS é necessário não só para a respiração mitocondrial geral 9, mas também para a progressão do ciclo celular bem sucedida 10. Unravelling os mecanismos subjacentes ao funcionamento deste complexo de enzima ligada à membrana na saúde e na doença pode permitir o desenvolvimento de novos processos de diagnóstico e estratégias terapêuticas avançadas. Em um estudo recente, verificou-se que o / complexo Cdk1 CyclinB1 transloca para dentro da mitocôndria na / (mitose) fase M (Gap 2) G2 e fosforila subunidades CI para aumentar a produção de energia mitocondrial, potencialmente, para compensar o aumento das necessidades energéticas das células durante celular ciclo de 11. Aqui nós showcase procedimentos experimentais e estratégias que podem ser usados ​​para estudar a translocação mitocondrial de cinases em contrário nucleares / citoplasmáticas, os seus substratos mitocondriais, bem como consequências funcionais da sua localização mitocondrial usando CyclinB1 / Cdk1 como um exemplo. A constatação de que o / complexo Cdk1 CyclinB1 transloca para dentro da mitocôndria, quando necessário solicitado os estudos de sobre-expressão específicas de mitocôndrias e knockdown deste complexo. Para conseguir a expressão específica de mitocôndrias de proteínas, pode-se adicionar uma sequência de direccionamento mitocondrial (MTS) no N-terminal da proteína de interesse. Mitocôndrias alvo sequências permitem a ordenação das proteínas mitocondriais na mitocôndria onde eles residem normalmente 12. Temos usado uma sequência de direccionamento 87 mitocôndrias de base derivado do precursor do citocromo humano subunidade c oxidase 8A (COX8) e clonado-lo em Green Fluorescent Protein (GFP) tagged CyclinB1 ou vermelho fluorescenteProtein (RFP) tagged Cdk1 contendo plasmídeos no quadro. Este método permitiu-nos atingir CyclinB1 e Cdk1 na mitocôndria, que alterou a expressão mitocondrial destas proteínas sem afetar sua piscina nuclear. Por fluorescente marcação destas proteínas, fomos capazes de monitorar a sua localização em tempo real. Da mesma forma, nós introduzimos MTS em um plasmídeo contendo marcado-RFP dominante negativo Cdk1, o que nos permitiu bater especificamente para baixo a expressão mitocondrial e funções de Cdk1. É essencial para distinguir entre as funções mitocondriais e nucleares das quinases que têm localizações dupla como Cdk1. Engenharia MTS para o N-terminal destas cinases funcionais duais oferece uma grande estratégia que é fácil de ser utilizado e eficaz. Desde Cdk1 é uma quinase do ciclo celular, que é fundamental para determinar a progressão do ciclo celular quando Cdk1 está localizada dentro da mitocôndria. Para conseguir isso, nós utilizamos uma nova metod para monitorizar o conteúdo em ADN em células. Os métodos tradicionais incluem a utilização de BrdU (bromodesoxiuridina), um análogo da timidina sintética, que incorpora-se no ADN sintetizado de novo durante a fase S do ciclo celular para substituir timidina. Em seguida, as células que estão a replicar o seu DNA activamente pode ser detectada utilizando anticorpos anti-BrdU. Uma desvantagem deste método é que ele exige a desnaturação de ADN para fornecer acesso para o anticorpo BrdU por métodos agressivos como tratamento com ácido ou calor, o que pode resultar em inconsistência entre os resultados 13,14. Alternativamente, utilizou-se uma abordagem semelhante para monitorar as células que se dividem activamente com um análogo da timidina diferente, EdU. detecção EdU não requer desnaturação DNA dura como o tratamento detergente suave permite que o reagente de detecção para acessar o EdU no DNA recém-sintetizado. O método EdU provou ser mais confiável, consistente e com potencial para a análise de alto rendimento 15. Finalmente, to determinar os substratos mitocondriais de Cdk1, usamos uma ferramenta proteômica chamado 2D DIGE, que é uma versão avançada do eletroforese em gel clássico bidimensional. Electroforese bidimensional separa as proteínas de acordo com o seu ponto isoeléctrico na primeira dimensão e o peso molecular na segunda. Uma vez que as modificações pós-traducionais, tais como fosforilação afectar o ponto isoeléctrico e o peso molecular das proteínas, géis 2D podem detectar diferenças entre os estados de fosforilação de proteínas nas amostras diferentes. O tamanho (área e intensidade) de proteína acaba alterações com o nível de expressão de proteínas, permitindo a comparação quantitativa entre múltiplas amostras. Usando este método, fomos capazes de diferenciar as proteínas fosforiladas no tipo selvagem contra células que expressam CDK1 segmentada por mitocôndrias mutantes. Os spots de proteínas específicas que mostraram no tipo selvagem, mas estavam faltando no mutante preparação Cdk1 segmentada por mitocôndrias foram isoladas eidentificado através de espectrometria de massa. Nos geles 2D tradicionais, corantes de trifenilmetano são utilizados para visualizar as proteínas no gel. 2D DIGE usa rótulos proteína fluorescente com um efeito mínimo sobre a mobilidade eletroforética de proteínas. Diferentes amostras de proteínas podem ser marcados com diferentes corantes fluorescentes, misturados entre si e separadas por os géis idênticos, permitindo que a co-electroforese de amostras múltiplas com uma única gel 16. Isto minimiza as variações de gel-a-gel, o que é um problema crítico nos estudos proteómica à base de gel.

<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.

Localização sub-mitocondrial de CyclinB1 e Cdk1 extracção de carbonato de sódio é usado para determinar se uma proteína está localizada no interior da mitocôndria ou na superfície exterior, ou seja, da membrana externa. Uma vez que uma proteína é mostrada para localizar dentro da mitocôndria, nova determinação da localização sub-mitocondrial pode ser feita através de mitoplasting combinado co...

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.

Abordagens experimentais para estudar mitocondrial Localização e função de um ciclo celular Nuclear Kinase, Cdk1

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

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11.8K visualizações.  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. 

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

Experimental Approaches to Tissue Engineering

Abordagens experimentais de Engenharia de Tecidos

Biologia

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.

Visualizando Localização RNA em Xenopus Oócitos

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.

Usando a resposta optocinético para estudar a função visual de 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 ...

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.

Atividades de monitoramento quinases e fosfatases através do ciclo celular por raciométrica 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.

Análise da posição do ciclo celular em células de mamíferos

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.

Avaliação de funções mitocondriais e viabilidade celular em células renais Proteína Kinase C superexpressão Isozimas

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).

Imunofluorescência combinados e FISH DNA em 3D preservado núcleos interfásicos para estudar mudanças na organização nuclear 3D

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.

Um método de triagem e Validação de mutações resistentes aos inibidores da quinase

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.

A Free Cell Ensaio para estudar Chromatin descondensação no fim da 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.

Um ensaio-Free Cell Usando<em&gt; Xenopus laevis</em&gt; Embryo Extrai para estudar mecanismos de regulação Tamanho Nuclear

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 ...