Name: subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment
Uploaded: 2017-09-25T15:00:00.000+00:00
Duration: 7 min 55 s
Description: Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Este trabalho foi apoiado por uma comunhão de Ellison/AFAR pós-doutorado no programa de pesquisa de envelhecimento de SJK, e um prêmio da Fundação de Glenn e NIH concede ao PC (1P01AG034906, 1R01GM 090311, 1R01ES 020812). Todos os autores aparecem no filme.

1. tratamento de peptídeos às células <ol><li>placa 2 milhões SH-SY5Y ou células HEK293 (2 x 10 6) em um 10 cm do prato e cultivá-las por 2 dias</li> <li>(opcional) no outro dia, lave as células com Dulbecco isento de soro &#39; s modificada Águia mídia médio (DMEM) uma vez e incubar com soro DMEM livre durante a noite, se o tratamento deve ser feito em meios livres de soro.</li>
	<li>No dia 3, dissolver peptídeos de S14G-humanin em 0,2 µm filtrado, água destilada e reconstituí-los como solução estoque de 1mM. 
	Nota: Os peptídeos devem ser dissolvidos em solvente apropriado, que pode ser determinado pelas características sequência do peptide (por exemplo, carga geral) e pode ser fornecido pelo fabricante se os peptídeos são comercialmente disponíveis. Por exemplo, S14G-humanin, SHLP2 e SHLP6 e MOTS-c se dissolvem na água. Alíquota os peptídeos em um volume adequado (por exemplo, 50 μL) para evitar congelar e descongelar ciclos. Uma vez descongelado, não usar outra vez.</li>
	<li>Alíquota do soro-contendo pré-aquecido ou media serum-free em um Erlenmeyer de 50 mL do tubo e adicionar 1 mM temperatura de solução para torná-lo em uma concentração de trabalho (por exemplo, 1 µM, 10 µM).</li>
	<li>Substituir a mídia com 6 mL de solução de peptídeo da etapa 1.4. e incube as celulas para a quantidade adequada de tempo. 
	Nota: Escolher o tempo de incubação, dependendo de sua condição que ativa ERK.</li>
</ol> 2. Fraccionamento subcelular para o citoplasma e núcleo <ol><li>lavar as células duas vezes com 10 mL de PBS gelado, raspar as células no prato em 5 mL de PBS gelado e transferir a suspensão de células para um tubo cônico de 15 mL.</li>
	<li>Centrifugar as células a 500 x g por 5 min a 4 ° C.</li>
	<li>Aspire a PBS e ressuspender o sedimento em 200 µ l de tampão de fracionamento gelada, (pH HEPES 10mm = 7,6, 3 mM MgCl 2, 10 mM KCl, 5% (v/v) glicerol, 1% não iónico (C 13 H 22 O (C 2 H 4 O) n), inibidores da protease/fosfatase). 
	Nota: Para manter as proteínas no seu estatuto de fosforilação, o inibidor da fosfatase como bem como protease inibidor deve ser adicionado recentemente e as amostras devem ser mantidas no gelo em todos os tempos. Repetição de congelamento e degelo ciclos de amostras devem ser evitados. Amostras de alíquotas em pequena quantidade (por exemplo, 50 μg) antes de congelá-los a -80 ° C.</li>
	<li>Incubar a pelota re-suspensa no gelo por 15 min e em seguida centrifugar a 250 x g por 5 min a 4 ° C.</li>
	<li>Recolher o sobrenadante como a fração citoplasmática e manter a pelota para a fração nuclear.</li>
	<li>De centrifugação o sobrenadante para remover restos celulares e outros contaminantes a 18.000 x g durante 10 minutos a 4 ° C e depois transferir o sobrenadante para um novo tubo de microcentrifugadora. Esta é a fração do citosol.</li>
	<li>Resuspenda o pellet em tampão de lavagem gelada 200 μL (pH HEPES 10mm = 7,6, 1,5 mM de MgCl 2, 420 mM de NaCl, 25% (v/v) glicerol, 0,2 mM EDTA, inibidores da protease/fosfatase) e centrifugar 250 x g por 5 min a 4 ° C</li>
	<li>Remover o sobrenadante e ressuspender o precipitado em 100 μL gelada, nuclear solução tampão (pH HEPES 20 mM = 7,6, 1,5 mM de MgCl 2, 420 mM de NaCl, 25% (v/v) glicerol, 0,2 mM EDTA, inibidores da protease/fosfatase) e proceda à sonicação 10 vezes (5 s, 10 s fora, 30% amp.) na Ice.</li>
	<li>Centrifugar a 18.000 x g durante 10 minutos a 4 ° C.</li>
	<li>Transferir o sobrenadante para um novo tubo de microcentrifugadora. Esta é a fração nuclear.</li>
	<li>Quantify a quantidade de proteína usando um ensaio BCA</li></ol>
 3. fração mitocondrial bruto <ol><li>lavar as células em cada prato de 10 cm com 10 mL de PBS gelado, adicionar 5 mL PBS gelado e separar as células com uma espátula de celular. 
	Nota: Use três pratos de 10cm de células para obter um bom rendimento da mitocôndria para Western Bloting.</li>
	<li>Transferir a suspensão de células para um tubo cônico de 15 mL e combinar tudo a partir de três pratos de 10 cm em um tubo cônico de 15 ml.</li>
	<li>Centrifugar as células a 600 x g durante 10 minutos a 4 ° C.</li>
	<li>Aspire a PBS e ressuspender o sedimento em 1 mL de tampão de isolamento de mitocôndrias gelada (10 mM Tris-MOPS, 1 mM Tris/EGTA, 200 mM de sacarose, ajustar o pH = 7,4).</li>
	<li>Homogeneizar as células com 25 golpes de um homogeneizador de 2 mL com pilão revestida de politetrafluoretileno na Ice. 
	Nota: Este passo é fundamental para manter a integridade mitocondrial e maximizar o rendimento da fração mitocondrial. O número de traços deve ser otimizado para cada tipo de célula. Precool o homogenizador antes de iniciar o procedimento.</li>
	<li>Transferir o homogeneizado para um tubo de microcentrifugadora e centrifugar, a 600 x g durante 10 minutos a 4 ° C para remover núcleos e células ininterrupta.</li>
	<li>Recolher o sobrenadante, transferi-lo para um novo tubo de microcentrifugadora e centrifugá-lo em 7.000 x g durante 10 minutos a 4 ° C. 
	Nota: A pelota é solta, recolher o sobrenadante com cuidado e tente não perturbar a pelota.</li>
	<li>Remover o sobrenadante e ressuspender o sedimento com 200 μL de tampão de isolamento de mitocôndrias gelada, transferir a solução para um tubo de microcentrifugadora novo. Centrifugar o tubo a 7.000 x g durante 10 minutos a 4 ° C.</li>
	Repita o passo 3.8 para lavar o sedimento de <li>. Não é necessário transferir o sobrenadante para um novo tubo de microcentrifuga nesta etapa.</li>
	<li>Retire o pellet lavado o sobrenadante e ressuspender o sedimento contendo mitocôndria com 50 μL de tampão RIPA.</li>
	<li>Incubar a suspensão no gelo durante 10 min.</li>
	<li>Centrifugar a suspensão de 16.000 x g, durante 15 min a 4 ° C.</li>
	<li>Transferir o sobrenadante para um novo tubo de microcentrifugadora. Esta é a fração mitocondrial.</li>
	<li>Quantificar a quantidade de proteína usando um ensaio BCA.</li>
</ol> 4. Mancha ocidental para Phospho-específicas proteínas <ol><li>realizar uma eletroforese em gel de SDS-poliacrilamida (gel de 8-16% premade) e a proteína de transferência para uma membrana PVDF 4.</li>
	<li>Incubar a membrana com 5% BSA em TBST (0,1% polissorbato 20) por 30 min à temperatura ambiente para bloquear sites de fundo inespecificas. 
	Nota: Para a deteção de proteína fosforilada, bloquear a membrana com BSA não leite. O leite contém caseína, um phosphoprotein abundante, o que resulta em alto sinal inespecífico.</li>
	<li>Incubar a membrana com o anticorpo primário (antifosfo-ERK1/2) a 4 ° C durante a noite.</li>
	<li>No dia seguinte, lave a membrana com TBST (0,1% polissorbato 20) três vezes por 5 min à temperatura ambiente.</li>
	<li>Incubar a membrana com anticorpo secundário (anticoelho HRP) por 1h à temperatura ambiente.</li>
	<li>Lavar a membrana com TBST (0,1% polissorbato 20) três vezes por 5 min à temperatura ambiente.</li>
	<li>Incubar a membrana com solução ECL e imagem da membrana no analisador de imagem.</li>
	<li>Tira a membrana com descascamento de buffer para 15 min a temperatura e a lavagem da membrana com TBST três vezes durante 5 min.</li>
	<li>Repita os passos 4.2 a 4.7 usando anticorpo anti-total ERK1/2.</li>
	<li>Para verificar a pureza de cada fração, executar SDS-PAGE e executar immunoblots usando anticorpos reconhecendo marcadores para cada compartimento (por exemplo, B1 jesuina para núcleo, GAPDH para o citosol e TOM20 para as mitocôndrias).</li>
</ol>

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Pinchas Cohen é sócio e consultor para Kelvin CohBar, Inc., Yen atuou como consultor da CohBar, Inc.

Aqui, temos demonstrado que humanin mediada por peptídeo ERK1/2 ativação ocorre em dois diferentes tipos de células, e a Localização subcellular de ERK1/2 registrados pode ser diferente dependendo das condições (por exemplo, dose de peptídeo, ponto do tempo e célula tipo). Tem sido demonstrado que humanin sinais através de dois receptores diferentes22,23, que pode explicar as diferenças na sinalização entre as linhas de duas células, bem c...

Estudos emergentes mostram que peptídeos derivados mitocondriais (MDPs) desempenham importantes funções no metabolismo e cytoprotection1,2,3. Compreensão da via de transdução de sinal na presença de MDPs nos dá insight sobre o mecanismo pelo qual o MDPs modular várias funções. O primeiro PDM identificado, humanin, foi mostrado para aumentar a quinase de sinal-regulado extracellular 1/2 (ERK1/2) fosforilação através de seu receptor ligação4,5. No entanto, o efeito a jusante de ERK1/2 ativação é ainda underexplored.
A cascata de ERK1/2 serve como um mediador essencial em uma variedade de processos celulares, incluindo a proliferação, migração celular, metabolismo celular, sobrevivência e apoptose6,7,8. Para orquestrar todos estes processos celulares distintos, a atividade e Localização subcellular de ERK1/2 estão fortemente regulamentados por fosfatases e proteínas de andaime9,10. Além de modificação pós-traducional, o vaivém dinâmico de ERK1/2 também regula sua sinalização função, atividade e especificidade11,12. ERK1/2 é principalmente localizadas no citosol13. Um conjunto de proteínas de ancoragem e andaime ajudar a reter ERK1/2 elementos do citoesqueleto, na superfície das organelas, ou difusa no citoplasma13. Após a estimulação, ERK1/2 é fosforilada e torna-se dissociado de suas proteínas de ancoragem, permitindo a translocação de ERK1/2 para outros compartimentos subcelulares, incluindo o núcleo, mitocôndrias, Golgi e lisossomos14, 15 , 16.
Embora humanin seja conhecido para ativar a via de sinalização ERK1/2, a ativação de ERK1/2 só é observada no lisado celular total. Conforme descrito anteriormente, desde que a Localização subcellular ERK1/2 desempenha um papel crucial em seu efeito a jusante, a análise de tanto a Localização subcellular e os níveis totais de fosforilada ERK1/2 é necessário para fornecer uma compreensão abrangente de ativação induzida por humanin de ERK1/2 e a ativação dos destinos a jusante.
Para entender as organelas do alvo do ativado ERK1/2, realizou-se o fraccionamento subcelular seguido pela mancha ocidental para fosforilada ERK1/2. Este método pode ser implementado facilmente como ele utiliza reagentes e equipamentos laboratoriais padrão. Os compartimentos subcellular isolados são de alta pureza, permitindo que o resultado deve ser interpretado direta. Immunostaining de ERK1/2 pode produzir resultados semelhantes. No entanto, certos compartimentos subcellular são relativamente difíceis de Visualizar e requerem métodos especiais de fixação e permeabilização. ERK1/2 níveis variam em compartimentos subcelulares, e esta variação pode causar sinais falsos positivos e falsos negativos quando se olha para o lisado celular total. Portanto, um immunoblot usando compartimentos subcellular isolados dá-em uma melhor compreensão da localização ERK1/2.
A versatilidade do método permite modificações do protocolo para investigar os efeitos de outros estimulantes, incluindo outros MDPs ou translocação de outras moléculas de sinalização como STAT3. Recentemente descobertos pequenos humanin, como peptídeos (corsários) são codificados da região de rRNA 16S onde humanin é codificado, e eles têm propriedades semelhantes mas distintas em relação ao HN17. Por exemplo, SHLP2 e SHLP3 ativam ERK1/2 após 8 h embora humanin ativa ERK1/2 dentro de 5 min. Localização subcellular exame de ERK1/2, em resposta a diferentes peptídeos nos dará uma melhor compreensão da biologia destes peptides. Emergentes evidências mostraram que a Localização subcellular das moléculas de sinalização desempenha um papel crucial em seus efeitos a jusante. Por exemplo, STAT3 é tradicionalmente conhecida para ser localizada principalmente no citosol em células de descanso, e então ele translocates até ao núcleo para ativar a expressão gênica em resposta a citocinas18. STAT3 também translocates à mitocôndria e regula o ciclo TCA e de produção de ATP19. Sobre regulamento de autofagia, diferente Localização subcellular de STAT3 modula autofagia em várias maneiras20. Por exemplo, nuclear STAT3 transcricionalmente regula genes relacionados autofagia e atua como um modulador de autofagia. Citoplasmáticos STAT3 constitutivamente inibe a autofagia interagindo com moléculas de sinalização de autofagia. Mitocondrial STAT3 inibe e impede mitophagy suprimindo o estresse oxidativo induzido por autofagia. Portanto, esse método de isolamento do compartimento subcellular é crucial para compreender o papel da outra sinalização moléculas bem como ERK1/2.

<table><tbody><tr><td>p44/42 MAPK (ERK1/2)</td><td>Cell signaling</td><td>9102</td><td>Dilution 1:1,000</td></tr><tr><td>phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)</td><td>Cell signaling</td><td>4370</td><td>Dilution 1:1,000</td></tr><tr><td>Lamin B1</td><td>Cell signaling</td><td>12586</td><td>Nuclear Marker, Dilution 1:1,000</td></tr><tr><td>GAPDH</td><td>Cell signaling</td><td>5174</td><td>Cytoplasmic marker, Dilution 1:2,000</td></tr><tr><td>Tom20</td><td>Santa cruz</td><td>SC-17764</td><td>Mitochondria marker, Dilution 1:2,000</td></tr><tr><td>anti-Rabbit-HRP conjugated</td><td>Cell signaling</td><td>7074</td><td>Dilution 1:30,000</td></tr><tr><td>RIPA Lysis and Extraction Buffer</td><td>ThermoFisher SCIENTIFIC</td><td>89900</td><td></td></tr><tr><td>100 mm Culture Dish</td><td>ThermoFisher SCIENTIFIC</td><td>12556002</td><td></td></tr><tr><td>HNG peptide</td><td>Genescript</td><td></td><td></td></tr><tr><td>25mm sylinge filter</td><td>ThermoFisher SCIENTIFIC</td><td>09-719A</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>MgCl2</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G9012</td><td></td></tr><tr><td>Triton X-100</td><td>ThermoFisher SCIENTIFIC</td><td>BP151-100</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>3609</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>Tris-base</td><td>ThermoFisher SCIENTIFIC</td><td>BP152-1</td><td></td></tr><tr><td>HCL</td><td>Sigma</td><td>H1758</td><td></td></tr><tr><td>PBS</td><td>Lonza</td><td>17-512F</td><td></td></tr><tr><td>Cell Scraper</td><td>FALCON</td><td>353085</td><td></td></tr><tr><td>Halt&amp;trade; Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>ThermoFisher SCIENTIFIC</td><td>78440</td><td></td></tr><tr><td>Thomas Pestle Tissue Grinder Assemblies with Smooth Pestles</td><td>Thomas Scientific</td><td>3432S90</td><td></td></tr><tr><td>Tween-20</td><td>ThermoFisher SCIENTIFIC</td><td>BP337-500</td><td></td></tr><tr><td>BSA</td><td>ThermoFisher SCIENTIFIC</td><td>BP1600-100</td><td></td></tr><tr><td>8-16% Mini-PROTEAN TGX Precast Protein Gels</td><td>BIO RAD</td><td>4561104</td><td></td></tr><tr><td>Mini Trans-Blot Module</td><td>BIO RAD</td><td>1658030</td><td></td></tr><tr><td>Trans-Blot Turbo Transfer System</td><td>BIO RAD</td><td>1704150</td><td></td></tr><tr><td>Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td><td>BIO RAD</td><td>1704272</td><td></td></tr><tr><td>Clarity Western ECL Blotting Substrates</td><td>BIO RAD</td><td>1705060</td><td></td></tr><tr><td>Restore Western blot stripping buffer</td><td>ThermoFisher SCIENTIFIC</td><td>21059</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>ThermoFisher SCIENTIFIC</td><td>11965-092</td><td></td></tr><tr><td>Sonicator, Medel: FB120</td><td>ThermoFisher SCIENTIFIC</td><td>695320-07-12</td><td></td></tr></tbody></table>

subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment

Peptídeos derivados mitocondrial (MDPs) são uma nova classe de peptídeos que são codificados por frames de leitura abertos pequenos dentro de outros genes conhecidos do genoma mitocondrial. MDPs têm uma grande variedade de efeitos biológicos como protegendo os neurônios da apoptose, melhorando marcadores metabólicos e protegendo as células da quimioterapia. Humanin foi o MDP primeiro a ser descoberto e o peptídeo mais estudado entre a família MDP. Os receptores de membrana e vias de sinalização a jusante de humanin foram cuidadosamente caracterizadas. MDPs adicionais tais como MOTS-c e SHLP1-6 foram descobertos mais recentemente e os mecanismos de sinalização ainda precisa ser elucidado. Aqui nós descrevemos um método de cultura com base de célula para determinar a função destes peptides. Em particular, técnicas de fracionamento celular em combinação com mancha ocidental permitem a determinação quantitativa da ativação e translocação de importante molécula de sinalização. Embora existam outros métodos de fracionamento celular, o descrito aqui é um método fácil e simples. Estes métodos podem ser usados para elucidar ainda mais o mecanismo de ação desses peptídeos e outros agentes terapêuticos.

Usando o procedimento apresentado aqui, tratamos de células HEK293 e SH-SY5Y com 1 μM e 100 μM S14G-humanin, um potente humanin analógico21, respectivamente, em completa mídia para os períodos de tempo indicado (Figura 1A e Figura 1 B). em seguida examinamos a forma fosforilada e total de ERK1/2 em Thr202/Tyr204 de extratos de proteínas totais...

Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Este protocolo descreve como estimulam as células com peptídeos derivados mitocondriais e avaliar a cascata de sinalização e localização de fosfo-proteínas.

Fraccionamento subcelular para activação de ERK tratamento de peptídeo derivado mitocondrial

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

subcellular-fractionation-for-erk-activation-upon-mitochondrial

Research

JoVE Journal

Biology

7.8K visualizações.  University of Southern California. Este protocolo descreve como estimulam as células com peptídeos derivados mitocondriais e avaliar a cascata de sinalização e localização de fosfo-proteínas.

Vídeo: Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Ensaio de Atividade da Proteína Quinase com ATP Radiomarcado

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Bioquímica

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Sondagem para a Atividade Complexo mitocondrial em células estaminais embrionárias humanas

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Biologia

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

Espaciais e temporais Análise do Active ERK na<em&gt; C. elegans</em&gt; germinativa

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Biologia do Desenvolvimento

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

Fracionamento celular de células U937, na ausência de centrifugação de alta velocidade

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Avaliação da Localização da Proteína Subocondrial em Budding Levedura Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

Um protocolo eficiente para avaliar a modulação da atividade de ERK em modelos iniciais da síndrome de Noonan do peixe-zebra por meio de microscopia FRET ao vivo e imunofluorescência

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

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 ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Um ensaio funcional para juncional Exame; Eletroporação de aderentes em células no Índio-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

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.

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 ...

Otimização MATLAB para Análise de Redes Mitocondriais em Neurociência

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. 
To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. 
This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

Autor em destaque: Um método automatizado otimizado para investigar receptores de ácido retinóico em mitocôndrias neuronais

The mitochondrial network is extremely complex, making it very challenging to analyze. A novel MATLAB tool analyzes live confocal imaged mitochondria in timelapse images but results in a large output volume requiring individual manual attention. To address this issue, a routine optimization was developed, allowing for speedy file analysis.

Análise Automatizada Otimizada da Modulação da Homeostase das Mitocôndrias Neuronais Vivas por Receptores de Ácido Retinóico Isoform-Específicos

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in ...