This work was supported in part by NIH grants (1R01CA137494, R01CA132115, R01CA086072 to R.G.P.), the Kimmel Cancer Center NIH Cancer Center Core grant P30CA056036 (R.G.P.), a grant from the Breast Cancer Research Foundation, generous grants from the Dr. Ralph and Marian C. Falk Medical Research Trust (R.G.P.) and a grant from the Pennsylvania Department of Health (R.G.P.). In part this work was supported by an American Italian Cancer Foundation postdoctoral fellowship (G.D.) and Bioimaging Shared Resource of the Sidney Kimmel Cancer Center (NCI 5 P30 CA-56036).The Department specifically disclaims responsibility for an analysis, interpretations or conclusions. There are no conflicts of interest associated with this manuscript.

1. Eletroporação de Fibroblasto com Ambos Expression Vectors EYFP-Parkin e pDsRed2-Mito <ol><li> Cresça o rato imortalizada células de fibroblastos embrionários de 10 centímetros placa de cultura de tecidos, utilizando meio DMEM (meio de Eagle modificado por Dulbecco) suplementado com 10% de soro bovino fetal, 2 mmol / l de L-glutamina, 100 U / ml de penicilina, e 100 mg / mL estreptomicina, em atmosfera humidificada contendo 5% de CO2 a 37 ° C. <ol><li> Na confluência celular de 80%, descartar o meio DMEM completo por sucção estéril e adicionar 10 ml de solução tampão de fosfato de 1x estéril (PBS) (80 g de NaCl, 2,0 g de KCl, 14,4 g de Na 2 HPO 4, 2,4 g de KH 2 PO 4 e 1 L de H2O destilada, pH: 7,4). </li><li> Descartar o PBS por sucção estéril e adicionar 1 mL de Tripsina-EDTA a 0,25%. Incubar a placa a 37 ° C até as células são separadas (2-3 minutos). Adicione 4 ml de meio DMEM completo, voltar a suspender as células e tirar 10 _6; L da suspensão de células para a contagem em hemocitómetro. NOTA: O hemocitómetro é concebido de modo que o número de células em um conjunto de quadrados de canto 16 é equivalente ao número de células x 10 4 / ml. </li></ol></li><li> Semente de 1 x 10 6 células nas placas de 10 cm de cultura de tecidos de 24 horas antes do processo de electroporação. </li><li> Descartar o meio DMEM completo por sucção estéril e adicionar 10 ml de PBS estéril. Descartar o PBS por sucção estéril e adicionar 1 mL de Tripsina-EDTA a 0,25%. Incubar a placa a 37 ° C até as células são separadas (2-3 minutos). Adicionar 4 ml de meio DMEM completo e ressuspender as células em um tubo de 15 ml. </li><li> Rodar as células para baixo a 250 xg durante 5 min, utilizando a-centrifugadora refrigerada (4 ° C). Descartar o sobrenadante por aspiração estéril e ressuspender o sedimento em 1 ml de PBS estéril. Rodar as células para baixo a 250 xg durante 5 min a 4 ° C. </li><li> Descartar o sobrenadante por aspiração estéril, e adiciona-se 100 ul Of mix solução para eletroporação (82 ul de eletroporação Solução V + 18 ml de solução Suplementar 1) para o pellet celular e delicadamente ressuspender o sedimento pipetando (Para mais detalhes, consulte o manual do utilizador). Adicionar 2 ug de EYFP-Parkin (excitação / emissão de 514/527) e 1 ug pDsRed2-Mito (excitação / emissão de 565/620) a suspensão de células. </li><li> Transferir a solução para a cuvete estéril usando um pasteur descartável (ambas as ferramentas são fornecidos com o kit) e electroporate usando o programa pré-definido NIH / 3T3 U-030 (pulso único, tensão de 200 V, capacitância 960 mF, tempo de pulso de 20 milissegundos , número de pulsos: 1). </li><li> Imediatamente após a electroporação, adicionar 500 uL de meio DMEM completo pré-aquecido frescos e as sementes da célula em um-processamento de imagem de grau directo de 6 centímetros de cultura de tecidos de placa e incubar-los numa atmosfera humidificada contendo 5% de CO2 a 37 ° C durante 24 horas. </li></ol> 2. Time-Lapse Vídeo Microscopy <ol><li&gt; Ajustar a temperatura da câmara do microscópio a 37 ° C antes de usar. </li><li> Neste ponto, se preparar um meio específico de imagens ao vivo para prosseguir com o protocolo experimental. Prepare o suporte de imagens ao vivo da seguinte forma; misturar DMEM isento de fenol suplementado com 10% de soro bovino fetal, 2 mmol / l de L-glutamina, 100 U / ml de penicilina, e 100 mg / ml de estreptomicina. Pré-aquecer o meio de imagens ao vivo a 37 ° C num banho de água. </li><li> Descartar a forma das células electroporadas por sucção estéril e adicionar 1 ml de meio de imagens ao vivo pré-aquecido, usando uma pipeta P1000 e incubar a placa durante 30 minutos a 37 ° C. </li><li> Durante o período de incubação da placa, influxo de 5% de CO 2 na câmara do microscópio já a uma temperatura estável de 37 ° C. </li><li> Coloque a placa com as células electroporadas à câmara do microscópio evitar grandes movimentos ou oscilações. </li><li> Utilizando o software de interface, definir o microscópio, a fim de detectar tanto fluorésinais scence de as proteínas de fusão codificadas pelos vectores co-transfectados, EYFP-Parkin (gama Excitação 495-510 nm e faixa de emissão de 520-550 nm, verde) e o pDsRed2-Mito (excitação máximo 558 nm e de emissão de máximo 583 nm, vermelho). <ol><li> Abra o software de vídeo microscopia de lapso de tempo. No menu superior, selecione a FITC para EYFP-Parkin e Rodamina para pDsRed2-Mito. No menu superior, selecione a ampliação (20x). </li></ol></li><li> Usando a interface do software, procure a única célula expressando ambos os vectores de co-transfectadas e registrar a posição. Repita este passo até um mínimo de 10 células são coletadas para cada condição experimental (grupo experimental). <ol><li> Selecione o menu &quot;Aplicativos&quot; e clique em Aquisição multi-dimensional. Nas janelas de aquisição multi dimensional seleccionar os parâmetros necessários, tais como o número de aquisições, o intervalo de tempo entre cada uma das aquisições e a posição da célula registada. Clique em &quot;Adquirir&quot;Para iniciar o processo de aquisição. </li></ol></li><li> Comece a aquisição basal de ambos EYFP-Parkin e sinal fluorescente DsRed2-Mito recolher imagens a cada 5 minutos para um intervalo total de 15 min. </li><li> Prepare meio de imagem ao vivo pré-aquecido com uma concentração de FCCP duas vezes mais elevada do que a concentração final de trabalho (0,1-10 uM, dependentes de tipo de célula). </li><li> Interromper o processo de aquisição e adicionar suavemente 1 ml de meio de imagens ao vivo pré-aquecido com FCCP para a placa dentro da câmara do microscópio utilizando uma pipeta P1000. </li><li> Reiniciar o processo de aquisição, como anteriormente descrito, para um período total de 3 horas, utilizando a posição registada. Salve todas as imagens adquiridas, a fim de analisá-los e criar um vídeo do processo mitophagic quando a aquisição é longo. </li></ol> 3. Análise <ol><li> Recolha todas as imagens adquiridas em formato &quot;tiff&quot; em um computador pessoal </li><li> Abra as imagens com um pano macio gráficoWare e definem o intervalo de tempo ocorreu a partir da mitocondrial induzida por despolarização com FCCP para a primeira imagem que mostra o recrutamento de Parkin na membrana mitocondrial (as imagens são adquiridas a cada 5 minutos). Repetir esta análise para cada célula no grupo experimental. </li><li> Rotular a primeira célula da coluna, numa folha de cálculo com o nome do grupo experimental. Para cada grupo experimental, medir os intervalos temporais para Parkin recrutamento de um mínimo de 10 células. Realizar uma média dos intervalos temporais calculados para o recrutamento e Parkin definir o desvio padrão para cada grupo experimental (Média ± DP, n = 10). <ol><li> Organizar-se em colunas os intervalos temporais calculados individuais. Cada coluna contém o n = 10 medidas do grupo experimental. </li><li> Seleccione a função de &quot;médio&quot; a partir do menu Formula Builder. Seleccionar os dados na coluna. Seleccione a função &quot;Desvio Padrão&quot; no menu Formula Builder. SeleCT os dados na coluna. </li></ol></li><li> Usando uma abordagem estatística, comparar os grupos experimentais que aplicam a estatística apropriada, a fim de definir uma diferença significativa na dinâmica de mitophagy entre os grupos experimentais. (Por exemplo, dois grupos = t de Student, três ou mais grupos = ANOVA além de um teste post-hoc) </li></ol>

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The authors have nothing to disclose.

microscopia de lapso de tempo pode ser definido como a técnica que se estende imagem de células vivas a partir de uma única observação em tempo para a observação da dinâmica celular durante longos períodos de tempo. Esta metodologia é distinguida de uma simples confocal ou microscopia de células vivas porque permite que o observador identificar em tempo real, com uma única proteína fluorescente marcado com e seguir a sua dinâmica dentro de uma única célula viva. Na verdade, a microscopia confocal pode facilmente identificar proteínas marcadas com imuno utilizando anticorpo fluorescente, mas não permite a observação da célula e o evento molecular no seu ambiente real. A microscopia de lapso de tempo vídeo é usado principalmente em pesquisa monitoramento de processos moleculares em diferentes tipos de células, tais como HEK293 16, neurônios primários 17 e células Hela 18 mas também tem aplicação clínica tais como a gravidez e prever aneuploidia 19,20. microscopia de lapso de tempo fornece embryologistas com informações adicionais sobre o desenvolvimento do embrião para uma gravidez clínica, e é possível que esta informação pode ser usada para melhorar a nossa capacidade de selecionar embriões viáveis ​​para transferência. Outras vantagens do uso de microscopia de lapso de tempo incluem menor manipulação de embriões e os seus pratos para observações intermediárias, o que reduziria o risco de perda ou contaminação dentro do laboratório 19. As células são capazes de manter a homeostase mitocondrial saudável, degradando mitocôndrias danificadas e disfuncionais através mitophagy. Mitophagy defeituoso está ligado à doença de Parkinson 21, a apoptose 22, o envelhecimento 23 e câncer 24. Mitophagy é impulsionado pela ligase de ubiquitina e Parkin PINK1 21. Devido à importância deste processo para manter um status mitocondrial saudável, achamos que é importante estudar a dinâmica deste evento usando um microscópio de vídeo time-lapse. O pro propostaprotocolo irá fornecer evidências sobre como homeostase mitocôndria é afectada sob diferentes condições de celulares, incluindo mudanças nas funções de expressão de genes e proteínas. Neste estudo, foram transientemente transfectadas por electroporação fibroblastos. O método de electroporação aplica um impulso eléctrico com uma tensão optimizada durando alguns microssegundos para a suspensão de células, a fim de aumentar a permeabilidade da membrana celular por perturbação da bicamada de fosfolípido da membrana. Esta acção resulta na formação de poros temporários. A permeabilização da membrana permite que o ADN a ser introduzido para dentro da célula 25. Nós escolhemos este processo para transfectar transientemente fibroblastos, a fim de ter uma maior eficiência de transfecção de ambos os plasmídeos EYFP-Parkin e pDsRed2-Mito. De facto, o processo de electroporação é cerca de dez vezes mais eficaz do que a transfecção química 26. O processo de aquisição das imagens pode ser comprometida por tanto extefatores rnal; flutuações de temperatura, vibrações, humidade e fatores internos; pH, motilidade celular. A eletroporação e rastreamento fluorescentes são dois passos considerados os mais críticos para o sucesso deste protocolo experimental. Mesmo que a eficiência de transfecção é mais elevada do que outros métodos, a electroporação mata ~ 20% das células electroporadas. Outro grande problema é a qualidade de aquisição das imagens durante a etapa de monitorização. Uma vez que o ambiente celular é dinâmico, o plano de focagem precisa ajustamentos contínuos, a fim de manter o foco ao longo do processo de aquisição. dano mitocondrial pode ser provocada pela despolarização da membrana mitocondrial e stress oxidativo. Estes factores induzem uma resposta mitophagic. A prova de que as mitocôndrias despolarização pode induzir mitophagy seletiva vem de experimentos photodamage que levam à produção de ROS e lesão mitocondrial 27-30. De modo a induzir MEM mitocondrialdespolarização brana e resposta mitophagic, FCCP foi usado como protonophore 10. FCCP é um ionóforo que permite protões para atravessar bicamadas lipidicas que induzem a despolarização da membrana. Usando essa abordagem, induzida mitophagy e seguiu a dinâmica do marcador molecular. A concentração de FCCP é crucial, uma vez que pode ser citotóxico e promovem a morte celular. Uma vez que esta abordagem baseia-se na observação de células vivas, a concentração final de FCCP tem de ser ajustado de uma forma dependente do tipo de célula de modo a induzir mitophagy sem desencadear os mecanismos que conduzem à morte celular. No geral, nós pensamos que na sequência da dinâmica de mitophagy, usando EYFP-Parkin como um marcador fluorescente, irá fornecer novos insights sobre a função mitocondrial. A remoção selectiva de mitocôndrias através mitophagy é um processo crucial na manutenção da qualidade mitocondrial, a viabilidade celular e a homeostase. Esta abordagem ao vivo Imaging elelp para entender o processo mitophagic em condições saudáveis ​​e estressado. abordagens modernas estão ampliando ainda mais observações de microscopia de lapso de tempo para além de fazer filmes de dinâmica celular. Cada vez mais este limite é turva como técnicas de citometria estão sendo integradas com técnicas de imagem para o monitoramento e avaliação das actividades desenvolvidas dinâmicas de células e estruturas subcelulares 31.

Macro autophagy is an intracellular process that involves the catabolic degradation of both damaged and dysfunctional cellular components, such as organelles and proteins for the purpose of either recycling or energy production. To initiate this metabolic process, the cell engulfs the damaged cellular components into a double-membrane structure, known as an autophagosome, which fuses with a lysosome and its content is degraded and recycled 1,2. There are two major types of autophagy, the non-selective and selective. The non-selective autophagy process occurs when the cell is under nutrient deprivation conditions and needs to scavenge for both essential nutrients and energy. However, selective autophagy occurs to mediate the removal of both dysfunctional/damaged organelles and proteins that otherwise could be toxic. One of the most studied selective autophagy process is the removal of mitochondria, termed mitophagy 1,3-5.
Mitochondria are the central organelles for cell metabolism and the primary source of adenosine triphosphate (ATP) via oxidative phosphorylation through the electron transport chain, fatty acid oxidation, and tricarboxylic acid (TCA) cycle. Moreover, mitochondria regulate reactive oxygen species (ROS) production and release proteins that participate in cell death pathways 6-8.
PTEN-induced putative kinase 1 (PINK1) and Parkin RBR E3 ubiquitin ligase (Parkin) are the key proteins implicated in the mitophagy process. Parkin can protect against cell death by keeping the cell healthy through mitochondrial quality control9. Upon the loss of mitochondrial membrane potential, cytosolic Parkin is recruited to the mitochondria by PINK1. This recruitment triggers the sequential events of mitophagy 10. There is a broad range of evidence that mitophagy is a fundamental mitochondria quality control process and abnormalities in this process drive disease 7. For instance, autosomal recessive Parkinson's disease has been associated with mutations in the genes that encode for Parkin and PINK1 (PARK2 and PINK1, respectively) 11. The quality control of mitochondrial health is essential for the removal of mitochondria that contribute to the accumulation of ROS12. Excessive presence of intracellular ROS can lead to damage of both nuclear and mitochondrial DNA (DNA and mt DNA, respectively).
Herein, we show a time-lapse video microscopy approach to follow the aggregation of Parkin after the induction of Parkin-mediated mitophagy in immortalized mouse embryonic fibroblasts via in vitro administration of carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP), an uncoupling agent. FCCP disrupts ATP synthesis by short circuiting protons across the outer mitochondria membrane and hence uncoupling oxidative phosphorylation from the electron transport chain 13. Triggering the depolarization of the mitochondrial membrane leads to the disruption of mitochondria and selective Parkin-dependent removal. Therefore, transfecting the cells of interest with an expression vector encoding Parkin fused with a fluorescent marker (enhanced yellow fluorescent protein, EYFP) can be used as a fluorescent tag to follow the recruitment of Parkin during the mitophagic process. In order to visualize the mitochondria, we co-transfected pDsRed2-Mito, which encodes red fluorescent protein (DsRed2) that contains a mitochondrial targeting sequence of cytochrome c oxidase subunit VIII (Mito). pDsRed2-Mito is designed for fluorescent labeling of mitochondria14. The time required for Parkin translocation into the mitochondrial membrane can be measured and gives an indirect measure of cellular health. For example, we can say that if a cell line knocked-out for a particular gene of interest shows either a faster or slower recruitment of Parkin after the induction of mitophagy by FCCP, that gene product would be a key player in order to keep the metabolic rates of the cell at the physiological status and prevent the development of diseases. Therefore, the time-lapse video microscopy provides a very powerful tool for both basic and clinical research applications in following the dynamic of labeled proteins during their molecular processes and understanding how these processes are affected during a pathological condition.

<table border="0" cellpadding="0" cellspacing="0" width="1736">
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">DMEM</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">10-013-CV</td>
			<td style="width:369px;">Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">PHENOL-FREE DMEM</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">17-205-CV</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">FETAL BOVINE SERUM</td>
			<td style="width:357px;">Sigma-Aldrich</td>
			<td style="width:276px;">F2442</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">L-GLUTAMINE</td>
			<td style="width:357px;">Gibco</td>
			<td style="width:276px;">25030</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">PENICILLIN/ STREPTOMYCIN</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">30-002-CI</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">EYFP-PARKIN EXPRESSION VECTOR</td>
			<td style="width:357px;">Addgene</td>
			<td style="width:276px;">23955</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">pDsRed2-Mito EXPRESSION VECTOR</td>
			<td style="width:357px;">Clontech</td>
			<td style="width:276px;">632421</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">NUCLEOFECTOR 2B DEVICE</td>
			<td style="width:357px;">LONZA</td>
			<td style="width:276px;">AAD-1001S</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">NUCLEOFECTOR FOR KIT R NIH/3T3</td>
			<td style="width:357px;">LONZA</td>
			<td style="width:276px;">VCA-1001</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">ZEISS AXIOVERT 200M INVERTED MICROSCOPE</td>
			<td style="width:357px;">CARL ZEISS</td>
			<td style="width:276px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Carbonyl Cyanide 4-(trifluoromethoxy)-Phenylhydrazone (FCCP)</td>
			<td style="width:357px;">Sigma-Aldrich</td>
			<td style="width:276px;">C2920</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">MetaMorph</td>
			<td style="width:357px;">Molecular Devices</td>
			<td style="width:276px;">Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:735px;">ImageJ</td>
			<td style="width:357px;">National Institute of Health</td>
			<td style="width:276px;">Experimental Builder</td>
			<td></td>
		</tr>
	</tbody>
</table>

time-lapse video microscopy for assessment of eyfp-parkin aggregation as a marker for cellular mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Aqui, mostramos como microscopia de lapso de tempo vídeo é uma técnica poderosa que pode ser usado para acompanhar os acontecimentos moleculares de proteínas fluorescentes-marcado em uma única célula. Os resultados representativos também mostram como esta técnica permite a aquisição de imagens de alta qualidade. Quando as imagens dos processos moleculares, são obtidas, que têm a oportunidade de analisá-los de modos diferentes. Aqui, analisamos o intervalo de tempo entre o início do processo de mitophagy induzida, que é o processo fundamental que molecular manter a homeostase celular remover selectivamente as mitocôndrias danificadas. Esta técnica funda uma ampla gama de aplicações. Pode ser usado tanto para o acompanhamento macro processos celulares, tais como migração de células ou eventos moleculares micro, através da marcação das moléculas-chave envolvidas no processo que tem que ser estudado. o: manter-together.within-page = &quot;1&quot;&gt; Parkin mediada Mitophagy A remoção selectiva Parkin-mediada de processo mitocôndrias danificado e disfuncional está resumida na Figura 1, onde uma despolarização da membrana induzida pelo agente de desacoplamento FCCP desencadeia mitophagy selectiva Parkin-mediada, recrutando PINK1 para a membrana mitocondrial externa. A seguir, Parkin é recrutado para as mitocôndrias danificadas e interage com PINK1. Este complexo Parkin-PINK1 leva à ubiquitinação da organela danificado e sua incorporação em um autophagosome, que se funde com uma lisossoma. Este processo conhecido como mitophagy é considerado como um processo metabólico essencial, a fim de manter o ambiente celular saudável e permitir a regeneração das mitocôndrias funcionais 8,15. Protocolo experimental NT &quot;FO: manter-together.within-page =&quot; 1 &quot;&gt; A Figura 2 mostra uma representação esquemática do protocolo experimental e o microscópio set-up usado para seguir o recrutamento de EYFP-Parkin antes e após o processo de mitophagy induzida por FCCP. Parkin recrutamento para despolarizado Membrana Mitocondrial Na Figura 3A, mostramos a distribuição Parkin administração anterior e pós FCCP. Durante os pontos no tempo basal (BT5, BT10 e BT15 min) EYFP-Parkin (Verde) é difundido de forma homogénea por toda a célula e a rede de mitocôndrias (vermelho) parece ser bem interligado. Após a administração FCCP (FT = 0 min) (Figura 3B), observa-se o fraccionamento mitocôndrias (FT = 5 min). No entanto, EYFP-Parkin ainda está homogeneamente difundida embora fora do citoplasma. Mitophagy é estimulado a 55 min administração pós FCCP,EYFP-Parkin é recrutado nas membranas mitocondriais danificados para desencadear o processo mitophagy (FT = 55). <img alt="figura 1" src="/files/ftp_upload/53657/53657fig1.jpg" /> Figura 1. Representação esquemática da Mitophagy Parkin-mediada. Uma diminuição do potencial de membrana (Ψm) ocorre nas mitocôndrias danificados ou disfuncionais e pode ser induzida pelo agente de desacoplamento FCCP. A perda do potencial de membrana desencadeia mitophagy selectiva Parkin-mediada, recrutando PINK1 (vermelho) para a membrana mitocondrial externa. Parkin (azul) é recrutado para as mitocôndrias danificadas, interagindo diretamente com PINK1. A presença de Parkin leva à ubiquitinação (amarelo) da organela danificado e sua incorporação em um autophagosome, que se funde com uma lisossoma. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53657/53657fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura</a>. <img alt="Figura 2" src="/files/ftp_upload/53657/53657fig2.jpg" /> Figura 2. Microscópio Set-up e Representação do protocolo experimental. (A) Visão geral dos eventos experimentais descritos na seção de protocolo. Os eventos experimentais relatados permitem ao operador transitoriamente co-transfectar as células com EYFP-Parkin e pDsRed2-Mito por eletroporação, a fim de acompanhar o processo mitophagy induzida por FCCP usando um (B) set-up para time-lapse abordagem microscopia de vídeo. BT = ponto de tempo basal; FT = FCCP ponto de tempo. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53657/53657fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53657/53657fig3.jpg" /> Figura 3. celular de fibroblastos Mostrando Parkin Recruitme<html> nt depois de Membrana Mitocondrial despolarização induzida por EYFP-Parkin (verde) translocação para a mitocôndria (vermelho) FCCP. foi monitorada por microscopia de lapso de tempo vídeo durante o ponto de tempo basal (BT) e após a administração FCCP (FT). De BT = 0, EYFP-Parkin e fluorescência mitocondrial foram detectados a cada 5 minutos. (A) EYFP-Parkin é homogeneamente difundido em todo o período basal e a rede mitocondrial é intactos (setas brancas). (B) fracionamento mitocondrial devido à despolarização da membrana por FCCP é gravado em FT = 5 min (setas brancas) e recrutamento EYFP-Parkin às membranas mitocondriais danificados começou no FT = 55 min (setas brancas). As caixas brancas representam a área ampliada dos painéis (5x). Para mais detalhes sobre a aplicação deste protocolo experimental referem-se a referência 8. (Barra de escala: 10 mm).&gt; Por favor, clique aqui para ver uma versão maior desta figura.

Watch this Scientific Journal Video about Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy at JoVE.com

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-Lapse Vídeo Microscopia de Avaliação da EYFP-Parkin Agregação como um marcador para Cellular Mitophagy

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

time-lapse-video-microscopy-for-assessment-eyfp-parkin-aggregation-as

Nanyang Technological University

Research

JoVE Journal

Biology

7.1K visualizações.  Thomas Jefferson University, SKCC. Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Vídeo: Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

Monitoramento Actina Desmontagem com a Time-lapse Microscopia

Biologia

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Lapso de tempo de imagens de Mitose Depois de Transfection siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Um Ensaio de microscopia de fluorescência para Mitophagy Monitoramento no fermento Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Lapso de tempo Microscopia de embriogênese inicial em Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Análise Quantitativa de Migração aleatória de células usando microscopia de lapso de tempo vídeo

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

Uso de Microscopia Time Lapse para Visualize Anoxia induzida Suspended Animation em<em&gt; C. elegans</em&gt; Embriões

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualização de Desenvolvimento Craniofacial nas SOX10: Kaede Transgênicos Zebrafish Linha Usando Time-lapse microscopia confocal

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Métodos de estudo mudanças na agregação de proteínas inerente com a idade em Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Microscopia confocal revela agregação de receptores de superfície celular através de espectroscopia de correlação de imagem

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

Um método versátil para a montagem de Arabidopsis deixa para Intravital imagem de lapso de tempo

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...