Agradecemos ao Centro Tufts para Neuroscience Research, P30 NS047243 (Jackson), a mitocôndria Affinity Pesquisa Colaborativa (mtARC), apoiado pelo Centro de Investigação Biomédica Evans Interdisciplinar da Universidade de Boston Medical Campus, Medicine Corporation Link, e Zeiss para apoiar este trabalho.

1. Preparação Placa Imagem <ol><li> Cultura INS1 células em meio RPMI contendo 10% de soro fetal bovino padrão, 1% de penicilina-estreptomicina, 2 mM de L-glutamina, 50 mM 2-mercaptoetanol, 5 mM de NaHCO3, 2 mM de HEPES, 2 mM de ácido pirúvico, e 11 mM de glucose a 80% de confluência. </li><li> Trypsinize as culturas de células INS1 com tripsina a 0,05% ea placa-los em poli-D-lisina placas revestidas com lamela de fundo de imagem (% de confluência 30-40). </li><li> Permitir para placas de atingir confluência de 60-80% (~ 2 dias) e adicionar matriz mitocondrial alvo (COXVIII) adenoviral PA-GFP durante 24 horas (MOI = 5). Meios câmbio e permitem que as células a crescer por mais 2 dias antes de imagem. </li><li> No dia da imagem, adicionar 7-15 TMRE nM para as placas de imagem, e equilibrar durante pelo menos 45 min. </li><li> Durante este por sua vez, tempo na incubadora (fase superior incubadora tem sido usado aqui) e permitir que o microscópio para equilibrar a 37 ° C durante cerca de 1 hora. Ligue o 5% de CO 2. </li&gt; </ol> 2. Parâmetros de imagem para a LSM 710 microscópio confocal Zeiss <ol><li> Após o microscópio tem equilibrado, sob a guia de Aquisição, clique em &quot;Ferramentas manual mostram&quot;, abra a imagem criada painel e escolha &quot;modo de canal&quot; e mude acompanhar cada &quot;frame&quot;. </li><li> No painel caminho da luz, escolha LSM e modo de canal. Trabalhando a partir do ícone do espécime para cima, selecione &quot;traseiro&quot;, MBS 690 +, e MBS 488. Na parte inferior do painel, escolher &quot;T-PMT&quot;, para permitir a visualização do campo brilhante ou canal DIC. </li><li> Termine o ajuste dos parâmetros de caminho de luz, definindo o intervalo para o corante GFP para 490-540 nm e 580-700 nm para o corante TMRE. </li><li> No modo de aquisição, use um de 512 × 512 pixels campo de leitura, 4x médios, e escolher um fator de zoom (que você pode querer manter consistente para fins de calibração). </li></ol> 3. Otimizar os parâmetros de imagem <ol><li> Usando o Apochromat plano 100x (1,4 NA) lente objetiva, focnos em suas células com a luz halógena não, luz fluorescente, para proteger as mitocôndrias de fototoxicidade. </li><li> Tela para PA-GFP células que expressam através de um orifício aberto e máxima de varredura para encontrar as células que são mais brilhantes verde. </li><li> Encontrar a mais baixa potência do laser 2-fotão necessária para activar a PA-GFP e optimizar os parâmetros de imagem que assegurem que o sinal não saturar o detector. Verificar que o sinal PA-GFP colocalizes com o sinal de TMRE para poucos z-série. Perda do sinal de TMRE indica despolarização mitocondrial ou fototoxicidade, e as células apresentando estas características não deve ser utilizado para a análise. </li><li> Encontre uma célula PA-GFP expressar e especificar um fator de zoom. </li><li> Defina o intervalo de z-série para coletar 6 fatias (este intervalo será necessário para satisfazer todas as 10 células a menos que um específico z foco é definida em cada posição). </li><li> Salve o método de imagem para cada célula (célula 1, célula 2 etc.) </li></ol> 4. Ajuste o AutomateParcela d do Programa e designar as 10 células a serem seguidas para o 1 ensaio de fusão Hora mitocondrial <ol><li> No painel esquerdo da janela multitime, selecione a opção &quot;salvar&quot; do painel, e especifique o local onde os arquivos serão salvos. </li><li> Em a &quot;Aquisição&quot; do painel, carregue o método de aquisição salvo para a célula 1 na configuração de digitalização e marque a caixa de pilha z. Além disso, escolha &quot;marcado Z-meio da pilha z&quot;. </li><li> No &quot;blocos&quot; do painel, selecione &quot;bloco único em cada local&quot;. Clique em &quot;Adicionar&quot; blocos para cada intervalo a ser medido. </li><li> No &quot;timing&quot; do painel, selecione &quot;esperar intervalo&quot; e digite &quot;0&quot; no &quot;intervalo de espera antes de bloco no primeiro local só&quot;. Blocos 2-4 terá &quot;15 minutos&quot; nesta seção. </li><li> No painel Local, escolha &quot;mover o foco para carregar posição entre os locais&quot; e &quot;carga varredura config quando &#39;mover-se para loc&quot; ou &quot;próximo loc&#39; clicado. Sob a&quot; lista de locais de edição &quot;escolher&quot; limpar tudo &quot;e selecione&quot; múltipla locais motorizada palco &quot;. </li><li> Under o &quot;Bleach&quot; do painel, clique em &quot;branquear&quot; caixa, e designar o arquivo de configuração no &quot;config&quot; no menu suspenso, como designado na janela do software principal. Em seguida, no &quot;branqueamento&quot; janela, salve o método de fotoativação apropriado. No painel regiões, escolha o ROI dessa célula em particular, e adicionar este ROI para o multitime escolhendo &quot;região atual adicionar à lista de ROI&quot; janela. Certifique-se de selecionar o mesmo local na &quot;ROI&quot; no menu suspenso. </li></ol> Para a temporização para funcionar correctamente e para cada célula de ter um intervalo de 15 minutos, dois métodos necessitam de ser guardada. Na lista de blocos, o primeiro terá a configuração de fotoativação &quot;real&quot;, eo resto vai ter uma configuração &quot;mock&quot; que não usa o laser de dois fótons. O primeiro bloco também será o único bloco que não tem um atraso (BKIntv = 0). Portanto, o método começa com um exame inicial, seguido de uma verificação de fotoactivação. O resto dos blocos têm um 900 seg BkIntv, eem cada ponto de tempo, existem dois exames assim como em tempo de 0 segundos, para manter a consistência de temporização. <ol start="7"><li> Para cada uma das 10 células a serem seguidos ao longo do tempo, realizar esta sequência: Encontre um PAGFP expressar células salvar seu método de imagem Localização do painel de marcar a posição etapa, especificar o método de imagem Principal ROI painel de escolher uma região de interesse para ser Bleach fotoativado painel de salvar ROI específico e também carregá-lo na caixa de ROI: Apague o ROI e redefinir o zoom varredura a 1 Encontrar a próxima célula e definir o zoom Repetir o processo para os próximos 9 células </li></ol> Verifique se cada local palco e todos os blocos têm o método de imagem adequado (scan configuração) associado. Também certifique-se que o primeiro bloco em cada local corresponde ao método de fotoativação apropriado, enquanto o resto conter um método &quot;mock&quot;. Finalmente, selecione &quot;executar&quot;. 5. Analisar a intensidade do sinal PA-GFP <ol><li&gt; Para as células em que o sinal fotoactivável-GFP colocalizes com o sinal de TMRE vermelho, subtrair o fundo nas imagens PAGFP (aqui usamos metamorph). </li><li> Em seguida, exportar os dados para o Excel e calcular a intensidade média de z-pilhas em cada momento. Descartar secções ópticas que não têm nenhum sinal. </li><li> Medir a diluição do sinal em cada pilha z-através do cálculo da percentagem do sinal original fotoativado. </li></ol> Cada ponto de dados é digitalizado e registada duas vezes, resultando em informações z pilha duplicado para cada ponto de tempo. Verifique se os valores z-stack concordar. Se não, o que é indicativo de um problema (por exemplo, o foco do movimento das células,). 6. Os resultados representativos Quando um evento de fusão ocorre entre uma activado e não-activado PA-GFP mitocôndria, o PAGFP dentro da matriz mitocondrial mistura-se com a matriz não-etiquetados e torna-se diluída sobre uma área maior, diminuindo o sinalintensidade (Figura 1A). Na célula INS1, uma diminuição significativa da intensidade do sinal ocorre a cada 15 minutos, até que o equilíbrio de fusão mitocondrial foi atingido (aproximadamente 1 hora). Note-se que a célula na Figura 1B apresenta colocalização quase completo de PA-GFP e os sinais de TMRE. Nestes ensaios uma concentração muito baixa de TMRE (15 nM) é usado para ajudar a atingir o fotoactivação de PAGFP, e também para controlar a saúde célula. As células com uma abundância de mitocôndrias despolarizada terá colocalização incompleta de PAGFP e TMRE e não devem ser analisados, porque isso indica quer fototoxicidade, ou células num estado de morrer. O tempo de equilíbrio para a fusão mitocondrial é geralmente de 1 hora em INS1 células, quando ~ 15% do volume mitocondrial é activado. Às vezes, mesmo se uma pequena área é iluminada, a maioria das mitocôndrias tornar-se fotoativados devido a ser altamente rede, caso em que a fusão adicional é difícil de detectar. Outros tipos de células podem apresentar tempos de equilíbrio diferentes, e devem ser testados em intervalos mais curtos e mais de um período mais longo para caracterizar a dinâmica mitocondriais. Para inibir a fusão mitocondrial, as células podem ser colocadas dentro de um ambiente lipotoxic. Foi previamente demonstrado que 0,4 mM palmitato fragmentos mitocôndrias, e inibe a fusão mitocondrial 9. Este efeito pode ser visto na Figura 2, onde as mitocôndrias são fragmentados, mas a intensidade do sinal da mtPAGFP não muda, tanto quanto sob condições normais (Figura 1). Portanto, a diluição do sinal PA-GFP é provável que seja devido à fusão mitocondrial em vez de fissão. Em outros tipos de células, sugerimos utilizando outros métodos conhecidos para induzir a fragmentação mitocondrial, tais como o silenciamento OPA1 que é necessária para a fusão mitocondrial 14. <img alt="A Figura 1" src="/files/ftp_upload/3991/3991fig1.jpg" /> Figura 1. </strong&gt; diluição típica do sinal PA-GFP após fotoactivação durante um ensaio de fusão mitocondrial. A. PA-GFP se torna progressivamente mais escura devido a eventos de fusão mitocondriais que conduzem à diluição da proteína sobre uma área crescente como pode ser visto nestas imagens projectadas de 6 secções ópticas quantificado em intervalos de 15 minutos. B. TMRE com PA-GFP mostra que as mitocôndrias são ativos e não despolarizada. <a href="http://www.jove.com/files/ftp_upload/3991/3991fig1large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img alt="A Figura 2" src="/files/ftp_upload/3991/3991fig2.jpg" /> Figura 2. Fusão mitocondrial inibida com 0,4 mM de palmitato diminui a diluição de PA-GFP. A. Projecções de 6 secções ópticas mostrando mitocôndrias curto com uma intensidade de sinal imutável ao longo do tempo. B. colocalização de PA-GFP com TMRE mostra que as mitocôndrias não são depolarizada. <a href="http://www.jove.com/files/ftp_upload/3991/3991fig2large.jpg" target="_blank">Clique aqui para ver maior figura</a> .

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Produção e acesso gratuito a este artigo é patrocinado pela Zeiss, Inc.

Este método permite a criação de imagens de cerca de 10 células de cada vez, se a aquisição ocorre a cada 15 minutos após a fotoactivação. O número exato de células dependerá da rapidez com que um é capaz de localizar e marcar as células que expressam mtPAGFP dentro da placa de cultura, e quão rapidamente pode-se configurar todos os parâmetros de software. Para fazer a automação de correr suavemente uma camada uniforme de células deve ser usado porque os designados z-stack margens será aplicada para todas as células. O tamanho desta área fotoactivação inicial regerá o tempo de equilíbrio. Para ser capaz de medir a fusão mitocondrial, é importante para a fotoativação apenas 10-20% da rede, de tal modo que a propagação do sinal para o resto da rede pode ser monitorizado ao longo do tempo. Se for muito grande parte da rede é fotoativados, é possível que a fusão completa irá ocorrer muito rapidamente, eo evento não será capturada. Cuidado extremo deve ser levado paraajustar a potência do laser do laser de dois fotões, bem como a concentração TMRE para evitar fototoxicidade o que leva a despolarização mitocondrial. Garantir que o sinal mtPAGFP colocalizes com o sinal TMRE pode ajudar a avaliar a fototoxicidade e de saúde geral das células 8,15. Iluminação com luz epifluoerescent deve ser evitada. Embora a pesquisa de células que expressam mtPAGFP, o pinhole deve ser maximamente aberto durante a digitalização com uma excitação de baixa potência 488 nm. Ajustando a potência do laser de dois fotões para a fotoativação suficiente PA-GFP para medir o sinal ao longo de 1 hora, mas não para oversaturate qualquer uma das células pode ser complicado 8. Entretanto, o tempo deve ser gasto nesta etapa de otimização porque o programa uma vez automatizado é iniciado, ele é entediante para pará-lo, para escolher mais células, e currículo. Para controle de qualidade, a aquisição de um contraste de interferência diferencial (DIC) imagem (ou luz transmitida) para monitorar o foco sobre as células podem ser muitoútil e também uma boa maneira de detectar bolhas formadas no óleo de imersão durante a digitalização, o que por vezes acontece a partir dos movimentos do palco. Apesar de usar esse método mtPAGFP reúne dados sobre o movimento unidirecional de proteínas da matriz mitocondrial da mitocôndria fotoativado para aqueles que não estão rotulados, é concebível para utilizar esta técnica para estudar outros processos. Por exemplo, fluorocromos específicos pode ser ligado a proteínas de membrana para observar o seu movimento específico durante eventos de fusão, como foi mostrado para ABC-me, a fusão de que ocorre numa escala de tempo diferente a partir da mistura de proteínas da matriz solúveis 15.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> COXIII-adenoviral PA-GFP </td><td> Lippincott-Schwartz </td><td></td></tr><tr><td> TMRE </td><td> Invitrogen </td><td> T669 </td></tr><tr><td> Zeiss LSM 710 confocal </td><td> Zeiss </td><td></td></tr></tbody></table>

a faster, high resolution, mtpa-gfp-based mitochondrial fusion assay acquiring kinetic data of multiple cells in parallel using confocal microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Watch this Scientific Journal Video about A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy at JoVE.co

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Fusão mitocondrial foi medida seguindo o equilíbrio da GFP matriz segmentada photoconverted através da rede mitocondrial ao longo do tempo. Até agora, apenas uma célula possa ser sujeita a uma análise de longo horas cinética de cada vez. Nós apresentamos um método que mede simultaneamente várias células, acelerando assim o processo de coleta de dados.

Uma resolução mais rápida, alta, MTPA-GFP baseada em ensaio de fusão mitocondrial aquisição de dados cinéticos de várias células em paralelo usando microscopia confocal

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

a-faster-high-resolution-mtpa-gfp-based-mitochondrial-fusion-assay

Research

JoVE Journal

Biology

16.4K visualizações.  Tufts School of Medicine. Fusão mitocondrial foi medida seguindo o equilíbrio da GFP matriz segmentada photoconverted através da rede mitocondrial ao longo do tempo. Até agora, apenas uma célula possa ser sujeita a uma análise de longo horas cinética de cada vez. Nós apresentamos um método que mede simultaneamente várias células, acelerando assim o processo de coleta de dados.

Vídeo: A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Fluorescência baseada em Medição da loja operada entrada de cálcio em células vivas: a partir de células cancerosas cultivadas em fibra muscular esquelética

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Biologia

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

Análise de SNARE mediada por fusão de membrana usando um ensaio de fusão celular enzimática

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo Clonal Rastreamento de Stem hematopoiéticas e células progenitoras Marcado por cinco proteínas fluorescentes usando confocal e microscopia multifotônica

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

De alta resolução de imagem Time-lapse e análise automatizada de microtúbulos Dynamics em Viver umbilical humano Células Endoteliais da Veia

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

De alta resolução Respirometria para avaliar a função mitocondrial em permeabilizadas e células intactas

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Combinando células mitóticas sincronização e microscopia Confocal de alta resolução para estudar o papel das proteínas Multifunctional ciclo celular durante a mitose

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

Respirometria de alta resolução para medir a função mitocondrial das células-Beta intacto na presença de compostos naturais

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Ensaio cinético de molécula única de alta produtividade paralela para decote de DNA específico do local

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Análise em Tempo Real da Bioenergética em Células Epiteliais Pigmentares Pigmentares Humanas Primárias Usando Respirometria de Alta Resolução

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Quantificação do potencial da membrana mitocondrial e dos níveis de superóxido

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

Autor em destaque: Quantificação baseada em fluorescência do potencial da membrana mitocondrial e níveis de superóxido usando imagens ao vivo em células HeLa  

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

Quantificação Baseada em Fluorescência do Potencial de Membrana Mitocondrial e Níveis de Superóxido Usando Imagem Viva em Células HeLa

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...