Este trabalho foi suportado por concessões do Deutsche Forschungsgemeinschaft (DFG) BL567/3-1 ea Friedrich-Baur-Stiftung. Gostaríamos de agradecer Roger Y. Tsien, Howard Hughes Medical Institute Laboratories na Universidade da Califórnia, em San Diego por nos fornecer as Tag-RFP-T2. Nós felizmente reconhecer David Baltimore, do Instituto de Tecnologia da Califórnia em Pasadena, e Didier Trono, da Universidade de Genebra, Genebra, por nos fornecer o lentiviral plasmídeos FUGW e psPAX2/pMD2.G.

<ol>
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Os autores declaram que não têm interesses financeiros concorrentes.

As vantagens e desvantagens do método TED foram amplamente discutidas em publicações recentes 34,35. Em comparação com os outros métodos descritos acima, o TED contorna o problema da interrupção e perfundir as células. Além disso, dois aspectos precisam ser enfatizados. O princípio de TED para ER imagem requer cálcio (1.) A expressão de um alvo carboxilesterase activo no lúmen do ER, com a ajuda de TED construções de vectores e (2.) A libertação eficiente e preferencial de baixa afinidade si...

A fim de resolver as respostas fisiológicas de cálcio do ER, desenvolvemos uma nova estratégia para melhorar a retenção de corantes sensíveis cálcio sintéticos para o ER. O método permite o monitoramento em tempo real direto, sem interrupções de livre ER cálcio na presença de cálcio extracelular. Função e sinalização de cálcio ER Sinais de cálcio são encontradas em diferentes tipos de células, células musculares, por exemplo, neurónios e células gliais e a sua gama de funções de mediar a contração do músculo a um envolvimento na transmissão sináptica na aprendizagem e na memória 1,2. As alterações na concentração de cálcio livre são de interesse científico porque o cálcio está envolvido na regulação da transcrição de genes, a proliferação celular, a excitabilidade neuronal, morte celular e outros eventos de sinalização celular 1-7. Todos estes sinais de cálcio celulares estão funcionalmente ligados e contribuir para cálcio intracelularloja dinâmica 8-10. Uma característica comum a todos os sinais de cálcio é o fluxo de cálcio entre o espaço extracelular, o citosol e organelas, principalmente, o ER e mitocôndrias. Isto faz com que as alterações dinâmicas na concentração de cálcio dentro destes organelos, que são detectados pelos diferentes componentes de sinalização. Em geral, a concentração de cálcio em gamas de RE entre 100 a 800 | iM, no citosol da concentração de cálcio é de cerca de 100 nM, e no espaço extracelular, a concentração é de cerca de 1-2 mM. Assim, não existe uma força motriz maior para o fluxo químico cálcio para o citosol 2,9,10. Os sinais de cálcio ER derivados mais comumente investigados dependem da estimulação dos receptores acoplados à proteína G (GPCR), que, em seguida, activam a fosfolipase C (PLC). PLC em vez produz o inositol 1,4,5-trifosfato (IP 3) 1. Após a ligação de IP 3 à sua conseptor (IP 3-Rec, Figura 1) na ER-membrana, os íons cálcio são liberados a partir do lúmen ER. Historicamente, a liberação de cálcio 3 mediada IP do ER foi o primeiro - mesmo que indiretamente - medido em células pancreáticas acinares por Streb et al, em 1983, 11.. Esta publicação sugeriu pela primeira vez que uma cascata de sinalização que envolvem a acetilcolina, a fosfolipase C e IP3. Esta forma de libertação de cálcio é geralmente denominado libertação de cálcio induzida por IP 3 (IiCR) (Figura 1). Ativação da quinase dependente de fosfolipase Cy por receptores tirosina quinases liga a ação de fatores de crescimento e fatores neurotróficos para ER cálcio sinalização após IP 3 elevação 12. Além IiCR, elevação de cálcio podem ser a entrada de cálcio mediada por receptores ionotrópicos, por exemplo através de canais de cálcio dependentes da voltagem (Ca V), e subsequente libertação de cálcio induzida por cálcio (CICR) por re rianodinaceptors (RyR). IiCR e CICR são fisiologicamente ligado a entrada de cálcio loja operado (SOCE). SOCE inclui a ação do STIM (molécula interagindo estroma), que é um sensor para a liberação ER cálcio. STIM foi mostrada para estimular a entrada de cálcio extracelular através de canais de receptores transientes potenciais (Trp) 13, Orai canais de cálcio de 14 e até mesmo dos canais de cálcio dependentes da voltagem 15 (Figura 1). Perda de ER cálcio é dinamicamente resgatado pela ação do Sarco-retículo endoplasmático cálcio ATPase (SERCA), que ativamente bombas de cálcio de volta para o ER. O bloqueio da SERCA com drogas tais como tapsigargina revela uma perda contínua de cálcio RE para o compartimento citosólico. Este requisito de cálcio &quot;fuga&quot; é causado por ER intramembrane complexos de poros, tais como o complexo de proteína de 16,17 Sec61 (Figura 1). Em 1998, Berridge publicou um modelo, o &quot;neurônio dentro de um modelo de neurônio&quot;, which sugere um papel fisiológico princípio da ER na integração de cálcio neuronal 5. Este modelo considera a existência de um sistema de membrana ER contínuo formando uma &quot;imagem&quot; intracelular da membrana plasmática neuronal 5. Este sistema de membranas eucarióticas binário foi reivindicada a ser um pré-requisito básico para a integração temporal e espacial dos sinais de cálcio rápidas e lentas em neurônios. Sinais de cálcio que ocorrem ou concomitantemente ou posteriormente em diferentes espinhos ou dendritos de um mesmo neurônio são conferidos a soma ou núcleo através da ER, onde se resumem 5,18 da célula. Então, a soma pode ter efeitos sobre a excitabilidade do neurônio, a regulação da transcrição do gene ou a integração de cascatas de sinalização. Assim, o ER suporta a integração de sinais de cálcio. Um pré-requisito para este conceito é a continuidade do ER de uma única célula, o que foi reivindicado em diversos estudos e que foi provado pelo menos para somato-dáreas endritic e curta distância projeções axonais 19-21. Se há continuidade ER dentro projeções axonais longas é uma questão de debate. Estratégias para medir o fluxo de cálcio livre sobre a membrana ER Sinais de cálcio são mais frequentemente monitorados no citosol 22,23. Portanto, não pode ser facilmente distinguida se Ca 2 + a fluir para o citosol a partir extracelular ou de reservas intracelulares 6,24. Para superar esta limitação, foram desenvolvidas estratégias metodológicas para a imagem latente de cálcio direto ER. Em resumo, são utilizadas as seguintes estratégias: (1) ER-alvo geneticamente modificadas indicadores de proteína-25-27 de baixa afinidade de Ca 2 + os indicadores baseados Proteína utilizar a proteína bioluminescente aequorina GFP ou em combinação com uma proteína. Sensível ao cálcio. Estes Ca 2 + indicadores geneticamente modificados (Gecis) podeser orientada para o ER, com a ajuda de um péptido de sinal e são activamente mantido no ER e de retenção utilizando um padrão de recuperação. Comum ER Ca 2 + indicadores de base no princípio da Cameleon e são o Cameleon YC4.3 26,28; Cameleon divisão YC7.3ER 29, e Cameleon D1 30. (2) direto carregamento de AM-éster de baixa afinidade Ca 2 tintura baseada em indicadores esterase + 31,32. Dos corantes indicadoras (Mag-Fura2-AM, Mag-Fluo4-AM ou Fluo5N-AM) derivativos AM passar o membranas biológicas em um estado de cálcio-insensitive lipofílico. Em seguida, no citosol, bem como no ER, esterases endógenas de clivar o grupo de AM-éster e libertar o indicador de Ca2 +, deixando para trás uma certa quantidade de corante activa no citossol e no ER. Por conseguinte, esta abordagem é útil em condições de uma elevada concentração de cálcio no ER, enquanto a concentração de cálcio citosólico permanece bem inferior ao nível delimite de protecção dos indicadores de baixa afinidade, em particular durante os sinais característicos de cálcio (por exemplo, de baixo nM até uM). (3) Carga AM-éster, em combinação com a permeabilização da membrana do plasma 32. Qualquer citosólica de Ca2 + indicador remanescente é removido por permeabilização da membrana do plasma com quantidades pequenas de um detergente &quot;leve&quot; (por exemplo, saponina) num tampão intracelular artificial. Assim, as membranas intracelulares podem ser estimuladas, por exemplo, com IP 3 intracelular no tampão, directamente através de &quot;poros&quot; da membrana plasmática. (4) Diálise do citosol sob configuração de célula inteira e medições simultâneas de Ca 2 + no lúmen ER e citosol 32,33. Uma célula é carregado pela primeira vez com uma baixa afinidade Ca 2 + indicador (por exemplo, Mag-Fura2- AM, raciométrica, UV-luz). Depois, com a ajuda de uma pipeta de remendo, qualquer cit remanescenteosolic baixa afinidade de Ca 2 + indicador é dialisado para fora do citosol com um tampão contendo um indicador de Ca2 + de elevada afinidade (por exemplo, Fluo-3, luz visível). Esta estratégia permite a gravação simultânea de sinais derivados citosólicas e ER. (5) Targeted-esterase induzida corante de carga de 8,34. Carboxilesterase (CES) está voltado para o lúmen do RE e proporciona uma elevada actividade de esterase de aprisionamento eficaz da forma de AM-éster de baixa afinidade de Ca 2 + indicadores. Targeted-esterase induzida corante de carregamento (TED) Para melhorar o direcionamento de baixa afinidade de Ca 2 + indicadores para o lúmen ER, TED foi desenvolvido. TED requer a superexpressão de uma carboxilesterase alvo rato ER (CES2) (Figura 2), o que é conseguido por meio de construções de expressão. As células que expressam uma construção recombinante CES-são incubadas com a forma de AM-éster de cálcio emcorante dicator (Fluo5N-AM, a Figura 2). Em seguida, no ER, o corante é convertido para o Ca2 + sensíveis, a membrana impermeável de Ca 2 + complexo indicador (Fluo5N/Ca 2 +), pela elevada actividade de esterase, aprisionando, por conseguinte, o corante numa concentração elevada no lúmen ER 8 , 34. O método é especialmente útil para investigar ER-libertação de cálcio através dos IiCR-vias, por exemplo por meio de purinérgicos ou receptores de glutamato metabotrópicos, 8,34 e visualizar a depleção de cálcio através da &quot;ER&quot; canais de vazamento, por exemplo, directamente após o bloqueio da 17 SERCA , 34. Para nossa experiência, a baixa afinidade Ca 2 + indicador Fluo5N-AM é atualmente o melhor indicador disponível para usar com a estratégia de carregamento TED corante. Fluo5N-AM tem uma baixa afinidade pelo Ca 2 + (constante de dissociação K D ~ 90 mM, figura 2), é quase não-fluorescente em seu AM-forma, mas oferece emissão de fluorescência de alta sobre calciu8,34 m de ligação. O Fluo5N/Ca 2 + complexo pode ser excitado com uma fonte de luz padrão de ~ 490 nm, o que corresponde a corantes convencionais, tais como FITC, Alexa 488, ou eGFP. Sinais de cálcio citosólico dificilmente atingir uma concentração na gama uM baixo e são, portanto, apenas detectada por Fluo5N no citosol 35. Para melhorar o desempenho TED, várias construções de vectores recombinantes foram desenvolvidos (Figura 3). Originalmente, os vectores de TED baseados na sequência de codificação de CES2 (RefSeq número de acesso NM_145603, CES2c) e melhor desempenho TED é observado com a expressão estável de CES construtos. Novos vetores TED expressar um elemento central da CES2 fundido à proteína fluorescente vermelha TagRFP-T2 36. Estes vectores possuem a vantagem de que eles podem ser utilizados para identificar as células transduzidas e utilizar a fluorescência vermelha como um controlo interno para a normalização de alterações na Fluo5N/Ca 2 + fluorescência. A fluorescência vermelha também oferece a possibilidade de visualizar a distribuição estrutural do ER e as alterações na dinâmica do ER sob condições de estimulação.

<table border="1">
	 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>(S)-3,5-DHPG (Dihydroxyphenylglycine)</td>
 <td>Tocris</td>
 <td>0805</td>
 <td></td>
 </tr>
 <tr>
 <td>5';-ATP-Na2, ATP disodium salt hydrate</td>
 <td>Sigma</td>
 <td>A26209-1G</td>
 <td></td>
 </tr>
 <tr>
 <td>B-27 supplement,50x</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td></td>
 </tr>
 <tr>
 <td>Carbamoylcholine chloride (Charbachol)</td>
 <td>Sigma</td>
 <td>C4382-1g</td>
 <td></td>
 </tr>
 <tr>
 <td>Cyclopiazonic acid (CPA)</td>
 <td>Ascent scientific</td>
 <td>Asc-300</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethylsulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM with Glutamax</td>
 <td>Invitrogen-Gibco</td>
 <td>31966-047</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's PBS without Ca/Mg 1x</td>
 <td>PAA</td>
 <td>H15-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal calf serum</td>
 <td>Invitrogen-Gibco</td>
 <td>10270-106</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-5N-AM, 10 x 50 &mu;g</td>
 <td>Invitrogen</td>
 <td>F14204</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamate</td>
 <td>Ascent scientific</td>
 <td>Asc-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamax</td>
 <td>Invitrogen</td>
 <td>35050-038</td>
 <td></td>
 </tr>
 <tr>
 <td>Ham's F12 nutrients mixture</td>
 <td>Invitrogen-Gibco</td>
 <td>21765-029</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's BSS(1x) without Ca,without Mg, with Phenol Red, HBSS</td>
 <td>PAA Laboratories</td>
 <td>H15-010</td>
 <td></td>
 </tr>
 <tr>
 <td>Horse serum</td>
 <td>SHD3250YK</td>
 <td>Linearis</td>
 <td></td>
 </tr>
 <tr>
 <td>Ionomycin Ca2+ salt</td>
 <td>Ascent scientific</td>
 <td>Asc-116</td>
 <td></td>
 </tr>
 <tr>
 <td>murine EGF </td>
 <td>PreproTech</td>
 <td>315-09</td>
 <td></td>
 </tr>
 <tr>
 <td>N2 supplement 100x</td>
 <td>Invitrogen</td>
 <td>17502-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Neurobasal medium</td>
 <td>Invitrogen-Gibco</td>
 <td>21103-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127, low UV absorbance,2g</td>
 <td>Invitrogen</td>
 <td>P6867</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-DL-ornithine hydrobromide PORN</td>
 <td>Sigma</td>
 <td>P8638</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-D-lysine hydrobromide</td>
 <td>Sigma</td>
 <td>P7280</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-L-Lysin</td>
 <td>Sigma</td>
 <td>P2636</td>
 <td></td>
 </tr>
 <tr>
 <td>Thapsigargin</td>
 <td>Calbiochem</td>
 <td>586005-1mg</td>
 <td></td>
 </tr>
 <tr>
 <td>TryLE Express</td>
 <td>Invitrogen</td>
 <td>12605-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Worthington</td>
 <td>LS-003707</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsininhibitor from Glycine MAX (soybean)</td>
 <td>Sigma</td>
 <td>T6522</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Materials</td>
 </tr>
 <tr>
 <td>Confocal system: inverted laser scanning microscope: FV1000 with IX81</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: laser combiner FV10 and diode lasers (405, 473, 559, 635)</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: scan head FV10-SPD with spectraldetector</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Heater controller TC-344B</td>
 <td>Warner Instruments</td>
 <td>64-0101</td>
 <td>to control in line solution heater</td>
 </tr>
 <tr>
 <td>NIH ImageJ software</td>
 <td>WS Rasband, ImageJ, US National Institutes of Health, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>, 1997--2006</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UAPON20XW340 NA 0,7 WD 0,35</td>
 <td>Olympus</td>
 <td>N2709100</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLFLN40XO</td>
 <td>Olympus</td>
 <td>N1478700</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLSAPO60XO/1.35, WD=0.15</td>
 <td>Olympus</td>
 <td>N1480700</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, inverse</td>
 <td>custom-made</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, RC-49FS</td>
 <td>Warner Instruments</td>
 <td>W4 64-1709 </td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion tube: PT-49</td>
 <td>Warner Instruments</td>
 <td>64-1730</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Peristaltic pump MiniPlus 3 (4 channels)</td>
 <td>Gilson</td>
 <td>n.a. </td>
 <td>perfusion pump</td>
 </tr>
 <tr>
 <td>Single inline solution heater SH-27B</td>
 <td>Warner Instruments</td>
 <td>64-0102</td>
 <td>controlled by heater controller</td>
 </tr>
 <tr>
 <td>Suction tubes: ST3R</td>
 <td>Warner Instruments</td>
 <td>64-1407</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Heating insert: Tempocontrol 37-2 digital 2-channel</td>
 <td>Pecon</td>
 <td>n.a. </td>
 <td></td>
 </tr>
	 </tbody>
 </table>

direct imaging of er calcium with targeted-esterase induced dye loading (ted)

Visualização da dinâmica do cálcio é importante para entender o papel do cálcio na fisiologia celular. Para examinar a dinâmica de cálcio, Ca 2 + indicadores fluorescentes sintéticos tornaram-se populares. Aqui demonstramos TED (= targeted-esterase induzida corante de carga), um método para melhorar a libertação de Ca2 + corantes indicadoras no lúmen do ER de diferentes tipos de células. Até à data, o TED foi utilizado em linhas de células, células gliais e neurónios, in vitro. TED sobre bases eficientes, recombinante alvo de uma atividade de alto carboxilesterase para o lúmen ER usando vetores construções que carboxilesterases rápidas (CES). Os últimos vectores TED contendo um elemento de núcleo de CES2 fundida com uma proteína fluorescente vermelha, permitindo assim imagiologia simultânea de duas cores. A dinâmica do cálcio livre na ER são gravadas em uma cor, enquanto a estrutura ER correspondente aparece em vermelho. No início do processo, as células foram transduzidas com um lentivírus. Posteriormente, as células infectadas are semeadas em lamínulas para finalmente permitir imagens de células vivas. Em seguida, as células vivas são incubadas com o éster acetoximetílico (AM-éster) forma de baixa afinidade Ca 2 + indicadores, por exemplo Fluo5N-AM, Mag-Fluo4-AM, ou Mag-Fura2-AM. A actividade de esterase nos cliva fora do ER cadeias laterais hidrófobas da forma AM do indicador de Ca 2 + e de um corante fluorescente / Ca 2 + complexo hidrófilo é formado e preso no lúmen do ER. Depois de corante de carga, as células foram analisadas num microscópio confocal laser scanning invertido. As células são continuamente perfundido com soluções de Ringer-like e as dinâmicas ER cálcio são diretamente visualizados por imagem time-lapse. Libertação de cálcio a partir do ER é identificado por uma redução na intensidade de fluorescência em regiões de interesse, enquanto que o reenchimento da loja ER cálcio produz um aumento na intensidade de fluorescência. Finalmente, a mudança na intensidade de fluorescência ao longo do tempo é determinada pelo cálculo da Af / F 0.

Este protocolo fornece uma abordagem sem interrupções para a imagem direta de livre ER cálcio. Baixa afinidade sintético Ca2 + indicadores são libertados e preso no lúmen do ER, com a ajuda de um ER alvo, a actividade da enzima esterase recombinante. Melhoria carregamento dos Ca 2 + indicador de corantes para o lúmen ER permite que uma imagem direta e rápida de dinâmicas armazenam cálcio ER. Tipos de células para TED A viabilidade do...

Watch this Scientific Journal Video about Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED at JoVE.com

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induzida corante de carregamento (TED) suporta a análise da dinâmica intracelular de cálcio loja por imagens de fluorescência. O método baseia-se na segmentação de uma carboxilesterase recombinante para o retículo endoplasmático (ER), onde se melhora o desmascaramento do local sintético de baixa afinidade Ca<sup&gt; 2 +</sup&gt; Corantes indicador no lúmen ER.

Imagem direta de cálcio ER com Targeted-Esterase Induzida Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

direct-imaging-er-calcium-with-targeted-esterase-induced-dye-loading

Research

JoVE Journal

Biology

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

19.1K visualizações.  University of Wuerzburg. Targeted-esterase induzida corante de carregamento (TED) suporta a análise da dinâmica intracelular de cálcio loja por imagens de fluorescência. O método baseia-se na segmentação de uma carboxilesterase recombinante para o retículo endoplasmático (ER), onde se melhora o desmascaramento do local sintético de baixa afinidade Ca 2 + Corantes indicador no lúmen ER.

Vídeo: Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Carregando Drosophila terminais nervosos com indicadores de cálcio

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Biologia

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

Vivo em 2-Photon Imagens de cálcio na camada 2 / 3 dos Ratos

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

Imagem de cálcio dos neurônios corticais usando Fura-02:00

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Montagem, de carga, e alinhamento de uma célula Analytical ultracentrífuga Amostra

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Imagens ao vivo de cálcio celular Combinado com siRNA silenciamento gênico mediado Identifica Ca 2 + Canais de vazamento na membrana ER e seus mecanismos de regulação

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Expressão e purificação de partículas semelhantes a vírus para a vacinação

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

Monitorização da libertação de cálcio ER / SR com o Ca<sup&gt; 2+</sup&gt; Captador<sup-benzóico.</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

Filamentos intermediários e microtúbulos com microscopia 2-dimensional direta estocástico reconstrução óptico de imagem

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

Recuperação de fluorescência após p62 fotobranqueamento de amarelo fluorescente proteína marcados em Aggresome induzida por estruturas

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Dinâmica de cálcio de imagem em subpopulações de células de ilhotas pancreáticas do rato

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...