The authors are deeply indebted to all the members of the scientific community working on pH and ion transport, who have originated and improved the measurements described here. They particularly thank Dr. Jacques Pouyss&#233;gur who originated the H+-killing selection technique used here. They acknowledge the University of Nice-Sophia Antipolis, the CNRS, the ANR (JCJC SVSE1 NHEint) and the ICST Labex for support.

1. H + Matar Seleção <ol><li> As linhas celulares <ol><li> Estavelmente NHE-transfectar células deficientes (por exemplo, a linha celular derivada de CCL39 PS120 16) usando qualquer combinação de vector de expressão de mamífero e transfecção método que produza rendimentos eficientes de transfecção e selecção numa linha de células de fibroblastos. NOTA: Para muitos anos de Fosfato de Cálcio precipitação 19 foi utilizado com bons rendimentos de transfecção. Este foi substituída mais recentemente por reagentes comerciais, tais como Lipofectamine2000, as transfecções ser realizadas seguindo as instruções do fabricante. </li></ol></li><li> Soluções <ol><li> Preparar três soluções estéreis descritos como abaixo. Esteriliza-se estas soluções por meio de filtração (0,22 um). <ol><li> Prepara-se uma NH4 + Carregando solução (pH 7,4) composto por 50 mM de NH4Cl, 70 mM de cloreto de colina, 5 mM de KCl, 1 mM de MgCl2, 2 mM de CaCl 2, 5 mM de glucose e 15 mM de MOPS a pH 7,4. </li><li> Prepara-se uma solução de lavagem (pH 7,0) composta por 120 mM de cloreto de colina, 5 mM de KCl, 1 mM de MgCl2, 2 mM de CaCl 2, 5 mM de glicose e 15 mM de HEPES a pH 7,0. </li><li> Prepara-se uma solução de recuperação (pH 7,4) composto por NaCl 120 mM, KCl 5 mM, MgCl2 1 mM, 2 mM de CaCl 2, 5 mM de glicose e 15 mM de HEPES a pH 7,4. </li></ol></li></ol></li><li> Antes de iniciar a selecção, obter uma incubadora de cultura de células que pode ser utilizado a 37 ° C sem adicional de CO 2 a partir do tanque externo (denominado CO 2 - incubadora &quot;livre&quot;), como 5% de CO 2 resultará na memória intermédia que pode prejudicar o acidificação. Amplificar a linha celular que expressa o NHE de interesse até cerca de 2 x 10 8 células (tipicamente cerca de 20 100 placas confluentes mm). </li><li> H + matando procedimento: <ol><li> Incubar as células que expressam o NHE intracelular de interesse durante 1 hora em solução a carregar, em 37 ° C na incubadora de CO 2 -livre. </li><li> Aspirar a solução de carga e, em seguida, lave-os duas vezes na solução descrita-rinse acima. Executar esta etapa eficiente para eliminar o residual extracelular NH4Cl que prejudicaria a criação de um gradiente de NH 4 + íngreme que irá gerar a acidificação. </li><li> Elimine o meio por aspiração e lavagem incubar as células durante uma hora em solução de recuperação de novo a 37 ° C na incubadora de CO 2 livre. </li><li> Substituir o meio de recuperação com meio de cultura normal (tipicamente DMEM com 7,5% de FCS) e as células crescer em condições de cultura padrão (37 ° C numa atmosfera humidificada de 5% de CO 2 e 95% de ar). </li></ol></li><li> Repetir este ciclo de selecção, duas vezes por semana, até surgir clones estáveis. NOTA: Tome especial cuidado relativo à cultura de células e condições de seleção. Meses de trabalho pode ser arruinada por qualquer contaminação que é mais propenso a oCCur após um longo período de cultura de células e manipulações repetidas. <ol><li> Aqui, escolha se deseja ampliar os clones e caracterizá-los individualmente conforme descrito nas seções 2-3, ou reuni-los em conjunto para gerar uma população celular antes de caracterização. </li></ol></li><li> Aplicar a selecção ocasional (cerca de uma vez a cada duas semanas) para reter as células resistentes à acidificação por contra-selecionar aqueles que podem ter revertido para o sensível ao ácido fenótipo parental (Figura 1B). </li></ol> 2. As medições de pH intracelular 2.1) Imagem de Fluorescência pH <ol><li> Use a ratiometric sensíveis ao pH de corante fluorescente BCECF / AM, que é sensível ao pH quando excitado a 490 nm e possui uma nm ponto isosbestic 445, seguindo os protocolos do fabricante. <ol><li> Em alternativa, utilizar outras sondas sensíveis ao pH, seguindo os protocolos do fabricante. </li></ol></li><li> Use um con imagem definidasisting de um microscópio invertido acoplado a uma câmera de vídeo de alta sensibilidade. Equipar este conjunto com 450 nm e 490 nm filtros de interferência de banda estreita emparelhados com quartzo adequado filtros de densidade neutra para a excitação. <ol><li> Se possível, use o conjunto adequado de filtros e condições de iluminação para configuração valores de fluorescência comparáveis ​​para λ1 e λ2 para que a relação se aproxima de 1. Além disso, evite valores de fluorescência basais que são definidas quer saturar ou muito baixa, tanto para λ1 e / ou λ2. Isso pode produzir artefatos importantes como então a relação pode não foi detectável embora pH intracelular está mudando. NOTA: Este sistema deve habilitar perfusão rápida, a excitação de lapso de tempo para os dois comprimentos de onda acima mencionados e aquisição no comprimento de onda de emissão própria (535 nm para BCECF). Equipar o sistema com software que permite a aquisição de dados automática e armazenamento. </li></ol></li></ol> 2.2) A medição do NHE Atacante Atividade (Extrusão dePrótons do citosol) <ol><li> Acidifica-se as células por uma incubação de 1 h na solução de NH 4 + Carregando (ver passo 1.4), na ausência de CO 2. </li><li> Adicionar BCECF-AM (5 mM concentração final) para a solução para os últimos cinco minutos e, em seguida, lave-o com NH 4 + solução de carga para eliminar a sonda extracelular. </li><li> Montar as células no microscópio e tomar várias imagens para gravar uma linha de base estável. Perfundir a solução de lavagem (ver acima). Isto resulta numa queda da fluorescência correspondente à acidificação. </li><li> Após a estabilização do pH, perfuse qualquer uma das soluções a seguir para medir as taxas de recuperação de pH mediadas por Li + / H +, Na + / H + ou K + / H + câmbio. Cada solução contém um potencial catião de acoplamento para ser testado para a troca de H +. Se um desses é transportado, isto irá resultar em um aumento de fluorescência a 495 nm que correspgundo a um aumento no pH intracelular: LiCl 120 mM, KCl 5 mM, MgCl2 1 mM, 2 mM de CaCl 2, 5 mM de glicose e 15 mM de HEPES a pH 7,4 120 mM de NaCl, 5 mM de KCl, 1 mM de MgCl2, 2 mM de CaCl 2, 5 mM de glicose e 15 mM de HEPES a pH 7,4 KCl 125 mM, MgCl2 1 mM, 2 mM de CaCl 2, 5 mM de glicose e 15 mM de HEPES a pH 7,4 </li></ol> 2.3) A medição do NHE Atividade Reversa NOTA: O princípio aqui é inverter os gradientes iônicos transmembrana. <ol><li> Antes da medição, carregar as células com o catião intracelular de interesse (ver abaixo). </li><li> Incubar-los por 5 min com BCECF / AM (veja o passo 2.2.2); lavá-los, montá-los no microscópio vídeo. </li><li> Perfundir a solução de carga e tomar várias imagens para gravar uma linha de base estável. </li><li> Depois da estabilização da linha de base, a imagem das variações de pH quando as células são perfundidos com um meio ácido extracelular contendo 120 mCloreto de colina M, 5 mM de KCl, 1 mM de MgCl 2, CaCl 2 2 mM, 5 mM de glicose e 15 mM de MES a pH 6,5. </li><li> Para LiCl carregamento, as células incubar durante 2 horas em uma solução composta de LiCl 120 mM, KCl 5 mM, MgCl2 1 mM, 2 mM de CaCl 2, 5 mM de glicose e 15 mM de HEPES a pH 7,4. Lítio intracelular medido por espectroscopia de absorção atómica gera um valor de cerca de 60 mM. </li><li> Para carregamento de Na +, incubar as células durante duas horas numa solução composta por 120 mM de NaCl, 5 mM de KCl, 1 mM de MgCl2, 2 mM de CaCl 2, 5 mM de glicose e 15 mM de HEPES a pH 7,4 na presença de 1 mM de ouabaina para bloquear a bomba de Na / K ATPase. Nestas condições, a concentração intracelular de Na + está na gama de 40 mM. NOTA: K + de carga não é necessário, como K + citosólico concentração está na gama de 140 mM e um gradiente dirigido exteriormente-K +, por conseguinte, é fácil de conseguir. </li></ol> 2.4) Tratamento e calibração de dados: NOTA: Este passo é válido tanto para as medições frente e verso. <ol><li> No final de cada experiência, perfundir as células com uma solução de KCl 140 mM, HEPES 20 mM e 5 uM nigericina ajustado a valores de pH entre 6,5 e 7,4. </li><li> Coletar dados como medições de fluorescência a partir das imagens (níveis de cinza que representam os níveis de intensidade) e exportação nos termos do formato de texto e tratar como explicado abaixo usando um programa de planilha. </li><li> Calcular os valores de pH intracelular para cada célula ou região de interesse utilizando a seguinte equação individual: pH = pKa + (Log (R-R min) / (R max -R)) x F min (λ2) / F max (λ2) </li><li> Utilizar a min F (λ2) / F x ma (λ2) proporção, se houver fuga de branqueamento ou sonda, resultando numa diminuição na fluorescência a 450 nm ao longo das experiências. Esta é no entanto muito raramente observada nas condições utilizadas no desexperimentos crita. </li><li> À medida que os dados de calibração estão presentes no final de cada medição de pH, verificar se os valores calculados que correspondem diferentes dos valores de pH utilizados para a calibração. Se for o caso, em seguida, ajustar todos os valores de pH experimentalmente-determinados para a calibração utilizando o seguinte procedimento: <ol><li> Calcula-se o seguinte quociente (Q, a diferença entre os valores medidos / diferença entre os valores de calibração). Multiplique todo o conjunto de dados por Q. fazer o ajuste final por adição ou subtração de modo que os valores calculados serão iguais aos valores de calibração correspondentes. </li></ol></li></ol> 3. Medidas de taxas iniciais do NHE7 pelo FAST Li + Captação <ol><li> Semear as células em placas de múltiplos poços (6 a 24 poços) e acidificam-los usando a técnica de NH 4 + carregamento descrito acima ou, alternativamente, o / albumina de soro bovino (BSA) a acidificação nigericina descrito em 20. </li><li&gt; placas de enxaguamento duas vezes rapidamente com a solução de lavagem acima descrito e, em seguida, incubar as células em soluções de absorção que contêm 1-10 mM de lítio (ligeiramente abaixo) os valores Km e as concentrações desejadas dos catiões e / ou inibidores de interesse para a quantidade desejada de tempo. </li><li> Manter durações de captação de curto e consistentes (tipicamente um minuto ou abaixo) para garantir que o transporte opera em condições de taxas iniciais. Também certifique-se de que todas as soluções utilizadas para a captação são isotônicas. </li><li> No final do tempo de absorção, eliminar cuidadosamente a forma absorção e lavar as células quatro vezes com solução salina de fosfato gelada tamponada (PBS). Realizar estas lavagens tão rápido quanto possível (menos de 10 seg para os quatro deles é ideal) para evitar o efluxo de lítio. </li><li> Lisar as células em 25% de ácido nítrico. Aplicar 250 ul por poço de 25% de ácido nítrico e deixá-lo assentar durante pelo menos 1 h, em seguida, cada poço de sucata com a extremidade da ponta da pipeta e transferir toda a suspensão bem em um novo mitubo crocentrifuge. </li><li> Transferir o lisado em tubos de microcentrífuga de 1,5 ml, centrifugar-los durante 5 minutos a 15.000 x g (temperatura ambiente), para remover os detritos celulares. </li><li> Medir a quantidade de lítio do sobrenadante por espectroscopia de absorção atômica 21. NOTA: Diferentes empresas fornecem espectrometria de absorção atômica, que têm de ser equipado com uma lâmpada de cátodo oco de lítio. Siga as instruções do fabricante como essas máquinas são muito precisos, mas também bastante delicado. </li></ol>

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The authors have nothing to disclose.

Este protocolo descreve como para seleccionar células que expressam intracelular de Na + / H + permutadores na membrana plasmática, sem alterar a sua sequência primária por mutagénese dirigida ao local. Estes trocadores de agora pode ser caracterizado. Este método baseia-se na toxicidade celular de protões intracelulares para seleccionar linhas celulares que expressam vesicular de Na + / H + permutadores na membrana plasmática. Pode ser pos...

A maioria dos compartimentos intracelulares exibir um pH luminal ácida, o que é um parâmetro essencial para a maturação, o tráfico, a reciclagem de proteínas ou hormônios e neurotransmissores que carregam. Tem sido demonstrado que o gradiente de pH entre o teor de citosol e vesicular é gerado por vacuolar H + ATPase 1, acoplado aos transportadores vesiculares cloreto clc 2. Tanto em camundongos knock-out (KO) e pacientes humanos, a importância desses transportadores tem sido destacada pelos fenótipos pesados ​​causadas por mutações em seus genes 3-6. Os membros da família SLC9A permutadores de sódio a hidrogénio, também denominado por NHE de Na + / H + permutadores, demonstraram ser importantes efectores no pH intracelular e regulação do volume celular, bem como no transporte vectorial de equivalentes de ácido-base entre epitélios . Além dos NHE da membrana plasmática, três altamente conservada de Na + / H + permutadores de NHE 6, 7e 9 são fixados em rede trans-Golgi e no início endosomes 7. As mutações em seus genes têm sido associados com Angelman-like ou Christianson Síndromes 8-9, autismo base familiar 10 e Attention Deficit Disorder Hiperatividade 11-12. Estes trocadores também foram envolvidos em problemas neurodegenerativas, como Alzheimer susceptibilidade à doença 13 e retardo mental genes contíguos X-ligados síndromes 14. Tomados em conjunto, estes estudos destacam a importância desses NHEs intracelulares no desenvolvimento e / ou a função cerebral. A localização intracelular destes trocadores impede medições precisas de sua seletividade ion, direcção de transporte, parâmetros cinéticos, e regulamentação. Como é o caso para todos os transportadores expressos em compartimentos intracelulares, que é extremamente difícil de avaliar as suas actividades bioquímicas e, consequentemente, para compreender totalmente os seus papéis fisiológicos e o mechanismos subjacente suas implicações patológicas. Para a concentração de K + cytosolic alta, a hipótese mais comumente aceita era de que eles estavam trabalhando como K + acoplados transportadores de efluxo de prótons. A existência de uma tal fuga de protões tinham sido implicado, como pode contrabalançar bombeamento de protões pelos V-ATPase, a fim de manter um pH vesicular estado estacionário. O objectivo deste artigo visual é (i) para demonstrar um método que permite a selecção genética de linhas celulares que expressam estes transportadores vesiculares na sua membrana plasmática, e (ii) mostram duas abordagens independentes para medir as funções destes transportadores. Três décadas atrás, Pouysségur e Franchi foram pioneiras na abordagem genética que permitiu a clonagem molecular e caracterização dos membros da família NHE 15. Este baseou-se na toxicidade de protões intracelulares, como um método de rastreio. O primeiro passo foi a obtenção de linhas celulares deficientesem qualquer troca + / H + Na expresso na membrana plasmática, usando a reversibilidade deste transportador. Os fibroblastos (linha celular CCL39) foram pré-carregado com Na + ou Li + e, em seguida, colocado num meio extracelular ácida (pH 6,5) durante 2 h. Isto levou à morte de células que expressam um funcional de Na + / H + e para a troca de selecção de células antiportador-deficiente (linha celular PS120 16). Quando cultivado em meio isento de bicarbonato, estas células são muito sensíveis à acidificação intracelular aguda. Por conseguinte, a expressão de qualquer mecanismo de efluxo de protões funcional na membrana de plasma vai ser seleccionadas positivamente (ver 17), se tais células são submetidas a acidifications intracelulares agudas. Tais técnicas de acidificação pode ser utilizado para isolar as linhas celulares com defeitos tráfico que permitam a expressão forçada de NHE intracelulares WT na membrana plasmática. Como eucariota Na + / H+ Trocadores são electroneutral, eles não são mensuráveis ​​pelas abordagens eletrofisiológicos que têm sido utilizados com grande sucesso para medir canais. Assim, o presente artigo propõe demonstrar como medir a atividade desta trocador por medições de pH intracelulares e rápida cinética de absorção de lítio. Como os conceitos subjacentes são o mesmo, é interessante notar que muitos dos processos desenvolvidos para a secção de selecção, também são utilizados directamente para medições funcionais. Curiosamente, observou-se que o defeito tráfico presentes nas linhas de células seleccionadas utilizando a abordagem descrita neste manuscrito conduz a uma maior expressão de outras proteínas vesiculares na membrana do plasma, tais como o canal de potássio vesicular TWIK1 18. Isso aponta para a seleção de um mecanismo geral defeito de retenção de proteínas transmembrana vesiculares. Assim, este processo de selecção e as células que gera Maioconstituem uma ferramenta promissora para a comunidade científica que trabalha em proteínas da membrana de compartimentos intracelulares. Como bem as técnicas de medição apresentados aqui podem ser aplicáveis ​​para o estudo de outros transportadores não-electrogénico.

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functional characterization of na+/h+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

Acidificação endossomal é crítica para uma vasta gama de processos, tais como a reciclagem e degradação de proteínas, dessensibilização do receptor e neurotransmissor em vesículas sinápticas carregamento. Esta acidificação é descrito para ser mediada por ATPases de protões, acoplados a transportadores de cloreto clc. Altamente conservada prótons electroneutral transportadores, do Na + / H + trocadores (NHE) 6, 7 e 9 também são expressos nestes compartimentos. As mutações nos seus genes foram ligados com doenças cognitivas e doenças neurodegenerativas humanas. Paradoxalmente, seus papéis são ainda imperceptíveis, como a sua localização intracelular impediu a caracterização funcional detalhado. Este manuscrito apresenta um método para resolver este problema. Esta consiste na selecção de linhas de células mutantes, capazes de sobreviver a acidificação citosólica aguda retendo NHE intracelular na membrana plasmática. Em seguida, ele mostra dois protocolos complementares para medir a seletividade e atividade iondestes permutadores de: (i) uma base em medições de pH intracelular por meio de microscopia de fluorescência de vídeo, e (ii) uma base na cinética de absorção rápida de lítio. Esses protocolos podem ser extrapolados para medir outros transportadores não-electrogénico. Além disso, o processo de selecção aqui apresentada gera células com um fenótipo de retenção intracelular defeituosa. Por conseguinte, estas células expressam também outras proteínas da membrana vesicular na membrana plasmática. A estratégia experimental descrito aqui pode, portanto, constituir um instrumento potencialmente poderoso para estudar outras proteínas intracelulares que será, então, expressa na membrana plasmática vesicular, juntamente com o Na + / H + permutadores utilizados para a selecção.

Seleção: A selecção H + matando baseia-se na difusão da amónio base fraca, tal como representado na Figura 1A. O efeito de bases fracas e ácidos difusão no pH intracelular foi iniciada por Walter Boron e colaboradores 22. A idéia elegante para usar este fenômeno para produzir uma acidificação letal para a seleção genética positiva foi então desenvolvido por Jacques Pouysségur 17. De acordo com um protocolo deste tipo, o pH in...

Watch this Scientific Journal Video about Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane at JoVE.c

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Caracterização funcional de Na<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Trocadores de intracelular compartimentos usando a Seleção Proton-matança de expressá-las na membrana plasmática

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

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JoVE Journal

Biology

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

9.2K visualizações.  Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics. The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers. 

Vídeo: Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Medição Perto membrana plasmática e global Dynamics cálcio intracelular em astrócitos

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Biologia

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Cristalizando Proteínas de Membrana para determinação da estrutura usando mesofases lipídico

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Medição quantitativa do GLUT4 translocação para a membrana plasmática por citometria de fluxo

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Identificar os efeitos das mutações BRCA1 em Recombinação Homóloga usando células que expressam BRCA1 tipo selvagem endógena

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

A utilização de um robô para alta capacidade de cristalização de proteínas de membrana em mesofases lipídicas

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Colheita e Cryo-resfriamento cristais de proteínas de membrana crescida no mesofases lipídicas para a determinação da estrutura de Cristalografia Macromolecular

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopia intravital para Estruturas de imagem Subcelulares em ratos vivos Expressando fluorescentes Proteínas

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

Imagem Plasma Membrane Deformações Com pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Investigando os prejudiciais efeitos de baixa pressão Plasma esterilização na sobrevivência de esporos de Bacillus subtilis usando Live celular microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...