Este trabalho foi financiado pela Fundação Memorial Uehara do Japão (a ES), bem como a Fundação Whitehall, do Instituto Nacional de Distúrbios Neurológicos e Derrame e um Stein-Oppenheimer Award Endowment (a BSK).

Procedimentos experimentais O procedimento experimental consiste de duas partes principais que são descritas em uma maneira passo sábio abaixo. Parte 1: PREPARAÇÃO cultura de astrócitos hipocampais Brevemente, misturado astrócitos do hipocampo neurônio-culturas foram preparadas usando um protocolo bem estabelecido 1,2,3. Nós otimizamos o procedimento para produzir saudável astrócitos em cultura. Todos os procedimentos listados abaixo devem ser realizadas em um ambiente estéril, como uma capela de fluxo laminar. Lamínulas preparar <ul><li> 22 lamínulas mm (VWR, 48380-068) </li><li> Poli-D-lisina (PDL, Sigma P0899), alíquotas (1 mg / ml) </li><li> Laminina alíquotas (20 mcg / ml): adicione 49 ml de água estéril a 1 mg / ml solução laminina (Sigma L2020), fazer 1,2 ml alíquotas e armazenar a -20 ° C </li></ul><ol><li> PDL dissolver em água estéril (50 mcg / ml) </li><li> Autoclave as lamelas, enxágüe com água estéril e colocar em PDL (20 ml para 100 lamínulas). Deixar à temperatura ambiente durante a noite. </li><li> Substituir PDL com água estéril (3 lavagens extensa). Em seguida, seque as lamelas e armazenar a 4 ° C. </li><li> O dia antes do plaqueamento os astrócitos lamínulas, lugar individual em um poço estéril em uma placa de 6 poços (com vários poços de fundo plano Placas com tampas, estéril de BD Bioscience). Coloque 400 mL de laminina em cada lamela e incubar durante a noite, na incubadora para evitar a evaporação. </li></ol> Dissecação Media dissecção: <ul><li> 500 ml de Earle solução salina balanceada (EBSS) (1X), líquido (Invitrogen, 14155-063) </li><li> 5 ml solução HEPES (Sigma, H0887) </li></ul> Media do hipocampo: <ul><li> 412,5 ml Médio Mínimo Essencial (MEM) (1X), líquido Contém sais de Earle, mas nenhum vermelho L-glutamina ou de fenol (Invitrogen, 51200-038) </li><li> 10 ml de glicose (Sigma, D-(+) glicose-ANIDRO SIGMA ULTRA G7528) 1 M em MEM </li><li> 5 ml Penicilina-estreptomicina (Invitrogen, 15140-122) </li><li> 5 ml de solução de piruvato de sódio (Sigma, S8636) </li><li> 12,5 ml de solução 1 M HEPES (Sigma, H0887) </li><li> 5 ml Suplemento N-2 (100X), líquido (Invitrogen, 17502-048) </li><li> 50 ml de soro do cavalo, inativado pelo calor (Invitrogen, 26050-088) </li></ul> Solução de papaína: <ul><li> Adicionar 5 ml de mídia dissecção em Worthington frasco papaína-022 (LK003178; concentração final, 20 U / ml) e incubar a 37 ° C por 60 min. </li></ul><ol><li> Decapitar filhotes de ratos com idades entre P1 (geralmente 2 filhotes) seguindo as diretrizes analisados ​​e aprovados por normas nacionais e seu Animal Care locais Institucional e Comitê de uso. </li><li> Retire a pele e crânio e do cérebro lugar na placa de Petri cheia de mídia dissecção fria. </li><li> Removido meninges, dissecar ambos os hemisférios e dissecar hipocampo. </li><li> Pique o hipocampo em ~ 1X1 peças mm e digerir papaína em solução a 37 ° C por min 11-13. </li><li> Uma vez que as peças de tecido se instalaram, remova cuidadosamente papaína e adicionar 5 ml de meio do hipocampo para remover todos os vestígios da enzima. Repita este passo e ressuspender em meio hipocampo (2 ml). </li><li> Triturar as células ~ 5 vezes até que não haja aglomerações esquerda, com três chama-polido pipetas de furos progressivamente menores (1000 M, ~ 500 mm e ~ 300 mm). </li><li> Uma vez triturado, passe as células através de um filtro de células (tamanho dos poros, 70 mM, BD Bioscience). Contar as células em 10 ml de suspensão. Ajustar o volume do meio do hipocampo, a fim de ter 400 mil células / ml. </li><li> Aspirar o laminina off as lamelas, e antes do cobre pode secar, placa de 200 mL da suspensão de células por lamínula. </li><li> Deixá-los para anexar por 60 min na incubadora, e depois adicionar 2 ml de meio de hipocampo por poço. </li><li> No dia seguinte, aspire média de idade do hipocampo para remover células mortas e detritos e adicionar 2 ml de pré-aquecido meio fresco do hipocampo. </li></ol> Manutenção Media Neurobasal: <ul><li> 500 ml neurobasal (Invitrogen, 12348-017) </li><li> 10 ml de soro livre suplemento B27 (Invitrogen, 17504-044) </li><li> 5 ml Penicilina-estreptomicina (Invitrogen, 15140-122) </li><li> 1,25 ml L-glutamina (Invitrogen, 25030-149) </li></ul> Alimentar a cultura de astrócitos neurônio-duas vezes por semana com o meio neurobasal, começando quatro dias após o plaqueamento. Pré-incubação da media cerca de 30 min na incubadora em um frasco ventilado para equilibrar a temperatura e CO 2. Parte 2: IMAGING CÁLCIO Buffer de gravação do hipocampo: 110 mM NaCl, 5,4 mMKCl, 1,8 mM CaCl 2, 0,8 mM MgCl 2, 10 mM D-glicose, 10 mM HEPES (Todos os produtos químicos da Sigma) pH 7,4 (ajustado com NaOH). Corante indicador de carga de cálcio em astrócitos <ol><li> Lugar a cultura em um poço em uma placa de seis bem-cheia com 2 ml de buffer de gravação contendo 2,5 mM hipocampo Fluo-4, AM (Invitrogen, F-14217) e 0,05% PLURONIC solução ® F-127 20% em DMSO (Invitrogen, P-3000MP) e incubar à temperatura ambiente por 10-30 min. </li><li> Remover Fluo-4 solução e lavar 3 vezes com lamínulas buffer de gravação do hipocampo e incubar em temperatura ambiente por 30 min. </li><li> Coloque a lamínula na câmara, e limpar o outro lado da lamela com o líquido de limpeza da lente. </li><li> Coloque uma pequena gota de óleo de imersão (imersão DF TIPO Óleo de Cargille) sobre o objectivo e colocar câmara (câmara aberta por 25 mm de lamela rodada Warner Instruments) no palco do microscópio (Olympus IX71). </li><li> Olhe para as células usando a luz de transmissão para ver como as pilhas olham, e levá-los em foco. Em seguida, iluminar as células com 488 nm de um monocromador (Polychrome V da TILL Vision) e determinar se o sinal de fluorescência de Fluo-4 é uniforme e detectável em astrócitos. </li><li> Use EPI e TIRF iluminação para medir o cálcio nos astrócitos. </li></ol> Microscopia TIRF Resumidamente, nós usamos um microscópio Olympus IX71 equipado com uma câmera DV887DCS ixon Andor EMCCD. O controle de excitação e de aquisição de imagem é conseguido usando software TILLVision. As vigas de 454/488/515 nm Argônio (100 mW) e 442 nm de estado sólido (45 mW) lasers são combinados e controlados com um ATÉ Polyline combinador de laser, TIRF condensador porta dupla e filtro sintonizável acoustoptical e controlador (AOTF; tudo a partir de ATÉ Photonics) e alimentado em uma fibra banda larga Kineflex para a entrada no condensador TIRF. Usamos uma lente Olympus 60X 1,45 NA alcançar TIRF. O ganho da câmera é ajustada para cada astrócitos para fornecer o melhor sinal em imagens de ruído. O fundo e os princípios da microscopia TIRF foram avaliados recentemente 4, 5. A maioria dos componentes ópticos que usamos foram comprados da TILL Photonics, que agora faz parte da Agilent Technologies (http://www.till-photonics.com/). A profundidade de penetração TIR pode ser calculada a partir das equações abaixo. <img style="float:left;" src="/files/ftp_upload/1142/equation.png" alt="Equação" /> d = profundidade de penetração n1 = índice de refração do vidro n2 = índice de refração do celular a = ângulo de incidência NAi abertura = numérica de incidência A fim de garantir o laser é alinhado de maneira ideal para TIRF achamos útil observar 100 nm fluorescentes talão (Invitrogen, F8803). Apresentamos ainda os quadros e vídeos de contas com EPI e microscopia TIRF. Quando em TIRF, observa-se um aumento dramático no sinal-ruído e os cordões de mostrar difusão browniano. Encontramo-lo útil para observar o comportamento de 100 nm com contas TIRF microscopia em uma base regular (~ uma vez por semana) para ter certeza de que TIRF ideal ocorre, ao invés de a iluminação comprometida oblíqua que ocorreria se o ângulo crítico não foram iguais a α (ver Fig. 1). Aplicação da proteína G-agonistas do receptor acoplado Astrócitos expressam uma variedade de Gq-coupled receptors 6, 7, incluindo receptores de glutamato metabotrópicos e receptores P2Y (agonista, ATP, ADP). Ativação desses receptores leva a aumentos significativos nos níveis de cálcio intracelular dentro de astrócitos. Por exemplo, se pode observar elevações de cálcio intracelular durante a aplicação da ATP (30 mM) para 10/08 astrócitos. Usamos uma solução rápida switcher da Warner Instruments chamado de VC-77SP sistema de perfusão Fast-Step (http://www.warneronline.com/index.cfm). Com este soluções método pode ser aplicado em menos de ~ 10 ms. <img src="/files/ftp_upload/1142/Fig 1a.jpg" alt="Figura 1" /> <img src="/files/ftp_upload/1142/fig1c.jpg" alt="" /> FIGURA 1. Dos desenhos animados e fotografias da imagem criada. A. mostra uma fotografia do microscópio montado em um airtable, enquanto que (B) mostra uma fotografia do conjunto do laser, os controladores e as caixas de feixe. C. Mostra uma foto do estágio do microscópio com a câmara montada para a imagem latente. Do lado esquerdo do dispositivo de perfusão rápida pode ser visto (junto com o motor de passo e tubos theta). À direita o headstage de um amplificador 200A Axopatch é visto. O cartoon esquematiza o caminho da luz no configurar e como TIRF é alcançado. O laser é focalizada sobre o plano focal posterior da lente objetiva 60X 1,45 NA e sua posição é ajustada para fora do centro, para que ela emerge para o óleo de imersão na α ângulo crítico. Neste ponto, o feixe sofre reflexão interna total e decai com uma distância λ (ver equação no texto principal) no meio de menor índice de refração. Neste caso, este é o buffer de gravação em torno do astrócitos e os astrócitos si. O resultado é excitação óptica (e, portanto, de imagem) de moléculas dentro de ~ 100 nm da membrana plasmática. No desenho da célula isso é mostrado como verde &quot;animado&quot; receptores de membrana, enquanto que aqueles dentro da célula ou na superfície superior da célula não está animado. Um relato completo da microscopia TIRF foi fornecido por Steyer e Almers 4. <img src="/files/ftp_upload/1142/fig2.png" alt="Figura 2" /> FIGURA 2. Imagens de 100 nm grânulos fluorescentes adquiridos com EPI e microscopia TIRF. A. Mostra imagens EPI de um campo de visão com várias dezenas de contas de 100 nm fluorescente. O ponto de setas vermelhas para contas que se instalaram sobre a lamínula de vidro, enquanto que as setas azuis apontam para contas que estão difundindo na água. B. Mostra uma imagem TIRF do mesmo campo de visão, como mostrado na A. Neste visualizar apenas as contas aderente mostrado por setas vermelhas são visíveis. Isto porque estas se tinham estabelecido para a coverlsip de vidro e foram, portanto, dentro do campo nm ~ 100 evanescente. As contas mostrado em A por setas azuis não estão dentro desta região e são, portanto, invisível nas imagens TIRF. As parcelas inferiores mostram renderização em 3D das imagens. É claro que um grande aumento no sinal-ruído ocorre para contas dentro do campo evanescente quando observado por microscopia TIRF. De fato, para estas imagens o sinal-ruído para o PAV foi de 7,1 ± 0,6, enquanto que para TIRF foi 20 ± 0,7. <img src="/files/ftp_upload/1142/fig3.png" alt="Figura 3" /> FIGURA 3. Imagens de astrócitos carregado com Fluo-4 corante indicador de cálcio adquiridos com EPI e microscopia TIRF. A. EPI imagens de um campo de visão com cinco astrócitos. B. Uma imagem TIRF do mesmo campo de visão mostrada na B. Note-se que as imagens em A e B são significativamente diferentes. Isto porque com TIRF iluminação apenas as regiões da membrana plasmática em aposição perto da lamela de vidro são gravadas. Os painéis inferiores mostram ATP evocadas transientes de cálcio intracelular fotografada com EPI e microscopia TIRF.

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É bem estabelecido que os astrócitos mostrar elevações de cálcio intracelular. Estas ocorrem de forma espontânea, pode ser desencadeada pela atividade neuronal ou por aplicação de agonistas para ativar receptores na superfície de astrócitos 11. Uma questão importante e controversa é se astrócitos elevações de cálcio intracelular pode desencadear a liberação de moléculas de sinalização que ativam os receptores nos neurônios 11, 12. Este é controverso, porque tem havido evidências a favor e contra...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>VWR&#174; Micro Cover Slips, Round, No. 1</td>
<td>Tool</td>
<td>VWR</td>
<td>48380-068</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0899</td>
<td>&nbsp;</td>
<tr>
<td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L2020</td>
<td>&nbsp;</td>
<tr>
<td>Earle&#x2019;s Balanced Salt Solution (EBSS) (1X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14155-063</td>
<td>&nbsp;</td>
<tr>
<td>Minimum Essential Medium (MEM) (1X), liquid Contains Earle's salts, but no L-glutamine or phenol red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>51200-038</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate solution</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>HEPES solution 1 M</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H0887</td>
<td>&nbsp;</td>
<tr>
<td>N-2 Supplement (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>&nbsp;</td>
<tr>
<td>Horse Serum, Heat-Inactivated</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>26050-088</td>
<td>&nbsp;</td>
<tr>
<td>PAPAIN-022</td>
<td>Reagent</td>
<td>Worthington</td>
<td>LK003178</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal&#x2122; Medium (1X) Liquid without Phenol Red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>12348-017</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Serum-Free Supplement (50X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine-200 mM (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25030-149</td>
<td>&nbsp;</td>
<tr>
<td>Cell Strainers</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>21108</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M2670</td>
<td>&nbsp;</td>
<tr>
<td>HEPES free acid</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G7528</td>
<td>&nbsp;</td>
<tr>
<td>Fluo-4, AM 1 mM solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F-14217</td>
<td>&nbsp;</td>
<tr>
<td>Pluronic&#174; F-127 20% solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>P-3000MP</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil TYPE DF</td>
<td>Microscope</td>
<td>Cargille</td>
<td>16242</td>
<td>&nbsp;</td>
<tr>
<td>Open chamber for 25 mm round coverslips, 100 &mu;l volume</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0362 (RC-21BDW)</td>
<td>&nbsp;</td>
<tr>
<td>P-2 platform for Series 20 chambers, non-heater</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0278 (P-2)</td>
<td>&nbsp;</td>
<tr>
<td>FluoSpheres carboxylate-modified microspheres, 0.1 &mu;m, yellow-green fluorescent (505/515) 2% solids</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F8803</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measuring near plasma membrane and global intracellular calcium dynamics in astrocytes

O cérebro contém células da glia. Astrócitos, um tipo de célula glial, tem sido conhecido por fornecer um papel passivo de apoio aos neurônios. No entanto, evidências crescentes sugerem que os astrócitos também podem participar ativamente na função cerebral por meio de interações funcionais com os neurônios. No entanto, muitos aspectos fundamentais da biologia astrócitos permanecem controversas, pouco clara e / ou experimentalmente inexplorado. Uma questão importante é a dinâmica dos transientes de cálcio intracelular em astrócitos. Isso é relevante porque o cálcio é bem estabelecido como um importante mensageiro segundo e porque tem sido proposto que a elevação de cálcio astrócitos pode desencadear a liberação de transmissores de astrócitos. No entanto, não houve qualquer descrição detalhada ou satisfatória de cálcio perto da membrana plasmática de sinalização em astrócitos. Reflexão interna total de fluorescência microscopia (TIRF) é uma ferramenta poderosa para analisar fisiologicamente relevantes eventos de sinalização dentro de cerca de 100 nm da membrana plasmática de células vivas. Aqui, usamos a microscopia TIRF e descrever como monitor perto de membrana plasmática e global a dinâmica do cálcio intracelular quase simultaneamente. O aperfeiçoamento e aplicação sistemática desta abordagem tem o potencial de informar sobre os detalhes precisos de sinalização de cálcio astrócitos. Um entendimento detalhado da dinâmica de cálcio astrócitos pode fornecer uma base para entender se, como, quando e por que astrócitos e neurônios sofrem de cálcio dependentes de interações funcionais.

Watch this Scientific Journal Video about Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes at JoVE.com

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Descrevemos como medir perto da membrana e global a dinâmica do cálcio intracelular em astrócitos cultivados utilizando reflexão interna total e microscopia de epifluorescência.

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, ...

measuring-near-plasma-membrane-global-intracellular-calcium-dynamics

Research

JoVE Journal

Biology

13.0K visualizações.  David Geffen School of Medicine, University of California, Los Angeles. Descrevemos como medir perto da membrana e global a dinâmica do cálcio intracelular em astrócitos cultivados utilizando reflexão interna total e microscopia de epifluorescência.

Vídeo: Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Taxas de medição de plasma Proteína de membrana endocítica por Biotinilação reversível

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Biologia

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Fluxos de medição de cálcio rápido no Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

Mapeamento de Difusão Molecular na membrana plasmática por Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim 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, ...

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 ...

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

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

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 ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

Determinação da membrana plasmática de particionamento para proteínas associadas perifericamente

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.

Caracterização da ruptura de membrana de Plasma MLKL-mediada em Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Medições simultâneas de cálcio intracelular e potencial de membrana no endotélio Cerebral rato recém isoladas e intacta

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

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 ...