Name: "phagosome closure assay" to visualize phagosome formation in three dimensions using total internal reflection fluorescent microscopy (tirfm)
Uploaded: 2016-08-26T15:00:00.000+00:00
Duration: 10 min 7 s
Description: Watch this Scientific Journal Video about "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM at JoVE.com

We thank Dr. Alexandre Benmerah (Institut Imagine Necker, Paris, France) for initial discussions on the experimental approach and Dr. Jamil Jubrail for reading the manuscript. Nad&#232;ge Kambou and Susanna Borreill are acknowledged for performing experiments with the method in our laboratory. This work was supported by grants from CNRS (ATIP Program), Ville de Paris and Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM INE20041102865, FRM DEQ20130326518 including a doctoral fellowship for FMA) to FN, and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales" (ANRS) including a doctoral fellowship for JM.

Nota: O plasmídeo utilizado Lifeact-mCherry é um dom tipo de Dr. Guillaume Montagnac, Institut Curie, Paris, gerada após 17. 1. Células e Transfecção Nota: RAW264.7 de macrófagos são crescidas até à sub-confluência em meio completo (meio RPMI (Roswell Instituto Parque Memorial) 1640, HEPES 10 mM, piruvato de sódio 1 mM, 50 ^ M β-mercaptoetanol, 2 mM de L-glutamina e 10% de FCS ( soro fetal de vitelo)) em uma placa de 100 mm. Eles são transf ectadas com plasmídeos que codificam para proteínas marcadas fluorescentemente por electroporação. Rotineiramente aproximadamente 5-6 x 10 6 células são transfectadas com 20 ug ou 10 ug de plasmídeo para cada transfecção e co-transfecção, respectivamente. Note-se que outros meios de transfecção à base de electroporação ou lipofecção podem ser utilizados como abordagens alternativas. <ol><li> Raspar as células com um levantador de células e ressuspender-los em 10 ml de meio de cultura por pipetagem para cima e para baixo várias vezes. </li><li> Centrifugar asuspensão de células 300 x g, 5 min a RT no rotor de ângulo oscilante. </li><li> Pré-aqueça 10 ml de meio completo suplementado com 10 ug / ml de gentamicina, a 37 ° C. </li><li> A seguir à centrifugação, desprezar o sobrenadante e ressuspender o sedimento em 3 ml de &quot;tampão de lavagem A&quot; a partir do kit de electroporação. </li><li> Centrifugar a suspensão de células a 300 xg, 5 min a temperatura ambiente no rotor de ângulo oscilante. </li><li> Enquanto isso preparar a mistura de ADN à temperatura ambiente: 120 ul de 2x tampão B, 20 ^ g de ADN plasmidico que codifica para a proteína de interesse, qsp 240 mL de H 2 O. </li><li> A seguir à centrifugação, eliminar todo o sobrenadante. </li><li> Ressuspender o sedimento de células na mistura de ADN e transferência em cuvetes de electroporação de 4 mm. </li><li> Incubar à temperatura ambiente durante 3 min. </li><li> Electroporate a 250 V, 900 uF. </li><li> Imediatamente ressuspender as células no pré-aquecido (37 ° C) de meio completo suplementado com gentamicina e a chapam em uma placa de 100 mm. </li><li> Incubam-se as células transfectadas durante a noite a 37 ° C, 5% de CO 2. </li><li> Na manhã seguinte, substituir o meio completo com 10 ml de meio de microscopia isento de soro (RPMI 1640 sem vermelho de fenol, 10 mM de HEPES, 1 mM de piruvato de sódio, 50 uM β-mercaptoetanol, 2 mM de L-glutamina). </li></ol> 2. Opsonização de glóbulos vermelhos Nota: Como um modelo de meta de partículas para os macrófagos, células do sangue ovelhas vermelhas (SRBCs) são utilizados. Normalmente, cerca de 7 x 10 6 SRBCs por 35 milímetros prato fundo de vidro é usado. <ol><li> Lavar os SRBCs com 100 ul de 1x PBS (fosfato salino tamponado) /0.1% de BSA (albumina de soro bovino) e centrifugação (600 xg, 4 min). </li><li> A seguir à centrifugação, desprezar o sobrenadante e ressuspender as SRBCs em 100 ul de 1x PBS / 0,1% BSA e centrifugação (600 xg, 4 min). </li><li> A seguir à centrifugação, desprezar o sobrenadante e ressuspender as SRBCs em 1X PBS / BSA a 0,1% com IgG de coelho anti-SRBCsna sub-aglutinar concentração. Utilizar 500 ul de solução contendo anticorpo / 5 ul de SRBCs. Nota: A concentração sub-aglutinação de anticorpos corresponde à concentração mais baixa que não induziu aglutinação detectada como a formação de uma rede. Em geral, a concentração sub-aglutinação é de 8,2 ug / ml. <ol><li> Determinar a concentração de anti-IgG de SRBCs necessários para opsonizar os SRBCs por um teste de neutralização. Serialmente dilua a anti-IgG de SRBCs (estoque a 13,1 mg / ml) entre 1/50 - 1 / 25.600 de uma microplaca. Adicionar 2 x 10 6 SRBCs em cada poço. Incubar a placa no escuro à temperatura ambiente durante várias horas. </li></ol></li><li> Incubar à temperatura ambiente durante 30 minutos com rotação lenta. </li><li> Após duas lavagens em 1X PBS / BSA a 0,1% como descrito acima, ressuspender os SRBCs-IgG (IgG-opsonizadas SRBCs) em meio isento de soro microscopia pré-aquecido (2 ml / prato). </li></ol> 3. Revestimento de poli-lisina de lamelas <ol><li> Enquanto isso, Tratar pratos 35 milímetros de vidro de fundo com 2 ml de 0,01% de poli-L-lisina durante 30 min à temperatura ambiente. </li><li> Lavar os pratos duas vezes com 2 ml de PBS 1x. </li></ol> 4. Fixação não-covalente de SRBCs em pratos com fundo de vidro <ol><li> Despeje 2 ml de SRBCs suspensão por 35 milímetros prato de vidro de fundo. </li><li> Centrifugar com um rotor girando a 500 xg durante 2 min para 35 milímetros de vidro pratos de fundo. </li><li> Remover o sobrenadante e lavar uma vez com 2 ml de PBS 1X / BSA a 10%. </li><li> Incubam-se as partículas durante 30 min com 2 ml de PBS 1X / BSA a 10% por placa de Petri. </li><li> Lavar os pratos três vezes com 2 ml de PBS 1x. </li><li> Substituir 1x PBS com 2 ml de meio pré-aquecido (37 ° C) sem soro microscopia. </li></ol> 5. A fagocitose visualizado por TIRFM <ol><li> Microscópio Nota: TIRFM foi realizada usando um microscópio equipado com uma objectiva de imersão em óleo (N 100X, NA1.49), uma câmara de aquecimento com CO 2, e duas cantele câmeras de detecção de fótons EMCCD (Electron Multiplicando Charge Coupled Device) acoplado com uma lente de 1.5X. <ol><li> O dia antes da sessão de microscópio TIRF, ligar a câmara de aquecimento a 37 ° C para permitir um aquecimento homogéneo da platina do microscópio. </li></ol></li><li> Determinação ângulo crítico Nota: software ImageJ Color Profiler é usado para processar fluxos de imagem TIRF. <ol><li> Coloque um 35 milímetros prato de vidro de fundo contendo SRBC opsonizado com meio de microscopia sem soro sob o microscópio. </li><li> Raspar as células e ressuspender-los na forma antes de os adicionar (100 - 500 ul) no prato. </li><li> Utilizar o software &quot;Aquisição vivo&quot; para controlar o microscópio e executar de excitação com um 491 nm e / ou um laser de 561 nm para identificar as células que expressam as proteínas marcadas fluorescentemente. </li><li> Identificar uma célula que expressa as proteínas marcadas fluorescentemente. </li><li> Coloque-o no meio do campo e adquirir 500 imas idades em um comprimento de onda de excitação, com ângulos diferentes a partir de 0º até 5º, com um incremento de 0.01º (Figura 2A). Os ângulos são automaticamente alterada pelo sistema de microscópio. </li><li> Determinar o ângulo crítico permitindo que a luz incidente seja totalmente reflectida na interface vidro / médio e gerar a onda evanescente. Usando o software ImageJ Color Profiler, abra a sequência de imagens, clicando em &quot;Arquivo&quot;, &quot;Abrir&quot; e selecione o arquivo. </li><li> Seleccionar uma região de interesse (ROI), com a ferramenta rectangular, na célula com uma fluorescência uniforme (Figura 2B amarelo 1). </li><li> Traçar o &quot;perfil do eixo Z&quot; significa intensidade de fluorescência medida no ROI com a função dos ângulos no eixo x, clicando na guia &quot;Imagem&quot;, &quot;Pilhas&quot; no menu suspenso e &quot;Plot-eixo Z perfil&quot; para baixo (Figura 2B vermelho 2). Nota: O ângulo crítico é o ângulo que conduz à mafluorescência ximum antes de uma queda acentuada na fluorescência. </li><li> Usar qualquer valor de ângulo sobre o eixo x superior ao ângulo crítico durante a sessão de microscopia para obter um sinal TIRF como exemplo 2.00 (Figura 2C). </li></ol></li><li> aquisições <ol><li> Usando o software vivo Aquisição, configurar os parâmetros de aquisição com o módulo &quot;Protocolo Editor&quot; (Figura 3A). <ol><li> Criar um protocolo com um &quot;loop&quot; compreendendo &quot;Canais Multi&quot; aquisição para adquirir sinal fluorescente a partir de proteínas de interesse na região de TIRF. Introduzir o ângulo TIRF (por exemplo 2,00), o tempo de exposição (por exemplo, 50 ms) e a intensidade do laser (por exemplo, 50%) (Figura 3B 1). </li><li> Introduzir um &quot;movimento Z&quot; da mm objectivo dos 3 acima da região TIRF para obter a aquisição de sinal na epifluorescência com o &quot;Canais Multi&quot; ferramenta. Insira um ângulo abaixo tele ângulo crítico (por exemplo 1,00), o tempo de exposição (50 ms) e a intensidade do laser (50%) (Figura 3B, 2 e 3). </li><li> Em seguida adicione um &quot;z move&quot; da mm objectiva 3 abaixo, para retornar na região de TIRF (Figura 3B 4). Adicionar um &quot;instantâneo&quot; da célula em LED (Light-Emitting Diode) (Figura 3B 5) luz brilhante. </li></ol></li><li> Encontrar uma célula de interesse, com um nível moderado de expressão de proteína marcada com fluorescência que inicia a fagocitose de SRBC por membrana plasmática que se estende em torno da partícula. </li><li> Coloque-o no meio do campo. </li><li> Iniciar a transmissão de aquisição de 500 - 1000 quadros. Na aba &quot;contagem de loop&quot;, digite &quot;750 frames&quot; (Figura 3B 1). Nota: software ImageJ Color Profiler é usado para processar fluxos de imagem TIRF. </li><li> Abra as imagens da sequência de programa Image J clicando&quot;Arquivo&quot;, &quot;Abrir&quot; e escolher o arquivo. </li><li> Separar os canais clicando na guia &quot;Imagem&quot;, &quot;Hyperstacks&quot; no menu suspenso e em &quot;Stack para hyperstack&quot; função. Conclua a janela apareceu: Ordem: xyctz; Channel (c): número de canais (ex: &quot;2&quot; se você tem dois fluorocromos); Slices (z): número de fatias em eixo z (ex: &quot;1&quot; se não há movimento no eixo Z); Frames (t): número de imagens divididas por número de canais; Modo de exibição: escala de cinza. Nota: Duas sequências de imagem separados são gerados, correspondendo aos dois canais diferentes. </li></ol></li></ol>

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The authors have nothing to disclose.

The experimental protocol described here proposes an unprecedented method to follow in real time and in living cells, with high-resolution, the formation of a phagosome and in particular its closure. Several technical aspects have to be discussed. Firstly, the assay is very sensitive to temperature. It is very important to check that the heating chamber is at 37 &#176;C and that all media, devices or cells are kept within the chamber to avoid temperature changes that could impair the efficiency of phagocytosis. We notice...

Fagocitose é uma função de célula grande que começa com o reconhecimento e ligação de material para a superfície receptores, que depois leva à internalização e degradação do material ingerido. Enquanto eucariotas unicelulares como o Dictyostelium discoideum molde e amebas usar fagocitose para a alimentação de bactérias, organismos superiores evoluíram com células profissionais. Os macrófagos ou células dendríticas são a primeira linha de defesa contra agentes patogénicos em vários tecidos e órgãos, e são cruciais para activar o sistema imunitário adaptativo através de apresentação de antigénio e produção de citocinas 1-4. Sob certas circunstâncias a fagocitose pode ser realizada por células fagocíticas não profissionais, por exemplo, células endoteliais e epiteliais. Este processo é importante para manter a homeostase durante o desenvolvimento e na vida adulta de renovação do tecido normal e de remodelação. fagócitos Finalmente, especializados, tais como células de Sertoli nos testículos ou da retinanas células epiteliais pigmentares são extremamente potentes fagócitos 5. A formação de um fagossoma onde a degradação de microrganismos ou fragmentos celulares ocorre começa com o agrupamento de receptores fagocíticos na superfície da célula fagocítica. eventos de sinalização a jusante seguintes agrupamento de receptores opsónicos, tais como receptores Fc (FcR) ou receptores de complemento (CRS) têm sido bem caracterizadas. No entanto, também existem numerosas receptores não-opsónicos incluindo receptores semelhantes a Toll (TLRs), lectinas, receptores de manose e receptores scavenger. Estes receptores reconhecem determinantes sobre a superfície das partículas, tais como resíduos de manose ou fucose, fosfatidilserina, e lipopolissacarídeos 1,6-9. Agente patogénico ou fragmentos de células de reconhecimento envolve a ligação ao agrupamento e de vários tipos de receptores de fagocitária, que, em seguida, conduzir a uma intensa e transiente remodelação da actina. Em exocitose paralelo, focal de compartmen intracelularTS contribui para a libertação da tensão da membrana e é importante para a fagocitose eficiente de grandes partículas. Os eventos de sinalização que levam à deformação de polimerização e membrana actina foram dissecados em modelos experimentais que desencadearam um único receptor fagocítica. Durante a fagocitose FcR-mediada, há intensa polimerização de actina, que é regulada por pequenas GTPases (Rac, Cdc42). Entre os seus efectores a jusante, a proteína síndroma de Wiskott-Aldrich (WASP) conduz à activação da proteína relacionada com a actina 2/3 complexo (Arp2 / 3) que nucleia 1,2,4,10 filamentos de actina. A produção local de fosfatidilinositol-4, 5-bifosfato (PI (4,5) P 2) é crucial para a polimerização de actina inicial que dirige a formação pseudópode. Sua conversão em PI (3,4,5) P 3 é necessário para a extensão pseudopod e fechamento fagossomo 11. Diversas vias de contribuir para o desaparecimento de PI (4,5) P 2. Em primeiro lugar destacamento de fosfatidilinositol kina fosfatoses (PIPKIs) do PI prisões fagosoma (4,5) P 2 síntese. Em segundo lugar, pode ser fosforilada e consumido por PI3K de classe I-quinases (PI3K) e convertido em PI (3,4,5) P 3 12. Um papel para fosfatases e fosfolipases também tem sido implicado em PI (4,5) P 2 hidrólise e remoção de F-actina durante a fagocitose em células de mamífero e em Dictyostelium 13,14. O δ fosfolipase C (PLC) hidrolisa PI (4,5) P 2 em diacilglicerol e fosfato de tris-inositol-1,4,5-. O OCRL PI (4,5) P 2 e PI (3,4,5) P 3 fosfatase (síndrome oculocerebrorrenal de Lowe) também tem sido implicado na formação do fagossoma. formação local preciso da F-actina e sua despolimerização é estreitamente regulada no espaço e no tempo e temos demonstrado que o recrutamento de compartimentos intracelulares é importante para entregar localmente a fosfatase OCRL, contribuindo assim para a despolimerização de actina locais na base do copo fagocitária <sup&gt; 13,15. Para isso, utilizou-se o conjunto experimental descrita aqui. Os jogadores mecanismo e moleculares necessários para o encerramento fagossomo e cisão da membrana permanecem pouco conhecidos devido às dificuldades em visualizar e monitorar o local de encerramento fagossomo. Até recentemente, a fagocitose foi observado em células fixas ou vivos que internalizam partículas na face dorsal ou em seus lados, tornando a visualização em tempo útil do local de fechamento fagossomo difícil. Além disso, métodos de fixação pode causar retração de membranas e enviesar os resultados na extensão pseudópodos e encerramento. Em contrapartida, o ensaio que criámos e descrever aqui nos permite visualizar a extensão pseudopod ea etapa de encerramento da fagocitose em células vivas 13, com base em microscopia interna total reflexão (TIRFM) 16. Esta técnica utiliza óptica uma onda evanescente para excitar fluoróforos numa zona fina na interface entre um modo transparentetampa (lamela) e um (meio de cultura de células) de líquido. A espessura da profundidade de excitação é de cerca de 100 nm a partir da superfície sólida, permitindo a visualização de eventos moleculares perto da membrana plasmática. TIRFM permite uma elevada relação de sinal-para-fundo, e limita a fluorescência para fora de foco recolhida e a citotoxicidade devido à iluminação de células. Tomando vantagem de TIRFM, foi desenvolvido o &quot;ensaio de encerramento fagossoma&quot; em que lamelas são activados com poli-lisina e, em seguida, revestida com as células vermelhas do sangue de IgG-IgG-opsonizados (SRBCs). Macrófagos que expressam proteínas transitoriamente fluorescente etiquetado de interesse são, então, autorizado a engolir as IgG SRBCs. Enquanto as células separar as partículas que são alvo não-ligado de forma covalente à superfície do vidro, as pontas dos pseudopods podem ser observadas e registadas no modo de TIRF. aquisições TIRF são combinados com aquisições no modo de epifluorescência, após o deslocamento da uM fase 3 acima, o que permite a visualization da base do copo fagocítica. A adição de drogas farmacológicas, tais como os da inibição da actina ou dinamina durante o processo também é possível, para dissecar ainda mais o processo, a nível molecular. O protocolo descrito em pormenor aqui é para uma linha celular de macrófagos de murídeo e RAW264.7 partículas opsonizadas, mas virtualmente, ele pode ser adaptado a qualquer outra célula fagocítica e com outros objectivos, tais como esferas. Este método permitirá uma melhor caracterização da regulação no tempo e no espaço dos jogadores moleculares envolvidos na extensão pseudopod e fechamento fagossoma durante vários processos fagocíticas.

<table><tbody><tr><td>Anti-sheep red blood cells IgG</td><td>MP Biomedicals</td><td>55806</td><td></td></tr><tr><td>Bovine Serum Albumin heat shock fraction, pH 7, &amp;ge;98%&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Cell lifter</td><td>Corning</td><td>3008</td><td></td></tr><tr><td>Cuvettes 4 mm</td><td>Cell project</td><td>EP104</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fischer Scientific</td><td>14190-094</td><td>Room temperature</td></tr><tr><td>Electrobuffer kit</td><td>Cell project</td><td>EB110</td><td></td></tr><tr><td>100 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Gene X-cell pulser</td><td>Biorad</td><td>165-2661</td><td></td></tr><tr><td>Gentamicin solution</td><td>Sigma</td><td>G1397</td><td></td></tr><tr><td>Glass Bottom Dishes 35 mm uncoated 1.5</td><td>MatTek corporation</td><td>P35G-1.5-14-C Case</td><td></td></tr><tr><td>iMIC</td><td>TILL Photonics</td><td></td><td>Oil-immersion objective (N 100X, NA1.49), heating chamber with CO&lt;sub&gt;2&lt;/sub&gt;, a camera single photon detection EMCCD (Electron Multiplying Charge Coupled Device) and a 1.5X lens</td></tr><tr><td>Poly-L-Lysine Solution 0.1%</td><td>Sigma</td><td>P8920-100ml</td><td>Dilution at 0.01% in water&amp;nbsp;</td></tr><tr><td>RPMI 1640 medium GLUTAMAX&amp;nbsp; Supplement</td><td>Life technologies</td><td>61870-010</td><td></td></tr><tr><td>RPMI 1640 medium, no phenol red (10 x 500 ml)</td><td>Life technologies</td><td>11835-105</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Sheep red blood cells (SRBCs)</td><td>Eurobio</td><td>DSGMTN00-0Q</td><td>Conserved in Alsever buffer at 4 &amp;deg;C before use</td></tr></tbody></table>

"phagosome closure assay" to visualize phagosome formation in three dimensions using total internal reflection fluorescent microscopy (tirfm)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

O sistema experimental descrito neste manuscrito está esquematicamente representada na Figura 1. Macrófagos RAW264.7 transfectadas que expressam as proteínas de interesse fundida com uma marcação fluorescente são colocados em contacto com glóbulos vermelhos de ovelha IgG-opsonizado (SRBCs) que eram não-covalentemente fixo sobre a lamela. Os macrófagos pode destacar o SRBC da lamela para afundá-lo. O microscópio utilizado TIRF permite a aquisição concomitante...

Watch this Scientific Journal Video about "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM at JoVE.com

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Fagossomo Encerramento Ensaio&quot; para Visualize Formação fagossomo em Três Dimensões Usando Total Internal Reflection Microscopia fluorescente (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

phagosome-closure-assay-to-visualize-phagosome-formation-three

Research

JoVE Journal

Immunology and Infection

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

6.4K visualizações.  Université Paris Descartes, Sorbonne Paris Cité. We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Vídeo: "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-célula Microscopia vídeo de Fagocitose patógeno fúngico

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Imunologia e Infecção

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Estudo da fagolisossomo Biogenesis em macrófagos ao vivo

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Técnica High Throughput Fluorometric de Avaliação da macrófago fagocitose e actina Polimerização

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Medindo phagosome pH Raciometrico por microscopia de fluorescência

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Imaging o neutrófilos fagossoma e citoplasma utilizando um indicador de pH Ratiometric

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Time-Lapse imagem em 3D da fagocitose por macrófagos de rato

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Visualizando Endocitose clatrina mediada por receptores acoplados à proteína a resolução de evento único através de microscopia TIRF

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Biologia

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

Visualizando os estágios iniciais da fagocitose

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

<meta charset="utf8"></head><body>Fluorescência ao vivo, imagem inversa de frisação celular e macropinocitose

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Fluorescência ao vivo, imagem inversa de frisação celular e macropinocitose

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

"Fagossomo Encerramento Ensaio" para Visualize Formação fagossomo em Três Dimensões Usando Total Internal Reflection Microscopia fluorescente (TIRFM)

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