“吞噬体封闭试验”可视化吞噬体形成的三个维度利用全内反射荧光显微镜（TIRFM）
Summary
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.
Abstract
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.
Introduction
吞噬作用是，随着识别和表面受体，其然后导致摄入材料的内化和降解材料的结合开始的主要细胞功能。而单细胞真核生物如模具粘菌和阿米巴使用吞噬的细菌喂食，高等生物进化与专业的细胞。巨噬细胞或树突状细胞是针对在各种组织和器官的病原体防御的第一线，并且通过抗原呈递和细胞因子的产生1-4激活适应性免疫系统的关键。在某些情况下吞噬作用可以由非专业的吞噬细胞， 例如，内皮细胞和上皮细胞中进行。这个过程是非常重要的开发过程中，并在成年后正常组织转化和重塑保持动态平衡。最后，专门吞噬细胞如支持细胞中的睾丸或视网膜色素上皮细胞是非常有效的吞噬细胞5。
一个吞噬体的形成，其中的微生物或细胞碎片的降解发生与吞噬受体的吞噬细胞的表面上的聚类开始。如Fc受体（FCR）或补体受体（CRS）以下调理受体集群下游信号事件已得到很好的特点。然而，也有许多非调理性受体，包括Toll样受体（TLRs），凝集素，甘露糖受体和清道夫受体。这些受体识别的粒子表面上的决定因素如甘露糖或岩藻糖残基，磷脂酰丝氨酸，和脂多糖1,6-9。
病原体或细胞碎片的识别包括结合和多种类型的吞噬受体，然后引起强烈的和短暂的肌动蛋白重塑的群集。在细胞内compartmen并行，焦胞吐TS有助于膜张力的释放而为大颗粒的有效细胞吞噬重要。导致肌动蛋白聚合和膜变形的信号事件在实验模型触发一个吞噬受体进行了解剖。期间的FcR介导的吞噬作用，存在由小GTP酶（外消旋，Cdc42的）调节的强烈的肌动蛋白聚合。在他们的下游效应，所述Wiskott-Aldrich公司综合征蛋白（WASP）导致了成核肌动蛋白微丝1,2,4,10的肌动蛋白相关蛋白2/3复合体（的Arp2 / 3）的激活。当地生产磷脂4,5二磷酸（PI（4,5）P 2）的为驱动伪足形成初始肌动蛋白聚合的关键。其转化为PI（3,4,5）P 3需要伪足扩展和吞噬体封闭11。几个途径有助于PI（4,5）P2的消失。首先磷脂酰肌醇磷酸基那支队SES（PIPKIs）从吞噬逮捕PI（4,5）P2的合成。其次，它可以被磷酸化并通过类消耗我PI3K激酶（PI3K）和PI（3,4,5）P 3 12转换。一种用于磷酸酶和磷脂酶作用也已在PI（4,5）P2水解和F-肌动蛋白去除在哺乳动物细胞和在盘基网柄 13,14吞噬期间暗示。磷脂酶C（PLC）δ水解的PI（4,5）P 2为二酰基甘油和肌醇1,4,5- 三磷酸盐。该PI（4,5）P2和PI（3,4,5）P 3磷酸OCRL（罗威oculocerebrorenal综合征）也被牵连的吞噬小体的形成。 F-肌动蛋白和其解聚的精确局部形成在空间和时间被严格调控，我们已经表明细胞内隔室的招募到本地传递OCRL磷酸酶，从而在吞噬杯的底部有助于当地肌动蛋白解聚是重要<sup> 13,15。对于这一点，我们使用这里所描述的实验设置的。
对吞噬体闭合和膜断裂所需的机制和分子玩家仍定义不清，因为在可视化和监测吞噬体封闭的部位的困难。直到最近，观察到在该上其背面或在其两侧内在粒子固定或活细胞的吞噬能力，使得吞噬体闭合困难的部位的及时可视化。此外，固定方法可能会导致膜和偏见的伪足延伸和闭合的结果回缩。相比之下，我们已经建立并在这里描述的实验使我们能够可视化伪足延伸，并在活细胞13，吞噬关闭一步基于全内反射显微镜（TIRFM）16。该光学技术使用渐逝波在一个透明的界面，以激发在薄区域的荧光团，以便盖（盖玻片）和液体（细胞培养基）。激发深度的厚度为约100纳米的从固体表面，从而允许接近质膜分子事件的可视化。 TIRFM允许高的信号 – 背景比率，并且限制所收集的失焦荧光和细胞毒性由于细胞的照明。
趁着TIRFM，我们开发了“吞噬体闭合测定法”，其中盖玻片用聚赖氨酸活化，然后涂上的IgG调理的红血细胞（IgG的SRBCs）。然后表达的兴趣瞬时荧光标记蛋白的巨噬细胞被允许吞没的IgG-SRBCs。而细胞分离该非共价结合于玻璃表面上的目标粒子，所述伪足的尖端可观察并记录在TIRF模式。 TIRF收购相结合，与在落射荧光模式采集，移位上述阶段3微米，这允许相后吞噬杯的基部的ualization。的药理学药物，例如那些在此过程中抑制肌动蛋白或dynamin上加成，也可以在分子水平上，以进一步解剖过程。
在这里详细描述的协议为RAW264.7小鼠巨噬细胞系和调理粒子，但实际上，它可以适用于任何其他吞噬细胞，并与其他目标，如珠。这种方法将使监管更好的表征在时间和在各种吞噬过程中涉及伪足延伸和吞噬封闭分子球员空间。
Protocol
Representative Results
Discussion
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 °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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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. Nadège 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 Médicale (FRM INE20041102865, FRM DEQ20130326518 including a doctoral fellowship for FMA) to FN, and Agence Nationale de Recherche sur le SIDA et les hépatites virales” (ANRS) including a doctoral fellowship for JM.
Materials
| Anti-sheep red blood cells IgG | MP Biomedicals | 55806 | |
| Bovine Serum Albumin heat shock fraction, pH 7, ≥98% | Sigma | A7906 | |
| Cell lifter | Corning | 3008 | |
| Cuvettes 4mm | Cell project | EP104 | |
| DPBS, no calcium, no magnesium | Thermo Fischer Scientific | 14190-094 | Room temperature |
| Electrobuffer kit | Cell project | EB110 | |
| 100mm TC-Treated Cell Culture Dish | Corning | 353003 | |
| Gene X-cell pulser | Biorad | 165-2661 | |
| Gentamicin solution | Sigma | G1397 | |
| Glass Bottom Dishes 35 mm uncoated 1.5 | MatTek corporation | P35G-1.5-14-C Case | |
| iMIC | TILL Photonics | Oil-immersion objective (N 100x, NA1.49.), heating chamber with CO2, a camera single photon detection EMCCD ( Electron Multiplying Charge Coupled Device) and a 1.5X lens | |
| Poly-L-Lysine Solution 0.1% | Sigma | P8920-100ml | Dilution at 0.01% in water |
| RPMI 1640 medium GLUTAMAX Supplement | Life technologies | 61870-010 | |
| RPMI 1640 medium, no phenol red (10×500 ml) | Life technologies | 11835-105 | Warm in 37°C water bath before use |
| Sheep red blood cells (SRBCs) | Eurobio | DSGMTN00-0Q | Conserved in Alsever buffer at 4°C before use |
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