哺乳动物细胞细菌挑战后应激颗粒形成的免疫荧光分析
Summary
我们描述了在用细菌和许多不同的应力攻击细胞后，哺乳动物细胞中应激颗粒形成的定性和定量分析的方法。该方案可用于研究广泛的宿主细菌相互作用中的细胞应激颗粒反应。
Abstract
细胞成分的荧光成像是调查宿主 – 病原体相互作用的有效工具。病原体可以影响感染细胞的许多不同特征，包括细胞器超微结构，细胞骨架网络组织，以及细胞过程如应激颗粒（SG）形成。病原体如何颠覆宿主过程的特征是发病机理领域的重要和组成部分。虽然可变表型可能容易可见，但是由病原体攻击诱导的细胞结构中的定性和定量差异的精确分析对于定义实验和对照样品之间的统计学显着性差异至关重要。 SG形成是进化上保守的应激反应，导致抗病毒反应，长期以来使用病毒感染进行了研究1 。 SG形成也影响信号级联，可能还有其他未知的结果适应2 。对病毒以外的病原体如细菌病原体的这种应激反应的表征目前是一个新兴的研究领域3 。目前，即使在病毒系统中，SG形成的定量和定性分析尚未被常规使用。在这里，我们描述了诱导和表征未感染细胞中SG形成和细胞感染细胞病原体的细胞的简单方法，其影响响应于各种外源胁迫的SG的形成。 SG形成和组成的分析通过使用许多不同的SG标记和ICY的点检测器插件（开源图像分析工具）来实现。
Introduction
在细胞水平上可视化宿主 – 病原体相互作用是获得对致病性策略和识别关键细胞途径的见解的有力方法。事实上，病原体可以用作确定重要细胞靶点或结构的工具，因为病原体已经发展为颠覆中枢细胞过程，作为其自身存活或繁殖的策略。可以通过重组表达荧光标记的宿主蛋白来实现细胞成分的可视化。虽然这允许进行实时分析，但具有特异标记宿主蛋白质的细胞系的产生是非常费力的并且可能导致不期望的副作用。使用特异性抗体检测细胞因子更方便，因为可以同时分析多个宿主因子，并且不限于特定的细胞类型。缺点是只有静态视图可以被捕获，因为免疫荧光分析需要宿主细胞修复离子。然而，免疫荧光成像的一个重要优点是其易于定性和定量分析。这又可用于获得统计学显着的差异，以提供对宿主 – 病原体相互作用的新见解。
荧光图像分析程序是执行3D和4D分析的强大的分析工具。然而，软件及其维护成本高昂使得基于免费开源软件的方法更具吸引力。使用生物分析软件的仔细图像分析是有价值的，因为它证实了视觉分析，并且在分配统计学意义时，增加了给定表型的正确性的置信度。以前，使用免费的ImageJ软件分析SG，这需要手动识别个人SG 4 。在这里，我们提供了一个在细菌SG形成诱导和分析方案使用免费的开源生物图像分析软件ICY（http://icy.bioimageanalysis.org）进行真实感染。生物图像分析软件具有高度适用于SG分析的内置点检测程序。它允许在指定的感兴趣区域（ROI）中对自动检测过程进行微调。这克服了对个别SG的手动分析的需要，并消除了抽样偏差。
许多环境胁迫引起SGs的形成，SG是直径为5 – 6μm的相间致密的胞质结构，非膜结构为0.2-5μm。这种细胞反应在酵母，植物和哺乳动物中进化上保守，并且当全球蛋白质翻译被抑制时发生。它涉及停滞的翻译起始复合物聚集到SG中，其被认为是用于翻译失活的mRNA的保留位置，允许细胞mRNA的子集的选择性翻译。消除压力后，SGs溶解，蛋白质合成的全球速率恢复。 SG由翻译延伸起始因子，涉及RNA代谢的蛋白质，RNA结合蛋白质以及支架蛋白质和参与宿主细胞信号传导的因子2组成 ，尽管精确的组成可以根据施加的应力而变化。诱导SG形成的环境因素包括氨基酸饥饿，紫外线照射，热休克，渗透压休克，内质网应激，缺氧和病毒感染2,7,8 。在理解病毒如何诱导和颠覆SG形成方面取得了许多进展，而关于其他病原体（如细菌，真菌或原生动物病原体）如何影响细胞应激反应1,7尚不清楚。
师哥lla flexneri是人类的革兰氏阴性兼性细胞溶质病原体和严重腹泻或志贺氏菌病的致病因子。志贺氏菌病是一个主要的公共卫生负担，每年导致5岁至9岁的儿童死亡28 000人。 S. flexneri感染结肠上皮，并通过劫持宿主的细胞骨架成分11,12扩散细胞对细胞。上皮细胞的感染支持了胞内溶胶酶的复制，但被感染的巨噬细胞通过炎性细胞死亡过程称为焦灼死亡。感染导致大量招募中性粒细胞和严重的炎症，伴随着热，氧化应激和组织破坏。因此，虽然被感染的细胞受到感染引起的内部应激，例如高尔基体破坏，遗传毒性应激和细胞骨架重排感染细胞也由于炎症过程而受到环境压力。
使用许多SG标记物表征S. flexneri感染对细胞对环境胁迫反应能力的影响已经证明，感染导致SG组成3的定性和定量差异。然而，其他细菌病原体知之甚少。在这里，我们描述了一种感染宿主细胞与细胞溶质病原体S. flexneri ，不同环境胁迫的细胞的应激，SG组分的标记，以及感染SG的形成和组成的定性和定量分析的方法和非感染细胞。该方法广泛适用于其他细菌病原体。此外，SG形成的图像分析可以用于病毒或其他病原体的感染。可用于分析SG感染后的形成或感染对SG形成的影响以响应外源性应激。
Protocol
Representative Results
Discussion
本文概述的方案描述了在存在或不存在外源胁迫的情况下，非感染细胞和感染细胞溶质病原体柔毛霉的细胞中的SG的诱导，定位和分析。使用免费的成像软件，协议允许对SG形成进行精确的定性和定量分析，以鉴别和统计学地解决给定表型的差异。
在感染，SG感应和成像部件的协议中有几个关键步骤。对于感染，重要的是与宿主细胞的细菌相互作用，如本文中概述的方?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
PS是比尔和梅琳达·盖茨大挑战奖牌OPP1141322的收件人。 PV由瑞士国家科学基金会早期博士后移动奖学金和Roux-Cantarini博士后研究金支持。 PJS由HHMI授权和ERC-2013-ADG 339579解密支持。
Materials
| Primary Antibodies | |||
| eIF3b (N20), origin goat | Santa Cruz | sc-16377 | Robust and widely used SG marker. Cytosolic staining allows cell delineation. Dilution 1 in 300 |
| eIF3b (A20), origin goat | Santa Cruz | sc-16378 | Same target as eIF3b (N20) and in our hands was identical to eIF3b (N20). Dilution 1 in 300 |
| eIF3A (D51F4), origin rabbit (MC: monoclonal) | Cell Signaling | 3411 | Part of multiprotein eIF3 complex with eIF3b . Dilution 1 in 800 |
| eIF4AI, origin goat | Santa Cruz | sc-14211 | Recommended by (Ref # 13). Dilution 1 in 200 |
| eIF4B, origin rabbit | Abcam | ab186856 | Good stress granule marker in our hands. Dilution 1 in 300 |
| eIF4B, origin rabbit | Cell Signaling | 3592 | Recommended by Ref # 13. Dilution 1 in 100 |
| eIF4G, origin rabbit | Santa Cruz | sc-11373 | Widely used SG marker. (Ref # 13): may not work well in mouse cell lines. Dilution 1 in 300 |
| G3BP1, origin rabbit (MC: monoclonal) | BD Biosciences | 611126 | Widely used SG marker. Dilution 1 in 300 |
| Tia-1, origin goat | Santa Cruz | sc-1751 | Widely used SG marker. Can also be found in P bodies when SG are present (Ref # 13). Dilution 1 in 300 |
| Alexa-conjugated Secondary Antibodies | |||
| A488 anti-goat , origin donkey | Thermo Fisher | A-11055 | Cross absorbed. Dilution 1 in 500 |
| A568 anti-goat, origin donkey | Thermo Fisher | A-11057 | Cross absorbed. Dilution 1 in 500 |
| A488 anti-mouse, origin donkey | Thermo Fisher | A-21202 | Dilution 1 in 500 |
| A568 anti-mouse, origin donkey | Thermo Fisher | A10037 | Dilution 1 in 500 |
| A647 anti-mouse, origin donkey | Thermo Fisher | A31571 | Dilution 1 in 500 |
| A488 anti-rabbit, origin donkey | Thermo Fisher | A-21206 | Dilution 1 in 500 |
| A568 anti-rabbit, origin donkey | Thermo Fisher | A10042 | Dilution 1 in 500 |
| Other Reagents | |||
| Shigella flexneri | Available from various laboratories by request | ||
| Tryptone Casein Soya (TCS) broth | BD Biosciences | 211825 | Standard growth medium for Shigella, application – bacterial growth |
| TCS agar | BD Biosciences | 236950 | Standard growth agar for Shigella, application – bacterial growth |
| Congo red | SERVA Electrophoresis GmbH | 27215.01 | Distrimination tool for Shigell that have lost the virulence plasmid, application – bacterial growth |
| Poly L lysine | Sigma-Aldrich | P1274 | Useful to coating bacteria to increase infection, application – infection |
| Gentamicin | Sigma-Aldrich | G1397 | Selective killing of extracellular but not cytosolic bacteria, application – infection |
| HEPES | Life Technologies | 15630-056 | PH buffer useful when cells are incubated at room-temperature, application – cell culture |
| DMEM | Life Technologies | 31885 | Standard culture medium for HeLa cells, application – cell culture |
| Fetal calf serum | Biowest | S1810-100 | 5% supplementation used for HeLa cell culture medium, application – cell culture |
| Non-essential amino acids | Life Technologies | 11140 | 1/100 dilution used for HeLa cell culture medium, application – cell culture |
| DMSO | Sigma-Aldrich | D2650 | Reagent diluent, application – cell culture |
| Sodium arsenite | Sigma-Aldrich | S7400 | Potent stress granule inducer (Note: highly toxic, special handling and disposal required), application – stress inducer |
| Clotrimazole | Sigma-Aldrich | C6019 | Potent stress granule inducer (Note:health hazard, special handling and disposal required), application – stress inducer |
| PFA | Electron Microscopy Scences | 15714 | 4% PFA is used for standard fixation of cells, application – fixation |
| Triton X-100 | Sigma-Aldrich | T8787 | Used at 0.03% for permeabilizationof host cells before immunofluorescent staining, application – permeabilization |
| A647-phalloidin | Thermo Fisher | A22287 | Dilution is at 1/40, best added during 2ary antibody staining, application – staining |
| DAPI | Sigma-Aldrich | D9542 | Nucleid acid stain used to visualize both the host nucleus and bacteria, application – staining |
| Parafilm | Sigma-Aldrich | BR701501 | Paraffin film useful for immunofluorescent staining of coverslips, application – staining |
| Prolong Gold | Thermo Fisher | 36930 | Robust mounting medium that works well for most fluorophores , application – mounting |
| Mowiol | Sigma-Aldrich | 81381 | Cheap and robust mounting medium that works well for most fluorophores, application – mounting |
| 24-well cell culture plate | Sigma-Aldrich | CLS3527 | Standard tissue culture plates, application – cell culture |
| 12-mm glass coverslips | NeuVitro | 1001/12 | Cell culture support for immunofluorescent applications, application – cell support |
| forceps | Sigma-Aldrich | 81381 | Cheap and obust mounting medium that works well for most fluorophores, application – mounting |
| Programs and Equipment | |||
| Prism | GraphPad Software | Data analysisand graphing program with robust statistical test options, application – data analysis | |
| Leica SP5 | Leica Microsystems | Confocal microsope, application – image acquisition | |
| Imaris | Bitplane | Professional image analysis program, application – data analysis | |
| Excel | Microsoft | Data analysis and graphing program, application – data analysis | |
Referenzen
- Reineke, L. C., Lloyd, R. E. Diversion of stress granules and P-bodies during viral infection. Virology. 436 (2), 255-267 (2013).
- Kedersha, N., Ivanov, P., Anderson, P. Stress granules and cell signaling: more than just a passing phase. Trends Biochem Sci. 38 (10), 494-506 (2013).
- Vonaesch, P., Campbell-Valois, F. -. X., Dufour, A., Sansonetti, P. J., Schnupf, P. Shigella flexneri modulates stress granule composition and inhibits stress granule aggregation. Cell Microbiol. 18 (7), 982-997 (2016).
- Kolobova, E., Efimov, A., et al. Microtubule-dependent association of AKAP350A and CCAR1 with RNA stress granules. Exp Cell Res. 315 (3), 542-555 (2009).
- Anderson, P., Kedersha, N. Stress granules: the Tao of RNA triage. Trends in biochemical sciences. 33 (3), 141-150 (2008).
- Anderson, P., Kedersha, N. Stressful initiations. J Cell Sci. 115, 3227-3234 (2002).
- Mohr, I., Sonenberg, N. Host translation at the nexus of infection and immunity. Cell Host Microbe. 12 (4), 470-483 (2012).
- Hu, S., Claud, E. C., Musch, M. W., Chang, E. B. Stress granule formation mediates the inhibition of colonic Hsp70 translation by interferon- and tumor necrosis factor. Am J Physiol Gastointest Liver Physiol. 298 (4), 481-492 (2010).
- Barry, E. M., Pasetti, M. F., Sztein, M. B., Fasano, A., Kotloff, K. L., Levine, M. M. Progress and pitfalls in Shigella vaccine research. Nat Rev Gastroenterol Hepatol. 10 (4), 245-255 (2013).
- Lanata, C. F., Fischer-Walker, C. L., et al. Global Causes of Diarrheal Disease Mortality in Children. PloS One. 8 (9), 72788 (2013).
- Ashida, H., Ogawa, M., et al. Shigella deploy multiple countermeasures against host innate immune responses. Curr Opin Microbiol. 14 (1), 16-23 (2011).
- Ashida, H., Ogawa, M., Mimuro, H., Kobayashi, T., Sanada, T., Sasakawa, C. Shigella are versatile mucosal pathogens that circumvent the host innate immune system. Curr Opin Immunol. 23 (4), 448-455 (2011).
- Kedersha, N., Anderson, P. Mammalian stress granules and processing bodies. Methods Enzymol. 431, 61-81 (2007).
- Ray, K., Marteyn, B., Sansonetti, P. J., Tang, C. M. Life on the inside: the intracellular lifestyle of cytosolic bacteria. Nat Re. Micro. 7 (5), 333-340 (2009).
