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Fluorescence Microscopy Analysis of Drug Effect on Autophagosome Formation
荧光显微法分析药物对自噬体形成的影响   

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Abstract

The autophagy protein, LC3 represents a reliable characteristic marker for autophagosomal structures. The initial LC3 is processed by the cysteine protease autophagy-related gene 4 (Atg4) at its C terminus in order to create LC3-I generally localized in the cytoplasm. Afterwards LC3-I is conjugated with phosphatidylethanolamine (PE) to become LC3-PE or LC3-II predominantly localised on the autophagosomal membranes (outer and inner). Autolysosomal content of LC3-II is very low as upon autophago/lysosomal fusion it is either cleaved off from the outer membrane by Atg4 or degraded together with the inner membrane by the lysosomal activity. Therefore GFP-LC3 and mCherry-GFP-LC3 might be visualized by conventional or confocal fluorescence microscopy (FM). In this situation mCherry-GFP-LC3 or GFP-LC3 cytoplasmic pool is visualized as a homogeneously dispersed signal and mCherry-GFP-LC3-II or GFP-LC3-II containing autophagosomes are detected as punctae formations. The number of punctae may be used as marker of autophagosomal abundance. In general we recommend counting the average number of GFP-LC3 punctae per cell.

Keywords: Autophagy(自噬), MCherry-GFP-LC3(mcherry-gfp-lc3), GFP-LC3(GFP-LC3), Fluorescence Microscopy(荧光显微镜), Autophagic flux(自噬潮)

Materials and Reagents

  1. Cell lines of interest (HepG2, HUH7, CMK, K562 etc.) stably expressing GFP-LC3
    We recommend the following commercially available plasmids: pBABEpuro GFP-LC3 (plasmid 22405) and pBABE-puro mCherry-EGFP-LC3B (plasmid 22418) generated by Jayanta Debnath from Addgene to be inserted into retroviral constructs and used for cell transduction
  2. Eagle's minimal essential medium (EMEM) (ATCC, catalog number: 30-2003 ) containing 10% fetal bovine serum (FBS) with 100 U/100 μg/ml penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
  3. RPMI 1640 with L-glutamine (Lonza, catalog number: BE12-702F ) containing 10% fetal bovine serum (FBS) with 100 U/100 μg/ml penicillin/streptomycin
  4. Fetal Bovine Serum (FBS) (Biochrom, catalog number: S0615 )
  5. Dulbecco’s Phosphate Buffered Saline (PBS) (Biochrom, catalog number: L1825 )
  6. 1x 0.05% Trypsin-EDTA (phenol red) (Life Technologies, catalog number: 25300 )
  7. Hanks Balanced Salt Solution (HBSS) (Life Technologies, Gibco®, catalog number: 14025 ) containing 6 mM glucose (starvation medium)
  8. Rapamycin from Streptomyces hygroscopicus (1-5 µmol/L) (Sigma-Aldrich, catalog number: R0395 )
  9. PP242 hydrate (1-5 µmol/L) (Sigma-Aldrich, catalog number: P0037 )
  10. 3-methyladenine (3-MA) (3-10 mmol/L) (Sigma-Aldrich, catalog number: M9281 )
  11. Wortmannin (30-100 nmol/L) (Sigma-Aldrich, catalog number: W3144 )
  12. LY294002 (7-20 µmol/L) (Sigma-Aldrich, catalog number: L9908 )
  13. Nocodazole (12-50 µmol/L) (Sigma-Aldrich, catalog number: M1404 )
  14. Vinblastine (12-50 µmol/L) (Sigma-Aldrich, catalog number: V1377 )
  15. Ammonium chloride (NH4Cl) (10-20 mmol/L) (Sigma-Aldrich, catalog number: A0171 )
  16. Hydrohychloroquine sulphate (HCQ) (5-10 µmol/L) (Sigma-Aldrich, catalog number: H0915 )
  17. Chloroquine (CQ) (5-10 µmol/L) (Sigma-Aldrich, catalog number: C6628 )
  18. Dimethyl sulfoxide DMSO (Sigma-Aldrich, catalog number: D8418 )

Equipment

  1. 37 °C, 5% CO2 humidified incubator
  2. Centrifuge
  3. Olympus IX81 instrument and analySIS (Soft Imaging System GmbH) or analogous equipment

Procedure

  1. Maintained the cells under standard tissue culture conditions at 37 ºC, 5% CO2 in a humidified incubator. Keep cell density below 1 x 106/ml and analyse at subconfluent stages.
    Caution: Prior to analysis cell should be kept for several hours in fresh medium to avoid potential activation of autophagy due to nutrients exhaustion. Generally culture medium contains autophagy affecting substances: amino acids, glucose, growth factors, hormones…etc. Take care when comparing autophagic flux in different conditions to normalize for all the necessary factors. Normalize also for the solvent used when analysing the effect of different substances on autophagy – for example DMSO, ethanol etc might affect autopagic flux.
  2. Incubate the cells for the desired time and under the conditions of interest.
    Caution: When analysing prolonged periods of time and/or under conditions potentially affecting cells numbers or viability, differences in nutrient consumption and therefore abundance might occur and influence your results as autophagic activity is highly related to the nutritional status.
  3. Positive control [rapamycin (1-5 µmol/L), PP242 (1-5 µmol/L), Hanks’ Balanced Salt Solution containing 6 mmol/L glucose (starvation medium)] and negative control [3-methyladenine (3-MA) (3–10 mmol/L), wortmannin (30-100 nmol/L), LY294002 (7-20 µmol/L), nocodazole (12-50 µmol/L), vinblastine (12-50 µmol/L), ammonium chloride (NH4Cl) (10-20 mmol/L), hydrohychloroquine (HCQ) or chloroquine (CQ) (5-10 µmol/L)] may be also included. Positive controls activate autophagy and should result in an initial increase in the number of punctae per cell. Negative controls inhibit different stages of autophagic process. Inhibitors of autophagosome formation are expected to decrease the number of punctae per cell as inhibitors of autophagosomal maturation or downstream events should result in an increased number of punctae per cell.
  4. At the end of incubation period transfer the cell on microscopic glass and take fluorescent images for each separate treatment condition, from several cells belonging to a number of randomly chosen fields with a GFP filter.
    Notes:
    1. When analysing the effect of a certain substance or condition on autophagy we recommend the evaluation of potential changes in autophagosomal numbers per cell and comparison to the effects of established activators and inhibitors.
    2. Furthermore it would be informative to perform a range of co incubations with the above mentioned conditions and to use the provided Table 1 for rough orientation. (Caution: It is imperative to rule out experimental artefacts potentially arising from GFP-LC3 aggregates formation. Use GFP-LC3 cells with stable expression of the fusion protein. Select cellular clones with levels of GFP-LC3 expression associated with minimum aggregate formation. Although FM is useful to determine the autophagosome number per cell, it is not a good indicator for the estimation of cellular autophagic activity.).
    3. Autophagosome accumulation may be due to both autophagy induction with increased generation of autophagosomes as well as suppressed autophagosomal maturation or downstream events and inability to complete the autophagic pathway. There are several approaches to distinguish between these situations. We recommend the following simplistic model presented in Table 1.

      Table 1. Analysis of unknown substance X
      X plus:  
      Medium    
      Activator  
      iAF  
      iAM or iAA
      I   
      →   
      ↑↑   
      → or ↓   
      ↑↑
      II   
      ↑↑   
      ↑↑↑   
      → or ↑ or ↓   
      ↑↑↑
      III   
      → or ↓   
      → or ↑ or ↓   
      →    

      IV   
      ↑↑   
      ↑↑↑   
      ↑   
      ↑↑

      Caution: Generally pharmacological inhibitors of autophagy lack specificity and therefore some cell type specific effects might occur. We also recommend analysing the effect of a new substance not only on basic, but also on rapamycin, PP242 or starvation activated autophagic flux. Table 1 may be used to facilitate the interpretation of the results.
      Notes:
      1. →↑↓: Change in the number of FM visualized autophagosomes.
      2. (Activator): autophagy induction [rapamycin (1-5 µmol/L), PP242 (1-5 µmol/L) or starvation medium].
      3. (iAF): Inhibition of autophagosom formation [3-methyladenine (3-MA) (3–10 mmol/L), wortmannin (30–100 nmol/L), LY294002 (7–20 µmol/L)].
      4. (iAM): Inhibition of autopagosomal maturation [nocodazole (12–50 µmol/L) and vinblastine (12–50 µmol/L)].
      5. (iAA): Inhibition of autophagosomal acidification [ammonium chloride (NH4Cl) (10–20 mmol/L), hydrohychloroquine (HCQ) and chloroquine (CQ) (5–10 µmol/L)].
      6. The analyses of unknown substance X should include co incubation with: Activator, iAF, iAM and/or iAA. Table 1 illustrates four possible scenarios. a) Scenario (I) the unknown substance X does not impact cellular autophagic activity; b) Scenario (II) X is an activator; c) Scenario (III) X is an early inhibitor of autophagosome formation; d) Scenario (IV) X inhibits autophagy at the level of autophagosomal maturation or at some downstream stage.
      7. Caution: The suggested interpretation of FM data should be used with care as by itself FM is prone to many limitations and the results obtained with this method must be verified using other methodological approaches with not overlapping limitations such as electron microscopy (EM), flow cytometry, western blotting (WB) etc.
      8. Example data:


        Figure 1. Fluorescent microscopy analysis of autophagic punctae formation in 3T3-F442A cells stably expressing LC3-GFP fusion protein incubated with autophagy inhibitor 3MA (10 mM) for 6 h in the presence or absence of autophagy activator PP242 for the last 4 h

    Acknowledgments

    This work was supported in part by the German Research Foundation (KFO 250, TP1) and an unrestricted grant from Abbott. Leverkus, M. was supported by the German National Academic Foundation (LE-953/5-1 and LE-953/6-1). Behrens, G. was supported by the Excellence Cluster EXC 62/1.

    References

    1. Mizushima, N., Yoshimori, T. and Levine, B. (2010). Methods in mammalian autophagy research. Cell 140(3): 313-326.
    2. Sinicrope, F. A., Sirko, A., Siu, P. M. et al. (2012). Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8(4): 445-544.
    3. Stankov, M. V., El Khatib, M., Kumar Thakur, B., Heitmann, K., Panayotova-Dimitrova, D., Schoening, J., Bourquin, J. P., Schweitzer, N., Leverkus, M., Welte, K., Reinhardt, D., Li, Z., Orkin, S. H., Behrens, G. M. and Klusmann, J. H. (2014). Histone deacetylase inhibitors induce apoptosis in myeloid leukemia by suppressing autophagy. Leukemia 28(3): 577-588.
    4. Stankov, M. V., Panayotova-Dimitrova, D., Leverkus, M., Schmidt, R. E. and Behrens, G. M. (2013). Thymidine analogues suppress autophagy and adipogenesis in cultured adipocytes. Antimicrob Agents Chemother 57(1): 543-551.

简介

自噬蛋白,LC3代表自噬体结构的可靠特征标记。最初的LC3由半胱氨酸蛋白酶自噬相关基因4(Atg4)在其C末端处理,以产生通常位于细胞质中的LC3-I。之后LC3-I与磷脂酰乙醇胺(PE)缀合以变成主要定位在自噬体膜(外部和内部)上的LC3-PE或LC3-II。 LC3-II的自溶酶体内含物非常低,因为在自噬/溶酶体融合时,其被Atg4从外膜切割或与内膜一起通过溶酶体活性降解。因此,GFP-LC3和mCherry-GFP-LC3可能通过常规或共聚焦荧光显微镜(FM)可视化。在这种情况下,mCherry-GFP-LC3或GFP-LC3细胞质池被可视化为均匀分散的信号,并且含有mCherry-GFP-LC3-II或GFP-LC3-II的自噬体被检测为点状形成。斑点数可以用作自噬体丰度的标志物。一般来说,我们建议计数每个细胞的GFP-LC3斑点的平均数。

关键字:自噬, mcherry-gfp-lc3, GFP-LC3, 荧光显微镜, 自噬潮

材料和试剂

  1. 稳定表达GFP-LC3的感兴趣的细胞系(HepG2,HUH7,CMK,K562等) 我们推荐以下可商购的质粒:由来自Addgene的Jayanta Debnath产生的pBABEpuro GFP-LC3(质粒22405)和pBABE-puro mCherry-EGFP-LC3B(质粒22418)插入逆转录病毒构建体中并用于细胞转导
  2. 包含含有100U /100μg/ml青霉素/链霉素(Life Technologies,Gibco)的10%胎牛血清(FBS)的Eagle's最小必需培养基(EMEM)(ATCC,目录号:30-2003) ,目录号:15140-122)
  3. 含有含100U /100μg/ml青霉素/链霉素的10%胎牛血清(FBS)的L-谷氨酰胺RPMI 1640(Lonza,目录号:BE12-702F)
  4. 胎牛血清(FBS)(Biochrom,目录号:S0615)
  5. Dulbecco's磷酸盐缓冲盐水(PBS)(Biochrom,目录号:L1825)
  6. 1x 0.05%胰蛋白酶-EDTA(酚红)(Life Technologies,目录号:25300)
  7. 含有6mM葡萄糖(饥饿培养基)的Hanks平衡盐溶液(HBSS)(Life Technologies,Gibco ,目录号:14025)
  8. 来自吸湿链霉菌(1-5μmol/L)(Sigma-Aldrich,目录号:R0395)的雷帕霉素
  9. PP242水合物(1-5μmol/L)(Sigma-Aldrich,目录号:P0037)
  10. 3-甲基腺嘌呤(3-MA)(3-10mmol/L)(Sigma-Aldrich,目录号:M9281)
  11. 渥曼青霉素(30-100nmol/L)(Sigma-Aldrich,目录号:W3144)
  12. LY294002(7-20μmol/L)(Sigma-Aldrich,目录号:L9908)
  13. 诺可达唑(12-50μmol/L)(Sigma-Aldrich,目录号:M1404)
  14. 长春花碱(12-50μmol/L)(Sigma-Aldrich,目录号:V1377)
  15. 氯化铵(NH 4 Cl)(10-20mmol/L)(Sigma-Aldrich,目录号:A0171)
  16. 硫酸氢氯喹(HCQ)(5-10μmol/L)(Sigma-Aldrich,目录号:H0915)
  17. 氯喹(CQ)(5-10μmol/L)(Sigma-Aldrich,目录号:C6628)
  18. 二甲基亚砜DMSO(Sigma-Aldrich,目录号:D8418)

设备

  1. 37℃,5%CO 2湿润培养箱
  2. 离心机
  3. 奥林巴斯IX81仪器和analySIS(软成像系统有限公司)或类似设备

程序

  1. 在湿润的培养箱中在37℃,5%CO 2下在标准组织培养条件下保持细胞。保持细胞密度低于1×10 6/sup/ml,并在亚汇合阶段进行分析。
    注意:在分析之前,细胞应该在新鲜培养基中保持几个小时,以避免由于营养物耗尽而导致的自噬的潜在激活。一般培养基包含自噬影响物质:氨基酸,葡萄糖,生长因子,激素... 等 。在比较不同条件下的自噬通量时要小心,以便对所有必要因素进行归一化。在分析不同物质对自噬的影响时,例如DMSO,乙醇等,可能会影响自动通量。
  2. 孵育细胞所需的时间和在感兴趣的条件下。
    注意:当分析延长的时间段和/或潜在影响细胞数量或活力的条件下时,营养物消耗和因此丰度的差异可能发生并影响您的结果,因为自噬活性与营养状态高度相关。
  3. 阳性对照[雷帕霉素(1-5μmol/L),PP242(1-5μmol/L),含6mmol/L葡萄糖的Hanks平衡盐溶液(饥饿培养基)]和阴性对照[3-甲基腺嘌呤),枸杞素(30-100nmol/L),LY294002(7-20μmol/L),诺考达唑(12-50μmol/L),长春花碱还可以包括氯化铵(NH 4 Cl)(10-20mmol/L),氢氯喹(HCQ)或氯喹(CQ)(5-10μmol/L)]。阳性对照激活自噬,并应导致每个细胞的斑点数的初始增加。阴性对照抑制自噬过程的不同阶段。预期自噬体形成的抑制剂减少 作为自噬体成熟或下游事件抑制剂的每个细胞的斑点数应导致每个细胞的斑点数量增加。
  4. 在孵育期结束时,将细胞转移到显微镜玻璃上,并对于每种单独的处理条件,从属于许多随机选择的具有GFP滤膜的细胞的几个细胞取荧光图像。
    注意:
    1. 当分析某种物质或条件对自噬的影响时,我们建议评价每个细胞的自噬体数量的潜在变化,并与建立的激活剂和抑制剂的作用进行比较。
    2. 此外,在上述条件下进行一系列共孵育并且使用所提供的表1用于粗略取向是有益的。 (注意:必须排除GFP-LC3聚集体形成可能引起的实验假象,使用具有稳定表达融合蛋白的GFP-LC3细胞,选择具有与最小聚集体形成相关的GFP-LC3表达水平的细胞克隆。 FM对于确定每个细胞的自噬体数目是有用的,它不是细胞自噬活性估计的良好指示剂。
    3. 自噬体积累可能是由于自噬诱导与自噬体的产生增加以及抑制自噬体成熟或下游事件和不能完成自噬途径。有几种方法可以区分这些情况。我们建议在表1中提供以下简单化模型。

      表1.未知物质X的分析
      X加:  
      中    
      激活者  
      iAF  
      iAM或iAA
      I   
      →   
      ↑↑   
      →或↓   
      ↑↑
      II   
      ↑↑   
      ↑↑↑   
      →或↑或↓   
      ↑↑↑
      III   
      →或↓   
      →或↑或↓   
      →    

      IV   
      ↑↑   
      ↑↑↑   
      ↑   
      ↑↑

      注意:通常自噬的药理学抑制剂缺乏特异性,因此可能发生一些细胞类型特异性效应。 我们还建议分析一种新物质不仅对碱性,而且对雷帕霉素,PP242或饥饿激活的自噬通量的影响。 表1可用于方便解释结果。
      注意:
      1. →↑↓:FM可视化自噬体数目的变化。
      2. (激活剂):自噬诱导[雷帕霉素(1-5μmol/L),PP242(1-5μmol/L)或饥饿培养基]。
      3. (iAF):自噬体形成的抑制[3-甲基腺嘌呤(3-MA)(3-10mmol/L),渥曼青霉素(30-100nmol/L),LY294002(7-20μmol/L)]。
      4. (iAM):抑制自噬体成熟[诺考达唑(12-50μmol/L)和长春碱(12-50μmol/L)]。
      5. (iAA):抑制自噬体的酸化[氯化铵(NH 4 Cl)(10-20mmol/L),氢氯喹(HCQ)和氯喹(CQ)(5-10μmol/L) br />
      6. 未知物质X的分析应包括与活化剂,iAF,iAM和/或iAA的共孵育。表1示出了四种可能的情况。 a)情况(I)未知物质X不影响细胞自噬活性; b)情况(II)X是活化剂; c)情况(III)X是自噬体形成的早期抑制剂; d)情况(IV)X抑制自噬体水平的自噬体成熟或 在某些下游阶段。
      7. 注意:FM数据的建议解释应该谨慎使用,因为它本身FM容易有许多限制,并且使用这种方法获得的结果必须使用其他方法方法进行验证,没有重叠的限制,如电子显微镜(EM) ,流式细胞术,免疫印迹(WB)等。
      8. 示例数据:


        图1.荧光显微镜分析在自噬抑制剂3MA(10mM)孵育6h的稳定表达LC3-GFP融合蛋白的3T3-F442A细胞中自噬噬斑形成在存在或不存在自噬激活物PP242的情况下的最后4天 h

    致谢

    这项工作得到了德国研究基金会(KFO 250,TP1)的部分支持,以及来自雅培的不受限制的资助。 Leverkus,M.由德国国家学术基金会(LE-953/5-1和LE-953/6-1)支持。 Behrens,G.获得EXC 62/1卓越集群的支持。

    参考文献

    1. Mizushima,N.,Yoshimori,T。和Levine,B。(2010)。 哺乳动物自噬研究中的方法 140(3):313-326。
    2. Sinicrope,F.A.,Sirko,A.,Siu,P.M.等人。 (2012)。 使用和解释用于监测自噬的测定法。自噬 8(4):445-544。
    3. Stankov,MV,El Khatib,M.,Kumar Thakur,B.,Heitmann,K.,Panayotova-Dimitrova,D.,Schoening,J.,Bourquin,JP,Schweitzer,N.,Leverkus,M.,Welte,K 。,Reinhardt,D.,Li,Z.,Orkin,SH, Behrens,G.M.和Klusmann,J.H。(2014)。 组蛋白脱乙酰酶抑制剂通过抑制自噬在骨髓性白血病中诱导凋亡。 >白血病 28(3):577- 588。
    4. Stankov,M.V.,Panayotova-Dimitrova,D.,Leverkus,M.,Schmidt,R.E.and Behrens,G.M。(2013)。 胸苷类似物抑制培养的脂肪细胞中的自噬和脂肪形成。抗微生物剂化疗 57(1):543-551。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Stankov, M., Panayotova-Dimitrova, D., Leverkus, M. and Behrens, G. (2014). Fluorescence Microscopy Analysis of Drug Effect on Autophagosome Formation. Bio-protocol 4(7): e1089. DOI: 10.21769/BioProtoc.1089.
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