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Analysis of Autophagic Activity Using ATG8 Lipidation Assay in Arabidopsis thaliana
通过分析ATG8脂化解析拟南芥的自噬活性   

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Yasin  Dagdas Yasin Dagdas
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本实验方案简略版
Proceedings of the National Academy of Sciences of the United States of America
Jan 2017

Abstract

As a fundamental metabolic pathway to degrade and recycle cellular cargos, autophagy is highly induced upon stress, starvation and senescence conditions in plants. A double-membrane structure named autophagosome will form during this process for cargo sequestration and delivery into the vacuole.

A number of regulators have been characterized in plants, including the autophagy-related (ATG) proteins and other plant-specific proteins. Among them, ATG8 will undergo a lipidation process to become a membrane-bound ATG8-phosphatidylethanolamine form and mark the growing autophagosomal membrane as well as the completed autophagosome. Therefore, ATG8 has been regarded as a marker for autophagosomes; and biochemical detection of the membrane-associated form of ATG8 is used as one of the principal methods for measurement of autophagic activity. Here, we describe an ATG8 lipidation assay for detection of the ATG8-PE form using Arabidopsis thaliana seedlings.

Keywords: ATG8 (ATG8), ATG8-PE (ATG8-PE), Lipidation (脂化), Autophagy (自噬), Autophagosome (自噬体)

Background

Autophagy is an essential metabolic process which mediates the bulk degradation of the damaged organelles and unwanted cellular contents. During autophagy, a double-membrane structure called autophagosome will form and deliver the cargos into the vacuole for degradation. The autophagy-related (ATG) proteins are required to regulate the autophagic activity (Liu and Bassham, 2012). Among them, two conjugation systems, including ATG8 conjugate and ATG5-ATG12 conjugate, are involved for autophagosome formation. Upon autophagic induction, the ATG5-ATG12 conjugate forms and functions as an E3-like enzyme to promote ATG8 lipidation for binding to the phosphatidylethanolamine (PE) on the autophagosome membrane (Ohsumi, 2001). Although ATG8-PE on the outer membrane will be recycled before the autophagosome fusion with the vacuole, ATG8-PE on inner membrane will traffic together with the cargo into the vacuole for degradation. Thus, the amount of ATG8-PE usually correlates with the number of punctate ATG8-positive structures as well as autophagic activity (Mizushima et al., 2010). Particularly, due to the high hydrophobicity of ATG8-PE, ATG8-PE migrates faster than ATG8 in SDS-PAGE gel, though the actual molecular weight of ATG8-PE is larger than the unconjugated ATG8 (Mizushima and Yoshimori, 2007). Accordingly, the amount of ATG8-PE from cell membrane fraction (CM) can be detected by immunoblotting with ATG8 antibodies. For example, in Arabidopsis atg5 mutant, the level of ATG8-PE is severely impaired upon autophagic induction, whereas no autophagosome structures labeled by ATG8 are formed (Chung et al., 2010). Therefore, biochemical detection of the ATG8 lipidation can serve as a useful method to access the autophagic activity when combined with different treatments, which has been applied in our previous study as well as others related to plant autophagy (Chung et al., 2010; Suttangkakul et al., 2011; Li et al., 2014; Zhuang et al., 2017). Here, we describe the protocol for ATG8 lipidation detection by ultracentrifuge separation of the membrane and cytosol fractions using acibenzolar-S-methyl (BTH)-treated seedlings (Zhuang et al., 2017).

Materials and Reagents

  1. 10 µl pipette tips (Thermo Fisher Scientific, catalog number: 3510 )
  2. 200 µl pipette tips (Wolf Laboratories, catalog number: 2100.YN )
  3. 1,000 µl pipette tips (Thermo Fisher Scientific, catalog number: 3580 )
  4. 1.5 ml microcentrifuge tubes (Corning, Axygen®, catalog number: MCT-150-C )
  5. PVDF membrane
  6. X-ray film (Advansta, catalog number: L-07014-100 )
  7. 5-day-old seedlings
  8. MS salt (Caisson, catalog number: MSP01-50LT )
  9. UltraPureTM Sucrose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15503022 )
  10. BTH (Acibenzolar-S-methyl) (Sigma-Aldrich, catalog number: 32820
  11. Methanol (VWR, BDH, catalog number: 10158 )
  12. Liquid nitrogen 
  13. PIC (Protease Inhibitor Mixture) (Roche Diagnostics, catalog number: 11873580001 )
  14. Tris Base (Caisson, catalog number: T041-1KG )
  15. NaCl (Alfa Aesar, USB, catalog number: J21618 )
  16. EDTA (Alfa Aesar, USB, catalog number: J15701 )
  17. SDS (Sodium dodecyl sulfate) (Alfa Aesar, USB, catalog number: J75819 )
  18. Triton X-100 (GE Healthcare, Amerrsham, catalog number: 17-1315-01 )
  19. Glycerol ultrapure (Alfa Aesar, USB, catalog number: J16374 )
  20. Bromophenol Blue (Sigma-Aldrich, catalog number: B5525 )
  21. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  22. Urea (Alfa Aesar, USB, catalog number: J75826 )
  23. 40% Acrylamide/Bis Solution (Bio-Rad Laboratories, catalog number: 161-0148 )
  24. TEMED (Bio-Rad Laboratories, catalog number: 161-0801 )
  25. APS (Ammonium persulfate) (USB, catalog number: US76322 )
  26. Non-fat milk powder
  27. ATG8 antibody (Agrisera, catalog number: AS14 2769 )
  28. cFBPase (Agrisera, catalog number: AS04 043 )
  29. Secondary antibody (Anti-rabbit IgG peroxidase conjugate, Sigma-Aldrich, catalog number: A6154
  30. Precision Plus ProteinTM Dual Color Standards (Bio-Rad Laboratories, catalog number: 161-0374 )
  31. Sodium hydrogen carbonate (NaHCO3) (VWR, catalog number: 144-55-8 )
  32. Sodium carbonate (Na2CO3) (USB, catalog number: 21602
  33. Sodium phosphate monobasic monohydrate (NaH2PO4·H2O) (USB, catalog number: 20233 )
  34. Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) (Sigma-Aldrich, catalog number: 04272 )
  35. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 31248 )
  36. Tween-20 (Sigma-Aldrich, catalog number: 63158 )
  37. MS liquid medium (see Recipes)
  38. 10 mM BTH stock (see Recipes)
  39. 25x PIC (see Recipes)
  40. 10% (v/v) Triton X-100 (see Recipes)
  41. 1 M Tris-HCl stock (pH 6.8 or pH 7.4) (see Recipes)
  42. 0.5 M EDTA (pH 8.0) (see Recipes)
  43. 5x extraction buffer (see Recipes)
  44. 1x extraction buffer containing 1x PIC and 1% (v/v) Triton X-100 (see Recipes)
  45. 5x sample loading dye (see Recipes)
  46. 30% APS (see Recipes)
  47. 3x separation buffer (see Recipes)
  48. 5x stacking buffer (see Recipes)
  49. 15% SDS-PAGE gel with 6 M urea (see Recipes)
    1. 15% Urea separating gel
    2. 5% Stacking gel
  50. Running buffer (see Recipes)
  51. Transfer buffer (see Recipes)
  52. PBS (see Recipes) 
  53. PBS-T (see Recipes)

Equipment

  1. Eppendorf Research® plus Pipette 0.5-10 µl (Eppendorf, catalog number: 3120000020 )
  2. Eppendorf Research® plus Pipette 10-100 µl (Eppendorf, catalog number: 3120000046 )
  3. Eppendorf Research® plus Pipette 100-1,000 µl (Eppendorf, catalog number: 3120000062 )
  4. Mortar and pestle
  5. Centrifuge (Eppendorf, model: 5430 )
  6. X-ray film cassette (Amersham Biosciences HypercassetteTM)
  7. Ultracentrifuge tube (7 x 20 mm)
  8. Ultracentrifuge (Beckman Coulter, model: OptimaTM MAX-XP )
  9. Western Blotting apparatus (Bio-Rad)
  10. Developer machine (Fujifilm FPM100A)

Procedure

  1. Incubate 0.2 g 5-day Arabidopsis thaliana seedlings in MS liquid medium with and without 100 µM BTH (10 mM stock in methanol) for 8 h (see Notes 1 and 2).
  2. Freeze the seedlings in the mortar using liquid nitrogen and grind the plants thoroughly.
  3. Add 2x extraction buffer containing PIC without detergent into the mortar on ice.
  4. After defrost slowly on ice, transfer the liquid to a 1.5 ml tube and centrifuge at 1,000 x g for 5 min, 4 °C.
  5. Transfer the supernatant to an ultracentrifuge tube (7 x 20 mm) and centrifuge at 100,000 x g for 45 min, 4 °C.
  6. Transfer the supernatant (Cell Soluble fraction, CS) into a labeled new Eppendorf tube and keep on ice.
  7. Wash the pallet (Cell Membrane fraction, CM) slightly with the 1x extraction buffer containing 1x PIC for 2-3 times.
    Note: In this step, add the 1x extraction buffer slightly without touching the surface of the pallet, and the centrifuge and re-suspend are not required. The main purpose of this step is washing out the remaining CS liquid on the CM surface.
  8. Resuspend the pallet using 1x extraction buffer containing 1x PIC and 1% (v/v) Triton X-100 to solubilize the membranes.
  9. Add the 5x sample loading dye to both of the CS and CM samples.
  10. Boil the samples at 100 °C for 10 min.
  11. Prepare the 15% SDS-PAGE gel with 6 M urea.
    Note: Only add the urea in the separating gel.
  12. Load the CM samples and run at 90 V for 3 h with 1x running buffer. Run for additional 30 min at 90 V after the blue band reaches the bottom of the gel (see Notes 3 and 4).
  13. Transfer the proteins from the gel to PVDF membrane at 55 V for 2.5 h with 1x transfer buffer.
  14. After blotting, soak the blotted membrane in 1x PBS with 5% milk powder for 1 h. Wash with PBS-T for 3 times.
  15. Incubate with ATG8 specific primary antibody (4 μg/μl in PBS-T) for 1 h. Wash with PBST for 3 times.
  16. Incubate with the secondary antibody (1:5,000 in PBS-T) for 1 h. Wash with PBS-T for 3 times.
  17. Add Western blotting detecting reagents onto the membrane, and expose it to the X-ray film or use an imaging machine. (Figure1)

Data analysis

As shown in Figure 1, in the wild-type (WT) background, the ATG8-PE from the CM sample increases after BTH induction, with a size about 12-15 kDa, indicating that autophagy is induced with the formation of autophagosomes. However, ATG8-PE is not detected in the ATG5 deficient mutant after autophagic induction, implying that autophagosome formation is inhibited. Differently, a higher level of ATG8-PE was detected in atg9 mutant, suggesting that autophagosome formation might be interrupted at a certain stage. There might be non-specific bands in the ATG8 antibody detection. Also, it should be pointed out that there are multiple isoforms of ATG8 in the Arabidopsis genome with different SDS-PAGE mobilities, resulting the detection of cross-reacting species with similar size to the ATG8-PE adducts and making the results contradictory (Chung et al., 2010). Therefore, it is critical to include both the WT and atg5 samples as the positive and negative controls respectively to identify the correct size of ATG8-PE, as atg5 mutant lacks ATG8-PE but accumulates a large amount of non-lipidated ATG8. Our further examinations under confocal and electron microscopy identified that abnormal tubules labeled by ATG8 accumulated in the atg9 mutant, thus demonstrating that ATG9 is required for autophagosome progression (Zhuang et al., 2017). Therefore, the ATG8 lipidation assay should combine with other approaches such as microscopy analysis to further assess autophagic activity.


Figure 1. ATG8 lipidation detection in Arabidopsis thaliana wild-type and atg mutants. The 5-day-old wild-type, atg5 and atg9 seedlings were incubated in the MS liquid medium with or without BTH treatment (+B and -B) for 8 hours respectively. Crude extracts were subjected to ultracentrifuge to collect the cell membrane fraction (CM), followed by immunoblotting with plant ATG8 antibody. cFBPase represents the loading control with immunoblotting cFBPase antibodies.

Notes

  1. It is critical to make sure that the seedlings are in good condition without any stress before the autophagic induction, otherwise stressed seedlings will have induced autophagic activity, which may obscure the results. 
  2. It is recommended to include known atg mutants defective in ATG8 lipidation (e.g., atg5 or atg7) as negative controls in the lipidation assay.
  3. When running the SDS-PAGE, the urea gel will generate a lot of heat and it should keep at a low voltage, so that the two forms of ATG8 can be well separated.
  4. ATG8-PE only occurs on the membrane fraction, therefore the CM samples are used for detecting the ATG8-PE adducts. The CS fraction with non-lipidated ATG8 could be included in the western blot as a control to distinguish the non-lipidated ATG8 and lipidated ATG8.

Recipes

  1. MS liquid medium (350 ml)
    Components
    Volume/Quantity
    MS salt
    1.515 g
    Sucrose
    3.5 g
    ddH2O
    up to 350 ml
    Adjust the pH to 5.7 (with KOH)
  2. 10 mM BTH stock (10 ml) 
    Components
    Volume/Quantity
    BTH
    0.021 g
    Methanol
    10 ml
  3. 25x PIC (2 ml)
    Components
    Volume/Quantity
    PIC
    one tablet
    ddH2O
    2 ml
  4. 10% (v/v) Triton X-100 (100ml)
    Components
    Volume/Quantity
    Triton X-100
    10 ml
    ddH2O
    90 ml
  5. 1 M Tris-HCl stock (pH 6.8 or pH 7.4) (100 ml) 
    Components
    Volume/Quantity
    Tris Base 12.114 g
    ddH2O
    to 100 ml
    Adjust pH to 6.8 or 7.4 with conc. HCl
  6. 0.5 M EDTA (pH 8.0) (100 ml) 
    Components
    Volume/Quantity
    EDTA 16.81 g
    ddH2O
    80 ml
    Adjust the pH to 8.0 with NaOH (~10 g of NaOH pellets) and add to 100 ml with ddH2O
  7. 5x extraction buffer (10ml)
    Components
    Volume/Quantity
    250 mM Tris-HCl (pH 7.4) 2.5 ml of 1 M Tris-HCl (pH 7.4)
    750 mM NaCl
    0.438 g
    5 mM EDTA 0.1 ml of 0.5 M stock
    ddH2O
    to 10 ml
  8. 1x extraction buffer containing 1x PIC and 1% (v/v) Triton X-100 (10 ml)
    Components
    Volume/Quantity
    1x extraction buffer 2.5 ml of 5x extraction buffer
    1x PIC 0.40 ml of 25x PIC
    1% (v/v) Triton X-100 1 ml of 10% (v/v) Triton X-100
    ddH2O
    to 10 ml
  9. Sample loading dye (5x, 50 ml)
    Components
    Volume/Quantity
    1 M Tris-HCl (pH 6.8) 12.5 ml
    SDS 5 g
    Glycerol 25 ml
    Bromophenol Blue
    0.25 g
    β-mercaptoethanol
    6.25 ml
    ddH2O
    to 50 ml
  10. 30% APS (10 ml) 
    Components
    Volume/Quantity
    APS 3 g
    ddH2O
    10 ml
  11. 3x separation buffer (1 L)
    Components
    Volume/Quantity
    Tris base 136.2 g
    SDS 3 g
    ddH2O
    up to 1 L
    Adjust the pH to 8.8 with conc. HCl (~10 ml)
  12. 5x stacking buffer (500 ml)
    Components
    Volume/Quantity
    Tris base 37.85 g
    SDS 2.5 g
    ddH2O
    up to 500 ml
    Adjust the pH to 6.8 with conc. HCl (~20 ml)
  13. 15% SDS-PAGE gel with 6 M urea 
    1. 15% Urea separating gel (10 ml)
      Components
      Volume/Quantity
      Urea 3.6036 g
      Acry-bis (40%) 3.75 ml
      3x separation buffer
      3.33 ml
      APS (30%) 20 μl
      TEMED 5 μl
      ddH2O
      up to 10 ml
    2. 5% Stacking gel (5 ml)
      Components
      Volume/Quantity
      Acry-bis (40%)
      0.625 ml
      5x stacking buffer
      1 ml
      APS (30%) 20 μl
      TEMED 5 μl
      ddH2O
      up to 5 ml
  14. Running buffer (10x, 1 L)
    Components
    Volume/Quantity
    Tris Base
    30.3 g
    Glycine
    144 g
    SDS 10 g
    ddH2O
    up to 1 L
  15. Transfer buffer (10x, 1 L)
    Components
    Volume/Quantity
    NaHCO3
    8.4 g
    Na2CO3
    3.2 g
    ddH2O
    up to 1 L
  16. PBS (10x, 2 L)
    Components
    Volume/Quantity
    NaCl
    80 g
    NaH2PO4·H2O
    2.3 g
    Na2HPO4·2H2O
    13.9 g
    KCl 2 g
    ddH2O
    up to 2 L
    Adjust the pH to 7.4 with 10 N NaOH
  17. PBS-T (1 L)
    Components
    Volume/Quantity
    1x PBS 1 L
    Tween-20
    0.5 ml

Acknowledgments

This protocol was adapted from Zhuang et al., 2017. Fundings from the Research Grants Council of Hong Kong (G-CUHK402/15, G-CUHK403/17, CUHK14130716, CUHK14102417, C4011-14R, C4012-16E, C4002-17G and AoE/M-05/12) and the National Natural Science Foundation of China (31470294 and 31670179) support this work. The authors declare no conflicts of interests.

References

  1. Chung, T., Phillips, A. R. and Vierstra, R. D. (2010). ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci. Plant J 62(3): 483-493.
  2. Li, F., Chung, T. and Vierstra, R. D. (2014). AUTOPHAGY-RELATED11 plays a critical role in general autophagy- and senescence-induced mitophagy in Arabidopsis. Plant Cell 26(2): 788-807.
  3. Liu, Y. and Bassham, D. C. (2012). Autophagy: pathways for self-eating in plant cells. Annu Rev Plant Biol 63: 215-237.
  4. Mizushima, N., Yoshimori, T. and Levine, B. (2010). Methods in mammalian autophagy research. Cell 140(3): 313-326.
  5. Mizushima, N. and Yoshimori, T. (2007). How to interpret LC3 immunoblotting. Autophagy 3(6): 542-545.
  6. Ohsumi, Y. (2001). Ubiquitin and proteasomes: Molecular dissection of autophagy: two ubiquitin-like systems. Nat Rev Mol Cell Biol 2(3): 211-216.
  7. Suttangkakul, A., Li, F., Chung, T. and Vierstra, R. D. (2011). The ATG1/ATG13 protein kinase complex is both a regulator and a target of autophagic recycling in Arabidopsis. Plant Cell 23(10): 3761-3779.
  8. Zhuang, X., Chung, K. P., Cui, Y., Lin, W., Gao, C., Kang, B. H. and Jiang, L. (2017). ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis. Proc Natl Acad Sci U S A 114(3): E426-E435.

简介

作为降解和回收细胞货物的基本代谢途径,自噬在植物的胁迫,饥饿和衰老条件下被高度诱导。 在这个过程中,将形成一个称为自噬体的双膜结构,用于货物隔离和输送到液泡中。

已经在植物中表征了许多调控因子,包括自噬相关(ATG)蛋白和其他植物特异性蛋白。 其中,ATG8将经历脂化过程以成为膜结合的ATG8-磷脂酰乙醇胺形式并标记日益增长的自噬体膜以及完成的自噬体。 因此,ATG8被认为是自噬体的标志; 并且膜结合形式的ATG8的生物化学检测被用作测量自噬活性的主要方法之一。 在这里,我们描述了使用拟南芥幼苗检测ATG8-PE形式的ATG8脂化测定法。

【背景】自噬是调节受损细胞器的大量降解和不需要的细胞内容物的基本代谢过程。在自噬过程中,称为自噬体的双膜结构将形成并将货物递送到液泡中以降解。自噬相关蛋白(ATG)需要调节自噬活性(Liu and Bassham,2012)。其中,包括ATG8偶联物和ATG5-ATG12偶联物在内的两种偶联体系参与自噬体形成。在自噬诱导后,ATG5-ATG12缀合物形成并且起E3样酶的作用以促进ATG8脂化以结合自噬体膜上的磷脂酰乙醇胺(PE)(Ohsumi,2001)。尽管外膜上的ATG8-PE在自噬体与液泡融合之前将被回收,但内膜上的ATG8-PE将与货物一起运输到液泡中进行降解。因此,ATG8-PE的量通常与点状ATG8阳性结构的数量以及自噬活性相关(Mizushima等人,2010)。特别地,由于ATG8-PE的高疏水性,尽管ATG8-PE的实际分子量大于未偶联的ATG8(Mizushima和Yoshimori,2007),但ATG8-PE在SDS-PAGE凝胶中的迁移速度比ATG8快。因此,可以通过用ATG8抗体进行免疫印迹来检测来自细胞膜部分(CM)的ATG8-PE的量。例如,在拟南芥atg5突变体中,ATG8-PE的水平在自噬诱导后严重受损,而没有形成由ATG8标记的自噬体结构(Chung等人, 2010)。因此,ATG8脂化作用的生物化学检测可以作为一种有用的方法,当与我们以前的研究中应用的不同处理组合时,进入自噬活性以及与植物自噬相关的其他处理(Chung等, 2010年; Suttangkakul等人,2011年; Li等人,2014年; Zhuang et al。,2017年)。在这里,我们描述了通过使用阿拉酸式苯-S-甲基(BTH)处理的幼苗超高速离心分离膜和胞质溶胶部分来检测ATG8脂化作用的方案(Zhuang等人,2017)。

关键字:ATG8, ATG8-PE, 脂化, 自噬, 自噬体

材料和试剂

  1. 10μl移液枪头(Thermo Fisher Scientific,目录号:3510)
  2. 200μl移液枪头(Wolf Laboratories,目录号:2100.YN)
  3. 1,000μl移液枪头(赛默飞世尔科技,产品目录号:3580)
  4. 1.5ml微量离心管(Corning,Axygen,目录号:MCT-150-C)
  5. PVDF膜
  6. X光胶片(Advansta,目录号:L-07014-100)
  7. 5日龄苗木
  8. MS盐(Caisson,目录号:MSP01-50LT)
  9. UltraPure TM蔗糖(Thermo Fisher Scientific,Invitrogen TM,目录号:15503022)
  10. BTH(Acibenzolar-S-甲基)(Sigma-Aldrich,目录号:32820) 
  11. 甲醇(VWR,BDH,目录号:10158)
  12. 液氮 
  13. PIC(蛋白酶抑制剂混合物)(Roche Diagnostics,目录号:11873580001)
  14. Tris底座(沉箱,目录号:T041-1KG)
  15. NaCl(Alfa Aesar,USB,目录号:J21618)
  16. EDTA(Alfa Aesar,USB,目录号:J15701)
  17. SDS(十二烷基硫酸钠)(Alfa Aesar,USB,目录号:J75819)
  18. Triton X-100(GE Healthcare,Amerrsham,目录号:17-1315-01)
  19. 甘油超纯(Alfa Aesar,USB,目录号:J16374)
  20. 溴酚蓝(Sigma-Aldrich,目录号:B5525)
  21. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  22. 尿素(阿法埃萨,USB,目录号:J75826)
  23. 40%丙烯酰胺/双溶液(Bio-Rad Laboratories,目录号:161-0148)
  24. TEMED(Bio-Rad Laboratories,目录号:161-0801)
  25. APS(过硫酸铵)(USB,目录号:US76322)
  26. 非脂肪奶粉
  27. ATG8抗体(Agrisera,目录号:AS14 2769)
  28. cFBPase(Agrisera,目录号:AS04 043)
  29. 二抗(抗兔IgG过氧化物酶缀合物,Sigma-Aldrich,目录号:A6154) 
  30. Precision Plus Protein TM双色标准品(Bio-Rad Laboratories,目录号:161-0374)
  31. 碳酸氢钠(NaHCO 3)(VWR,目录号:144-55-8)
  32. 碳酸钠(Na 2 CO 3)(USB,目录号:21602) 
  33. 磷酸二氢钠一水合物(NaH 2 PO 4·2H 2 O)(USB,目录号:20233)
  34. 磷酸氢二钠二水合物(Na 2 HPO 4·2H 2 O)(Sigma-Aldrich,目录号:04272)
  35. 氯化钾(KCl)(Sigma-Aldrich,目录号:31248)
  36. 吐温-20(Sigma-Aldrich,目录号:63158)
  37. MS液体培养基(见食谱)
  38. 10毫米BTH股票(见食谱)
  39. 25倍的PIC(见食谱)
  40. 10%(v / v)Triton X-100(见食谱)
  41. 1M Tris-HCl原液(pH 6.8或pH 7.4)(见食谱)
  42. 0.5 M EDTA(pH 8.0)(见食谱)
  43. 5倍提取缓冲液(见食谱)
  44. 含有1x PIC和1%(v / v)Triton X-100的1x提取缓冲液(见食谱)
  45. 5倍样品加载染料(见食谱)
  46. 30%APS(见食谱)
  47. 3倍分离缓冲液(见食谱)
  48. 5倍堆叠缓冲区(请参阅食谱)
  49. 含有6M尿素的15%SDS-PAGE凝胶(参见食谱)
    1. 15%尿素分离胶
    2. 5%堆叠凝胶
  50. 运行缓冲区(请参阅食谱)
  51. 传输缓冲区(请参阅食谱)
  52. PBS(见食谱) 
  53. PBS-T(见食谱)

设备

  1. Eppendorf Research 加移液管0.5-10μl(Eppendorf,产品目录号:3120000020)
  2. Eppendorf Research 加移液管10-100μl(Eppendorf,产品目录号:3120000046)
  3. Eppendorf研究加移液管100-1,000μl(Eppendorf,目录号:3120000062)
  4. 灰浆和杵
  5. 离心机(Eppendorf,型号:5430)
  6. X光胶片盒(Amersham Biosciences Hypercassette TM TM)
  7. 超速离心管(7 x 20 mm)
  8. 超速离心机(Beckman Coulter,型号:Optima TM MAX-XP)
  9. Western Blotting设备(Bio-Rad)
  10. 开发者机器(Fujifilm FPM100A)

程序

  1. 在含有或不含100μMBTH的MS液体培养基(10mM储液在甲醇中)中培养0.2g 5天的拟南芥幼苗8小时(见注1和注2)。
  2. 用液氮将砂浆中的幼苗冻结并彻底研磨植物。

  3. 将含有不含去污剂的PIC的2x提取缓冲液加入研钵中。
  4. 在冰上缓慢解冻后,将液体转移到1.5ml管中,并在1,000℃×g下离心5分钟,4℃。
  5. 将上清液转移到超速离心管(7×20mm)中并在100,000×gg下离心4分钟45分钟。
  6. 将上清液(细胞溶解部分,CS)转移到带有标记的新Eppendorf管中并保持在冰上。
  7. 用含有1x PIC的1x提取缓冲液轻轻洗涤托盘(细胞膜部分,CM)2-3次。
    注意:在此步骤中,轻轻地添加1x提取缓冲液,不要接触托盘表面,不需要离心机和重新悬浮。此步骤的主要目的是清洗CM表面上剩余的CS液体。
  8. 使用含有1x PIC和1%(v / v)Triton X-100的1x提取缓冲液重新悬浮托盘以溶解膜。
  9. 将5倍样品加载染料添加到CS和CM样品中。
  10. 将样品在100°C煮10分钟。
  11. 用6M尿素制备15%SDS-PAGE凝胶。
    注意:只能在分离胶中加入尿素。
  12. 加载CM样品并用1x运行缓冲液在90 V下运行3 h。
    在蓝色条带到达凝胶底部后,再以90V运行30分钟(见注3和4)。

  13. 使用1x转移缓冲液将蛋白质从凝胶转移至PVDF膜,电压为55 V.
  14. 印迹后,将印迹膜浸入含有5%奶粉的1x PBS中1小时。用PBS-T清洗3次。
  15. 用ATG8特异性一抗(PBS-T中4μg/μl)孵育1小时。用PBST洗涤3次。
  16. 与二抗(1:5,000在PBS-T中)孵育1小时。用PBS-T清洗3次。
  17. 将蛋白质印迹检测试剂添加到膜上,并将其暴露于X射线胶片或使用成像设备。 (图1)

数据分析

如图1所示,在野生型(WT)背景中,来自CM样品的ATG8-PE在BTH诱导后增加,大小约为12-15kDa,表明自噬是由自噬体形成诱导的。然而,在自噬诱导后ATG5缺陷突变体中未检测到ATG8-PE,这意味着自噬体形成受到抑制。不同的是,在atg9突变体中检测到更高水平的ATG8-PE,表明自噬体形成可能在某个阶段中断。 ATG8抗体检测中可能存在非特异性条带。此外,应该指出的是,在拟南芥属基因组中存在多种ATG8同种型,其具有不同的SDS-PAGE迁移率,导致与ATG8-PE加合物具有相似大小的交叉反应物种的检测,以及使得结果相互矛盾(Chung等人,2010年)。因此,分别将WT和 atg5 样品作为阳性和阴性对照以鉴定ATG8-PE的正确大小是非常关键的,因为atg5突变体缺乏ATG8- PE但积累了大量的非脂化ATG8。我们在共焦和电子显微镜下的进一步检查发现ATG8标记的异常小管在atg9突变体中积累,因此证明ATG9是自噬体进展所必需的(Zhuang等, 2017年)。因此,ATG8脂化测定应结合其他方法如显微镜分析来进一步评估自噬活性。


图1.在拟南芥中野生型和 atg 突变体中的ATG8脂化检测 5天龄的野生型将atg5和atg9幼苗分别在含有或不含BTH处理(+ B和-B)的MS液体培养基中温育8小时。对粗提取物进行超速离心以收集细胞膜部分(CM),随后用植物ATG8抗体进行免疫印迹。 cFBPase代表用免疫印迹cFBPase抗体的加样对照。

笔记

  1. 确保幼苗在自噬诱导之前没有任何压力的情况下保持良好状态是至关重要的,否则应激幼苗会诱导自噬活性,这可能会使结果模糊不清。 
  2. 推荐在ATG8脂化中包含已知的 atg 突变体( eg , atg5 或 atg7 )作为阴性对照在脂化测定中。
  3. 当运行SDS-PAGE时,尿素凝胶会产生大量热量,并且应保持在低电压下,以便两种形式的ATG8可以很好地分离。
  4. ATG8-PE仅出现在膜部分上,因此CM样品用于检测ATG8-PE加合物。具有非脂质化ATG8的CS级分可以包含在蛋白质印迹中作为区分非脂质化ATG8和脂质化ATG8的对照。

食谱

  1. MS液体培养基(350ml)
    组件
    体积/数量
    MS盐

    1.515克
    蔗糖
    3.5克
    ddH <2>
    高达350毫升
    调整pH值至5.7(含KOH)
  2. 10毫米BTH股票(10毫升)&nbsp;
    组件
    体积/数量
    BTH
    0.021克
    甲醇
    10毫升
  3. 25倍PIC(2毫升)
    组件
    体积/数量
    PIC
    一个平板电脑
    ddH <2>
    2毫升
  4. 10%(v / v)Triton X-100(100ml)
    组件
    体积/数量
    Triton X-100
    10毫升
    ddH <2>
    90毫升
  5. 1 M Tris-HCl储备液(pH 6.8或pH 7.4)(100 ml)&nbsp;
    组件
    体积/数量
    Tris基地
    12.114克
    ddH <2>
    到100毫升
    用浓水将pH调节至6.8或7.4。 HCl
  6. 0.5M EDTA(pH8.0)(100ml)&nbsp;
    组件
    体积/数量
    EDTA 16.81克
    ddH <2>
    80毫升
    用NaOH调节pH至8.0(〜10g氢氧化钠丸粒)并用ddH 2 O加入到100ml中。
  7. 5倍提取缓冲液(10ml)
    组件
    体积/数量
    250mM Tris-HCl(pH7.4) 2.5毫升1M Tris-HCl(pH 7.4)
    750 mM NaCl
    0.438克
    5mM EDTA 0.1毫升的0.5 M股票
    ddH <2>
    到10毫升
  8. 含1x PIC和1%(v / v)Triton X-100(10ml)的1x提取缓冲液。
    组件
    体积/数量
    1x提取缓冲液 2.5毫升的5倍提取缓冲液
    1x PIC 0.40毫升的25x PIC
    1%(v / v)Triton X-100 1ml 10%(v / v)Triton X-100
    ddH <2>
    到10毫升
  9. 样品加载染料(5x,50毫升)
    组件
    体积/数量
    1M Tris-HCl(pH6.8) 12.5毫升
    SDS 5克
    甘油 25毫升
    溴酚蓝
    0.25克
    β-巯基乙醇
    6.25毫升
    ddH <2>
    到50毫升
  10. 30%APS(10毫升)&nbsp;
    组件
    体积/数量
    APS 3克
    双蒸<子> 2 0
    10毫升
  11. 3倍分离缓冲液(1 L)
    组件
    体积/数量
    Tris碱 136.2克
    SDS 3克
    双蒸<子> 2 0
    最多1升
    用浓水调节pH值至8.8。 HCl(约10毫升)
  12. 5倍堆积缓冲液(500毫升)
    组件
    体积/数量
    Tris碱 37.85克
    SDS 2.5克
    双蒸<子> 2 0
    高达500毫升
    浓缩调节pH至6.8。 HCl(约20毫升)
  13. 含6M尿素的15%SDS-PAGE凝胶&nbsp;
    1. 15%尿素分离胶(10毫升)
      组件
      体积/数量
      尿素 3.6036克
      丙烯酸酯(40%) 3.75毫升
      3倍分离缓冲液
      3.33毫升
      APS(30%) 20微升
      TEMED 5微升
      双蒸<子> 2 0
      多达10毫升
    2. 5%堆叠凝胶(5毫升)
      组件
      体积/数量
      丙烯 - 二(40%)
      0.625毫升
      5倍堆栈缓冲区
      1毫升
      APS(30%) 20微升
      TEMED 5微升
      双蒸<子> 2 0
      多达5毫升
    3. 运行缓冲区(10x,1L)
      组件
      体积/数量
      Tris基地
      30.3克
      甘氨酸
      144克
      SDS 10克
      ddH <2>
      最多1升
    4. 传输缓冲区(10x,1L)
      组件
      体积/数量
      NaHCO 3
      8.4克
      Na 2 CO 3 3/2 3.2克
      ddH <2>
      最多1升
    5. PBS(10x,2L)
      组件
      体积/数量
      NaCl
      80克
      NaH 2 PO 4 4 H 2 O
      2.3克
      Na 2 HPO 4·2H 2 O O 2/2 13.9克
      氯化钾 2克
      ddH <2>
      最多2升
      用10N NaOH调节pH值至7.4
    6. PBS-T(1L)
      组件
      体积/数量
      1x PBS 1 L
      Tween-20
      0.5毫升

      致谢

      本议定书改编自2017年的Zhuang e t 。。Fundings来自香港研究资助局(G-CUHK402 / 15,G-CUHK403 / 17,CUHK14130716,CUHK14102417,C4011-14R,C4012-16E,C4002-17G和AoE / M-05/12)和国家自然科学基金中国基金会(31470294和31670179)支持这项工作。作者声明不存在利益冲突。

      参考

      1. Chung,T.,Phillips,A.R。和Vierstra,R.D。(2010)。 ATG8脂化和ATG8介导的自噬在 Arabidops i < 要求ATG12表示由差分控制的 ATG12 A 和 A T > 1 2 B 位点。 l a J 62(3):483-493。
      2. Li,F.,Chung,T。和Vierstra,R.D。(2014)。 AUTOPHAGY-RELATED11在自噬和衰老诱导的线粒体自噬中起关键作用 rabidop s s 。 Plant Cell 26(2):788-807。
      3. Liu,Y.和Bassham,D.C。(2012)。 自噬:植物细胞自我进食的途径 Annu Rev Plant Biol 63:215-237。
      4. Mizushima,N.,Yoshimori,T.和Levine,B。(2010)。 哺乳动物自噬研究方法 e l l 140(3):313-326。
      5. Mizushima,N。和Yoshimori,T。(2007)。 如何解读LC3免疫印迹 自体吞噬 3(6) ):542-545。
      6. Ohsumi,Y.(2001)。 泛素和蛋白酶体:自噬的分子解剖:两个泛素样系统 Nat Rev Mol Cell Bio l 2(3):211-216。
      7. Suttangkakul,A.,Li,F.,Chung,T。和Vierstra,R.D。(2011)。 ATG1 / ATG13蛋白激酶复合物既是一种调节剂,也是自噬循环的目标拟南芥。植物 l 23(10):3761-3779。
      8. Zhuang,X.,Chung,K. P.,Cui,Y.,Lin,W.,Gao,C.,Kang,B. H. and Jiang,L.(2017)。 ATG9调控 Arabidopsi 的内质网的自噬体进程。 Proc Natl Acad Sci USA 114(3):E426-E435。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Luo, M. and Zhuang, X. (2018). Analysis of Autophagic Activity Using ATG8 Lipidation Assay in Arabidopsis thaliana. Bio-protocol 8(12): e2880. DOI: 10.21769/BioProtoc.2880.
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