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In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis
秀丽隐杆线虫中的体内线粒体自噬监测以确定线粒体稳态   

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本实验方案简略版
Nature
Nov 2013

Abstract

Perturbation of mitochondrial function is a major hallmark of several pathological conditions and ageing, underlining the essential role of fine-tuned mitochondrial activity (Lopez-Otin et al., 2013). Mitochondrial selective autophagy, known as mitophagy, mediates the removal of dysfunctional and/or superfluous organelles, preserving cellular and organismal homeostasis (Palikaras and Tavernarakis, 2014; Pickrell and Youle, 2015; Scheibye-Knudsen et al., 2015). In this protocol, we describe a method for assessing mitophagy in the nematode Caenorhabditis elegans.

Keywords: Ageing (衰老), Autophagosome (自噬体), Autophagy (自噬), Caenorhabditis elegans (秀丽隐杆线虫), Lysosomes (溶酶体), Mitochondria (线粒体), Mitophagy (线粒体自噬), mtRosella (mtRosella)

Background

Mitochondria are characterized as cellular powerhouses of eukaryotic cells, since they are the major energy providers through oxidative phosphorylation (OXPHOS) and ATP generation. Moreover, their pivotal role in cellular homeostasis is highlighted by their contribution in the regulation of several fundamental cellular processes including calcium buffering, metabolite synthesis and apoptosis, among others. Deregulation of mitochondrial function is associated with the onset of several pathological conditions including ageing and age-related neurodegenerative diseases (Vafai and Mootha, 2012; Palikaras and Tavernarakis, 2014). Thus, eukaryotic organisms have evolved several complex and highly specialized molecular pathways to guard energy homeostasis (Pickrell and Youle, 2015; Scheibye-Knudsen et al., 2015). Mitophagy is a selective type of autophagy promoting the elimination of impaired mitochondria, and the major degradation pathway by which cells regulate mitochondrial content in response to intracellular and environmental signals (Palikaras et al., 2015; Schiavi et al., 2015; Fang et al., 2016). In this protocol, we describe two methods for monitoring mitophagy in C. elegans. We developed two composites, in vivo imaging systems to asses mitophagy based, first, on the Rosella biosensor (Rosado et al., 2008), which combines a fast-maturing pH-insensitive DsRed fused to a pH-sensitive GFP variant, and second, on a custom, dual-fluorescence reporter system that involves a mitochondria-targeted GFP, together with the autophagosomal marker LGG-1/LC3 fused to DsRed. These protocols facilitate non-invasive monitoring of mitophagy in live specimens.

Materials and Reagents

  1. Greiner Petri dishes (60 x 15 mm) (Greiner Bio One International, catalog number: 628161 )
  2. Microscope slides 75 x 25 x 1 mm (Marienfeld-Superior, catalog number: 10 006 12 )
  3. Microscope cover glass 18 x 18 mm (Marienfeld-Superior, catalog number: 01 010 30 )
  4. Use the following transgenic nematodes to monitor mitophagy: IR1631: N2;Ex003 [pmyo-3TOMM-20::Rosella; pRF4], R1284: N2;Is [pmyo-3mtGFP];Ex011 [plgg-1DsRed::LGG-1; pmyo-2GFP])
  5. Escherichia coli OP50 strain (obtained from the Caenorhabditis Genetics Center)
  6. 70% of EtOH
  7. Potassium dihydrogen phosphate (KH2PO4) (EMD Millipore, catalog number: 104873 )
  8. di-Potassium hydrogen phosphate (K2HPO4) (EMD Millipore, catalog number: 137010 )
  9. Sodium chloride (NaCl) (EMD Millipore, catalog number: 106404 )
  10. Peptone (BD, BactoTM, catalog number: 211677 )
  11. Streptomycin (Sigma-Aldrich, catalog number: S6501 )
  12. Agar (Sigma-Aldrich, catalog number: 05040 )
  13. Cholesterol stock solution (SERVA Electrophoresis, catalog number: 17101.01 )
  14. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  15. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  16. Nystatin stock solution (Sigma-Aldrich, catalog number: N3503 )
  17. di-Sodium hydrogen phosphate (Na2HPO4) (EMD Millipore, catalog number: 106586 )
  18. Levamisole (Sigma-Aldrich, catalog number: L9756 )
  19. Paraquat (Sigma-Aldrich, catalog number: 856177 )
  20. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, catalog number: C2759 )
  21. Dimethyl sulfoxide cell culture grade BC (DMSO) (AppliChem, catalog number: A3672,0250 )
  22. Phosphate buffer (1 M; sterile, see Recipes)
  23. Nematode growth medium (NGM) agar plates (see Recipes)
  24. M9 buffer (see Recipes)
  25. Levamisole (0.5 M, see Recipes)
  26. M9-levamisole solution (20 mM solution, see Recipes)
  27. Paraquat (0.5 M, see Recipes)
  28. Carbonyl cyanide m-chlorophenylhydrazone (49 mM; CCCP, see Recipes)

Equipment

  1. UV crosslinker (Vilber Lourmat, model: BIO-LINK – BLX-E365 )
  2. Zeiss AxioImager Z2 epifluorescence microscope (Zeiss, model: Zeiss AxioImager Z2 )
  3. Olympus DP71 CCD camera (Olympus, model: Olympus DP71 )
  4. Zeiss AxioObserver Z1 confocal microscope (Zeiss, model: Zeiss AxioObserver Z1 )
  5. Dissecting stereomicroscope (Olympus, model: SMZ645 )
  6. Incubators for stable temperature (AQUA®LYTIC incubator 20 °C)

Software

  1. Olympus CELL-A software
  2. Zeiss ZEN 2012 software
  3. Image J (https://imagej.nih.gov/ij/)
  4. Microsoft Office 2011 Excel (Microsoft Corporation, Redmond, USA)
  5. GraphPad Prism software package (GraphPad Software Inc., San Diego, USA)

Procedure

  1. Growth and synchronization of nematode population
    1. Select L4 larvae of transgenic animals, which express mitochondria-targeted Rosella (mtRosella) or co-express mitochondria-targeted GFP (mtGFP) together with the autophagosomal marker LGG-1 fused with DsRed in body wall muscle cells, on a freshly E. coli (OP50) seeded NGM plate. Use at least three plates for each nematode strain.
    2. Incubate the nematodes at the standard temperature of 20 °C.
    3. Four days later the plates contain mixed animals population.
    4. Synchronize nematodes by picking L4 transgenic larvae and transfer them onto separate freshly E. coli (OP50) seeded plates (Palikaras, K. and Tavernarakis, N. [2016]. Intracellular Assessment of ATP Levels in Caenorhabditis elegans. Bio-protocol 6(23): e2048; Palikaras, K. and Tavernarakis, N. [2016]. Measuring Oxygen Consumption Rate in Caenorhabditis elegans. Bio-protocol 6(23): e2049).
    5. Add 20 L4 larvae per plate. For each experimental condition, use at least five plates (see Note 1).

  2. Oxidative and mitochondrial stress assay
    1. Kill E. coli (OP50) bacteria seeded on NGM plates by using a UV crosslinker (UV irradiation for 15 min; 0.5 J).
    2. Add paraquat or CCCP to the top of seeded NGM plates at 8 mM and 15 μM final concentrations in the total agar volume respectively. Paraquat and CCCP are well-known inducers of mitophagy in mammalian cells (Narendra et al., 2010).
    3. Gently swirl the plates and allow each drug to spread to the entire surface.
    4. Let the plates dry at room temperature.
    5. Transfer 2- or 4-day-old of adulthood adult transgenic animals on paraquat- or CCCP-containing plates.
    6. Incubate the transgenic animals at 20 °C.
    7. Upon two days of exposure to each drug prepare the nematodes for microscopic examination.

  3. Mounting nematodes for imaging
    1. Add a droplet of 10 μl M9-levamisole buffer (20 mM final concentration, see Note 2) on 2% agarose pad (see Note 3).
    2. Collect transgenic animals with an eyebrow/eyelash hair and place them in M9-levamisole droplet immobilizing transgenic animals for imaging (see Note 4).
    3. Gently place a coverslip on the top press. Samples are ready for microscopic examination with either a Zeiss AxioImager Z2 epifluorescence microscope or a Zeiss AxioObserver Z1 confocal microscope.

  4. Imaging of transgenic nematodes
    1. Capture single transgenic animals or single body wall muscle cells using a camera attached to the microscope.
    2. Acquire (a) fluorescent images of whole transgenic nematodes expressing mtRosella in body wall muscle cells by using Zeiss AxioImager Z2 epifluorescence microscope or perform (b) z-stack method of an entire single body wall muscle cell expressing mtGFP together with autophagosomal marker DsRed::LGG-1 by using Zeiss AxioObserver Z1 confocal microscope.
    3. Imaging parameters such as microscope and camera settings (lens and magnifier used, filters exposure time, resolution, laser intensity, gain etc.) should be documented and kept the same during the imaging process.
    4. Save collected images from each method (a or b) and proceed to data analysis.

  5. Analyze data from imaging process
    1. Process images acquired from (a) method with ImageJ software to measure the average pixel intensity values and total area for each fluorescent image of transgenic worm. Focus on body wall muscle cells of the head region avoiding intestinal autofluorescence (Figures 1 and 2; see Note 5). To analyze the area of interest manually:
      1. Select the ‘split channel’ command via the ‘image’ and ‘colour’ drop-down menu to convert images.
      2. Use the ‘freehand selection’ tool to enclose the fluorescent area.
      3. Select the ‘measurement’ command via the ‘analyze’ drop-down menu to perform pixel intensity analysis.
      4. Normalize pixel intensity values by dividing with the selected area values by using the Microsoft Office 2011 Excel software package (Microsoft Corporation, Redmond, USA).
      5. Upon normalization calculate GFP to DsRed ratio.


        Figure 1. Mitophagy induction upon oxidative and mitochondrial stress. Transgenic nematodes expressing mtRosella in body wall muscle cells, were exposed to paraquat or CCCP. Mitophagy induction is signified by the reduction of the ratio between pH-sensitive GFP to pH-insensitive DsRed (n = 100; ***P < 0.001; one-way ANOVA). Arrowheads point out intestinal autofluorescence. Size bars denote 20 μm. Images were acquired using a 10x objective lens. Error bars denote SEM values.


        Figure 2. Image analysis by using ImageJ software. 1. Open an acquired GFP signal-image with ImageJ software; 2. Select split channel’ command via the ‘image’ and ‘colour’ drop-down menu to convert images; 3. Keep ‘green channel’ image; 4. Use the ‘freehabnd selection’ tool to enclose the fluorescent area (head region); 5. Select the ‘measurement’ command via the ‘analyze’ drop-down menu to perform pixel intensity analysis; 6. Perform the same analysis pathway with an acquired DsRed-signal image.

    2. Process images collected from (b) method with Zeiss ZEN 2012 software to find mitochondria engulfed by autophagosomes, known as mitoautophagosomes.
      1. Determine mitoautophagosomes number by manually counting the co-localization events between mitochondrial (mtGFP) and autophagosomal marker (DsRed::LGG-1) displayed in each stack of body wall muscle cell (Figure 3).
      2. Analyze the data by using the Microsoft Office 2011 Excel software package (Microsoft Corporation, Redmond, USA).


        Figure 3. Monitor mitoautophagosomes formation in vivo. Transgenic nematodes co-expressing a mitochondria-targeted GFP (mtGFP) in body wall muscle cells together with the autophagosomal protein LGG-1 fused with DsRed, were treated with paraquat or CCCP. Mitophagy stimulation is signified by co-localization of GFP and DsRed signals (for each group of images mitochondria are shown in green on top, autophagosomes in red below, with a merged image at the bottom). Increased number of mitoautophagosomes upon oxidative and mitochondrial stress (n = 50; ***P < 0.001; one-way ANOVA). Size bars denote 20 μm. Images were acquired using a 40x objective lens. Error bars denote SEM values. 

Data analysis

  1. For each strain or condition, use at least 100 animals or 50 body wall muscle cells to obtain more accurate results.
  2. Each assay should be repeated at least three (3) times.
  3. Use the Student’s t-test with a significance cut-off level of P < 0.05 for comparisons between two groups.
  4. Use the one-factor (ANOVA) variance analysis and correct by the post hoc Bonferroni test for multiple comparisons.

Notes

  1. Transfer the selected transgenic animals to freshly seeded NGM plates every two days to avoid progeny and prevent starvation due to lack of food. Calorie deprivation and starvation induce mitophagy. Thus, well-fed animals should be used.
  2. The use of M9 buffer instead of water ensures a favourable osmotic environment for the nematodes.
  3. Levamisole is a mild anaesthetics and is required to immobilize nematodes. Avoid anaesthetics that could interfere with metabolic processes, such as sodium azide. Sodium azide induces mitochondrial and oxidative stress through inhibition of mitochondrial respiratory chain and perturbation of energy production. Thus, sodium azide is likely to induce mitophagy.
  4. Take a toothpick and glue an eyebrow/eyelash hair to the tip of it. Let it dry at room temperature. Then, use this tool to pick nematodes. Before using the eyebrow/eyelash hair always sterilize it by using 70% of EtOH.
  5. Intestinal autofluorescence increases during ageing in C. elegans. Thus, body wall muscle cells close to the intestine should be avoided during the imaging process.

Recipes

  1. Phosphate buffer (1 M)
    1. For 1 L, dissolve 102.2 g KH2PO4 and 57.06 g K2HPO4 in distilled water and fill up to 1 L. This is a 1 M solution, pH 6.0
    2. Autoclave and keep at room temperature
  2. Nematode growth medium (NGM) agar plates
    1. Mix 3 g NaCl, 2.5 g Bacto peptone, 0.2 g streptomycin, 17 g agar and add 900 ml distilled water. Autoclave
    2. Let cool to 55-60 °C
    3. Add 1 ml cholesterol stock solution, 1 ml 1 M CaCl2, 1 ml 1 M MgSO4, 1 ml nystatin stock solution, 25 ml sterile 1 M phosphate buffer, pH 6.0, and distilled sterile water up to 1 L
    4. Pour about 8 ml medium per Petri dish and leave to solidify
    5. Keep the plates at 4 °C until used
  3. M9 buffer
    1. Dissolve 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl in 1 L distilled water. Autoclave
    2. Let cool and add 1 ml 1 M MgSO4 (sterile)
    3. Store M9 buffer at 4 °C
  4. Levamisole (0.5 M)
    1. Dissolve 1.2 g levamisole in 10 ml distilled water
    2. Store levamisole solution at 4 °C
  5. M9-levamisole (20 mM)
    1. Dilute 400 μl 0.5 M levamisole in 10 ml M9 buffer
    2. Store M9-levamisole solution at 4 °C
  6. Paraquat (0.5 M)
    1. Dissolve 1 g paraquat in 8 ml distilled water
    2. Prepare aliquots of 400 ml and store them at 4 °C
  7. Carbonyl cyanide m-chlorophenylhydrazone (49 mM; CCCP)
    1. Dissolve 100 mg CCCP in 10 ml of DMSO
    2. Prepare aliquots of 1 ml and store them at -20 °C

Acknowledgments

This work was funded by grants from the European Research Council (ERC), the European Commission 7th Framework Programme and Bodossaki Foundation Postdoctoral Research Fellowship. The protocol has been adapted from Palikaras et al. (2015), Nature 521, 525-528.

References

  1. Fang, E. F., Kassahun, H., Croteau, D. L., Scheibye-Knudsen, M., Marosi, K., Lu, H., Shamanna, R. A., Kalyanasundaram, S., Bollineni, R. C., Wilson, M. A., Iser, W. B., Wollman, B. N., Morevati, M., Li, J., Kerr, J. S., Lu, Q., Waltz, T. B., Tian, J., Sinclair, D. A., Mattson, M. P., Nilsen, H. and Bohr, V. A. (2016). NAD+ replenishment improves lifespan and healthspan in ataxia telangiectasia models via mitophagy and DNA repair. Cell Metab 24(4): 566-581.
  2. Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M. and Kroemer, G. (2013). The hallmarks of aging. Cell 153(6): 1194-1217.
  3. Narendra, D. P., Jin, S. M., Tanaka, A., Suen, D. F., Gautier, C. A., Shen, J., Cookson, M. R. and Youle, R. J. (2010). PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol 8: e1000298.
  4. Palikaras, K., Lionaki, E. and Tavernarakis, N. (2015). Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans. Nature 521(7553): 525-528.
  5. Palikaras, K. and Tavernarakis, N. (2014). Mitochondrial homeostasis: the interplay between mitophagy and mitochondrial biogenesis. Exp Gerontol 56: 182-188.
  6. Pickrell, A. M. and Youle, R. J. (2015). The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson's disease. Neuron 85(2): 257-273.
  7. Rosado, C. J., Mijaljica, D., Hatzinisiriou, I., Prescott, M. and Devenish, R. J. (2008). Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast. Autophagy 4(2): 205-213.
  8. Scheibye-Knudsen, M., Fang, E. F., Croteau, D. L., Wilson, D. M., 3rd and Bohr, V. A. (2015). Protecting the mitochondrial powerhouse. Trends Cell Biol 25(3): 158-170.
  9. Schiavi, A., Maglioni, S., Palikaras, K., Shaik, A., Strappazzon, F., Brinkmann, V., Torgovnick, A., Castelein, N., De Henau, S., Braeckman, B. P., Cecconi, F., Tavernarakis, N. and Ventura, N. (2015). Iron-starvation-induced mitophagy mediates lifespan extension upon mitochondrial stress in C. elegans. Curr Biol 25(14): 1810-1822.
  10. Vafai, S. B. and Mootha, V. K. (2012). Mitochondrial disorders as windows into an ancient organelle. Nature 491(7424): 374-383.

简介

线粒体功能的扰动是几种病理状况和衰老的主要标志,强调了线粒体活性调控的重要作用(Lopez-Otin等,2013)。 线粒体选择性自噬,称为嗜中性细胞因子介导功能障碍和/或多余的细胞器的去除,保留细胞和有机体内稳态(Palikaras和Tavernarakis,2014; Pickrell和Youle,2015; Scheibye-Knudsen等,2015)。 在这个协议中,我们描述了一种评估线虫秀丽隐杆线虫线粒体的方法。
【背景】线粒体被认为是真核细胞的细胞动力,因为它们是通过氧化磷酸化(OXPHOS)和ATP生成的主要能量提供者。此外,它们在细胞稳态中的关键作用突出表现在它们对几种基本细胞过程(包括钙缓冲,代谢物合成和凋亡等)调节的贡献。线粒体功能的放松规律与几种病理状况(包括衰老和年龄相关的神经变性疾病)的发病有关(Vafai和Mootha,2012; Palikaras和Tavernarakis,2014)。因此,真核生物已经发展了几种复杂和高度专门化的分子途径来保护能量稳态(Pickrell和Youle,2015; Scheibye-Knudsen等,2015)。 Mitophagy是一种选择性类型的自噬,促进线粒体受损的消除,以及细胞调节线粒体内源物质和环境信号的主要降解途径(Palikaras等,2015; Schiavi et al。,2015; Fang et al 。,2016)。在这个协议中,我们描述了两种监测线虫的线虫病的方法。我们开发了两个复合体内成像系统,以首先在Rosella生物传感器(Rosado et al。,2008)上进行基于mitophagy的研究,其结合了快速成熟的不敏感的DsRed融合到pH敏感GFP变体,第二,涉及涉及线粒体靶向GFP的定制双荧光报告系统,以及与DsRed融合的自噬体标志物LGG-1 / LC3。这些方案促进活体标本中的线粒体的非侵入性监测。

关键字:衰老, 自噬体, 自噬, 秀丽隐杆线虫, 溶酶体, 线粒体, 线粒体自噬, mtRosella

材料和试剂

  1. Greiner Petri菜(60 x 15毫米)(Greiner Bio One International,目录号:628161)
  2. 显微镜滑动75 x 25 x 1 mm(Marienfeld-Superior,目录号:10 006 12)
  3. 显微镜盖玻璃18 x 18毫米(Marienfeld-Superior,目录号:01 010 30)
  4. 使用以下转基因线虫来监测线粒体:IR1631:N2; Ex003 TOMM-20 :: Rosella; pRF4],R1284:N2; em> [p lgg-1 DsRed :: LGG-1; p myo-2 GFP])
  5. 大肠杆菌OP50菌株(从Caenorhabditis 遗传学中心获得)
  6. 70%的EtOH
  7. 磷酸二氢钾(KH 2 PO 4)(EMD Millipore,目录号:104873)
  8. 磷酸氢二钾(K 2 HPO 4)(EMD Millipore,目录号:137010)
  9. 氯化钠(NaCl)(EMD Millipore,目录号:106404)
  10. 蛋白胨(BD,Bacto TM ,目录号:211677)
  11. 链霉素(Sigma-Aldrich,目录号:S6501)
  12. 琼脂(Sigma-Aldrich,目录号:05040)
  13. 胆固醇储备溶液(SERVA Electrophoresis,目录号:17101.01)
  14. 氯化钙脱水(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C5080)
  15. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  16. 制霉菌素储备溶液(Sigma-Aldrich,目录号:N3503)
  17. 二磷酸氢钠(Na 2 HPO 4)(EMD Millipore,目录号:106586)
  18. 左旋咪唑(Sigma-Aldrich,目录号:L9756)
  19. 百草枯(Sigma-Aldrich,目录号:856177)
  20. 羰基氰基间氯苯腙(CCCP)(Sigma-Aldrich,目录号:C2759)
  21. 二甲基亚砜细胞培养级BC(DMSO)(AppliChem,目录号:A3672,0250)
  22. 磷酸盐缓冲液(1M;无菌,见食谱)
  23. 线虫生长培养基(NGM)琼脂平板(参见食谱)
  24. M9缓冲区(见配方)
  25. 左旋咪唑(0.5 M,见食谱)
  26. M9-左旋咪唑溶液(20mM溶液,参见食谱)
  27. 百草枯(0.5 M,见食谱)
  28. 羰基氰化物m-氯苯腙(49mM; CCCP,参见食谱)

设备

  1. 紫外线交联剂(Vilber Lourmat,型号:BIO-LINK-BLX-E365)
  2. 蔡司AxioImager Z2落射荧光显微镜(Zeiss,型号:Zeiss AxioImager Z2)
  3. 奥林巴斯DP71 CCD相机(Olympus,型号:Olympus DP71)
  4. 蔡司AxioObserver Z1共聚焦显微镜(Zeiss,型号:Zeiss AxioObserver Z1)
  5. 解剖立体显微镜(Olympus,型号:SMZ645)
  6. 温度稳定的孵化器(AQUA ® LYTIC培养箱20°C)

软件

  1. 奥林巴斯CELL-A软件
  2. 蔡司ZEN 2012软件
  3. Image J( https://imagej.nih.gov/ij/
  4. Microsoft Office 2011 Excel(Microsoft Corporation,Redmond,USA)
  5. GraphPad Prism软件包(GraphPad Software Inc.,San Diego,USA)

程序

  1. 线虫种群的增长和同步
    1. 选择L4转基因动物的L4幼虫,其表达线粒体靶向罗莎氏菌(mtRosella)或共同表达线粒体靶向GFP(mtGFP)以及与体内壁肌细胞中与DsRed融合的自身体标志物LGG-1, E.大肠杆菌(OP50)种子NGM板。每个线虫菌株至少使用三个板块。
    2. 在20°C的标准温度下孵育线虫。
    3. 四天后,板块含有混合动物种群。
    4. 通过挑选L4转基因幼虫并将其转移到单独的新鲜E上来同步线虫。大肠杆菌(OP50)接种板(Palikaras,K.and Tavernarakis,N。[2016]。细胞内ATP检测在秀丽隐杆线虫。 6(23):e2048; Palikaras,K.and Tavernarakis,N。[2016]。测量秀丽隐杆线虫中的氧消耗率生物方案6(23): e2049)
    5. 每板加入20只L4幼虫。对于每个实验条件,使用至少五块板(见注1)
  2. 氧化和线粒体应激测定
    1. E。E。大肠杆菌(OP50)细菌通过使用UV交联剂(UV照射15分钟; 0.5J)接种在NGM平板上。
    2. 将百草枯或CCCP分别以总琼脂体积的8mM和15μM终浓度添加到接种的NGM板的顶部。百草枯和CCCP是哺乳动物细胞中众所周知的线粒体诱导剂(Narendra等人,2010)。
    3. 轻轻地旋转板材,让每种药物均匀扩散到整个表面
    4. 让板在室温下干燥。
    5. 在含有百草枯或含CCCP的平板上转移成年成年转基因动物的2日龄或4日龄
    6. 在20°C孵育转基因动物
    7. 每天接触两天后,准备线虫进行显微镜检查
  3. 安装线虫成像
    1. 在2%琼脂糖垫上加入10μlM9-左旋咪唑缓冲液(20mM终浓度,见附注2)的液滴(见注3)。
    2. 收集具有眉毛/睫毛头发的转基因动物,并将其置于M9-左旋咪唑液滴中,固定转基因动物进行成像(见注4)。
    3. 轻轻地将盖玻片放在顶部的压机上。用Zeiss AxioImager Z2表面荧光显微镜或Zeiss AxioObserver Z1共焦显微镜对样品进行显微镜检查。

  4. 转基因线虫成像
    1. 使用连接到显微镜的相机捕获单个转基因动物或单体壁肌细胞。
    2. 获得(a)通过使用Zeiss AxioImager Z2表面荧光显微镜或执行(b)表达mtGFP的整个单体壁肌肉细胞与自身体标志物DsRed的(b)z堆叠方法,在体壁肌肉细胞中表达mtRosella的整个转基因线虫的荧光图像: LGG-1采用Zeiss AxioObserver Z1共焦显微镜。
    3. 应该记录成像参数,例如显微镜和相机设置(使用透镜和放大镜,曝光时间,分辨率,激光强度,增益等等),并在成像过程中保持相同。
    4. 从每种方法(a或b)保存收集的图像,并进行数据分析。

  5. 从成像过程分析数据
    1. 使用ImageJ软件从(a)方法获取的过程图像,以测量转基因蠕虫每个荧光图像的平均像素强度值和总面积。注意头部区域的体壁肌细胞,避免肠道自体荧光(图1和图2;见注5)。手动分析感兴趣区域:
      1. 通过'image'和'color'下拉菜单选择'split channel'命令来转换图像。
      2. 使用"手写选择"工具封闭荧光区域。
      3. 通过"分析"下拉菜单选择"测量"命令来执行像素强度分析。
      4. 通过使用Microsoft Office 2011 Excel软件包(Microsoft Corporation,Redmond,USA)将所选区域值除以规格化像素强度值。
      5. 在归一化时,计算GFP至DsRed比率。


        图1.氧化和线粒体应激的米氏感染。在体壁肌细胞中表达mtRosella的转基因线虫暴露于百草枯或CCCP。通过降低pH敏感性GFP与pH不敏感的DsRed(n = 100; *** <0.001 <单因素方差分析)之间的比率来表示远端诱导。箭头指出肠道自体荧光。尺寸棒表示20μm。使用10x物镜获得图像。误差条表示SEM值。


        图2.使用ImageJ软件进行图像分析 1.使用ImageJ软件打开获取的GFP信号图像; 2.通过'image'和'color'下拉菜单选择split channel'命令来转换图像;保持"绿色通道"的形象; 4.使用'freehabnd selection'工具包围荧光区域(头部区域); 5.通过"分析"下拉菜单选择"测量"命令进行像素强度分析; 6.使用获得的DsRed信号图像执行相同的分析途径。

    2. 使用Zeiss ZEN 2012软件从(b)方法收集的过程图像,以发现被自噬吞噬的线粒体,称为mitoautophagosomes。
      1. 通过手动计算线粒体(mtGFP)和体细胞标志物(DsRed :: LGG-1)之间的共定位事件来确定线粒体体细胞数量,显示在每个体壁肌细胞堆叠中(图3)。
      2. 使用Microsoft Office 2011 Excel软件包(Microsoft Corporation,Redmond,USA)分析数据。


        图3.在体内监测mitoautophagosomes形成。转基因线虫与体内壁肌肉细胞中的线粒体靶向GFP(mtGFP)一起共表达与自身吞噬蛋白LGG-1融合用DsRed,用百草枯或CCCP治疗。 Mitophagy刺激通过GFP和DsRed信号的共定位来表示(对于每组图像,线粒体在上面是绿色的,下面是红色的自噬体,底部的合并图像)。氧化和线粒体应激(n = 50; *** <0.001),单因素方差分析(mitoautophagosomes)的数量增加。尺寸棒表示20μm。使用40x物镜获得图像。误差条表示SEM值。 

数据分析

  1. 对于每个菌株或病症,使用至少100只动物或50只体壁肌细胞获得更准确的结果。
  2. 每个测定应重复至少三(3)次。
  3. 使用学生的 t 测试,具有 P 的重要性截止级别0.05用于两组之间的比较。
  4. 使用单因素(ANOVA)方差分析,并通过事后Bonferroni检验进行多次比较。

笔记

  1. 将选择的转基因动物每两天转移到新种植的NGM板上,以避免后代并防止由于缺乏食物而导致的饥饿。卡路里的剥夺和饥饿诱发线虫病。因此,应该使用充足的动物。
  2. 使用M9缓冲液而不是水确保线虫有利的渗透环境。
  3. 左旋咪唑是一种温和的麻醉剂,需要固定线虫。避免可能干扰代谢过程的麻醉剂,如叠氮化钠。叠氮钠通过抑制线粒体呼吸链和能量产生的扰动诱导线粒体和氧化应激。因此,叠氮化钠可能诱发丝裂霉菌
  4. 拿一个牙签,将眉毛/睫毛粘在头上。让它在室温下干燥。然后,使用此工具选择线虫。在使用眉毛/睫毛之前,请始终使用70%的EtOH对其进行消毒。
  5. 肠道自身荧光在C老化过程中增加。线虫。因此,在成像过程中应避免靠近肠道的体壁肌细胞

食谱

  1. 磷酸盐缓冲液(1M)
    1. 对于1L,在蒸馏水中溶解102.2g KH 2 PO 4和57.06g K 2 HPO 4,填充1 L.这是一个1M溶液,pH 6.0
    2. 高压灭菌并保持室温
  2. 线虫生长培养基(NGM)琼脂板
    1. 混合3g NaCl,2.5g细菌蛋白胨,0.2g链霉素,17g琼脂并加入900ml蒸馏水。高压灭菌器
    2. 让凉爽至55-60°C
    3. 加入1ml胆固醇储备溶液,1ml 1M CaCl 2,1ml 1M MgSO 4,1ml制霉菌素储备溶液,25ml无菌1M磷酸盐缓冲液,pH 6.0,蒸馏无菌水至1升
    4. 倾倒约8毫升培养基每培养皿,并离开巩固
    5. 将板保持在4°C直到使用
  3. M9缓冲区
    1. 将3g KH 2 PO 4,6g Na 2 HPO 4,5L NaCl的1L蒸馏水溶解水。高压灭菌器
    2. 冷却并加入1ml 1M MgSO 4(无菌)
    3. 在4°C存储M9缓冲液
  4. 左旋咪唑(0.5M)
    1. 将1.2 g左旋咪唑溶于10ml蒸馏水中
    2. 在4°C储存左旋咪唑溶液
  5. M9-左旋咪唑(20mM)
    1. 在10ml M9缓冲液中稀释400μl0.5M左旋咪唑
    2. 在4°C储存M9-左旋咪唑溶液
  6. 百草枯(0.5 M)
    1. 将1克百草枯溶于8毫升蒸馏水中
    2. 准备400毫升的等分试样,并将其储存在4°C
  7. 羰基氰基间氯苯腙(49mM; CCCP)
    1. 将100mg CCCP溶于10ml DMSO中
    2. 准备1ml的等分试样并将其储存在-20℃

致谢

这项工作由欧洲研究委员会(ERC),欧盟委员会7(支持)框架计划和博多基基基金会博士后研究奖学金资助。该协议已经从Palikaras等人改编。 (2015),Nature 521,525-528。

参考文献

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  2. Lopez-Otin,C.,Blasco,MA,Partridge,L.,Serrano,M.和Kroemer,G。(2013)。< a class ="ke-insertfile"href ="http://www.ncbi .nlm.nih.gov/pubmed/23746838"target ="_ blank">老化的特征。 细胞 153(6):1194-1217。
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  4. Palikaras,K.,Lionaki,E.和Tavernarakis,N。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25896323" target ="_ blank">在老化期间线粒体和线粒体生物发生的协调。电影。 自然 521(7553):525-528。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Palikaras, K. and Tavernarakis, N. (2017). In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis. Bio-protocol 7(7): e2215. DOI: 10.21769/BioProtoc.2215.
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