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Dec 2015

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Analysis of Mitochondrial Structure in the Body Wall Muscle of Caenorhabditis elegans
秀丽隐杆线虫体壁肌线粒体结构分析   

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Abstract

Mitochondrial function is altered in various pathologies, highlighting the crucial role mitochondria plays in maintaining cellular homeostasis. Mitochondrial structure undergoes constant fission and fusion in response to changing cellular environment. Due to this, analyzing mitochondrial structure could provide insight into the physiological state of the cell. In this protocol, we describe a method to analyze mitochondrial structure in body wall muscles in the nematode Caenorhabditis elegans, using both transgenic and dye-based approaches.

Keywords: C. elegans (秀丽隐杆线虫), Mitochondria (线粒体), Calcium (钙), Mitochondrial membrane potential (线粒体膜电位), TMRE (TMRE), Mitotracker (Mitotracker)

Background

itochondria are involved in ATP production, cellular respiration, calcium buffering and reactive oxidative species (ROS) metabolism (Brookes et al., 2004). Mitochondrial structure and function are dynamic and closely linked, therefore analyzing mitochondrial structure can provide clues to the status of mitochondrial health (Sarasija and Norman, 2015). We developed two sets of protocols to assess mitochondrial structure in the body wall muscle of Caenorhabditis elegans. In the first protocol, we used transgenic ccIs4251 strain in which GFP is targeted to the matrix of the body wall muscle mitochondria to visualize the mitochondria (Fire et al., 1998). In the second protocol, we used mitochondrially targeted dyes, MitoTrackerTM Red CMXRos and tetramethylrhodamine ethyl ester (TMRE) to determine the structural integrity of the body wall muscle mitochondria. Normally, animals used in in-vivo imaging are anesthetized, however anesthetizing the animals could lead to mitochondrial morphological changes (Han et al., 2012), complicating data analysis. Our protocols allow for the in-vivo imaging of mitochondrial structure in live, un-anaesthetized nematodes.

Materials and Reagents

  1. 100 mm, 60 mm Petri dishes (Kord-Valmark Labware Products, catalog numbers: 2900 , 2901 )
  2. Glass Pasteur pipettes (Krackeler Scientific, catalog number: 6-72050-900 )
  3. 15-ml centrifuge tubes (Globe Scientific, catalog number: 6285 )
  4. 22 x 22 mm coverslip (Globe Scientific, catalog number: 1404-10 )
  5. 1.5 ml Micro Centrifuge tube (CELLTREAT Scientific, catalog number: 229443 )
  6. 50 ml conical tubes (Corning, catalog number: 430829 )
  7. 15 ml conical tubes (Corning Centristar, catalog number: 430791 )
  8. C. elegans strains including strain SD1347, ccIs4251 [(pSAK2) myo-3p::GFP::LacZ::NLS + (pSAK4) myo-3p::mitochondrial GFP + dpy-20(+)] (Liu et al., 2009) and OP50 (Caenorhabditis Genetics Center (CGC), University of Minnesota)
  9. Deionized water (dH2O)
  10. Polybead polystyrene 0.10 μm microspheres (Polysciences, catalog number: 00876-15 )
  11. Agarose (RPI, catalog number: A20090-500.0 )
  12. Clear nail polish (generic)
  13. Carl ZeissTM ImmersolTM Immersion Oil (ZEISS, catalog number: 444960-0000-000 )
  14. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-10 )
  15. Agar (Fisher Scientific, catalog number: BP1423-2 )
  16. Bacto peptone (BD, BactoTM, catalog number: 211677 )
  17. Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: C79-500 )
  18. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Fisher Scientific, catalog number: BP213-1 )
  19. Cholesterol (Fisher Scientific, catalog number: C314-500 )
  20. Potassium phosphate dibasic (K2HPO4) (Fisher Scientific, catalog number: BP363-1 )
  21. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285-500 )
  22. Sodium phosphate dibasic anhydrous (Na2HPO4) (Fisher Scientific, catalog number: BP332-1 )
  23. Bleach (generic, plain)
  24. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-500 )
  25. Bacto tryptone (BD, BactoTM, catalog number: 211705 )
  26. Bacto yeast extract (BD, BactoTM, catalog number: 212750 )
  27. MitoTracker Red CMXRos (Thermo Fisher Scientific, InvitrogenTM, catalog number: M7512 )
  28. Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE) (Thermo Fisher Scientific, InvitrogenTM, catalog number: T669 )
  29. Standard worm (NGM) plates (see Recipes)
  30. Sterile solutions (see Recipes)
  31. Sterile stocks for NGM (see Recipes)
    1. 1 M CaCl2
    2. 1 M MgSO4
    3. 1 M K2HPO4
    4. 1 M KH2PO4
    5. 1 M KPO4 pH 6.0
  32. M9 buffer (1 L) (see Recipes)
  33. Bleach solution (see Recipes)
  34. 10 N NaOH (see Recipes)
  35. MitotrackerTM Red CMXRos stock (see Recipes)
  36. TMRE stock (see Recipes)

Equipment

  1. Single channel pipettes (Rainin, models: PR-10 , PR-20 , PR-200 , PR-1000 )
  2. Finnpipette II Multichannel pipettes (Fisher Scientific, model: FisherbrandTM FinnpipetteTM II, catalog number: 21377830 )
  3. 20 °C Incubator (Percival Scientific, model: I-41NL )
  4. Centrifuges (Eppendorf, models: 5415 D , 5415 R ; Thermo Fischer Scientific, Thermo ScientificTM, model: IEC Centra CL2 )
  5. Zeiss SteREO Discovery.V8 microscope with SCHOTT Ace® I light source for maintaining (ZEISS, model: SteREO Discovery.V8 )
  6. Zeiss SteREO Discovery.V12 microscope with SCHOTT Ace® I light source and X-Cite® Series 120 Fluorescence Illuminator for transgenic selection (ZEISS, model: SteREO Discovery.V12 )
  7. Zeiss AxioObserver microscope with Andor Clara CCD camera and X-Cite® Series 120 Fluorescence Illuminator for imaging (ZEISS, model: Axio Observer )
  8. PYREX® Griffin beakers (Corning, catalog number: 1000-PACK )
  9. PYREX® Reusable Media Storage Bottles (Fisher Scientific)

Software

  1. MetaMorph® Microscopy Automation & Image Analysis Software (Molecular Devices)
  2. ImageJ (https://imagej.nih.gov/ij/)
  3. Microsoft Office 2011 Excel (Microsoft Corporation, Redmond, USA)
  4. GraphPad Prism software package (GraphPad Software Inc., San Diego, USA)

Part I: Determining mitochondrial structure using transgenic lines

Procedure

  1. Growth and synchronization of nematode population
    1. Transfer ccIs4251 L4 transgenic larvae, which express mitochondria-targeted GFP (mtGFP), onto a fresh E. coli (OP50) seeded NGM plate. Use at least three 60 mm or two 100 mm plates for each strain.
    2. Incubate the animals at 20 °C for 3-4 days until plates contain a large number of eggs and gravid nematodes.
    3. Wash the eggs and nematodes off the plates and transfer them using glass Pasteur pipettes into individual 15-ml centrifuge tubes for each strain using about 4 ml of M9 per plate of nematodes. Spin these down for 3 min at 6,180 x g and aspirate out the M9, retaining just the worms and eggs pellet. Add 3-4 ml of the bleach solution to each tube and vortex at full speed for 15 sec every 2 min for 6 min. Add M9 to fill each tube and spin at 6,180 x g for 1 min. Repeat the wash with M9 three times and move the worm eggs/carcass pellet to a fresh half-filled 15 ml tube of M9 .
    4. Synchronize the hatchlings by nutating at 20 °C for anywhere between 16-48 h (make sure that within an experiment all strains are synchronized for similar amounts of time). Due to lack of food, all the animals will arrest in their growth at L1 stage of larval growth. Spin these tubes down at 6,180 x g for 1.5 min and transfer them to individual OP50 seeded NGM plates and keep at 20 °C. Once the L1 animals are on OP50 seeded NGM plates, these animals will reach the L4 larval stage at ~42 h, and day 1 of adulthood at ~66 h.
      Note: We have observed no difference in phenotypes between animals age synchronized by starving for 24 h vs. 48 h.

  2. Mounting animals for imaging (Figure 1A)
    1. Make 3% agarose solution in M9 and use this solution to make a ~18 mm diameter agarose pad on the top of a glass slide.
    2. Add a 2 μl drop of polystyrene beads solution to this pad and pick approximately 20-30 animals onto this drop.
    3. Gently place a coverslip on top of the animals and use a fingernail to gently nudge the coverslip about 0.5 mm to ‘roll’ the nematodes onto their back. Rolling the animals will allow them to be visualized dorsally or ventrally where a larger area of body wall mitochondria can be imaged versus when the animals are positioned laterally.
    4. Now seal the edges of the coverslip with clear nail polish and the slide is ready for imaging on the Zeiss AxioObserver microscope equipped with an Andor Clara CCD camera using Metamorph software. 


      Figure 1. Cartoon depicting placement of nematodes for (A) Part I and (B) Part II

  3. Imaging the mounted animals
    1. Locate nematodes on the slide using the 10x objective and then switch over to the 63x oil objective to capture images.
    2. Capture fluorescent images of body wall muscle mitochondria in the transgenic animals at 3 different points along their length.
      Note: Ignore the vulval area in all animals, as the vulva is prone to deformation of body wall muscle mitochondria.
    3. Imaging parameters such as the settings of the microscope and camera (magnification and exposure time (240 msec) should be kept the same during the imaging process).
    4. Save these images and use them for data analysis under blinded conditions.

Data analysis

  1. Have someone other than the assayer/analyzer scramble the image file names and folders to obtain blinded conditions.
  2. Open each image in Metamorph (ImageJ is a viable alternative) and characterize the mitochondria as linear, intermediate or fragmented, depending on the structural arrangement in > 50% of mitochondria (see Figure 2 for representative images).
  3. Calculate the percentage mitochondria that exist as linear, intermediate and fragmented and analyze in chi-square tests using GraphPad Prism.
    Notes:
    1. Please refer to Sarasija and Norman, 2015 for representative data.
    2. Analyze 15+ animals per strain for mitochondrial structure.
    3. Mitochondrial morphological changes can happen due to various factors such as aging, drug treatments and genetic backgrounds. 


      Figure 2. Representative images of the three major mitochondrial phenotypic classifications in C. elegans body wall muscle mitochondria. Scale bar represents 10 µm.

Part II: Determining mitochondrial structural and functional integrity using mitochondrial targeted fluorescent dyes

Procedure

  1. Growth and synchronization of nematode population
    Animals with mitoGFP, (ccIs4251) are grown and synchronized to L4 larval (for MitotrackerTM) or young adult (for TMRE) stage (~42 and ~54 h respectively, after putting down synchronized L1s on seeded NGM plates) for imaging as described in Part I Procedure A.

  2. Staining the animals with mitochondrially targeted dyes
    1. Wash animals off the plates along with OP50 into 15 ml centrifuge tubes.
    2. Add MitotrackerTM or TMRE to a final concentration of 1 μg/ml or 200 nM in M9, respectively.
    3. Nutate these animals in 1 μg/ml of MitotrackerTM or 200 nM of TMRE for 6 h at 20 °C in the dark.
    4. Wash these animals with M9 four times and put them down on OP50 seeded NGM plates using the protocol described above.
    5. Destain MitotrackerTM and TMRE by allowing the stained animals to forage on seeded plates overnight or for 1 h in the dark at 20 °C, respectively.
      Note: Stain and destain animals on a staggering timescale so that each strain experiences similar staining and destaining times.

  3. Mounting the animals for imaging (Figure 1B)
    1. Make 3% agarose solution in M9 and use it to make ~18 mm diameter agarose pad.
    2. Add five drops of polystyrene beads solution (~0.5 μl each drop); one at the center and 4 diametrically opposite to each other (Figure 1B).
    3. Add one worm to each of the drops for 5 nematodes/slide. This will limit the impact of photobleaching.
    4. Gently place a coverslip on top the animals and use a fingernail to gently nudge the coverslip about 0.5 mm to ‘roll’ the nematodes onto their back.
    5. Now seal the edges of the coverslip with clear nail polish and the slide is ready for imaging on the Zeiss AxioObserver microscope equipped with an Andor Clara CCD camera using Metamorph software. 

  4. Imaging the mounted animals
    1. Locate nematodes on the slide using the 10x objective and then switch over to the 63x oil objective to capture images.
    2. Capture the green fluorescent image of body wall muscle mitochondria in the transgenic animals at a point between the pharynx and vulva. Now switch over to the red fluorescence filter set and capture the dye staining in the same area. Each animal should have two images associated with it; the mitochondria as labeled by the transgenic mitochondrially targeted GFP and the mitochondria as labeled by the mitochondrially targeted dyes.
    3. Move clockwise and go to the next worm on the slide, repeating imaging protocol.
    4. Imaging parameters such as the settings of the microscope and camera (magnification and exposure time (240 msec) should be kept the same during the imaging process).
    5. Save these images and use them for data analysis under blinded conditions. 

Data analysis

  1. Assayer/analyst should be blinded as explained in Part I Procedure D.
  2. Open the images of animals stained with MitotrackerTM CMXRos in MetaMorph and TMRE in ImageJ.
  3. For the MitotrackerTM labeled animals, use a binary system and denote each image of the mitochondria as stained or unstained with the dye (see Figure 3 for representative images).


    Figure 3. Representative images of C. elegans body wall muscle mitochondria that are stained or unstained by MitoTrackerTM. Scale bar represents 10 µm.

  4. For the TMRE labeling, using the freehand tool in ImageJ, demarcate the boundaries of the mitochondria using the transgenic mitochondrial GFP image and measure the fluorescence intensity of the GFP signal. Now use the same region of interest in the TMRE labeled image and determine the fluorescence intensity of the TMRE dye labeling. Make sure to subtract the respective background fluorescence from both images. Now determine TMRE to GFP fluorescence intensity for each animal and normalize this to the average fluorescence intensity ratio of wild type animals (see Figure 4 for representative images).
  5. Analyze the distribution of MitotrackerTM stained vs. unstained animals using a Chi-square test and the TMRE to GFP fluorescence intensity ratios of each strain using one-way ANOVA with Tukey or Bonferroni post-test. Please refer to the paper (Sarasija and Norman, 2015) for any clarifications.


    Figure 4. Representative images of C. elegans body wall muscle mitochondria that are TMRE-labeled. Scale bar represents 10 µm.

Notes

  1. For further information on C. elegans growth and maintenance, please refer to the chapter on ‘Maintenance of C. elegans’ on WormBook available at http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.pdf.
  2. Visualize these nematodes every day until they are imaged to make sure that they are not at the risk of starving. If the bacterial lawn on the plates look thin, nematodes need to be promptly moved to new OP50 seeded NGM plates. Starved animals should never be used for assaying.
  3. The ccIs4251 strain expresses GFP that is targeted to the mitochondrial matrix and nucleus, which accounts for the presence of a large spherical structure in each muscle cell.
  4. These protocols can be adapted to visualize the mitochondria in other tissues, like in the neurons. For example, jsIs609, a GFP transgene that is expressed in the mitochondrial matrix of mechanosensory neurons of C. elegans, can be used for this purpose (Fatouros et al., 2012).
  5. This protocol can be adapted to image animals at various stages. However, if mitochondrial dyes are to be used, then the animals will need to be incubated in the dyes accordingly, at the right time point. Also, it should be noted that the polystyrene beads will not immobilize animals at L1-L2 larval stages efficiently. If older animals need to be imaged, their eggs could pose an issue which could potentially be circumvented by focusing on imaging the region where eggs will not be present. Another option is to sterilize the animals using by moving them to 5-Fluoro-2’-deoxyuridine (FUDR) containing NGM plates. The latter solution might present problems since there is some concern in the field regarding the impact of FUDR on mitochondrial structure and function.
  6. Please refer to Sarasija and Norman (2015) for representative data for dye staining analysis.

Recipes

Note: For additional information, please refer to He, 2011.

  1. Standard Nematode Growth Media (NGM) plates 

  2. Sterile solutions
    1. Autoclave on liquid cycle (45 min exposure), allow to cool to ~60 °C and then use sterile technique and add the following:

    2. Swirl to mix thoroughly after each addition. After all additions are made, pour plates
    3. To make liquid NGM, follow the same protocol but omit the addition of Bacto-agar (and obviously no need to pour into plates)
  3. Sterile stocks for NGM
    1. 1 M CaCl2
      In a beaker (sterile if possible), add:
      110.9 g CaCl2
      dH2O to 250 ml
      Dissolve thoroughly and use vacuum filtration to sterilize, label, and store at room temperature
    2. 1 M MgSO4
      In a beaker (sterile if possible), add:
      120.3 g MgSO4
      dH2O to 250 ml
      Dissolve thoroughly and use vacuum filtration to sterilize, label, and store at room temperature
    3. 1 M K2HPO4
      174.18 g bring up to 1 L with dH2O
    4. 1 M KH2PO4
      136.01 g bring up to 1 L with dH2O and heat
    5. 1 M KPO4 pH 6.0
      Measure out 132 ml of 1 M K2HPO4 and 868 ml of 1 M KH2PO4
      Combine, and autoclave on liquid cycle (45 min exposure)
      Store at room temperature
  4. M9 buffer (1 L) (store at room temperature)

    Split between 2 bottles: 500 ml each. Autoclave on liquid cycle (15 min exposure)
  5. Bleach solution (store at room temperature)

  6. 10 N NaOH
    20 g NaOH
    Bring up to 50 ml with dH2O
    Store at room temperature
    Notes:
    1. Exercise CAUTION: This reaction is EXOTHERMIC!
    2. 50 ml conical should be fine, but make sure lid is tight, keep ice around to cool tube down to prevent conical breakage (expansion by heat).
  7. MitotrackerTM Red CMXRos stock (make fresh/store at -20 °C for a week)
    1. Add 1 ml of dimethylsulfoxide (DMSO) to a tube containing 50 µg of MitotrackerTM Red CMXRos to make a 50 µg/ml stock of MitotrackerTM Red CMXRos
    2. Dilute this 50 fold in M9 for staining animals
  8. TMRE stock (make fresh/store at -20 °C for a week)
    1. Add 970.9 µl of DMSO to a tube containing 25 mg of TMRE to make a 50 mM stock solution
    2. Dilute this stock 250 fold in M9 to get the desired concentration of 200 nM

Acknowledgments

The authors would like to acknowledge J. T. Laboy for her help in ordering and preparing reagents, and P. McKeown-Longo, Y. Tang, M. Barroso and their lab members for support. Some nematode strains were provided by the Caenorhabditis Genetics Center, which is funded by National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40 OD010440). The Alzheimer’s Association (NIRG-09-132122) and NIH (GM088213) supported this work. The authors declare that there are no conflicts of interest or competing interests.

References

  1. Brookes, P. S., Yoon, Y., Robotham, J. L., Anders, M. W. and Sheu, S. S. (2004). Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol 287(4): C817-833.
  2. Fatouros, C., Pir, G. J., Biernat, J., Koushika, S. P., Mandelkow, E., Mandelkow, E. M., Schmidt, E. and Baumeister, R. (2012). Inhibition of tau aggregation in a novel Caenorhabditis elegans model of tauopathy mitigates proteotoxicity. Hum Mol Genet 21(16): 3587-3603.
  3. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669): 806-811.
  4. Han, S. M., Tsuda, H., Yang, Y., Vibbert, J., Cottee, P., Lee, S. J., Winek, J., Haueter, C., Bellen, H. J. and Miller, M. A. (2012). Secreted VAPB/ALS8 major sperm protein domains modulate mitochondrial localization and morphology via growth cone guidance receptors. Dev Cell 22(2): 348-362.
  5. He, F. (2011). Common worm media and buffers. Bio-protocol Bio101: e55.
  6. Liu, X., Long, F., Peng, H., Aerni, S. J., Jiang, M., Sanchez-Blanco, A., Murray, J. I., Preston, E., Mericle, B., Batzoglou, S., Myers, E. W. and Kim, S. K. (2009). Analysis of cell fate from single-cell gene expression profiles in C. elegans. Cell 139(3): 623-633.
  7. Sarasija, S. and Norman, K. R. (2015). A γ-secretase independent role for presenilin in calcium homeostasis impacts mitochondrial function and morphology in Caenorhabditis elegans. Genetics 201(4): 1453-1466.
  8. Stiernagle, T. (2006). Maintenance of C. elegans. WormBook (Ed.). The C. elegans Research Community: doi/10.1895/wormbook.1.101.1.

简介

线粒体功能在各种病理学中被改变,突出了线粒体在维持细胞稳态方面发挥的关键作用。 线粒体结构响应不断变化的细胞环境而经历不断的分裂和融合。 因此,分析线粒体结构可以提供对细胞生理状态的了解。 在这个协议中,我们描述了一种使用转基因和基于染料的方法来分析线虫秀丽隐杆线虫体壁细胞线粒体结构的方法。

【背景】线粒体参与ATP产生,细胞呼吸,钙缓冲和反应性氧化物(ROS)代谢(Brookes等人,2004)。线粒体结构和功能是动态和紧密联系的,因此分析线粒体结构可以为线粒体健康状况提供线索(Sarasija and Norman,2015)。我们制定了两套方案来评估秀丽隐杆线虫体壁细胞的线粒体结构。在第一个方案中,我们使用了转基因的ccIs4251株,其中GFP靶向体壁细胞线粒体的基质以显现线粒体(Fire等人,1998) 。在第二种方案中,我们使用线粒体靶向染料,MitoTracker TM Red CMXRos和四甲基罗丹明乙酯(TMRE)来测定体壁肌肉线粒体的结构完整性。通常,用于体内成像的动物是麻醉的,然而麻醉动物可能导致线粒体形态改变(Han等人,2012),使数据分析变得复杂。我们的协议允许活体非麻醉线虫体内线粒体结构的体内成像。

关键字:秀丽隐杆线虫, 线粒体, 钙, 线粒体膜电位, TMRE, Mitotracker

材料和试剂

  1. 100mm,60mm培养皿(Kord-Valmark Labware Products,目录号:2900,2901)
  2. 玻璃巴斯德吸液管(Krackeler Scientific,目录号:6-72050-900)

  3. 15-ml离心管(Globe Scientific,目录号:6285)

  4. 22 x 22毫米盖玻片(Globe Scientific,目录号:1404-10)
  5. 1.5 ml微量离心管(CELLTREAT Scientific,目录号:229443)

  6. 50毫升锥形管(Corning,目录号:430829)

  7. 15毫升锥形管(Corning Centristar,产品目录号:430791)
  8. ℃。线虫体系,包括菌株SD1347,ccIs4251 [em(pSAK2)myo-3p :: GFP :: LacZ :: NLS +(pSAK4)myo-3p ::线粒体GFP + dpy -20(+ )](Liu等人,2009)和OP50(Caenorhabditis 遗传中心(CGC),University明尼苏达州)
  9. 去离子水(dH 2 O)

  10. Polybead聚苯乙烯0.10μm微球(Polysciences,目录号:00876-15)
  11. 琼脂糖(RPI,目录号:A20090-500.0)
  12. 清除指甲油(通用)
  13. Carl Zeiss TM Immersol TM浸没油(ZEISS,目录号:444960-0000-000)
  14. 氯化钠(NaCl)(Fisher Scientific,目录号:BP358-10)
  15. 琼脂(Fisher Scientific,目录号:BP1423-2)
  16. 细菌蛋白胨(BD,Bacto TM,产品目录号:211677)
  17. 氯化钙二水合物(CaCl 2·2H 2 O)(Fisher Scientific,目录号:C79-500)
  18. 硫酸镁七水合物(MgSO 4·7H 2 O)(Fisher Scientific,目录号:BP213-1)
  19. 胆固醇(Fisher Scientific,目录号:C314-500)
  20. 磷酸二氢钾(KH 2 HPO 4)(Fisher Scientific,目录号:BP363-1)
  21. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:P285-500)
  22. 磷酸二氢钠无水(Na 2 HPO 4)(Fisher Scientific,目录号:BP332-1)
  23. 漂白(普通,普通)
  24. 氢氧化钠(NaOH)(Fisher Scientific,目录号:BP359-500)
  25. 细菌用胰蛋白胨(BD,BactoTM,目录号:211705)
  26. 细菌酵母提取物(BD,Bacto TM,目录号:212750)
  27. MitoTracker Red CMXRos(Thermo Fisher Scientific,Invitrogen TM,目录号:M7512)
  28. 四甲基罗丹明,乙酯,高氯酸盐(TMRE)(Thermo Fisher Scientific,Invitrogen TM,目录号:T669)
  29. 标准蠕虫(NGM)盘子(见食谱)
  30. 无菌解决方案(请参阅食谱)
  31. NGM的无菌库存(见食谱)
    1. 1 M CaCl 2 2
    2. 1M MgSO 4
    3. 1 M K 2 HPO 4
    4. 1 M KH 2 PO 4 4
    5. 1 M KPO 4 4 pH 6.0
  32. M9缓冲液(1 L)(见食谱)
  33. 漂白剂解决方案(请参阅食谱)
  34. 10 N NaOH(见食谱)
  35. Mitotracker TM Red CMXRos股票(见食谱)
  36. TMRE股票(见食谱)

设备

  1. 单通道移液器(Rainin,型号:PR-10,PR-20,PR-200,PR-1000)
  2. Finnpipette II多道移液器(Fisher Scientific,型号:Fisherbrand TM TM Finnpipette TM II,目录号:21377830)
  3. 20°C培养箱(Percival Scientific,型号:I-41NL)
  4. 离心机(Eppendorf,型号:5415 D,5415 R; Thermo Fischer Scientific,Thermo Scientific TM,型号:IEC Centra CL2)
  5. Zeiss SteREO Discovery.V8显微镜和SCHOTT Ace ®I光源用于维护(ZEISS,型号:SteREO Discovery.V8)
  6. Zeiss SteREO Discovery.V12显微镜,带有SCHOTT Ace I / II光源和X-Cite 120系列用于转基因选择的荧光照明器(ZEISS,型号:SteREO Discovery.V12) br />
  7. Zeiss AxioObserver显微镜配备Andor Clara CCD相机和X-Cite 120系列荧光成像灯(ZEISS,型号:Axio Observer)
  8. PYREX®格里芬烧杯(Corning,目录号:1000-PACK)
  9. PYREX ®可重复使用的介质储存瓶(Fisher Scientific)

软件

  1. MetaMorph ®显微镜自动化&图像分析软件(分子设备)
  2. ImageJ( https://imagej.nih.gov/ij/ )
  3. Microsoft Office 2011 Excel(Microsoft Corporation,Redmond,USA)
  4. GraphPad Prism软件包(GraphPad Software Inc.,圣地亚哥,美国)

第一部分:使用转基因品系确定线粒体结构

程序

  1. 线虫种群的增长和同步
    1. 将表达线粒体靶向GFP(mtGFP)的ccIs4251L4转基因幼虫转移到新鲜的E.coli上。 (OP50)接种NGM板。
      每个菌种至少使用三个60 mm或两个100 mm培养皿
    2. 在20°C孵育动物3-4天,直到板含有大量的卵和妊娠线虫。
    3. 将平板上的鸡蛋和线虫清洗掉,并使用玻璃巴斯德吸管将它们转移到每个菌株的单个15-ml离心管中,每个线虫平板使用约4ml M9。在6,180 em g下旋转3分钟,吸出M9,保留蠕虫和卵粒。向每个管中加入3-4ml漂白剂溶液并全速涡旋15秒,每2分钟6分钟。加入M9填充每个试管并以6,180×g <1 / min的速度旋转1分钟。用M9重复洗涤三次,并将蠕虫卵/屠体颗粒移至新鲜的半充满的15ml M9试管中。
    4. 通过在20°C下在16-48h之间的任何地方进行章动来同步孵出的幼体(确保在实验内所有菌株在同样的时间内同步)。由于缺乏食物,所有动物都会在幼虫生长的L1阶段停止生长。将这些管以6,180×g 速度旋转1.5分钟,并将它们转移到单个OP50接种的NGM板并保持在20℃。一旦L1动物接种OP50接种的NGM板,这些动物将在约42小时达到L4幼虫阶段,并在约66小时达到成年的第1天。
      注意:我们观察到动物之间的表型没有差异,因为饥饿24小时和48小时之间的时间同步。

  2. 装载用于成像的动物(图1A)
    1. 在M9中制备3%琼脂糖溶液,并使用该溶液在载玻片的顶部制备〜18mm直径的琼脂糖垫。
    2. 添加2微升聚苯乙烯珠溶液到这个垫,并选择约20-30动物到这个下降。
    3. 轻轻地将盖玻片放在动物的顶部,并用指甲轻轻地将盖玻片轻轻推动约0.5mm,以将线虫“卷”到它们的背部。滚动动物可以使它们在背侧或腹侧可视化,其中可以成像较大面积的体壁线粒体而不是侧向放置动物。
    4. 现在使用明确的指甲油密封盖玻片的边缘,并使用Metamorph软件将载玻片准备好在配备安道尔克拉拉CCD相机的Zeiss AxioObserver显微镜上成像。&nbsp;


      (A)部分I和(B)部分II

  3. 成像安装的动物
    1. 使用10倍物镜在幻灯片上找到线虫,然后切换到63x油目标以捕捉图像。

    2. 在转基因动物的3个不同点上捕捉体壁肌肉线粒体的荧光图像。
      注意:忽略所有动物的外阴区域,因为外阴很容易发生体壁肌肉线粒体的变形。
    3. 成像参数,例如显微镜和相机的设置(放大倍率和曝光时间(240毫秒))应该在成像过程中保持不变。
    4. 保存这些图像,并将其用于盲法条件下的数据分析。

数据分析


  1. 。让测试人员/分析人员以外的其他人加扰图像文件名称和文件夹以获得不知情的情况。
  2. 在Metamorph中打开每个图像(ImageJ是一个可行的选择),并将线粒体表征为线性,中间或片段化,这取决于&gt;中的结构排列。线粒体的50%(见图2代表图像)。
  3. 使用GraphPad Prism计算线性,中间和片段存在的线粒体百分比,并在卡方检验中进行分析。
    注意:
    1. 请参阅2015年Sarasija和Norman的代表性数据。
    2. 每个菌株分析15个以上的动物线粒体结构。
    3. 线粒体形态学变化可能由于诸如衰老,药物治疗和遗传背景等多种因素而发生。&nbsp;


      图2.线粒体三种表型分类的代表性图像。线虫体壁肌肉线粒体。 比例尺表示10μm。

第二部分:使用线粒体靶向荧光染料测定线粒体结构和功能完整性

程序

  1. 线虫种群的增长和同步
    使用mitoGFP( ccIs4251 )的动物与L4幼虫(用于Mitotracker TM)或年轻成人(用于TMRE)阶段(分别约42和约54小时)如同在第一部分程序A中所描述的,在放下的NGM板上放置同步的L1之后)进行成像。

  2. 用线粒体靶向染料染色动物

    1. 将动物与OP50一起清洗到15 ml离心管中。
    2. 在M9中分别加入Mitotracker TM TM或TMRE至终浓度为1μg/ ml或200nM。
    3. 将这些动物在20°C在黑暗中以1μg/ ml的Mitotracker TM或200nM的TMRE处理6小时。
    4. 使用M9将这些动物洗四次,并使用上述方案将它们放在OP50种植的NGM板上。
    5. Destain Mitotracker TM TM和TMRE,通过允许染色的动物在接种的平板上分别过夜或在20℃在黑暗中1小时进行研磨。
      注意:在惊人的时间尺度上染色和脱色动物,以便每种菌株都经历类似的染色和脱色时间。

  3. 装配动物进行成像(图1B)
    1. 在M9中制备3%琼脂糖溶液,并用它制作直径约18mm的琼脂糖垫。
    2. 加入5滴聚苯乙烯珠溶液(每滴约0.5μl);一个在中心,四个在直径方向上彼此相对(图1B)。
    3. 为每个5个线虫/幻灯片添加一个蠕虫。这将限制光漂白的影响。
    4. 轻轻地将盖玻片放在动物的顶部,并用指甲轻轻地将盖玻片轻推约0.5mm,以将线虫“滚动”到它们的背部。
    5. 现在使用明确的指甲油密封盖玻片的边缘,并使用Metamorph软件将载玻片准备好在配备安道尔克拉拉CCD相机的Zeiss AxioObserver显微镜上成像。&nbsp;

  4. 成像安装的动物
    1. 使用10倍物镜在幻灯片上找到线虫,然后切换到63x油目标以捕捉图像。
    2. 在咽部和外阴之间的某个点捕获转基因动物体壁肌肉线粒体的绿色荧光图像。现在切换到红色荧光过滤器组并捕获同一区域的染料染色。每只动物应该有两张相关的图像;由转基因线粒体靶向的GFP标记的线粒体和由线粒体靶向的染料标记的线粒体。

    3. 顺时针移动并转到幻灯片上的下一个蠕虫,重复成像协议。
    4. 成像参数,例如显微镜和相机的设置(放大倍率和曝光时间(240毫秒))应该在成像过程中保持不变。
    5. 保存这些图像并将其用于盲法条件下的数据分析。&nbsp;

数据分析

  1. 如第一部分程序D所述,分析者/分析员应该被蒙蔽。
  2. 打开MetaMorph中的Mitotracker TM CMXRos和ImageJ中的TMRE染色的动物图像。
  3. 对于Mitotracker TM标记的动物,使用二元系统,并用染料将线粒体的每个图像表示为染色或未染色(代表性图像见图3)。


    图3. C的代表性图像。线虫体壁细胞线粒体被MitoTracker™TM染色或未染色。比例尺表示10μm。

  4. 对于TMRE标记,使用ImageJ中的徒手工具,使用转基因线粒体GFP图像划定线粒体的边界并测量GFP信号的荧光强度。现在使用TMRE标记图像中相同的感兴趣区域并确定TMRE染料标记的荧光强度。确保从两幅图像中减去相应的背景荧光。现在确定每只动物的TMRE至GFP荧光强度并将其归一化为野生型动物的平均荧光强度比(参见图4代表性图像)。
  5. 使用Chi-square检验分析Mitotracker TM染色与未染色动物的分布以及使用具有Tukey或Bonferroni事后检验的单向ANOVA的每种菌株的TMRE至GFP荧光强度比。请参阅该论文( 2015年Sarasija and Norman )澄清。


    图4.代表性图像线虫线虫体内壁肌肉线粒体被TMRE标记。 比例尺表示10μm。

笔记

  1. 有关 C的更多信息。线虫生长和维护,请参阅“维护 C”一章。线虫'可在 http://www.wormbook.org/chapters上找到/www_strainmaintain/strainmaintain.pdf 。
  2. 每天观察这些线虫,直到它们成像,以确保它们没有挨饿的危险。如果平板上的细菌草坪看起来很薄,线虫需要立即移动到新的OP50种子NGM板上。
    不应该使用饥饿的动物进行化验。
  3. ccIs4251 菌株表达针对线粒体基质和细胞核的GFP,这说明了每个肌细胞中存在大球形结构。
  4. 这些协议可以适应可视化其他组织线粒体,如在神经元。例如, jsIs609 ,一种GFP转基因,表达于机械感受神经元线粒体基质中。 elegans 可用于此目的(Fatouros et al。,2012)。
  5. 该协议可以适应各种阶段的动物图像。但是,如果要使用线粒体染料,则需要在正确的时间点将染料与染料一起温育。此外,应该指出的是,聚苯乙烯珠粒不会有效地将动物固定在L1-L2幼虫阶段。如果需要对较老的动物进行成像,它们的卵可能会成为一个问题,可能会通过将成像重点放在鸡蛋不会出现的区域进行成像。另一种选择是通过将它们移动到含有NGM板的5-氟-2'-脱氧尿苷(FUDR)来使动物消毒。后一种解决方案可能存在问题,因为在该领域中有一些关于FUDR对线粒体结构和功能的影响的问题。
  6. 请参阅Sarasija和Norman(2015)了解染料染色分析的代表性数据。

食谱

注意:有关其他信息,请参阅 2011,

  1. 标准线虫生长培养基(NGM)培养板&nbsp;

  2. 无菌解决方案
    1. 液体循环高压灭菌(暴露45分钟),使其冷却至〜60°C,然后使用无菌技术并添加以下物质:

    2. 每次添加后都要充分搅拌。所有添加完成后,倒入平板
    3. 要制造液态NGM,请遵循相同的协议,但省略添加细菌琼脂(并且显然不需要倒入板)
  3. NGM的无菌库存
    1. 1 M CaCl 2 2
      在烧杯中(如果可能,无菌),请添加:
      110.9克氯化钙2
      dH 2 O至250毫升

      彻底溶解并使用真空过滤进行消毒,贴标签和室温保存
    2. 1M MgSO 4
      在烧杯中(如果可能,无菌),请添加:
      120.3克MgSO 4。
      dH 2 O至250毫升

      彻底溶解并使用真空过滤进行消毒,贴标签和室温保存
    3. 1 M K 2 HPO 4
      174.18克用dH <2:O>调至1升
    4. 1 M KH 2 PO 4 4
      使用dH 2 O和加热至136.01 g使其升至1 L
    5. 1 M KPO 4 4 pH 6.0
      测量132ml的1M K 2 HPO 4和868ml的1M KH 2 PO 4 4 />
      在液体循环(暴露45分钟)下结合并高压灭菌 在室温下储存
  4. M9缓冲液(1 L)(在室温下储存)

    分装2瓶:每瓶500毫升。液体循环高压灭菌(暴露15分钟)
  5. 漂白剂溶液(在室温下储存)

  6. 10 N NaOH
    20克NaOH
    用dH 2 O O将其置于50ml中 在室温下储存
    备注:
    1. 练习小心:这个反应是EXOTHERMIC!
    2. 50毫升锥形应该很好,但要确保盖子是紧的,保持周围冰块以冷却管以防止锥形破裂(通过加热膨胀)。
  7. Mitotracker TM Red CMXRos库存(在-20°C下保存一周)
    1. 向含有50μgMitotracker TM Red CMXRos的管中加入1ml二甲基亚砜(DMSO)以制备50μg/ ml的Mitotracker TM Red CMXRos储存液。
    2. 在M9中稀释这个50倍,用于染色动物
  8. TMRE库存(在-20°C下保鲜一周)
    1. 加入970.9μlDMSO到含有25 mg TMRE的试管中,制成50 mM储备液。
    2. 在M9中稀释此库存250倍,以获得200 nM所需的浓度

致谢

作者要感谢J. T. Laboy在订购和制备试剂方面的帮助,以及P. McKeown-Longo,Y. Tang,M. Barroso和他们的实验室成员的支持。一些线虫菌株由美国国立卫生研究院(NIH)研究基础设施项目办公室(P40 OD010440)资助的Caenorhabditis遗传中心提供。阿尔茨海默病协会(NIRG-09-132122)和美国国立卫生研究院(GM088213)支持这项工作。作者声明不存在利益冲突或利益冲突。

参考

  1. Brookes,P. S.,Yoon,Y.,Robotham,J.L.,Anders,M.W。和Sheu,S.S。(2004)。 钙,ATP和ROS:一种线粒体爱恨三角形 Am J Physiol Cell Physiol 287(4):C817-833。
  2. Fatouros,C.,Pir,G.J.,Biernat,J.,Koushika,S.P.,Mandelkow,E.,Mandelkow,E.M。,Schmidt,E。和Baumeister,R。(2012)。 抑制tau蛋白聚集新型线虫模型的tau蛋白减少蛋白毒性。 Hum Mol Genet 21(16):3587-3603。
  3. Fire,A.,Xu,S.,Montgomery,M.K。,Kostas,S.A.,Driver,S.E。和Mello,C.C。(1998)。 秀丽隐杆线虫中双链RNA的有效和特异性遗传干扰。 Nature 391(6669):806-811。
  4. Han,S.M.,Tsuda,H.,Yang,Y.,Vibbert,J.,Cottee,P.,Lee,S.J.,Winek,J.,Haueter,C.,Bellen,H.J.and Miller,M.A。(2012)。 分泌型VAPB / ALS8主要精子蛋白结构域通过生长锥诱导受体调节线粒体定位和形态学。 a> Dev Cell 22(2):348-362。
  5. 他,楼(2011年)。 常见的蠕虫介质和缓冲区 Bio-protocol Bio101:e55。< br />
  6. Liu,X.,Long,F.,Peng,H.,Aerni,SJ,Jiang,M.,Sanchez-Blanco,A.,Murray,JI,Preston,E.,Mericle,B.,Batzoglou,S., Myers,EW和Kim,SK(2009)。 分析单细胞基因表达谱在 C中的细胞命运。 elegans 。 Cell 139(3):623-633。
  7. Sarasija,S.和Norman,K. R.(2015)。 早老素的γ分泌酶独立作用在钙稳态中影响线粒体功能和形态Caenorhabditis elegans 。 Genetics 201(4):1453-1466。
  8. Stiernagle,T.(2006)。 维护 C。线虫。 WormBook (Ed。)。 C。线虫研究社区:doi / 10.1895 / wormbook.1.101.1。
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引用:Sarasija, S. and NORMAN, K. R. (2018). Analysis of Mitochondrial Structure in the Body Wall Muscle of Caenorhabditis elegans. Bio-protocol 8(7): e2801. DOI: 10.21769/BioProtoc.2801.
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