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Measurement of Lysosomal Size and Lysosomal Marker Intensities in Adult Caenorhabditis elegans
成年秀丽隐杆线虫中溶酶体大小和溶酶体标志物信号强度测定   

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Genetics
Feb 2016

Abstract

Assays have been developed to study trafficking in various tissues of Caenorhabditis elegans. Adult C. elegans intestinal cells are large and have extensive endocytic networks, thus making them a good system for deciphering the endocytic pathway using live imaging techniques. However, the presence of auto-fluorescent gut granules in adult intestine can interfere with the signals of endocytic compartment reporters, like GFP. Here we demonstrate a protocol adapted from the original method developed by the Grant laboratory to identify signals from reporters in adult intestinal cells. The goal of this protocol is to identify endocytic compartments tagged with fluorescent markers without any confounding effects of background autofluorescent gut granules in adult intestinal cells of Caenorhabditis elegans.

Keywords: C. elegans (秀丽隐杆线虫), Intestine (小肠), Endocytosis (胞吞作用), Gut granule (肠道颗粒), Confocal microscopy (共聚焦显微镜技术), Lysosome (溶酶体)

Background

Caenorhabditis elegans is a multicellular organism that has been used to study endocytic trafficking. Originally, assays were developed to study endocytosis in C. elegans oocytes, embryos, and coelomocytes (scavenger cells). Briefly, the assays in oocytes and embryos were performed by measuring the intensities and sizes of compartments containing a yolk protein-green fluorescent protein reporter (VIT-2::GFP) in intestinal compartments at the comma to ‘1.5 fold’ stages of development (Grant and Hirsh, 1999; Schaheen et al., 2006a). In adults, the intensities and sizes of compartments containing GFP (secreted from body wall muscle cells into the psuedocoelom and endocytosed by coelomocytes) were measured in the coelomocytes of transgenic adult C. elegans expressing Pmyo-3::ssGFP (signal sequence-GFP fusion protein) (Treusch et al., 2004). These assays have been used to identify and to elucidate functions of mediators of the endocytic pathway (Fares and Greenwald, 2001a and 2001b; Schaheen et al., 2006b; Huynh et al., 2016).

Here, we present an assay that can be used to study endocytosis in another C. elegans tissue. Adult intestinal cells of C. elegans are large and are thus also a great system for deciphering the endocytic pathway using live imaging techniques. The functions of intestinal cells include food assimilation and synthesis, storage of macromolecules, stress response, and host-pathogen interactions (McGhee, 2007). However, one of the main challenges of studying endocytic transport by live imaging in adult intestinal cells is the prevalence of auto-fluorescent gut granules that interfere with the unambiguous determination of bona fide endocytic compartment reporter (like GFP) signals and therefore bias qualitative and quantitative studies (Clokey and Jacobson, 1986). We therefore adapted a method developed by the Grant laboratory to conclusively identify signals from reporters in adult intestinal cells (Gleason et al., 2016; Huynh et al., 2016).

Materials and Reagents

  1. Microscope slides (VWR, catalog number: 48382-171 )
  2. Coverslips for microscope slides (Fisher Scientific, Fisherbrand, catalog number: 12-541A )
  3. 60 mm plates (Fisher Scientific, Fisherbrand, catalog number: FB0875713A )
  4. 100 mm plates (Fisher Scientific, Fisherbrand, catalog number: FB0875713 )
  5. Labeling tape (Fisher Scientific, Fisherbrand, catalog number: 15-901-5K )
  6. Glass pipette
  7. Aluminum foil
  8. Autoclave tape
  9. Inoculating loops
  10. Pipette tips
  11. C. elegans experimental strain:
    RT258: unc-119(ed3); pwIs50[lmp-1::GFP, unc-119]
    Note: LMP-1 is the orthologue of mammalian Lamp1 that localizes to lysosomes in Caenorhabditis elegans (Kostich et al., 2000)
  12. C. elegans control strain: N2
  13. Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: C79-500 )
  14. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Fisher Scientific, catalog number: M63-500 )
  15. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number P285-500 )
  16. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
  17. EtOH (Merck, catalog number: EX0276-4 )
  18. LE agarose for making 2.2% agarose (BioExpress, GeneMate, catalog number: E-3120 )
  19. Levamisole (Sigma-Aldrich, catalog number: 31742 )
  20. C. elegans culture
    1. Platinum wire-pick to transfer C. elegans
    2. Making NGM plates (Brenner, 1974; He, 2011) (see Recipes)
      Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3 )
      Bacto peptone (BD, BactoTM, catalog number: 211677 )
      Bacto agar (BD, BactoTM, catalog number: 214030 )
      Double distilled water
      Cholesterol 5 mg/ml in 95% EtOH (see Recipes)
      1 M CaCl2 sterile (see Recipes)
      1 M MgSO4 sterile (see Recipes)
      1 M KH2PO4 pH 6.0 sterile (see Recipes)
    3. Making 2x YT + OP50 for spotting NGM plates
      OP50 frozen stock
      2x YT agar plate
    4. 2x YT agar plate (see Recipes)
      Bacto tryptone (BD, BactoTM, catalog number: 211705 )
      Yeast extract (Fisher Scientific, catalog number: BP1422-500 )
      Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3 )
      Bacto agar (BD, catalog number: 214030 )
      Double distilled water
    5. 2x YT medium (see Recipes)
    6. 2x YT medium + OP50 (see Recipes)
  21. 2.2% agarose pad (see Recipes)
  22. 9 mM levamisole/1x PBS (see Recipes)

Equipment

  1. 20 °C Incubator for C. elegans storage (VWR, manufactured by Sheldon Manufacturing, model: Model 2020 )
  2. 4 L flask
  3. Stir bar
  4. Stir plate
  5. Autoclave
  6. 2 L beaker
  7. 1 L bottle
  8. 37 °C Incubator (VWR, manufactured by Sheldon Manufacturing, model: 5025 T )
  9. Microwave
  10. Heating block
  11. 5-100 ml bottle
  12. Microscope (Carl Zeiss, model: STEMI SV 6 )
  13. Zeiss LSM 510 Meta confocal microscope (Zeiss, model: LSM 510 ):
    1. 63x lens
    2. Argon 488-nm laser
    3. Helium neon 543-nm laser

Software

  1. MetaMorph® Microscopy Automation & Image Analysis Software (Sunnyvale, CA)

Procedure

Note: Before starting the protocol: Ensure that 60 mm NGM plates (spotted with OP50) have been prepared.
Day 1

  1. Set up three plates with three adult hermaphrodites (one day post-L4 stage) on each spotted 60 mm NGM plate for each C. elegans experimental and control strain.
  2. Leave plates in a 20 °C incubator for three days for the hermaphrodites to lay eggs.

Day 4
Move 50-100 L4 stage C. elegans of each experimental strain onto new OP50-spotted 60 mm NGM plates (the offspring of the P0 hermaphrodites). This ensures that these animals would be fairly synchronized young adults for imaging on the following day.

Day 5: Preparation for microscopy

  1. Prepare the imaging slide by first making the agarose pad. See steps 1-4 in Figure 1 for instructions.
  2. Next, add 7 μl of 9 mM levamisole/1x PBS (see Recipes) to the center of the agarose pad (Figure 1, S5).
  3. Pick 20-30 adult worms of one C. elegans experimental strain and submerge them in the 9 mM levamisole/1x PBS (Figure 1, S5).
  4. Put a cover slip on top of the agarose pad, ensuring that it fully covers the C. elegans and the 9 mM levamisole/1x PBS (Figure 1, S6).
  5. Repeat Steps 1-5 for all experimental and control strains used.
  6. Do microscopy to image LMP-1::GFP (or marker of interest) and focus on the intestine right below the pharynx, using the 63x objective. Take images that show the desired signal while avoiding saturation of the signal.
  7. Image C. elegans wild type strain RT258 first with both the Argon 488-nm laser and the Helium neon 543-nm laser. RT258 should be imaged first because it is the wild type strain which measurements from other experimental strains should be compared against.
  8. Take images of the intestines of approximately 5-10 adult C. elegans for the wild type RT258 strain.
  9. Repeat Steps 7 and 8 for each control and experimental strain. Make sure that images of all strains are taken using the same exposure and magnification as RT258.


    Figure 1. Preparation of imaging slides

Data analysis

  1. MetaMorph version 7.5.0.0 was used to analyze confocal microscopy images, though other software could also be used.
  2. All confocal microscopy images were converted to ‘.tif’ format for use with MetaMorph.
  3. Identify the compartments that ONLY have green fluorescence (excited by the 488-nm laser). These compartments are LMP-1::GFP-positive that marks lysosomes. Compartments that are yellow (fluoresce with both 488-nm and 543-nm lasers) are gut granules, which will also be seen in C. elegans control strain N2: these gut granule compartments should not be measured or included for determining sizes of bona fide LMP-1::GFP compartments (Figure 2). We do not know whether the LMP-1::GFP signal decreases in older adults; if it does, this protocol may not be effective at differentiating bona fide GFP signal from autofluorescence.


    Figure 2. Representative images of intestinal images. Compartments that fluoresce with both 543-nm and 488-nm excitation are granules while compartments that fluoresce with only 488-nm excitation are LMP-1::GFP-positive compartments. Arrows indicate examples of LMP-1::GFP-positive compartments. Arrowheads indicate examples of gut granules. Yellow circles indicate clumped compartments. Scale bar = 10 μm.

  4. For each LMP-1::GFP positive compartment, use MetaMorph to measure the intensity and area of the selected compartment. See Figure 3 for a pictorial guide (Figure 3).
    1. Go to Measure in the tool bar and select ‘Show Region Statistics…’
    2. Use the trace region tool to select the LMP-1::GFP positive compartment.
    3. Double click on the selected region.
    4. To get measurements of LMP-1::GFP-positive compartments’ intensities, use the ‘Average’ Gray Level value. This value is already normalized by the area size.
    5. To get measurements of LMP-1::GFP-positive compartment size, use the ‘Area’ value.


      Figure 3. Using MetaMorph to measure LMP-1::GFP-positive compartment size and marker intensity

    6. Collect measurements of all the LMP-1::GFP-positive compartments for each adult C. elegans intestine, for each strain, from each image. Do not include clumped compartments because it is difficult to get accurate quantitations (due to ambiguity in the location of each individual compartment). The result is approximately 10-20 clearly defined compartments per image.
    7. Average all the LMP-1::GFP-positive intensity measurements using Excel to obtain the average LMP-1::GFP-positive intensity for each strain. Average all of the LMP-1::GFP positive size measurements to obtain the average compartment size for each strain. Figure 4 depicts results obtained when comparing the wild type strain with a cup-5(null) strain (CUP-5 is a cation channel; loss of CUP-5 protein results in lysosomal dysfunction) (Fares and Greenwald, 2001b; Hersh et al., 2002). Loss of CUP-5 resulted in an increase in LMP-1::GFP-positive intensity, but no significant change in lysosomal size.

Note: We have successfully used this protocol on strains carrying various mutations or following RNAi treatment.




Figure 4. Representative images of LMP-1::GFP intensity and compartment size data obtained. * indicates P < 0.05.

Notes

  1. The higher the ‘Average’ Gray Level, the larger the lysosomal compartment size.
  2. If imaging multiple strains with the same marker, take all images using identical confocal settings, preferably during the same microscopy session. This would allow comparisons of compartment sizes and intensities between strains/genotypes. Additionally, though not always technically possible, when feasible, strains to be compared should be imaged on the same slide to reduce slide-to-slide variation during microscopy.
  3. Repeat this experiment multiple times to determine reproducibility and statistical significance.
  4. This method can be used to analyze markers for other organelles than lysosomes in adult intestinal cells, distinguishing marker signals from gut granule autofluorescence.

Recipes

  1. Nematode Growth Medium (NGM) agar (3 L)
    1. Add the following to a 4-L flask:
      NaCl (9.0 g)
      Bacto peptone (7.5 g)
      Bacto agar (51 g)
      Double distilled water (2,925 ml)
    2. Stir bar
    3. Autoclave for 1 h
    4. Place flask on stir plate, set on low stir, and let cool to 55 °C
    5. Once cool, add the following:
      CaCl2 1 M sterile (3 ml)
      MgSO4 1 M sterile (3 ml)
      KH2PO4 1 M pH 6.0 sterile (75 ml)
      Cholesterol 5 mg/ml in 95% EtOH (3 ml)
    6. Pour 8 ml into each 60 mm plate
    7. Let dry for one night before storing unspotted plates at 4 °C or spotting with 2x YT + OP50
  2. 2x YT agar (1 L)
    1. Add the following into a 2-L (or larger) flask:
      NaCl (5.0 g)
      Bacto tryptone (16 g)
      Yeast extract (10 g)
      Bacto agar (15.0 g)
      Double distilled water (Up to 1 L)
    2. Stir bar
    3. Autoclave for 1 h
    4. Place flask on stir plate, set on low stir, and let cool 55 °C
    5. Pour 18 ml into each 100 mm plate
    6. Wait two days for plates to dry and then store at 4 °C
  3. 2x YT medium (1 L)
    1. Add the following to a 2-L beaker
      Tryptone (16 g)
      Yeast extract (10 g)
      NaCl (5 g)
      Fill to 1 L with double distilled water
    2. Add a stir bar into the beaker and let the mixture stir on a stir plate until homogenous
    3. Pour into 1 L bottles
    4. Autoclave for 30 min
    5. Store at room temperature
  4. 2x YT medium + OP50 (1 L)
    1. Day 1: Use a pipette tip to spread OP50 frozen stock across a 2x YT agar plate. Place plate in a 37 °C incubator and leave it overnight
    2. Day 2: Use an inoculating loop to pick one colony from the 2x YT agar plate and transplant the colony to a 1 L bottle of 2x YT Medium. Place the 1 L bottle into the 37 °C incubator and leave it overnight
    3. Day 3: Remove bottle from the 37 °C incubator and store at 4 °C until use
  5. 2.2% agarose pad
    1. Add the following to a flask:
      Agarose (0.22 g)
      Double distilled water (10 ml)
    2. Microwave flask for 60 sec. If the agarose and double distilled water solution is not homogenous, microwave for another 30 sec
    3. Store in a tube that is in a heating block (68 °C)
  6. 9 mM levamisole/1x PBS
    1. Mix the following in a tube:
      9 ml of 10 mM levamisole (Sigma-Aldrich)
      1 ml of 10x PBS
    2. Store at room temperature
  7. Cholesterol (5 mg/ml)
    1. Add the following to a 500 ml beaker
      Cholesterol (2.5 g)
      100% EtOH (475 ml)
      Double distilled water (25 ml)
    2. Add a stir bar in the beaker and let the mixture stir on a stir plate until homogenous
    3. Pour 100 ml into five 100 ml bottle (please confirm this description)
  8. 1 M CaCl2
    1. In a beaker, dissolve CaCl2 (73.5 g) into 500 ml double distilled water
    2. Add a Stir bar into the beaker and let the mixture stir on a stir plate until homogenous
    3. Pour into a 500 ml bottle
    4. Autoclave for 30 min
  9. 1 M MgSO4
    1. In a beaker, dissolve MgSO4 (123.25 g) into 500 ml double distilled water
    2. Add a Stir bar into the beaker and let the mixture stir on a stir plate until homogenous
    3. Pour into a 500 ml bottle
    4. Autoclave for 30 min
  10. 1 M KH2PO4 pH 6.0
    1. In a beaker, dissolve KH2PO4 (68.05 g) into 450 ml with double distilled water
    2. Add a Stir bar into the beaker and let the mixture stir on a stir plate until homogenous
    3. Adjust pH to 6.0 with NaOH
    4. Fill up to 500 ml with double distilled water
    5. Pour into a 500 ml bottle
    6. Autoclave for 30 min

Acknowledgments

This protocol was adapted from Huynh et al. (2016). This work was supported by a Microscopy Society of America grant (to J.M.H) and by National Science Foundation grant 3004290 (to H.F.). The authors have no conflicts of interest or competing interests.

References

  1. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77(1): 71-94.
  2. Clokey, G. V. and Jacobson, L. A. (1986). The autofluorescent “lipofuscin granules” in the intestinal cells of Caenorhabditis elegans are secondary lysosomes. Mech Ageing Dev 35(1): 79-94.
  3. Fares, H. and Greenwald, I. (2001a). Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants. Genetics 159(1): 133-145.
  4. Fares, H. and Greenwald, I. (2001b). Regulation of endocytosis by CUP-5, the Caenorhabditis elegans mucolipin-1 homolog. Nat Genet 28(1): 64-68.
  5. Gleason, A. M., Nguyen, K. C., Hall, D. H. and Grant, B. D. (2016). Syndapin/SDPN-1 is required for endocytic recycling and endosomal actin association in the C. elegans intestine. Mol Biol Cell.
  6. Grant, B. and Hirsh, D. (1999). Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte. Mol Biol Cell 10(12): 4311-4326.
  7. He, F. (2011). Common worm media and buffers. Bio-protocol Bio101: e55.
  8. Hersh, B. M., Hartwieg, E. and Horvitz, H. R. (2002). The Caenorhabditis elegans mucolipin-like gene cup-5 is essential for viability and regulates lysosomes in multiple cell types. Proc Natl Acad Sci U S A 99(7): 4355-4360.
  9. Huynh, J. M., Dang, H., Munoz-Tucker, I. A., O'Ketch, M., Liu, I. T., Perno, S., Bhuyan, N., Crain, A., Borbon, I. and Fares, H. (2016). ESCRT-dependent cell death in a Caenorhabditis elegans model of the lysosomal storage disorder mucolipidosis type IV. Genetics 202(2): 619-638.
  10. Kostich, M., Fire, A. and Fambrough, D. M. (2000). Identification and molecular-genetic characterization of a LAMP/CD68-like protein from Caenorhabditis elegans. J Cell Sci 113 ( Pt 14): 2595-2606.
  11. McGhee, J. D. (2007). The C. elegans intestine. WormBook: 1-36.
  12. Schaheen, L., Dang, H. and Fares, H. (2006a). Basis of lethality in C. elegans lacking CUP-5, the Mucolipidosis Type IV orthologue. Dev Biol 293(2): 382-391.
  13. Schaheen, L., Patton, G. and Fares, H. (2006b). Suppression of the cup-5 mucolipidosis type IV-related lysosomal dysfunction by the inactivation of an ABC transporter in C. elegans. Development 133(19): 3939-3948.
  14. Treusch, S., Knuth, S., Slaugenhaupt, S. A., Goldin, E., Grant, B. D. and Fares, H. (2004). Caenorhabditis elegans functional orthologue of human protein h-mucolipin-1 is required for lysosome biogenesis. Proc Natl Acad Sci U S A 101(13): 4483-4488.

简介

已经开发了用于研究线虫的各种组织中的贩运的测定法。 成人 C。 线虫肠细胞很大,具有广泛的内吞网络,因此使它们成为使用实时成像技术破译内吞途径的良好系统。 然而,成年肠道中存在自身荧光的肠道颗粒会干扰吞噬细胞记者的信号,如GFP。 在这里,我们展示了一个协议,从格兰特实验室开发的原始方法改编,以确定记者在成人肠细胞的信号。 该方案的目标是识别标记有荧光标记物的内吞室,而在秀丽隐杆线虫成虫肠细胞中没有背景自发荧光消化道颗粒的混杂效应。

【背景】秀丽隐杆线虫(Caenorhabditis elegans)是一种多细胞生物,已被用于研究内吞运输。最初开发了用于研究内吞的试验。线虫卵母细胞,胚胎和体细胞(清道夫细胞)。简而言之,卵母细胞和胚胎中的测定通过在逗号至1.5倍发育阶段测量肠隔室中含有蛋黄 - 蛋白 - 绿色荧光蛋白报道分子(VIT-2 :: GFP)的区室的强度和大小来进行Grant和Hirsh,1999; Schaheen等人,2006a)。在成年人中,在转基因成虫的体细胞中测量了含有GFP的隔室的强度和大小(从体壁肌细胞分泌进入囊泡并由体细胞胞吞)。表达肌肉myo-3 :: ssGFP(信号序列-GFP融合蛋白)的秀丽隐杆线虫(Treusch等人,2004)。这些测定已被用于鉴定和阐明内吞途径的介质的功能(Fares和Greenwald,2001a和2001b; Schaheen等人,2006b; Huynh等人,,2016)。

在这里,我们提出一个可用于研究在另一个C内吞作用的测定。 elegans 组织。成年人的肠道细胞elegans 很大,因此也是使用实时成像技术破译内吞途径的一个很好的系统。肠细胞的功能包括食物同化和合成,大分子储存,应激反应和宿主 - 病原体相互作用(McGhee,2007)。然而,在成人肠细胞中通过活体显像研究内吞转运的主要挑战之一是自发荧光的肠道颗粒的发生,其干扰了真正的内吞室报道分子(如GFP)的明确测定,信号,因此偏向定性和定量研究(Clokey和Jacobson,1986)。因此,我们采用Grant实验室开发的方法来确定成人肠细胞中记者的信号(Gleason等,2016; Huynh等人,2016)。

关键字:秀丽隐杆线虫, 小肠, 胞吞作用, 肠道颗粒, 共聚焦显微镜技术, 溶酶体

材料和试剂

  1. 显微镜幻灯片(VWR,目录号:48382-171)
  2. 显微镜幻灯片的盖玻片(Fisher Scientific,Fisherbrand,目录号:12-541A)
  3. 60毫米板(Fisher Scientific,Fisherbrand,目录号:FB0875713A)
  4. 100毫米板(Fisher Scientific,Fisherbrand,目录号:FB0875713)
  5. 标签胶带(Fisher Scientific,Fisherbrand,目录号:15-901-5K)
  6. 玻璃移液器
  7. 铝箔
  8. 高压灭菌器胶带
  9. 接种循环
  10. 移液器提示
  11. ℃。线虫实验菌株:
    RT258: unc-119(ed3); pwIs50 [lmp-1 :: GFP,unc-119]
    注:LMP-1是哺乳动物Lamp1的直系同源物,定位于秀丽隐杆线虫中的溶酶体(Kostich et al。,2000)。
  12. ℃。线虫控制菌株:N2
  13. 氯化钙二水合物(CaCl 2•2H 2 O)(Fisher Scientific,目录号:C79-500)
  14. 七水合硫酸镁(MgSO 4•7H 2 O)(Fisher Scientific,目录号:M63-500)
  15. 磷酸二氢钾(KH2PO4)(Fisher Scientific,目录号P285-500)
  16. 胆固醇(Sigma-Aldrich,目录号:C8667)
  17. EtOH(Merck,目录号:EX0276-4)
  18. LE琼脂糖用于制备2.2%琼脂糖(BioExpress,GeneMate,目录号:E-3120)
  19. 左旋咪唑(Sigma-Aldrich,目录号:31742)
  20. ℃。线虫文化
    1. 白金线采摘转移线虫
    2. 制作NGM板材(Brenner,1974; He,2011)(见食谱)
      氯化钠(NaCl)(Fisher Scientific,目录号:S271-3)
      细菌蛋白胨(BD,Bacto TM,目录号:211677)
      细菌琼脂(BD,Bacto TM,目录号:214030)
      双蒸水
      胆固醇5毫克/毫升在95%乙醇(见食谱)
      1 M CaCl 2无菌(见食谱)
      1M MgSO 4无菌(参见食谱)
      1 M KH 2 PO 4 pH6.0无菌(见食谱)
    3. 制作2个YT + OP50来检测NGM板
      OP50冷冻库存
      2x YT琼脂平板
    4. 2x YT琼脂平板(见食谱)
      细菌用胰蛋白胨(BD,Bacto TM,目录号:211705)
      酵母提取物(Fisher Scientific,目录号:BP1422-500)
      氯化钠(NaCl)(Fisher Scientific,目录号:S271-3)
      细菌琼脂(BD,目录号:214030)
      双蒸水
    5. 2x YT中等(见食谱)
    6. 2x YT中等+ OP50(见食谱)
  21. 2.2%琼脂糖垫(见食谱)
  22. 9 mM左旋咪唑/ 1x PBS(见食谱)

设备

  1. 20°C孵化器 C。 elegans存储(VWR,由Sheldon Manufacturing制造,型号:2020型)
  2. 4升烧瓶
  3. 搅拌棒
  4. 搅拌盘
  5. 高压灭菌器
  6. 2升烧杯
  7. 1升瓶
  8. 37℃培养箱(VWR,由Sheldon Manufacturing制造,型号:5025T)
  9. 微波炉
  10. 加热块
  11. 5-100毫升瓶
  12. 显微镜(卡尔蔡司,型号:STEMI SV 6)
  13. 蔡司LSM 510元共聚焦显微镜(Zeiss,型号:LSM 510):
    1. 63倍镜头
    2. 氩488纳米激光
    3. 氦氖543纳米激光

软件

  1. MetaMorph ®显微镜自动化&amp;图像分析软件(Sunnyvale,CA)

程序

注意:在开始协议之前:确保准备好60毫米的NGM板(点缀OP50)。
第1天

  1. 在每个有斑点的60mm NGM板上,每个板上用三个成年雌雄同体(L4后一天)设置三个板。线虫实验和控制应变。
  2. 将盘子置于20°C培养箱中三天,使雌雄同体产卵。

第4天
移动50-100 L4阶段 C。每个实验菌株的线虫到新的OP50斑点的60mm NGM板(P0雌雄同体的后代)上。这样可以确保这些动物在第二天成为相当同步的年轻成年人进行成像。

第5天:准备显微镜

  1. 准备成像幻灯片首先制作琼脂糖垫。请参阅图1中的步骤1-4以获得说明。
  2. 接下来,加入7微升9毫米左旋咪唑/ 1x PBS(见食谱)到琼脂糖垫的中心(图1,S5)。
  3. 挑一个C的20-30成虫。线虫实验性菌株并将其浸入9mM左旋咪唑/ 1x PBS中(图1,S5)。
  4. 将盖玻片放在琼脂糖垫上,确保它完全覆盖线虫和9mM左旋咪唑/ 1x PBS(图1,S6)。
  5. 对所有使用的实验和对照菌株重复步骤1-5。
  6. 做显微镜成像LMP-1 :: GFP(或感兴趣的标记),并使用63x物镜重点在咽下面的肠。拍摄显示所需信号的图像,同时避免信号饱和。
  7. 图像线虫野生型RT258首先与氩488 nm激光和氦氖543 nm激光。应首先对RT258进行成像,因为它是野生型菌株,应与其他实验菌株的测量结果进行比较。
  8. 为野生型RT258菌株拍摄大约5-10只成虫线虫的肠的图像。
  9. 对每个对照和实验菌株重复步骤7和8。确保使用与RT258相同的曝光和放大率拍摄所有菌株的图像。


    图1.成像载玻片的制备

数据分析


  1. 使用MetaMorph 7.5.0.0版分析共焦显微图像,但也可以使用其他软件
  2. 所有的共聚焦显微镜图像被转换为'.tif'格式,用于MetaMorph。
  3. 识别仅具有绿色荧光的隔室(由488nm激光激发)。这些区域是标记溶酶体的LMP-1 :: GFP阳性。黄色的隔室(用488nm和543nm激光发出的荧光)是肠道颗粒,这也将在中看到。线虫对照菌株N2:不应测量或包括这些肠道颗粒区以确定LMP-1 :: GFP区室的大小(图2)。我们不知道LMP-1 :: GFP信号在老年人中是否下降;如果是这样,这个协议可能无法有效区分真正的GFP信号和自发荧光。


    图2.肠道图像的代表性图像。用543nm和488nm激发荧光的隔室是颗粒,而仅用488nm激发荧光的隔室是LMP-1 :: GFP-隔室。箭头表示LMP-1 :: GFP阳性隔室的例子。箭头表示肠道颗粒的例子。黄色圆圈表示成群的隔间。比例尺= 10微米。

  4. 对于每个LMP-1 :: GFP阳性区室,使用MetaMorph来测量所选区室的强度和面积。参见图3的图片指南(图3)。
    1. 转到工具栏中的“测量”,然后选择“显示区域统计...”
    2. 使用跟踪区域工具选择LMP-1 :: GFP阳性室。
    3. 双击所选区域。
    4. 要获得LMP-1 :: GFP阳性隔室强度的测量值,请使用“平均”灰度值。
    5. 要获得LMP-1 :: GFP阳性室大小的测量值,请使用“面积”值。


      图3.使用MetaMorph测量LMP-1 :: GFP阳性区室大小和标记强度

    6. 从每个图像收集每个成年线虫的所有LMP-1 :: GFP阳性区室的测量结果。不包括成团的隔室,因为难以获得准确的定量(由于每个隔室的位置不明确)。结果是每个图像大约有10-20个明确的隔间。
    7. 使用Excel平均所有LMP-1 :: GFP阳性强度测量结果以获得每种菌株的平均LMP-1 :: GFP阳性强度。平均所有的LMP-1 :: GFP正面大小测量结果以获得每个菌株的平均隔室大小。图4描述了比较野生型菌株与杯-5(空)株(CUP-5是阳离子通道; CUP-5蛋白质的损失导致溶酶体功能障碍)时获得的结果(Fares和Greenwald,2001b; Hersh等人,2002)。 CUP-5的丢失导致LMP-1 :: GFP阳性强度增加,但是溶酶体大小没有显着变化。

注意:我们已经成功地在携带各种突变或RNAi治疗后的菌株中使用这个方案。


图4.获得的LMP-1 :: GFP强度和区室大小数据的代表性图像 *表示

笔记

  1. “平均”灰度级越高,溶酶体室大小越大。
  2. 如果使用相同的标记对多个菌株进行成像,则使用相同的共焦设置拍摄所有图像,最好是在相同的显微镜检查过程中。这将允许比较菌株/基因型之间的隔室大小和强度。此外,虽然在技术上并不总是可行的,但是在可行的情况下,要比较的菌株应该在同一张载玻片上成像,以减少显微镜下的载玻片变化。

  3. 重复这个实验多次,以确定重复性和统计显着性
  4. 该方法可用于分析成人肠细胞中溶酶体以外的其他细胞器的标记物,区分来自消化道颗粒自发荧光的标记信号。

食谱

  1. 线虫生长培养基(NGM)琼脂(3L)
    1. 将以下内容添加到4-L烧瓶中:
      NaCl(9.0克)
      细菌蛋白胨(7.5克)
      细菌琼脂(51克)
      双蒸水(2,925毫升)
    2. 搅拌棒
    3. 高压灭菌1小时
    4. 将烧瓶置于搅拌盘上,低速搅拌,放冷至55°C
    5. 一旦很酷,请添加以下内容:
      CaCl 2 2M无菌(3ml)
      无菌硫酸镁(3ml)
      KH 2 PO 4 4M pH 6.0无菌(75ml)
      胆固醇5毫克/毫升在95%乙醇(3毫升)
    6. 每个60毫米的平板倒入8毫升
    7. 在干燥的状态下放置4°C,或者用2x YT + OP50
      点样
  2. 2x YT琼脂(1 L)
    1. 将以下内容添加到2升(或更大)的烧瓶中:
      NaCl(5.0克)
      细菌用胰蛋白胨(16克)
      酵母提取物(10克)
      细菌琼脂(15.0克)
      双蒸馏水(最多1升)
    2. 搅拌棒
    3. 高压灭菌1小时
    4. 将烧瓶置于搅拌盘上,置于低速搅拌下,放冷至55°C
    5. 每个100毫米的平板倒入18毫升
    6. 等待两天,盘子干燥,然后在4°C储存
  3. 2x YT培养基(1 L)
    1. 添加以下2升烧杯
      胰蛋白胨(16克)
      酵母提取物(10克)
      NaCl(5克)
      用双蒸水填充至1L
    2. 添加一个搅拌棒到烧杯中,让搅拌板上的混合物搅拌,直到均匀
    3. 倒入1升的瓶子
    4. 高压灭菌30分钟
    5. 在室温下储存
  4. 2x YT培养基+ OP50(1 L)
    1. 第1天:使用移液枪头将OP50冷冻的原料分散在2x YT琼脂平板上。将培养板置于37℃的培养箱中,放置一夜。
    2. 第2天:使用接种环从2x YT琼脂平板挑取一个菌落,并将菌落移植到1L 2X YT培养基的瓶中。将1L瓶放入37°C的培养箱中,放置一夜。
    3. 第3天:将瓶子从37°C的培养箱中取出,并在4°C保存,直到使用
  5. 2.2%琼脂糖垫
    1. 将以下内容添加到烧瓶中:
      琼脂糖(0.22克)
      双蒸水(10毫升)
    2. 微波烧瓶60秒。如果琼脂糖和双蒸水溶液不均匀,再微波30秒
    3. 储存在加热块(68°C)的管中
  6. 9mM左旋咪唑/ 1x PBS
    1. 将以下内容混合在一个管中:
      9毫升10mM左旋咪唑(Sigma-Aldrich)
      1毫升的10倍PBS
    2. 在室温下储存
  7. 胆固醇(5毫克/毫升)
    1. 添加以下500毫升烧杯
      胆固醇(2.5克)
      100%EtOH(475毫升)
      双蒸水(25毫升)
    2. 在烧杯中加入搅拌棒,搅拌均匀,直至均匀
    3. 将100毫升倒入5个100毫升瓶中(请确认此说明)
  8. 1 M CaCl 2 2
    1. 在烧杯中,将CaCl 2(73.5g)溶于500ml双蒸水中。
    2. 添加一个搅拌棒到烧杯中,让搅拌板上的混合物搅拌,直到均匀
    3. 倒入一个500毫升的瓶子
    4. 高压灭菌30分钟
  9. 1M MgSO 4
    1. 在烧杯中,将MgSO 4(123.25g)溶解在500ml双蒸水中。
    2. 添加一个搅拌棒到烧杯中,让搅拌板上的混合物搅拌,直到均匀
    3. 倒入一个500毫升的瓶子
    4. 高压灭菌30分钟
  10. 1M KH 2 PO 4 PO 4 6.0
    1. 在烧杯中,用双蒸水将KH2PO4(68.05g)溶解在450ml中。
    2. 添加一个搅拌棒到烧杯中,让搅拌板上的混合物搅拌,直到均匀
    3. 用NaOH调节pH至6.0
    4. 用双蒸水加注至500毫升
    5. 倒入一个500毫升的瓶子
    6. 高压灭菌30分钟

致谢

该协议是由Huynh等人改编的(2016)。这项工作得到了美国显微镜学会(J.M.H)和国家科学基金会(National Science Foundation)授予的3004290(致H.F.)的支持。作者没有利益冲突或利益冲突。

参考

  1. Brenner,S。(1974)。 秀丽隐杆线虫(Caenorhabditis elegans)的遗传学。 遗传学 77(1):71-94。
  2. Clokey,G.V。和Jacobson,L.A。(1986)。 秀丽隐杆线虫肠道细胞中的自发荧光“脂褐素颗粒”是次级溶酶体。老年痴呆症 35(1):79-94。
  3. Fares,H.和Greenwald,I.(2001a)。 秀丽隐杆线虫(Caenorhabditis elegans)胞吞作用的遗传分析:体细胞摄取缺陷型突变体<遗传学159(1):133-145。
  4. Fares,H.和Greenwald,I.(2001b)。 通过CUP-5调节内吞作用,秀丽隐杆线虫(Caenorhabditis elegans)粘球蛋白-1 homolog。 Nat Genet 28(1):64-68。
  5. Gleason,A.M.,Nguyen,K.C.,Hall,D.H。和Grant,B.D。(2016)。 Syndapin / SDPN-1是内吞循环和内体肌动蛋白结合所必需的。 elegans 肠。 Mol Biol Ce ll。
  6. Grant,B。和Hirsh,D。(1999)。 秀丽隐杆线虫卵母细胞中受体介导的内吞作用。分子生物细胞 10(12):4311-4326。
  7. 他,楼(2011年)。 常见的蠕虫媒体和缓冲区。 Bio-protocol Bio101:e55。
  8. Hersh,B.M.,Hartwieg,E。和Horvitz,H.R。(2002)。 秀丽隐杆线虫(Caenorhabditis elegans)粘球蛋白样基因cup-5对生存能力至关重要并在多种细胞类型中调节溶酶体。


    美国国家科学院院刊 99(7):4355-4360。

  9. Huynh,JM,Dang,H.,Munoz-Tucker,IA,O'Ketch,M.,Liu,IT,Perno,S.,Bhuyan,N.,Crain,A.,Borbon,I.和Fares,H. (2016)。 秀丽隐杆线虫的ESCRT依赖性细胞死亡溶酶体贮积模型IV型粘液脂肪病。 202(2):619-638。
  10. Kostich,M.,Fire,A.和Fambrough,D.M.(2000)。 秀丽隐杆线虫(Caenorhabditis elegans)的LAMP / CD68样蛋白的鉴定和分子遗传鉴定。 J Cell Sci 113(Pt 14):2595-2606。
  11. McGhee,J.D。(2007)。 C。 elegans 肠。 WormBook :1-36。
  12. Schaheen,L.,Dang,H.和Fares,H。(2006a)。 在 C的致命基础。线虫缺乏CUP-5,IV型粘蛋白病直系同源基因。“Dev Biol”293(2):382-391。
  13. Schaheen,L.,Patton,G。和Fares,H。(2006b)。 通过灭活ABC转运蛋白抑制杯状粘蛋白病IV型溶酶体功能障碍℃。 elegans 。 开发 133(19):3939-3948。
  14. Treusch,S.,Knuth,S.,Slaugenhaupt,S.A.,Goldin,E.,Grant,B.D。和Fares,H。(2004)。 秀丽隐杆线虫人类蛋白质h-粘蛋白-1的功能性直向同源物是必需的用于溶酶体生物合成。


    美国国家科学院院刊 101(13):4483-4488。

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Huynh, J. M., Dang, H. and Fares, H. (2018). Measurement of Lysosomal Size and Lysosomal Marker Intensities in Adult Caenorhabditis elegans. Bio-protocol 8(3): e2724. DOI: 10.21769/BioProtoc.2724.
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