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Dec 2017
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Detection of Apoptosis-like Cell Death in Ustilago maydis by Annexin V-FITC Staining
膜联蛋白V-FITC染色检测玉米黑粉菌凋亡样细胞死亡   

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Abstract

Programmed cell death (PCD) guides the transition between key developmental stages in many organisms. PCD also remains an important fate for many organisms upon exposure to different stress conditions. Therefore, an insight into the progression of PCD during the execution of a biological phenomenon can yield significant details of the underlying mechanism. Apoptosis, as well as apoptosis-like programmed cell death, constitutes one of the forms of PCD in higher and lower eukaryotes respectively. Flipping of phosphatidylserine (PS) from the inner leaflet of the plasma membrane to the outer leaflet is among the different hallmarks of apoptosis/apoptosis-like PCD that marks the initiation of the said cell death event. This flipping can be detected through staining of the target cells using annexin V-FITC that binds specifically to PS. In Ustilago maydis the staining of the externally exposed PS by annexin V-FITC is difficult due to the presence of cell wall. The key to such staining, therefore, relies on the gentle removal of the cell wall without significantly altering the underlying plasma membrane architecture/topology. This protocol highlights the dependence of the PS staining on the extent of protoplastation of the stressed cells in Ustilago maydis.

Keywords: Apoptosis (凋亡), Ustilago maydis (玉米黑粉菌), Phosphatidylserine externalization (磷脂酰丝氨酸外翻 ), Annexin V-FITC staining (膜联蛋白V-FITC染色), Protoplast (原生质体), Plasma membrane (质膜)

Background

PS externalization constitutes one of the hallmarks of apoptosis-like PCD that can be detected very early (Martin et al., 1995). Hence the appearance of PS on the outer leaflet of the plasma membrane marks the onset of an apoptotic cell death phenomenon. Ustilago maydis is a biotrophic plant pathogen and infects host plant Zea mays. The lifecycle of U. maydis has been demonstrated to comprise of primarily two morphological forms namely the non-pathogenic haploid sporidial form and the pathogenic diploid filamentous form. The transition from the haploid to the diploid takes place through the mating of the compatible haploid strains on plant surfaces (Kahmann and Kamper, 2004). This leads to the generation of an infectious structure called appressoria that further penetrates the host plant as the filamentous form of the pathogen. Within the plant cells, the filamentous form of U. maydis again undergoes several transitions between morphologically distinct phases leading to sporulation. PCD has been demonstrated to play a significant role in the morphological transformations of different cell types primarily in higher eukaryotes (Buss et al., 2006; Suzanne and Steller, 2013). Also in some phytopathogenic fungi, PCD has been evidenced to be absolutely essential for the generation of appressoria (Veneault-Fourrey et al., 2006). Besides aiding in the switching between distinct morphological forms, PCD is also an end result of a harsh environmental stress response (Phillips et al., 2003). During penetration of the host plant the pathogens are exposed to a number of host defense response derived stress conditions. Among them, exposure to an increasingly oxidative environment is the most common. The primary reason behind this is the increased production of reactive oxygen species by the host in response to pathogen invasion (Torres et al., 2006). Assaying PCD in the fungal pathogen under each of these conditions mentioned can give significant insights into the pathogenic development as well as stress response of U. maydis. This protocol described the steps in detail to stain U. maydis sporidia under axenic culture conditions with annexinV-FITC to detect onset of any apoptosis-like cell death event upon exposure to adverse environmental conditions. However, it doesn’t include the staining of filamentous hyphae of U. maydis during its growth in-planta. Therefore this protocol is only applicable to the U. maydis sporidial cell suspension.

Materials and Reagents

  1. Plastic Petri-dishes (Tarsons, catalog number: 460020-90MM )
  2. 50 ml centrifuge tubes (Tarsons, catalog number: 546041 )
  3. 15 ml centrifuge tubes (Tarsons, catalog number: 546021 )
  4. 250 ml conical flasks (Fisher Scientific, FisherbrandTM, catalog number: 15429103 )
  5. Culture tubes (BOROSIL, catalog number: 9800U08 , 25 x 150 mm, 55 ml) with plastic caps (Tarsons, catalog number: 020070 )
  6. 10 ml disposable syringe (Dispo Van, Hindustan Syringes and Medical Devices Limited)
  7. 0.22 μm syringe filter (Sartorius, catalog number: 16532-K )
  8. Microscope slides (Polar Industrial, Blue Star, catalog number: PIC-1 , 75 x 25 mm)
  9. Cover slips (Blue Star, 22 mm square, 0.13-0.16 mm thick)
  10. Pipette tips (Tarsons, 0.2-10 μl, catalog number: 521000 ; 2-200 μl, catalog number: 521010 ; 200-1,000 μl, catalog number: 521020 )
  11. Inoculation loop
  12. Kimwipes (Tarsons, catalog number: 370080 , 11.17 x 21.3 cm)
  13. Sterile scalpel blades
  14. Organisms: Ustilago maydis solopathogenic strain SG200 (Kamper et al.,2006)
  15. Yeast extract powder (HiMedia Laboratories, catalog number: RM027 )
  16. Peptone, bacteriological (HiMedia Laboratories, catalog number: RM001 )
  17. Sucrose, A.R. (HiMedia Laboratories, catalog number: RM3063 )
  18. Potato Dextrose Broth (HiMedia Laboratories, catalog number: M403 )
  19. D-Sorbitol (Sigma-Aldrich, catalog number: S1876 )
  20. Tri-sodium citrate dihydrate (Merck, catalog number: 1.93619.0521 )
  21. Citric acid monohydrate (Merck, catalog number: 1.93011.0521 )
  22. Calcium chloride dihydrate (SRL Sisco Research Laboratories, catalog number: 0344317 )
  23. Tris (hydroxymethyl) aminomethane (Merck, Calbiochem, catalog number: 9210-OP )
  24. Hydrochloric acid about 37% (Merck, catalog number: 1.93001.0521 )
  25. Hydrogen peroxide 30% (Merck, catalog number: 1.93007.0521 )
  26. Lysing Enzymes from Trichoderma harzianum (Sigma-Aldrich, catalog number: L1412 )
  27. Annexin V-FITC apoptosis detection kit (Sigma-Aldrich, catalog number: APOAF )
  28. YEPSL media (see Recipes)
  29. Potato Dextrose Agar (PD agar) media (see Recipes)
  30. SCS buffer (see Recipes)
  31. Lysing enzymes in SCS (see Recipes)
  32. STC buffer (see Recipes)
  33. 10x binding buffer (see Recipes)

Equipment

  1. Pipette (0.5-10 μl, 2-20 μl, 20-200 μl, 100-1,000 μl)
  2. Weighing balance (Shimadzu, model: BL220H )
  3. Shaking incubator
  4. Double beam spectrophotometer (Hitachi High-Technologies, model: U-2910 )
  5. Light Microscope (ZEISS, model: Axioskop 40 )
  6. Confocal microscope (Leica Microsystems, model: Leica TCS-SP7 )
  7. Cold centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall RC 6 Plus , catalog number: 36-101-0816)
  8. Laminar air flow
  9. Autoclave

Procedure

  1. Growing the fungal cells
    1. Day 1: Inoculate 5 ml of YEPSL media (see Recipes) in a culture tube with the U. maydisSG200 strain from a PD-agar plate (take about a loopful of inoculum) and incubate overnight in a shaking incubator at 28 °C and 180 rpm.
      Note: U. maydis usually forms a uniform lawn of cells in the PD agar plate if inoculated in higher density. However, when plated at lower density U. maydis single colonies are quite easily spotted on a PD agar plate.
    2. Day 2: Inoculate 50 ml of YEPSL media in a 250 ml conical flask with the overnight grown culture at an OD600 of 0.2 and incubate at 28 °C and 180 rpm. Pellet down the cells by centrifugation in a 50 ml centrifuge tube at 4 °C, 1,070 x g for 5 min when the OD600 of the culture reaches 0.8 (it usually takes 2.5 to 3 h for the OD600 to reach 0.8).

  2. Induction of apoptosis (Day 2)
    Resuspend the pellet from the previous step in 50 ml YEPSL media supplemented with 10 mM H2O2 and incubate the resulting cell suspension in a shaking incubator for 1 h at 28 °C and 180 rpm.

  3. Induction of protoplast formation (Day 2)
    1. Pellet H2O2-treated U. maydis cells by transferring the fungal culture to a 50 ml centrifuge tube followed by centrifugation at 4 °C, 1,070 x g for 10 min.
    2. Discard the supernatant and resuspend the resulting cell pellet in 25 ml SCS buffer (see Recipes) and centrifuge for 10 min at 4 °C, 1,070 x g.
    3. Discard the supernatant and resuspend the pellet in 2 ml of SCS buffer supplemented with 12.5 mg/ml Lysing enzyme (see Recipes) and incubate on ice.
    4. Start monitoring the protoplast formation from approximately 10 min of incubation in the Lysing enzyme solution. Take an aliquot of the cells every 3 min to observe protoplast formation under a light microscope at 40x magnification (Figure 1). Stop the reaction by adding 10 ml of SCS buffer to the cell suspension as soon as about 50% of the cells show early stage protoplastation.
    5. Centrifuge the resulting suspension at 4 °C, 470 x g for 15 min.
    6. Discard the supernatant and wash the pellet that now contains the early stage protoplasts 3 times with 10 ml SCS buffer and 1 time with 10 ml STC buffer (see Recipes) 10 min after every wash by centrifugation at 4 °C, 470 x g, 10 min.
      Note: During the washing steps, resuspend cells well in SCS/STC buffer by gently rotating the cell suspension in 50 ml centrifuge tube by hand; use of pipette and microtip may damage the protoplasts.


      Figure 1. Light microscopic image of U. maydis sporidia after incubation with lysing enzymes. Magnification used is 40x. The emerging protoplasts are indicated with black arrows.

  4. Staining with Annexin V-FITC and Propidium Iodide (Day 2)
    1. Resuspend the pellet from the previous step in 500 μl of Annexin V-FITC binding buffer. (Prepare 1x binding buffer by diluting the 10x binding buffer (see Recipe 6) provided with the kit with autoclaved water). At this point the cells tend to clump (Figures 2B and 2D). Cut the edge of a 1 ml tip with a sterile scalpel blade to increase the cross sectional diameter of the edge. Try to dissolve the clumps by pipetting up and down the protoplast suspension using this tip.
      Notes:
      1. It is important to break the clumps as much as possible to ensure even staining of the protoplasts.
      2. Clumping of protoplast is a common problem and the degree of clumping has been shown to be directly dependent on the density of the cell suspension from where the protoplastation is induced (Wallin et al., 1974). Possibly exposed plasma membrane following removal of cell wall exhibits a sticky nature and can therefore interact with other protoplasts leading thereby to the formation of the protoplast aggregates or clumps.


      Figure 2. Comparative view of U. maydis cell suspension before and after protoplastation. A. Cell suspension containing U. maydis sporidia before protoplastation; B. Protoplast suspension (Note the clumps that formed due to removal of the cell wall); C-D. Bright field images of the cell suspensions in (A) and (B) respectively. Scale bars for (C) and (D) are indicated within the figure. Yellow arrows point the visible cell clumps noticed following protoplastation.

    2. Add 0.5 μg/ml Annexin V-FITC conjugate and 2 μg/ml Propidium iodide to the cell suspension.
      Note: Direct contact with PI should be avoided as it may cause inflammation of skin, irritation of eyes and respiratory tract. Gloves and protective glasses should always be worn while handling PI solution.
    3. Incubate the cell suspension for 10 min in the dark at room temperature and then analyze the cells using a confocal microscope.

  5. Preparing the microscope slides (Day 2)
    1. Clean the slides by rubbing well with kimwipes to remove any dirt-particle on the glass surface.
    2. Drop 5-6 μl of cell suspension in the middle of the glass surface.
    3. Carefully place a cover-slip over the drop of the cell suspension and seal it to the slide using a suitable coverslip sealant.
    4. Observe under a confocal microscope.

Data analysis

Staining of PS externalization in U. maydis using annexin V-FITC is quite heterogeneous. In some cases, the fluorescence from FITC could be noticed primarily at the periphery of the protoplasts which is often seen in PS staining of higher eukaryotic cells. However, this peripheral localization of the fluorescence signal in case of U. maydis protoplasts has hardly any uniformity in terms of signal intensity throughout the periphery. For example, Figures 3A-3C show single (A and B) or multiple (C) intense spots at specific locations within the periphery of the stained protoplasts. On the contrary, some stained protoplasts show complete staining rather than a specific peripheral staining (Figures 3D-3F). Moreover, the complete staining is not always associated with late-stage apoptosis where the plasma membrane is already permeabilizedand protoplasts lack PI staining. Nevertheless, there are also instances where complete staining of protoplasts with both annexin V-FITC and PI could be noticed indicating a late stage of apoptosis (Figure 4). In none of the cases where peripheral FITC staining in U. maydis protoplasts could be seen, an internal PI staining could be noticed. Therefore, if protoplasts in U. maydis were stained in both the forms–peripheral staining as well as complete staining, it can be considered at the early stage of apoptosis. However, protoplasts that show both complete stainings with annexin V-FITC as well as PI should not be considered in their early stage of apoptosis. This is because these protoplasts might represent artifact associated with the protoplast preparation that led to the damage of the plasma membrane of sporidium and subsequent permeabilization to both PI and annexin V.


Figure 3. Annexin V-FITC stained U. maydis protoplasts. A-C. Typical peripheral localization of the fluorescence signals. Note the single or multiple intense spots (red arrows) in the otherwise uniformly stained periphery (Yellow arrows). D-F. Complete staining of the protoplasts. Note that no discrete peripheral staining is visible. The protoplast boundary is marked with dashed lines. Scale bars represent 4 μm. The settings for visualizing the fluorescence of FITC and Propidium Iodide are enlisted in Table 1.


Figure 4. Dual staining of U. maydis protoplasts with Annexin V-FITC and PI. Only protoplasts with fully compromised plasma membrane showed fluorescence signals from both the fluorophores. Dual stained protoplast is marked with yellow arrows. Scale bars represent 4 μm.

Table 1. The laser settings for visualizing FITC and Propidium Iodide fluorescence

Notes

The limiting step for annexin V-FITC staining in U. maydis is the protoplastation condition. The cells are best stained with a typical peripheral staining marking the externalized PS only when the protoplasts have either just come out of the cell walls and not completely dissociated from them (STAGE I) or those that have freshly left out the cell wall (STAGE II). We believe that at this point the integrity of surface topology and architecture of the cells remain compatible for annexin V-FITC staining. The same samples when incubated for longer periods in the protoplast inducing buffer, either showed cytosolic stains or could not be stained at all. These are the final stage protoplasts and are completely separated from their respective cell walls for quite some time (STAGE III) (Figure 5).


Figure 5. Different stages of protoplast formation in U. maydis. Stage I and II represent early stages protoplasts and Stage III represents late stage protoplasts with plasma membranes mostly incompatible for typical peripheral staining. Size bars represent 10 μm.

Recipes

  1. YEPSL media
    Yeast extract : 10 g
    Peptone, bacteriological: 4 g
    Sucrose: 4 g
    Dissolve in 1,000 ml of double distilled water, autoclave at 121 °C, 15 psi for 15 min
  2. Potato Dextrose Agar (PD agar) media
    1. Dissolve 2.4 g of Potato Dextrose broth in double distilled water and adjust the volume to 100 ml
    2. To this add 2 g of agar powder and autoclave the resulting media at 15 psi for 15 min
    3. Following autoclaving cool down the media to about 50 °C and do the plating
  3. SCS buffer
    1. To 100 ml of 20 mM Tri-sodium citrate dihydrate add D-sorbitol to a final concentration of 1 M
    2. Adjust pH with 1 M citric acid to 5.8, autoclave at 121 °C, 15 psi for 15 min and store at 4 °C
  4. Lysing enzymes in SCS (12.5 mg/ml)
    1. Dissolve 37.5 mg of Lysing enzymes in 3 ml of SCS buffer
    2. Pass the solution through a 0.22 μm syringe filter and keep on ice
  5. STC buffer
    1. To 50 ml of 10 mMTris (hydroxymethyl)aminomethane solution in distilled water add CaCl2 to a final concentration of 100 mM and D-sorbitol to a final concentration of 1 M
    2. Adjust the pH with 37% HCl to 7.5 and autoclave at 121 °C, 15 psi for 15 min and store at 4 °C
  6. 10x binding buffer
    100 mM HEPES/NaOH, pH 7.5
    1.4 M NaCl
    25 mM CaCl2

Acknowledgments

This work was funded by DST-INSPIRE Faculty grant IFA13-LSPA16 and research grant from Bose Institute. The authors thank Mr. Asim Kumar Poddar for his assistance in obtaining confocal images that are presented here. This protocol is adapted from Mukherjee et al.(2017). The authors declare that there are no conflicts of interest.

References

  1. Buss, R. R., Sun, W. and Oppenheim, R. W. (2006). Adaptive roles of programmed cell death during nervous system development. Annu Rev Neurosci 29: 1-35.
  2. Kahmann, R. and Kamper, J. (2004). Ustilago maydis: how its biology relates to pathogenic development. New Phytol 164(1): 31-42.
  3. Kamper, J., Kahmann, R., Bolker, M., Ma, L. J., Brefort, T., Saville, B. J., Banuett, F., Kronstad, J. W., Gold, S. E., Muller, O., Perlin, M. H., Wosten, H. A., de Vries, R., Ruiz-Herrera, J., Reynaga-Pena, C. G., Snetselaar, K., McCann, M., Perez-Martin, J., Feldbrugge, M., Basse, C. W., Steinberg, G., Ibeas, J. I., Holloman, W., Guzman, P., Farman, M., Stajich, J. E., Sentandreu, R., Gonzalez-Prieto, J. M., Kennell, J. C., Molina, L., Schirawski, J., Mendoza-Mendoza, A., Greilinger, D., Munch, K., Rossel, N., Scherer, M., Vranes, M., Ladendorf, O., Vincon, V., Fuchs, U., Sandrock, B., Meng, S., Ho, E. C., Cahill, M. J., Boyce, K. J., Klose, J., Klosterman, S. J., Deelstra, H. J., Ortiz-Castellanos, L., Li, W., Sanchez-Alonso, P., Schreier, P. H., Hauser-Hahn, I., Vaupel, M., Koopmann, E., Friedrich, G., Voss, H., Schluter, T., Margolis, J., Platt, D., Swimmer, C., Gnirke, A., Chen, F., Vysotskaia, V., Mannhaupt, G., Guldener, U., Munsterkotter, M., Haase, D., Oesterheld, M., Mewes, H. W., Mauceli, E. W., DeCaprio, D., Wade, C. M., Butler, J., Young, S., Jaffe, D. B., Calvo, S., Nusbaum, C., Galagan, J. and Birren, B. W. (2006). Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444(7115): 97-101.
  4. Martin, S. J., Reutelingsperger, C. P., McGahon, A. J., Rader, J. A., van Schie, R. C., LaFace, D. M. and Green, D. R. (1995). Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 182(5): 1545-1556.
  5. Mukherjee, D., Gupta, S., Saran, N., Datta, R. and Ghosh, A. (2017). Induction of apoptosis-like cell death and clearance of stress-induced intracellular protein aggregates: dual roles for Ustilago maydis metacaspase Mca1. MolMicrobiol 106(5): 815-831.
  6. Phillips, A. J., Sudbery, I. andRamsdale, M. (2003). Apoptosis induced by environmental stresses and amphotericin B in Candida albicans. Proc Natl Acad Sci U S A 100(24): 14327-14332.
  7. Suzanne, M. and Steller, H. (2013). Shaping organisms with apoptosis. Cell Death Differ 20(5): 669-675.
  8. Torres, M. A., Jones, J. D. and Dangl, J. L. (2006). Reactive oxygen species signaling in response to pathogens. Plant Physiol 141(2): 373-378.
  9. Veneault-Fourrey, C., Barooah, M., Egan, M., Wakley, G. and Talbot, N. J. (2006). Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science 312(5773): 580-583.
  10. Wallin, A., Glimelius, K. and Eriksson, T. (1974). The induction of aggregation and fusion of Daucus carota protoplasts by polyethylene glycol. Z Pflanzenphysiol 74(1): 64-80.

简介

程序性细胞死亡(PCD)指导许多生物体的关键发育阶段之间的过渡。 PCD在暴露于不同的胁迫条件下仍然是许多生物的重要命运。因此,在执行生物现象期间洞察PCD的进展可以产生潜在机制的重要细节。细胞凋亡以及凋亡样程序性细胞死亡分别构成高等和低等真核生物中PCD的一种形式。将磷脂酰丝氨酸(PS)从质膜的内部小叶翻转到外部小叶是凋亡/凋亡样PCD的不同标志之一,其标志着所述细胞死亡事件的开始。可以使用与PS特异性结合的膜联蛋白V-FITC通过染色靶细胞来检测这种翻转。在 Ustilago maydis 中,由于细胞壁的存在,膜联蛋白V-FITC对外露PS的染色是困难的。因此,这种染色的关键在于,在不显着改变下面的质膜结构/拓扑结构的情况下,温和地去除细胞壁。该协议强调了PS染色对 Ustilago maydis 中应激细胞原生质体的依赖性。

【背景】PS外化是早期可以检测到的凋亡样PCD的标志之一(Martin et al。,1995)。因此,质膜在细胞外膜上的出现标志着凋亡细胞死亡现象的发生。 Ustilago maydis 是一种生物营养植物病原体并感染寄主植物 Zea mays 。 U的生命周期。已证明maydis 主要包括两种形态形式,即非致病性单倍体孢子形式和致病性二倍体丝状形式。从单倍体到二倍体的转变通过相互作用的单倍体菌株在植物表面上的交配而发生(Kahmann和Kamper,2004)。这导致产生称为附着物的感染性结构,其进一步作为病原体的丝状形式穿透宿主植物。在植物细胞内, U的丝状形式。 maydis 再次在形态上不同的阶段之间经历几次转变,导致孢子形成。已经证明PCD在主要在高等真核生物中的不同细胞类型的形态转化中发挥重要作用(Buss 等人,2006; Suzanne和Steller,2013)。同样在一些植物致病真菌中,PCD已被证明对于附着物的产生是绝对必需的(Veneault-Fourrey 等人,,2006)。除了帮助在不同的形态形式之间切换之外,PCD也是恶劣环境应激反应的最终结果(Phillips et al。,2003)。在宿主植物的渗透过程中,病原体暴露于许多宿主防御反应导致的胁迫条件。其中,暴露于日益氧化的环境是最常见的。这背后的主要原因是宿主响应病原体入侵增加了活性氧的产生(Torres et al。,2006)。在所提到的每种条件下测定真菌病原体中的PCD可以对 U的致病性发展以及应激反应提供重要的见解。玉米小斑病。该协议详细描述了染色 U的步骤。在无菌培养条件下使用膜联蛋白V-FITC检测孢子虫以检测在暴露于不利环境条件下任何凋亡样细胞死亡事件的发生。但是,它不包括 U的丝状菌丝的染色。 maydis 在植物生长期间。因此,该协议仅适用于 U. maydis 孢子细胞悬浮液。

关键字:凋亡, 玉米黑粉菌, 磷脂酰丝氨酸外翻 , 膜联蛋白V-FITC染色, 原生质体, 质膜

材料和试剂

  1. 塑料培养皿(Tarsons,目录号:460020-90MM)
  2. 50毫升离心管(Tarsons,目录号:546041)
  3. 15毫升离心管(Tarsons,目录号:546021)
  4. 250毫升锥形瓶(Fisher Scientific,Fisherbrand TM ,目录号:15429103)
  5. 培养管(BOROSIL,目录号:9800U08,25 x 150 mm,55 ml),带塑料盖(Tarsons,目录号:020070)
  6. 10毫升一次性注射器(Dispo Van,Hindustan Syringes and Medical Devices Limited)
  7. 0.22μm注射器过滤器(Sartorius,目录号:16532-K)
  8. 显微镜载玻片(Polar Industrial,Blue Star,目录号:PIC-1,75 x 25 mm)
  9. 盖玻片(蓝星,22毫米见方,0.13-0.16毫米厚)
  10. 移液器吸头(Tarsons,0.2-10μl,目录号:521000;2-200μl,目录号:521010;200-1,000μl,目录号:521020)
  11. 接种循环
  12. Kimwipes(Tarsons,目录号:370080,11.17 x 21.3 cm)
  13. 无菌手术刀刀片
  14. 生物: Ustilago maydis 致病性菌株SG200(Kamper et al。,2006)
  15. 酵母提取物粉末(HiMedia Laboratories,目录号:RM027)
  16. 蛋白胨,细菌学(HiMedia Laboratories,目录号:RM001)
  17. 蔗糖,A.R。 (HiMedia Laboratories,目录号:RM3063)
  18. 马铃薯葡萄糖肉汤(HiMedia Laboratories,目录号:M403)
  19. D-山梨醇(Sigma-Aldrich,目录号:S1876)
  20. 柠檬酸三钠二水合物(Merck,目录号:1.93619.0521)
  21. 柠檬酸一水合物(默克,目录号:1.93011.0521)
  22. 二水氯化钙(SRL Sisco Research Laboratories,目录号:0344317)
  23. 三(羟甲基)氨基甲烷(Merck,Calbiochem,目录号:9210-OP)
  24. Hydrochloric acid about 37% (Merck, catalog number: 1.93001.0521)
  25. Hydrogen peroxide 30% (Merck, catalog number: 1.93007.0521)
  26. Lysing Enzymes from Trichoderma harzianum(Sigma-Aldrich, catalog number: L1412)
  27. Annexin V-FITC apoptosis detection kit (Sigma-Aldrich, catalog number: APOAF)
  28. YEPSL media (see Recipes)
  29. Potato Dextrose Agar (PD agar) media (see Recipes)
  30. SCS buffer (see Recipes)
  31. Lysing enzymes in SCS (see Recipes)
  32. STC buffer (see Recipes)
  33. 10x binding buffer (see Recipes)

Equipment

  1. Pipette (0.5-10 μl, 2-20 μl, 20-200 μl, 100-1,000 μl)
  2. Weighing balance (Shimadzu, model: BL220H)
  3. Shaking incubator
  4. Double beam spectrophotometer (Hitachi High-Technologies, model: U-2910)
  5. Light Microscope (ZEISS, model: Axioskop 40)
  6. Confocal microscope (Leica Microsystems, model: Leica TCS-SP7)
  7. Cold centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall RC 6 Plus, catalog number: 36-101-0816)
  8. Laminar air flow
  9. Autoclave

Procedure

  1. Growing the fungal cells
    1. Day 1: Inoculate 5 ml of YEPSL media (see Recipes) in a culture tube with the U. maydisSG200 strain from a PD-agar plate (take about a loopful of inoculum) and incubate overnight in a shaking incubator at 28 °C and 180 rpm.
      Note: U. maydis usually forms a uniform lawn of cells in the PD agar plate if inoculated in higher density. However, when plated at lower density U. maydis single colonies are quite easily spotted on a PD agar plate.
    2. Day 2: Inoculate 50 ml of YEPSL media in a 250 ml conical flask with the overnight grown culture at an OD600 of 0.2 and incubate at 28 °C and 180 rpm. Pellet down the cells by centrifugation in a 50 ml centrifuge tube at 4 °C, 1,070 x gfor 5 min when the OD600 of the culture reaches 0.8 (it usually takes 2.5 to 3 h for the OD600 to reach 0.8).

  2. 诱导细胞凋亡(第2天)
    将来自前一步骤的沉淀重悬于补充有10mM H 2 O 2 的50ml YEPSL培养基中,并将所得细胞悬浮液在振荡培养箱中于28°温育1小时。 C和180 rpm。
  3. 诱导原生质体形成(第2天)
    1. 沉淀H 2 O 2 处理的 U。通过将真菌培养物转移到50ml离心管中,然后在4℃,1,070 x g 离心10分钟,将maydis 细胞分离。
    2. 弃去上清液,将得到的细胞沉淀重悬于25ml SCS缓冲液中(见食谱),4℃,1070 x g 离心10分钟。
    3. 弃去上清液,将沉淀重悬于2 ml SCS缓冲液中,补充12.5 mg / ml裂解酶(见食谱)并在冰上孵育。
    4. 在Lysing酶溶液中孵育约10分钟开始监测原生质体形成。每3分钟取一等份细胞,在光学显微镜下以40x放大倍数观察原生质体的形成(图1)。一旦约50%的细胞显示出早期原代质体,就通过向细胞悬浮液中加入10ml SCS缓冲液来终止反应。
    5. 将得到的悬浮液在4℃,470 x g 下离心15分钟。
    6. 弃去上清液,用10ml SCS缓冲液洗涤含有早期原生质体的沉淀3次,用10ml STC缓冲液洗涤1次(参见配方)每次洗涤10分钟后,在4℃,470 xg离心,10分钟。
      注意:在洗涤步骤中,通过用手轻轻旋转50ml离心管中的细胞悬液,将细胞重悬于SCS / STC缓冲液中;使用移液管和微尖可能会损坏原生质体。


      图1. U的光学显微镜图像。与裂解酶一起孵育后的孢子虫 。使用的放大倍数是40倍。新兴的原生质体用黑色箭头表示。

  4. 用膜联蛋白V-FITC和碘化丙锭染色(第2天)
    1. 将前一步骤的沉淀重悬于500μl膜联蛋白V-FITC结合缓冲液中。 (通过用高压灭菌的水稀释试剂盒提供的10x结合缓冲液(参见配方6)制备1x结合缓冲液)。此时细胞倾向于结块(图2B和2D)。用无菌手术刀刀片切割1毫升尖端的边缘,以增加边缘的横截面直径。尝试通过使用此尖端向上和向下移取原生质体悬浮液来溶解团块。
      注意:
      1. 尽可能打破团块以确保原生质体均匀染色非常重要。
      2. 原生质体的结块是一个常见的问题,并且已显示结块程度直接取决于诱导原生质体的细胞悬浮液的密度(Wallin等,1974)。去除细胞壁后可能暴露的质膜显示出粘性,因此可以与其他原生质体相互作用,从而形成原生质体聚集体或团块。
    2. 向细胞悬液中加入0.5μg/ ml膜联蛋白V-FITC结合物和2μg/ ml碘化丙啶。
      注意:应避免直接接触PI,因为它可能导致皮肤发炎,刺激眼睛和呼吸道。处理PI溶液时,应始终佩戴手套和防护眼镜。
    3. 在室温下在黑暗中孵育细胞悬浮液10分钟,然后使用共聚焦显微镜分析细胞。

  5. 准备显微镜载玻片(第2天)
    1. 通过与kimwipes良好摩擦来清洁载玻片,以清除玻璃表面上的任何污垢颗粒。
    2. 将5-6μl细胞悬浮液滴在玻璃表面中间。
    3. 小心地将盖玻片放在细胞悬浮液滴上,并使用合适的盖玻片密封剂将其密封到载玻片上。
    4. 在共聚焦显微镜下观察。


      图2. U的比较视图。原生质体前后的maydis 细胞悬液。 A.含有 U的细胞悬液。原生质体前的maydis 孢子虫; B.原生质体悬浮液(注意由于去除细胞壁而形成的团块);光盘。 (A)和(B)中细胞悬浮液的明视野图像。 (C)和(D)的比例尺在图中表示。黄色箭头指向原生质体后注意到的可见细胞团块。

数据分析

U中PS外化的染色。使用annexin V-FITC的maydis 非常不同。在一些情况下,来自FITC的荧光可以主要在原生质体的外围被注意到,这通常在高等真核细胞的PS染色中看到。然而,在 U的情况下,荧光信号的这种外周定位。 maydis 原生质体在整个外围的信号强度方面几乎没有任何均匀性。例如,图3A-3C显示在染色的原生质体周围的特定位置处的单个(A和B)或多个(C)强点。相反,一些染色的原生质体显示完全染色而不是特异性外周染色(图3D-3F)。此外,完全染色并不总是与晚期细胞凋亡相关,其中质膜已经透化并且原生质体缺乏PI染色。然而,也存在这样的情况:可以注意到具有膜联蛋白V-FITC和PI的原生质体的完全染色,表明细胞凋亡的晚期(图4)。在 U中外周FITC染色的情况均不存在。可以看到maydis 原生质体,可以注意到内部PI染色。因此,如果 U中的原生质体。 maydis 染色形式 - 外周染色以及完全染色,可以在凋亡的早期阶段考虑。然而,在细胞凋亡的早期阶段,不应考虑用膜联蛋白V-FITC以及PI显示完全染色的原生质体。这是因为这些原生质体可能代表与原生质体制剂相关的伪影,导致孢子质质膜的损伤以及随后对PI和膜联蛋白V的透化。


图3.膜联蛋白V-FITC染色 U. maydis 原生质体。 A-C。荧光信号的典型外周定位。注意在其他方面均匀染色的周边(黄色箭头)中的单个或多个强点(红色箭头)。 d-F。原生质体完全染色。注意,没有看到离散的外周染色。原生质体边界用虚线标出。比例尺代表4μm。用于可视化FITC和碘化丙啶的荧光的设置列于表1中。


图4. U的双重染色。具有膜联蛋白V-FITC和PI的maydis 原生质体。只有具有完全受损的质膜的原生质体显示来自两种荧光团的荧光信号。双染原生质体用黄色箭头标记。比例尺代表4微米。

表1.用于可视化FITC和碘化丙啶荧光的激光设置

笔记

U中膜联蛋白V-FITC染色的限制步骤。 maydis 是原生质体疾病。只有当原生质体刚刚从细胞壁出来并且没有完全从它们中解离时(第一阶段)或那些刚刚遗漏细胞壁的细胞(STAGE II),细胞最好用标记外化PS的典型外周染色染色。 )。我们相信,此时表面拓扑结构和细胞结构的完整性仍然与膜联蛋白V-FITC染色相容。当在原生质体诱导缓冲液中孵育较长时间时,相同的样品显示细胞溶质染色或根本不染色。这些是最后阶段的原生质体,并且与它们各自的细胞壁完全分离了一段时间(第三阶段)(图5)。


图5. U中原生质体形成的不同阶段。 maydis 。 I期和II期代表早期原生质体,而III期代表晚期原生质体,质膜与典型的外周染色大多不相容。尺寸条代表10μm。

食谱

  1. YEPSL媒体
    酵母提取物:10克
    蛋白胨,细菌学:4克
    蔗糖:4克
    溶于1,000毫升双蒸水中,在121℃,15磅/平方英寸高压灭菌15分钟
  2. 马铃薯葡萄糖琼脂(PD琼脂)培养基
    1. 将2.4克马铃薯葡萄糖肉汤溶于双蒸水中,调节至100毫升
    2. 向其中加入2g琼脂粉末并在15psi下将所得培养基高压灭菌15分钟
    3. 高压灭菌后,将介质冷却至约50℃并进行电镀
  3. SCS缓冲区
    1. 向100ml 20mM柠檬酸三钠二水合物中加入D-山梨糖醇至终浓度为1M。
    2. 用1M柠檬酸调节pH至5.8,在121℃高压灭菌,15psi,15分钟,并在4℃下储存
  4. SCS中的裂解酶(12.5 mg / ml)
    1. 将37.5毫克裂解酶溶于3毫升SCS缓冲液中
    2. 将溶液通过0.22μm注射器过滤器并保持在冰上
  5. STC缓冲区
    1. 向蒸馏水中的50ml 10mM三(羟甲基)氨基甲烷溶液中加入CaCl 2 至终浓度为100mM,D-山梨糖醇至终浓度为1M。
    2. 用37%HCl调节pH至7.5并在121℃,15psi高压灭菌15分钟并在4℃下储存
  6. 10x结合缓冲区
    100mM HEPES / NaOH,pH 7.5
    1.4 M NaCl
    25mM CaCl 2,

致谢

这项工作由DST-INSPIRE教师资助IFA13-LSPA16和Bose研究所的研究资助资助。作者感谢Asim Kumar Poddar先生在获取此处提供的共聚焦图像方面提供的帮助。该协议改编自Mukherjee 等人(2017)。

参考

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引用:Mukherjee, D., Mitra, A. and Ghosh, A. (2018). Detection of Apoptosis-like Cell Death in Ustilago maydis by Annexin V-FITC Staining. Bio-protocol 8(15): e2948. DOI: 10.21769/BioProtoc.2948.
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