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Apr 2019

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Non-invasive Quantification of Cell Wall Porosity by Fluorescence Quenching Microscopy
细胞壁孔隙的荧光猝灭显微镜无创定量分析   

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Abstract

All bacteria, fungi and plant cells are surrounded by a cell wall. This complex network of polysaccharides and glycoproteins provides mechanical support, defines cell shape, controls cell growth and influences the exchange of substances between the cell and its surroundings. Despite its name, the cell wall is a flexible, dynamic structure. However, due to the lack of non-invasive methods to probe the structure, relatively little is known about the synthesis and dynamic remodeling of cell walls. Here, we describe a non-invasive method that quantifies a key physiological parameter of cell walls, the porosity, i.e., the size of spaces between cell wall components. This method measures the porosity-dependent decrease of the plasma membrane-localized fluorescent dye FM4-64 in the presence of the extracellular quencher Trypan blue. This method is applied to bacteria, fungi and plant cell walls to detect dynamic changes of porosity in response to environmental cues.

Keywords: Cell wall (细胞壁), Porosity (多孔性), Fluorescence quenching (荧光淬灭), Bacteria (细菌), Fungi (真菌), S. cerevisiae (酿酒酵母), Arabidopsis thaliana (拟南芥), Maize (玉米)

Background

Bacterial, fungal and plant cells are surrounded by a cell wall which has a multitude of different functions, including, defining size and shape, and controlling the exchange of substances within the environment. A key feature of cell walls is their flexibility. Long polysaccharides form a strong network and their structure is frequently adjusted, for example to allow for cell elongation, or to counter pathogen attack. In addition, cell wall porosity allows for molecular movement between the wall’s various components. Cell wall porosity is also a reliable indicator of digestibility and saccharification efficiency of cell wall material (Himmel et al., 2007; Ding et al., 2012; Tavares et al., 2015) and has been linked to anti-fungal drug uptake (Liu et al., 2019). Current methods for measuring cell wall porosity are based on relatively invasive methods, such as transmission electron microscopy (TEM) or field-emission scanning electron microscopy (FESEM) (Sugimoto et al., 2000; Xiao et al., 2016; Zheng et al., 2017), which alter the original cell wall structure and may lead to artifacts. Another method, cryo-FESEM, is less invasive but the resolution is limited to 20 nm (Derksen et al., 2011). Pore size distributions at sub-nanometer resolution can be obtained by gas adsorption, but this method also requires harsh sample pretreatment (Adani et al., 2011). Instead of determining pore sizes directly, other methods measure the capacity for molecular movement within the wall, which can be compatible with live-cell analysis. For example, the relative porosity of yeast cell walls assesses the measuring the effect of different-sized polycations on the leakage of UV-absorbing intracellular compounds (De Nobel et al., 1990). Similarly, the effect of fluorescent quenchers with different hydrodynamic radius on the autofluorescence of lignin can be measured to gain insight on differences in porosity between wood samples (Donaldson et al., 2015).

The method presented here makes use of the ability of quenchers to diffuse through the cell wall to determine relative porosity. In contrast to the approaches described above, this method works on living cells and can be applied to all accessible tissues and all organisms. It measures the fluorescence intensity of the dye FM4-64 located at the plasma membrane in the presence of extracellular quenchers. The quenching effect has been shown to depend on cell wall porosity (Liu et al., 2019). The method has been successfully used on bacteria, fungi and plants, for example to determine changes in wall porosity during drought-induced cell elongation in the model plant Arabidopsis (Liu et al., 2019).

The method described below uses Trypan blue as a quencher. In comparison with other quenchers, Trypan blue has a higher dynamic range (Liu et al., 2019), which results in more reliable results when analyzing cell walls with low porosity. Furthermore, Trypan blue is widely available, doesn’t require co-factors and is inexpensive. The protocol for yeast cells can be quickly adapted to any other microorganism, by choosing the right culture medium and growth conditions. Similarly, the protocols for Arabidopsis roots and Maize leaf samples can be adapted to other plant species. For root samples, however, growth on agar plates is recommended instead of cultivation on soil, because it can be difficult to clean all soil particles without damaging the root surface.

Materials and Reagents

  1. Pipette tips
  2. Microscope cover slips, 50 x 20 mm and 20 x 20 mm (CITOTEST, P/N: 80340-0630, 80340-3610)
  3. Microscope slide (CITOTEST, P/N: 80320-2140)
  4. 15 ml round bottom tubes
  5. 10 cm glass culture dish
  6. 1.5 ml Centrifuge tube
  7. Tape
  8. Razor blades
  9. Filter paper
  10. Yeast (Saccharomyces cerevisiae, BY4742)
  11. Arabidopsis seedlings (Col-0)
  12. Maize (Zea mays, B73)
  13. FM4-64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide) (Thermo Fisher Scientific, catalog number: T3166)
  14. DMSO (Dimethyl sulfoxide) (MP Biomedicals, catalog number: 196055)
  15. Trypan blue (Solarbio, catalog number: T8070)
  16. Murashige and Skoog (MS) salts (Phyto Technology Laboratories, catalog number: M519)
  17. Sucrose (Guangzhou Jinhuada Chemical Reagent, CAS: 57-50-1)
  18. Agar (Solarbio, catalog number: A8190)
  19. Tryptone (OXIOD, catalog number: LP0042)
  20. Yeast extract (OXIOD, catalog number: LP0021)
  21. NaCl (Guangzhou Jinhuada Chemical Reagent, CAS: 7647-14-5)
  22. Glucose (Guangzhou Jinhuada Chemical Reagent, CAS: 58367-01-4)
  23. Na2HPO4 (Guangzhou Jinhuada Chemical Reagent, CAS: 10039-32-4)
  24. KH2PO4 (Guangzhou Jinhuada Chemical Reagent, CAS: 7778-77-0)
  25. KCl (Guangzhou Jinhuada Chemical Reagent, CAS: 7447-40-7)
  26. KOH 
  27. YDP medium (see Recipes)
  28. Half strength MS medium (see Recipes)
  29. PBS solution (pH 7.2-7.4) (see Recipes)
  30. FM4-64 stock solution (see Recipes)
  31. Trypan blue stock solution (see Recipes)

Equipment

  1. Forceps
  2. Scissors
  3. Staining jars
  4. Pipettes, 10, 100, 1000 μl (Thermo Scientific, models: PZ31631, PZ69795, QZ06709)
  5. Centrifuge (Sigma, model: 1-14)
  6. Analytical balance (Adam Equipment, model: PWC 124)
  7. Autoclave sterilizer (ZEALWAY, model: GR60DP)
  8. Water purification systems (Synergy, model: SYNSVHF00)
  9. Air clean bench (AIRTECH, model: SW-CJ-2FD)
  10. Shaker incubator (Nanrong, model: NRY-200)
  11. Plant growth chamber (NINGBO SOUTHEAST INSTRUMENT, model: GDN-1000D-4)
  12. Confocal fluorescence microscope (Leica, model: DMi8) with ThorLabs module (model: ThorLabs-CLS)
    Note: The protocol has also been tested on fluorescence widefield microscope systems with sCMOS cameras. These can be used instead of a confocal microscope as long as a clearly focused section of plasma membrane can be visualized with high signal-to-noise ratio.
  13. Computer (Dell, model: U2717D)

Software

  1. ThorImageLS 3.0 (Thorlabs)
  2. ImageJ 1.52n (National Institutes of Health. USA, http://imagej.nih.gov/ij) (Schindelin et al., 2012)

Procedure

  1. Yeast sample
    1. Culture yeast cells in 5 ml YPD medium (see Recipes) in round bottom tubes in a shaker incubator at 30 °C for 24 h or according to the requirements of a specific strain or a specific experiment.
    2. Transfer 500 μl of the culture into a centrifuge tube, centrifuge for 1 min at 4,000 x g and discard the supernatant.
    3. Resuspend the cells in 1 ml PBS (pH 7.2-7.4, see Recipes), centrifuge for 1 min at 4,000 x g and discard the supernatant.
    4. Resuspend the cells in 50 μl PBS-based FM4-64 solution (50 μM) and incubate for 3 min at room temperature in the dark.
      Note: Keep FM4-64 solution in the dark and stock solution on ice. Samples that have been labeled with FM4-64 should be imaged within 10 min, as the dye will eventually be endocytosed. Endocytosis of FM4-64 is indicated by internal signal, typically occuring in the form of small spheres.
    5. Add 1 ml PBS solution and centrifuge for 1 min at 4,000 x g, discard the supernatant. Repeat this step one time.
    6. Transfer 2 μl stained cells onto a microscope slide and add 10 μl PBS, mix by stirring with the pipet tip and place a coverslip.
      Note: If there are issues with cell movement during imaging, yeast cells can be immobilized on slides coated with polylysine or an agarose pad. 
    7. Mount the slide on the microscope and locate cells using bright field mode with a 100x objective.
      Note: Especially with samples of microorganisms, it is important to minimize exposure to laser light, which will cause bleaching. Therefore, it is recommended to adjust focus in bright field mode before switching to confocal mode and immediately acquire an image.
    8. Switch to confocal acquisition with 488 nm or 514 nm laser and a 582/75 nm bandpass emission filter to detect FM4-64 emission (see Notes). Pinhole diameter should be below two airy discs (about 100 μm).
    9. Set imaging parameters (laser power, detector gain or exposure time) to get a bright signal without overexposure. 
    10. Acquire images. Typically, one image of 3-6 different areas per sample can be taken.
    11. Prepare additional samples following Steps A2 to A8, but instead of adding PBS to the slide in Step A6, add PBS-based Trypan blue solution, each time with a different concentration (1, 5, 10, 50 and 100 μM). Acquire images of each sample without changing any imaging parameters. 

  2. Arabidopsis thaliana seedling root sample
    1. Grow Arabidopsis seedlings in half-strength MS medium on plates (see Recipes) at 22 °C with 16 h light/8 h dark for 7 d (Figure 1).
    2. Put a whole seedling into a tube containing 20 μM PBS-based FM4-64 solution, incubate for 3 min and wash with PBS solution in a staining jar.
    3. Add 50 μl PBS onto a microscope slide, arrange the seedling on the slide and add cover slip onto the root part of the seedling (Figure 1).
    4. Find the target region, for example the elongation zone, using a 40x objective and bright filed mode. 
    5. Switch to confocal acquisition with 488 nm or 514 nm laser and a 582/75 nm bandpass emission filter to detect FM4-64 emission (see Notes). Pinhole diameter should be below two airy discs (about 100 μm).
    6. Set imaging parameters (laser power, detector gain or exposure time) to give a bright signal without overexposure. 
    7. Acquire images. Typically, one image of 1-3 different areas per sample can be taken.
    8. Prepare additional samples following Steps B2 to B5, but instead of adding PBS to the slide in Step B3, add PBS-based Trypan blue solution, each time with a different concentration (1, 5, 10, 50 and 100 μM). Acquire images of each sample without changing any imaging parameters.


      Figure 1. Preparation of Arabidopsis root samples. A. Seven-day-old seedlings grown on plates. B. Seedling with root submerged in FM4-64 solution. C. Seedling washed in PBS buffer in a staining jar. D. Seedling mounted on a slide with root covered by the cover slip.

  3. Maize leaf sample
  1. Put two layers of tape along the long edge of a large cover slip, leaving a groove in the middle (Figure 2). This facilitates the efficient exchange of quencher solution without moving the sample.
    Note: Although this operation is more complicated than the ones described for the other samples, it can lead to higher data reliability when the effect of all quencher concentrations was measured on the same cell. The whole protocol should be finished within 10 to 15 min to prevent FM4-64 endocytosis from influencing the results. 
  2. Make a cut into a maize leaf using a razor blade and start peeling the lower epidermis using forceps. Cut a small piece of leaf together with the peeled epidermis (Figure 2).
    Note: Separating the maize leaf epidermis from the rest of the leaf helps to provide more reliable results through minimization of chlorophyll fluorescence, whose spectrum overlaps with that of FM4-64.
  3. Stain sample with 50 μM PBS-based FM4-64 solution in a centrifuge tube for 3 min, wash with PBS buffer in a staining jar (Figure 2).
  4. Add 100 μl PBS to the groove of the large cover slip and arrange the maize leaf sample in it. Cover the sample with a square cover slip (Figure 2).
  5. Find the target region using a 40x objective in bright field mode. 
  6. Switch to confocal acquisition with 488 nm or 514 nm laser and a 582/75 nm bandpass emission filter to detect FM4-64 emission (see Notes). Pinhole diameter should be below two airy discs (about 100 μm).
  7. Set imaging parameters (laser power, detector gain or exposure time) to give a bright signal without overexposure. 
  8. Acquire one image of the target region. 
  9. Without removing the sample from the microscope, use filter paper to drain the buffer and immediately add PBS-based Trypan blue solution, starting with the lowest concentration (1 μM).
  10. Aquire one image and change to the next higher concentration as described in the step above. Repeat until images at all concentrations (1, 5, 10, 50, 100 μM) have been acquired.


    Figure 2. Preparation of maize leaf samples. A. Maize seedling that was used here as sample material. B. A stretch of epidermis (arrow) is peeled from the leaf using forceps and cut out together with a small piece of leaf. C. Sample submerged in FM4-64 solution. D. Sample washed in PBS in a staining jar. E. Sample mounted on a large coverslip on which a central groove was created by taping the edges. F. Sample after application of a small coverslip. G. Change of quencher solution without removal of the sample from the microscope.

Data analysis

  1. Export all image data in a format compatible with ImageJ (e.g., tif) from the imaging system and import into ImageJ. 
  2. Eliminate background signal by checking the background signal intensity in the space between cells (in case of yeast or bacterial cells) or the center of a cell (in case of larger cells), adjusting the black level to this value and pressing “apply” (Figure 3).


    Figure 3. Background subtraction through adjustment of black level. A. Arabidopsis root sample with region of interest drawn at an area not containing the plasma membrane. B. The mean value of this area is used as black level and intensity values for the image redistributed by pressing “Apply”.

  3. Draw a rectangular region of interest (ROI) covering the cell periphery of a cell (Figure 4) and measure mean grey value by pressing “Ctrl + M”. Do not change the size of the ROI between different images of one sample type. If the same region should be measured on several images, e.g., for the maize leaf sample, add the ROI to the ROI manager. 
  4. Measure at least 50 ROIs for each type of sample and each quencher concentration. In yeast samples define one ROI per cell. In plant samples, several ROIs can be defined on the different walls of the same cell.


    Figure 4. Exemplary regions of interest for the measurement of signal intensity. A. Yeast cells. B. Arabidopsis root cells. C. Maize leaf cells. Scale bars, 10 μm.

  5. Export measurement data to spreadsheet software and calculate averages.
  6. Calculate quenching efficiency according to Lehrer (1971) with:



    where, F0 is the fluorescence intensity of the sample in the absence of Trypan blue in the buffer; F is the fluorescence intensity of the sample in the presence of Trypan blue at the concentration of [Q];
    K is the quenching efficiency of this sample system, which could be derived from slope of the concentration [Q] (x axis) plotted against  (y axis).

    A higher quenching efficiency indicates better access of the quencher to the plasma membrane, which can serve as a good indicator of cell wall porosity (Liu et al., 2019). A detailed discussion of the relationship between quenching efficiency and cell wall structure can be found in Liu et al., (2019). The variation of results obtained from the same sample depends on the sample type (Figure 5).


    Figure 5. Exemplary quenching efficiency data measured on yeast and plant cells. Samples were exposed to chemical treatment or change in growth conditions for 12 h (yeast) or 24 h (plant). Variation within one treatment group was generally higher for plant samples than for yeast samples. 

Notes

The emission of FM4-64 shows broad peaks at around 640 nm and 750 nm. Trypan blue was shown to emit fluorescence when bound to certain cell wall components between 640 and 680 nm (Liesche et al., 2015). To avoid overlap with the potential Trypan blue emission, it is recommended to detect FM4-64 around 580 nm or with a longpass filter beyond 700 nm.

Recipes

Note: Prepare all media using ultrapure water (drawn from a purification system set to a resistivity of 18 MΩ cm at 25 °C).

  1. YDP medium
    2% (w/v) Tryptone
    1% (w/v) Yeast extract
    2% (w/v) glucose
    Note: The mixture of tryptone and yeast extract, as well as the glucose solution should be sterilized at 121 °C for 20 min and adjusted to the final concentration after cooling.
  2. Half strength MS medium
    0.22% (w/v) Murashige and Skoog (MS) salts
    1% (w/v) sucrose
    Adjust the pH to 5.8 with 2 M KOH
    0.8% (w/v) agar
    Sterilize at 121 °C for 20 min, pour 25 ml medium to each glass culture dish
  3. PBS solution (pH 7.2-7.4)
    NaCl 130 mM
    KCl 2.6 mM
    Na2HPO4 7 mM
    KH2PO4 1.2 mM
  4. FM4-64 stock solution
    Prepare 2 mM FM4-64 stock solution in DMSO
    Store in aliquots of 100 µl at -20 °C in the dark
  5. Trypan blue stock solution
    Prepare 10 mM Trypan blue stock solution in PBS, keep at 4 °C in the dark
    Note: Weigh with a balance placed inside a fume hood or under suction and wear a mask when handling the solid. Trypan blue is suspected of causing cancer, especially in the powdered form as it can easily be inhaled.

Acknowledgments

This protocol was adapted from our previous work (Liu et al., 2019). The work was supported by the National Natural Science Foundation China (project grant number 31700313), the Science Foundation of Shaanxi Province (100 Talent Program), and Villum Foundation (project grant number 022868).

Competing interests

The authors declare that no competing interests exist.

References

  1. Adani, F., Papa, G., Schievano, A., Cardinale, G., D'Imporzano, G. and Tambone, F. (2011). Nanoscale structure of the cell wall protecting cellulose from enzyme attack. Environ Sci Technol 45(3): 1107-1113. 
  2. De Nobel, J. G., Klis, F. M., Munnik, T., Priem, J. and van den Ende, H. (1990). An assay of relative cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe. Yeast (Chichester, England) 6(6): 483-490. 
  3. Derksen, J., Janssen, G. J., Wolters-Arts, M., Lichtscheidl, I., Adlassnig, W., Ovecka, M., Doris, F. and Steer, M. (2011). Wall architecture with high porosity is established at the tip and maintained in growing pollen tubes of Nicotiana tabacum. Plant J 68(3): 495-506. 
  4. Ding, S. Y., Liu, Y. S., Zeng, Y., Himmel, M. E., Baker, J. O. and Bayer, E. A. (2012). How does plant cell wall nanoscale architecture correlate with enzymatic digestibility? Science 338(6110): 1055-1060.
  5. Donaldson, L. A., Kroese, H. W., Hill, S. J. and Franich, R. A. (2015). Detection of wood cell wall porosity using small carbohydrate molecules and confocal fluorescence microscopy. J Microsc 259(3): 228-236.
  6. Himmel, M. E., Ding, S. Y., Johnson, D. K., Adney, W. S., Nimlos, M. R., Brady, J. W. and Foust, T. D. (2007). Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315(5813): 804-807.
  7. Liesche, J., Marek, M., Günther Pomorski, T. (2015). Cell wall staining with Trypan Blue enables quantitative analysis of morphological changes in yeast cells. Front Microbiol 6:107.
  8. Lehrer, S. S. (1971). Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry 10(17): 3254-3263.
  9. Liu, X., Li, J., Zhao, H., Liu, B., Günther-Pomorski, T., Chen, S. and Liesche, J. (2019). Novel tool to quantify cell wall porosity relates wall structure to cell growth and drug uptake. J Cell Biol 218(4): 1408-1421.
  10. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  11. Sugimoto, K., Williamson, R. E. and Wasteneys, G. O. (2000). New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis. Plant Physiol 124(4): 1493-1506.
  12. Tavares, E.Q., De Souza, A.P. and Buckeridge, M.S. (2015). How endogenous plant cell-wall degradation mechanisms can help achieve higher efficiency in saccharification of biomass. J Exp Bot 66(14): 4133-4143.
  13. Xiao, C., Zhang, T., Zheng, Y., Cosgrove, D. J. and Anderson, C. T. (2016). Xyloglucan deficiency disrupts microtubule stability and cellulose biosynthesis in Arabidopsis, altering cell growth and morphogenesis. Plant Physiol 170(1): 234-249.
  14. Zheng, Y., Cosgrove, D. J. and Ning, G. (2017). High-resolution field emission scanning electron microscopy (FESEM) imaging of cellulose microfibril organization in plant primary cell walls. Microsc Microanal 23(5): 1048-1054.

简介

所有细菌,真菌和植物细胞都被细胞壁包围。 这种多糖和糖蛋白的复杂网络提供机械支持,定义细胞形状,控制细胞生长并影响细胞与其周围环境之间的物质交换。 尽管它的名字,细胞壁是一个灵活,动态的结构。 然而,由于缺乏探测结构的非侵入性方法,关于细胞壁的合成和动态重塑的知识相对较少。 在这里,我们描述了一种非侵入性方法,量化细胞壁的关键生理参数,孔隙度即,细胞壁组分之间的空间大小。 该方法测量在细胞外猝灭剂台盼蓝存在下质膜定位荧光染料FM4-64的孔隙度依赖性降低。 该方法适用于细菌,真菌和植物细胞壁,以检测响应环境因素的孔隙度的动态变化。
【背景】细菌,真菌和植物细胞被细胞壁包围,细胞壁具有多种不同的功能,包括限定大小和形状,以及控制环境中物质的交换。细胞壁的一个关键特征是它们的灵活性。长多糖形成强网络,并且经常调节它们的结构,例如以允许细胞伸长,或抵抗病原体攻击。此外,细胞壁孔隙度允许壁的各种组分之间的分子运动。细胞壁孔隙度也是细胞壁材料消化率和糖化效率的可靠指标(Himmel et al。,2007; Ding et al。,2012; Tavares et al。,2015)并且与抗真菌药物摄取有关(Liu et al。,2019)。目前用于测量细胞壁孔隙率的方法基于相对侵入性的方法,例如透射电子显微镜(TEM)或场发射扫描电子显微镜(FESEM)(Sugimoto et al。,2000; Xiao et al。,2016; Zheng et al。,2017),它改变了原始细胞壁结构并可能导致伪影。另一种方法,cryo-FESEM,侵入性较小,但分辨率限制在20 nm(Derksen et al。,2011)。亚纳米分辨率的孔径分布可以通过气体吸附获得,但这种方法也需要严格的样品预处理(Adani et al。,2011)。其他方法不是直接测定孔径,而是测量壁内分子运动的能力,这可以与活细胞分析相容。例如,酵母细胞壁的相对孔隙度评估了测量不同大小的聚阳离子对吸收紫外线的细胞内化合物泄漏的影响(De Nobel et al。,1990)。类似地,可以测量具有不同流体动力学半径的荧光猝灭剂对木质素自发荧光的影响,以深入了解木材样品之间孔隙度的差异(Donaldson et al。,2015)。
这里给出的方法利用猝灭剂扩散通过细胞壁以确定相对孔隙率的能力。与上述方法相反,该方法适用于活细胞,并且可以应用于所有可接近的组织和所有生物。它在细胞外猝灭剂存在下测量位于质膜上的染料FM4-64的荧光强度。已显示猝灭效应取决于细胞壁孔隙率(Liu 等,,2019)。该方法已成功用于细菌,真菌和植物,例如用于确定模式植物拟南芥中干旱诱导的细胞伸长期间壁孔隙度的变化(Liu et al。,2019)。
下面描述的方法使用台盼蓝作为猝灭剂。与其他淬灭剂相比,台盼蓝具有更高的动态范围(Liu et al。,2019),这在分析低孔隙度的细胞壁时产生更可靠的结果。此外,台盼蓝广泛可用,不需要辅因子且价格低廉。通过选择正确的培养基和生长条件,酵母细胞的方案可以快速适应任何其他微生物。类似地,拟南芥根和玉米叶样品的方案可以适应其他植物物种。然而,对于根样品,建议在琼脂平板上生长而不是在土壤上培养,因为在不损坏根表面的情况下清洁所有土壤颗粒可能是困难的。

关键字:细胞壁, 多孔性, 荧光淬灭, 细菌, 真菌, 酿酒酵母, 拟南芥, 玉米

材料和试剂

  1. 移液器吸头
  2. 显微镜盖玻片,50 x 20 mm和20 x 20 mm(CITOTEST,P / N:80340-0630,80340-3610)
  3. 显微镜载玻片(CITOTEST,P / N:80320-2140)
  4. 15毫升圆底管
  5. 10厘米玻璃培养皿
  6. 1.5毫升离心管
  7. 胶带
  8. 剃刀片
  9. 过滤纸
  10. 酵母( Saccharomyces cerevisiae ,BY4742)
  11. 拟南芥幼苗(Col-0)
  12. 玉米( Zea mays ,B73)
  13. FM4-64(N-(3-三乙基铵丙基)-4-(6-(4-(二乙基氨基)苯基)六噻吩基)吡啶鎓二溴化物)(Thermo Fisher Scientific,目录号:T3166)
  14. DMSO(二甲基亚砜)(MP Biomedicals,目录号:196055)
  15. 台盼蓝(Solarbio,目录号:T8070)
  16. Murashige和Skoog(MS)盐(Phyto Technology Laboratories,目录号:M519)
  17. 蔗糖(广州金华达化学试剂,CAS:57-50-1)
  18. 琼脂(Solarbio,目录号:A8190)
  19. 胰蛋白胨(OXIOD,目录号:LP0042)
  20. 酵母提取物(OXIOD,目录号:LP0021)
  21. NaCl(广州金华达化学试剂,CAS:7647-14-5)
  22. 葡萄糖(广州金华达化学试剂,CAS:58367-01-4)
  23. Na 2 HPO 4 (广州金华达化学试剂,CAS:10039-32-4)
  24. KH 2 PO 4 (广州金华达化学试剂,CAS:7778-77-0)
  25. KCl(广州金华达化学试剂,CAS:7447-40-7)
  26. KOH 
  27. YDP培养基(见食谱)
  28. 半强度MS培养基(见食谱)
  29. PBS溶液(pH 7.2-7.4)(见食谱)
  30. FM4-64储备液(见食谱)
  31. 台盼蓝原液(见食谱)

设备

  1. 钳子
  2. 剪刀
  3. 染色罐子
  4. 移液器,10,100,1000μl(Thermo Scientific,型号:PZ31631,PZ69795,QZ06709)
  5. 离心机(Sigma,型号:1-14)
  6. 分析天平(Adam Equipment,型号:PWC 124)
  7. 高压灭菌器(ZEALWAY,型号:GR60DP)
  8. 水净化系统(Synergy,型号:SYNSVHF00)
  9. 空气洁净工作台(AIRTECH,型号:SW-CJ-2FD)
  10. 摇床培养箱(南荣,型号:NRY-200)
  11. 植物生长室(NINGBO SOUTHEAST INSTRUMENT,型号:GDN-1000D-4)
  12. 共聚焦荧光显微镜(徕卡,型号:DMi8,带ThorLabs模块,型号:ThorLabs-CLS)
    注意:该协议还在使用sCMOS相机的荧光宽场显微镜系统上进行了测试。这些可用于代替共聚焦显微镜,只要能够以高信噪比可视化清晰聚焦的质膜部分。
  13. 电脑(戴尔,型号:U2717D)

软件

  1. ThorImageLS 3.0(Thorlabs)
  2. ImageJ 1.52n(美国国立卫生研究院。美国, http://imagej.nih.gov/ij ) (Schindelin et al。,2012)

程序

  1. 酵母样品
    1. 将培养酵母细胞置于圆底管中的5ml YPD培养基(参见配方)中,在摇床培养箱中于30℃培养24小时或根据特定菌株或特定实验的要求。
    2. 将500μl培养物转移到离心管中,在4,000 x g 下离心1分钟并弃去上清液。
    3. 将细胞重悬于1ml PBS(pH 7.2-7.4,参见配方)中,在4,000 x g 下离心1分钟并弃去上清液。
    4. 将细胞重悬于50μl基于PBS的FM4-64溶液(50μM)中并在室温下在黑暗中孵育3分钟。
      注意:将FM4-64溶液置于黑暗中,并将储备液置于冰上。已经用FM4-64标记的样品应在10分钟内成像,因为染料最终将被内吞。内部信号表明FM4-64的内吞作用,通常以小球体的形式出现。
    5. 加入1 ml PBS溶液,在4,000 x g 下离心1分钟,弃去上清液。重复此步骤一次。
    6. 将2μl染色的细胞转移到显微镜载玻片上并加入10μlPBS,通过用移液管尖端搅拌混合并盖上盖玻片。
      注意:如果在成像过程中细胞运动存在问题,酵母细胞可以固定在涂有聚赖氨酸或琼脂糖垫的载玻片上。
    7. 将载玻片安装在显微镜上,使用100倍物镜的明场模式定位细胞。
      注意:特别是对于微生物样本,重要的是尽量减少激光照射,这会导致漂白。因此,建议在切换到共焦模式之前在明场模式下调整焦距并立即获取图像。
    8. 切换到使用488 nm或514 nm激光器和582/75 nm带通发射滤光片进行共焦采集,以检测FM4-64发射(参见注释)。针孔直径应低于两个透气盘(约100微米)。
    9. 设置成像参数(激光功率,探测器增益或曝光时间)以获得明亮的信号而不会过度曝光。 
    10. 获取图像。通常,可以拍摄每个样品3-6个不同区域的一个图像。
    11. 在步骤A2至A8之后制备另外的样品,但不是在步骤A6中向载玻片中添加PBS,而是添加基于PBS的台盼蓝溶液,每次使用不同浓度(1,5,10,50和100μM)。在不改变任何成像参数的情况下获取每个样品的图像。 

  2. 拟南芥 thaliana 幼苗根样品
    1. 在板上的半强度MS培养基中生长拟南芥幼苗(参见食谱),在22℃,16小时光照/ 8小时黑暗处理7天(图1)。
    2. 将整个幼苗放入含有20μM基于PBS的FM4-64溶液的管中,孵育3分钟并用染色罐中的PBS溶液洗涤。
    3. 将50μlPBS添加到显微镜载玻片上,将幼苗布置在载玻片上,并将盖玻片添加到幼苗的根部(图1)。
    4. 使用40x物镜和明场模式找到目标区域,例如伸长区域。 
    5. 切换到使用488 nm或514 nm激光器和582/75 nm带通发射滤光片进行共焦采集,以检测FM4-64发射(参见注释)。针孔直径应低于两个透气盘(约100微米)。
    6. 设置成像参数(激光功率,探测器增益或曝光时间),以提供明亮的信号而不会过度曝光。 
    7. 获取图像。通常,每个样品可以拍摄1-3个不同区域的图像。
    8. 在步骤B2至B5之后制备另外的样品,但是不是在步骤B3中向载玻片中添加PBS,而是添加基于PBS的台盼蓝溶液,每次使用不同浓度(1,5,10,50和100μM)。在不改变任何成像参数的情况下获取每个样品的图像。


      图1. 拟南芥根样品的制备。 A.在板上生长的7日龄幼苗。 B.将根浸没在FM4-64溶液中的幼苗。 C.在PBS缓冲液中在染色罐中洗涤幼苗。 D.幼苗安装在滑盖上,根部覆盖着滑盖。

  3. 玉米叶样品
  1. 沿着大盖玻片的长边放两层胶带,在中间留下一个凹槽(图2)。这有助于在不移动样品的情况下有效交换猝灭剂溶液。
    注意:尽管此操作比其他样品描述的操作更复杂,但是当在同一电池上测量所有猝灭剂浓度的影响时,它可以导致更高的数据可靠性。整个方案应在10到15分钟内完成,以防止FM4-64内吞作用影响结果。 
  2. 使用剃刀刀片切成玉米叶,并用镊子开始剥离下表皮。将一小片叶子与去皮的表皮一起切开(图2)。
    注意:将玉米叶表皮与叶片的其余部分分开,有助于通过最小化叶绿素荧光提供更可靠的结果,叶绿素荧光的光谱与FM4-64的光谱重叠。
  3. 在离心管中用50μM基于PBS的FM4-64溶液染色样品3分钟,用PBS缓冲液在染色罐中洗涤(图2)。
  4. 将100μlPBS加入大盖玻片的凹槽中,并将玉米叶样品放入其中。用方形盖玻片盖住样品(图2)。
  5. 在明场模式下使用40x物镜找到目标区域。 
  6. 切换到使用488 nm或514 nm激光器和582/75 nm带通发射滤光片进行共焦采集,以检测FM4-64发射(参见注释)。针孔直径应低于两个透气盘(约100微米)。
  7. 设置成像参数(激光功率,探测器增益或曝光时间),以提供明亮的信号而不会过度曝光。 
  8. 获取目标区域的一个图像。 
  9. 在不从显微镜中取出样品的情况下,使用滤纸排出缓冲液,立即加入基于PBS的台盼蓝溶液,从最低浓度(1μM)开始。
  10. 如上面的步骤所述,获取一个图像并改变到下一个更高的浓度。重复上述步骤,直至获得所有浓度(1,5,10,50,100μM)的图像。


    图2.玉米叶片样品的制备 A.玉米幼苗,此处用作样品材料。 B.使用镊子将一段表皮(箭头)从叶子上剥下,并与一小片叶片一起切出。 C.样品浸没在FM4-64溶液中。 D.样品在PBS中在染色罐中洗涤。 E.样品安装在大盖玻片上,通过粘贴边缘在其上形成中央凹槽。 F.应用小盖玻片后的样品。 G.在不从显微镜中取出样品的情况下改变猝灭剂溶液。

数据分析

  1. 从成像系统以与ImageJ兼容的格式(例如,tif)导出所有图像数据并导入ImageJ。 
  2. 通过检查细胞之间(如果是酵母或细菌细胞)或细胞中心(如果细胞较大的情况)中的背景信号强度消除背景信号,将黑色水平调整为此值并按“应用”(图3)。


    图3.通过调整黑色水平进行背景扣除。 A. 拟南芥根样品,在不含质膜的区域绘制感兴趣区域。 B.此区域的平均值用作按“应用”重新分配的图像的黑色电平和强度值。

  3. 画一个覆盖细胞周边的矩形感兴趣区域(ROI)(图4),按“Ctrl + M”测量平均灰度值。请勿更改一种样本类型的不同图像之间的ROI大小。如果应在多个图像上测量相同的区域,例如,对于玉米叶样本,将ROI添加到ROI管理器。 
  4. 为每种类型的样品和每种猝灭剂浓度测量至少50个ROI。在酵母样品中,每个细胞定义一个ROI。在植物样本中,可以在同一细胞的不同壁上定义几个ROI。


    图4.用于测量信号强度的示例性感兴趣区域。 A.酵母细胞。 B. 拟南芥根细胞。 C.玉米叶细胞。比例尺,10μm。

  5. 将测量数据导出到电子表格软件并计算平均值。
  6. 根据Lehrer(1971)计算淬火效率:



    其中,F 0 是缓冲液中没有台盼蓝的样品的荧光强度; F是在浓度为[Q]的台盼蓝存在下样品的荧光强度;
    K是该样品系统的猝灭效率,其可以从浓度的斜率[Q](x轴)得出,而不是(y轴)。

    较高的猝灭效率表明猝灭剂更容易进入质膜,这可以作为细胞壁孔隙度的良好指标(Liu et al。,2019)。关于猝灭效率和细胞壁结构之间关系的详细讨论可以在Liu 等人,,(2019)中找到。从同一样品中获得的结果变化取决于样品类型(图5)。


    图5.在酵母和植物细胞上测量的示例性猝灭效率数据。将样品暴露于化学处理或在生长条件下改变12小时(酵母)或24小时(植物)。植物样品中一个处理组内的变异通常高于酵母样品。 

笔记

FM4-64的发射在640nm和750nm附近显示出宽峰。台盼蓝在与640和680nm之间的某些细胞壁组分结合时显示出发出荧光(Liesche 等,,2015)。为避免与潜在的台盼蓝发射重叠,建议检测580 nm附近的FM4-64或超过700 nm的长通滤波器。

食谱

注意:使用超纯水准备所有介质(从25°C时设置为电阻率为18MΩcm的净化系统中提取)。

  1. YDP媒体
    2%(w / v)胰蛋白胨
    1%(w / v)酵母提取物
    2%(w / v)葡萄糖
    注意:胰蛋白胨和酵母提取物以及葡萄糖溶液的混合物应在121°C下灭菌20分钟,并在冷却后调整至最终浓度。
  2. 半强度MS培养基
    0.22%(w / v)Murashige和Skoog(MS)盐
    1%(w / v)蔗糖
    用2 M KOH将pH调节至5.8 0.8%(w / v)琼脂
    在121℃下灭菌20分钟,向每个玻璃培养皿中倒入25ml培养基
  3. PBS溶液(pH 7.2-7.4)
    NaCl 130 mM
    KCl 2.6 mM
    Na 2 HPO 4 7 mM
    KH 2 PO 4 1.2mM
  4. FM4-64库存解决方案
    在DMSO中制备2mM FM4-64储备液
    在-20°C下在黑暗中以100μl的等分试样储存
  5. 台盼蓝蓝色库存解决方案
    在PBS中制备10 mM锥虫蓝原液,在黑暗中保持4°C
    注意:在通风橱内或抽吸下将天平称重,并在处理固体时戴上口罩。台盼蓝被怀疑会导致癌症,特别是粉末状,因为它很容易被吸入。

致谢

该协议改编自我们以前的工作(Liu et al。,2019)。这项工作得到了国家自然科学基金(项目拨款号31700313),陕西省科学基金(100人才计划)和Villum基金会(项目拨款号022868)的支持。

利益争夺

作者声明不存在竞争利益。

参考

  1. Adani,F.,Papa,G.,Schievano,A.,Cardinale,G.,D'Imporzano,G。和Tambone,F。(2011)。 保护纤维素免受酶侵袭的细胞壁的纳米结构。 Environ Sci Technol 45(3):1107-1113。 
  2. De Nobel,J.G.,Klis,F.M.,Munnik,T.,Priem,J。和van den Ende,H。(1990)。 酿酒酵母中相对细胞壁孔隙度的测定 Kluyveromyces lactis 和 Schizosaccharomyces pombe 。 Yeast(Chichester,England) 6(6):483-490。 
  3. Derksen,J.,Janssen,G.J.,Wolters-Arts,M.,Lichtscheidl,I.,Adlassnig,W.,Ovecka,M.,Doris,F。和Steer,M。(2011)。 在尖端建立具有高孔隙度的墙体结构,并在生长的 Nicotiana花粉管中维持tabacum 。 Plant J 68(3):495-506。 
  4. Ding,S.Y.,Liu,Y.S。,Zeng,Y.,Himmel,M。E.,Baker,J。O.和Bayer,E.A。(2012)。 植物细胞壁纳米级结构如何与酶消化率相关? Science 338(6110):1055-1060。
  5. Donaldson,L.A.,Kroese,H.W.,Hill,S.J。和Franich,R.A。(2015)。 使用小碳水化合物分子和共聚焦荧光显微镜检测木材细胞壁孔隙度。 J Microsc 259(3):228-236。
  6. Himmel,M。E.,Ding,S。Y.,Johnson,D。K.,Adney,W。S.,Nimlos,M。R.,Brady,J。W. and Foust,T。D.(2007)。 生物质顽固性:用于生物燃料生产的工程植物和酶。 Science 315(5813):804-807。
  7. Liesche,J.,Marek,M.,GüntherPomorski,T。(2015)。 使用台盼蓝进行细胞壁染色可以定量分析酵母细胞的形态变化。 Front Microbiol 6:107。
  8. Lehrer,S。S.(1971)。溶质扰动蛋白质荧光。 通过碘离子淬灭模型化合物和溶菌酶的tryptophyl荧光。 Biochemistry 10(17):3254-3263。
  9. Liu,X.,Li,J.,Zhao,H.,Liu,B.,Günther-Pomorski,T.,Chen,S。和Liesche,J。(2019)。 量化细胞壁孔隙度的新工具将壁结构与细胞生长和药物摄取联系起来。 J Cell Biol 218(4):1408-1421。
  10. Schindelin,J.,Arganda-Carreras,I.,Frize,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B 。,Tinevez,JY,White,DJ,Hartenstein,V.,Eliceiri,K.,Tomancak,P。和Cardona,A。(2012)。 斐济:生物图像分析的开源平台。 Nat方法 9(7):676-682。
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  13. Xiao,C.,Zhang,T.,Zheng,Y.,Cosgrove,D。J. and Anderson,C。T.(2016)。 木葡聚糖缺乏会破坏拟南芥中的微管稳定性和纤维素生物合成,改变细胞生长和形态发生。 植物生理学 170(1):234-249。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Liu, X., Günther Pomorski, T. and Liesche, J. (2019). Non-invasive Quantification of Cell Wall Porosity by Fluorescence Quenching Microscopy. Bio-protocol 9(16): e3344. DOI: 10.21769/BioProtoc.3344.
  2. Liu, X., Li, J., Zhao, H., Liu, B., Günther-Pomorski, T., Chen, S. and Liesche, J. (2019). Novel tool to quantify cell wall porosity relates wall structure to cell growth and drug uptake. J Cell Biol 218(4): 1408-1421.
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