Determination of Local pH Differences within Living Salmonella Cells by High-resolution pH Imaging Based on pH-sensitive GFP Derivative, pHluorin(M153R)
采用基于pH敏感性GFP衍生物pHluorin(M153R)的高分辨率pH成像测定活体沙门氏菌细胞中局部pH值的差异   

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mBio
20-Dec 2016

 

Abstract

The bacterial flagellar type III protein export apparatus is composed of a transmembrane export gate complex and a cytoplasmic ATPase complex. The export apparatus requires ATP hydrolysis and the proton motive force across the cytoplasmic membrane to unfold and transport flagellar component proteins for the construction of the bacterial flagellum (Minamino, 2014). The export apparatus is a proton/protein antiporter that couples the proton flow with protein transport through the gate complex (Minamino et al., 2011). A transmembrane export gate protein, FlhA, acts as an energy transducer along with the cytoplasmic ATPase complex (Minamino et al., 2016). To directly measure the proton flow through the FlhA channel that is coupled with the flagellar protein export, we have developed an in vivo pH imaging system with high spatial and pH resolution (Morimoto et al., 2016). Here, we describe how we measure the local pH near the export apparatus in living Salmonella cells (Morimoto et al., 2016). Our approach can be applied to a wide range of living cells. Because local pH is one of the most important parameters to monitor cellular activities of living cells, our protocol would be widely used for diverse areas of life sciences.

Keywords: Intracellular pH (细胞内pH), Local pH (局部pH值), Fluorescence microscopy (荧光显微镜检查), Bacteria (细菌), Bacterial flagellum (细菌鞭毛), Proton motive force (质子动力), Type III protein export (III型蛋白质输出)

Background

The proton channel activities of transmembrane proton channel complexes have been detected as reduction in cytoplasmic pH of bacterial cells (Morimoto et al., 2010; Che et al., 2014; Furukawa et al., 2017). However, to measure the proton channel activity of membrane complexes in living cells in detail, precise measurements of the local cytoplasmic pH are required. A derivative of the green fluorescence protein (GFP), pHluorin, with excitation at wavelengths of 410 and 470 nm and emission at 508 nm is a useful probe to measure the cytoplasmic pH in living cells (Miesenböck et al., 1998). This probe enables us to measure the intracellular pH precisely and quantitatively because the fluorescent intensity ratio of the two excitation wavelengths R410/470 shows a remarkable pH dependence. pHluorin can be fused genetically to a target protein to monitor the local pH around the protein. However, proteolytic cleavage often removes pHluorin from its fusion protein, not only causing a poor signal-to-noise ratio but also leading to the wrong interpretations of the data. We have found that the mutation M153R in pHluorin significantly stabilizes its fusion products while retaining the marked pH dependence of the 410/470 nm excitation ratio of the fluorescence intensity (Morimoto et al., 2011). We therefore used pHluorin(M153R) to develop a method to measure the local cytoplasmic pH in living bacterial cells in a highly quantitative and precise manner (Morimoto et al., 2016).

Materials and Reagents

  1. 1.5 ml microcentrifuge tubes
  2. Glass test tubes (IWAKI, catalog number: A-18P )
  3. Double-sided tape (NICHIBAN, catalog number: NW-5 )
  4. 24 x 32 mm coverslips (thickness: 0.12-0.17 mm) (Matsunami Glass, catalog number: C024321 )
  5. 18 x 18 mm coverslips (thickness: 0.12-0.17 mm) (Matsunami Glass, catalog number: C018181 )
  6. Pipette tips
  7. Filter paper (Toyo Roshi Kaisha, ADVANTEC, catalog number: 00011090 )
  8. Salmonella YVM1004 strain (pHluorin(M153R)-fliG) (Morimoto et al., 2011)
  9. Salmonella YVM1049 strain (∆fliH-fliI flhB(P28T) pHluorin(M153R)-fliG) (Morimoto et al., 2016)
  10. Salmonella SJW1103 strain (wild type for motility and chemotaxis) (Yamaguchi et al., 1984)
  11. Salmonella SJW1368 strain (∆(cheW-flhD); flagellar master operon mutant) (Ohnishi et al., 1994)
  12. pYVM008 (pTrc99A/pHluorin(M153R)-FliG) (Morimoto et al., 2016)
  13. Purified pHluorin(M153R)-FliG-His6 protein in PBS
    Note: The pHluorin(M153R)-FliG-His protein is purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography from the soluble fractions of E. coli BL21(DE3) cells overexpressing pHluorin(M153R)-FliG-His as described by Minamino et al. (2000).
  14. Ampicillin sodium salt (Wako Pure Chemical Industries, catalog number: 014-23302 )
  15. Gramicidin (Thermo Fisher Scientific, catalog number: G6888 )
  16. Potassium benzoate (Wako Pure Chemical Industries, catalog number: 164-19342 )
  17. Bacto tryptone (BD, BactoTM, catalog number: 211705 )
  18. Yeast extract (BD, BactoTM, catalog number: 212750 )
  19. Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 192-13925 )
  20. Bacto agar (BD, BactoTM, catalog number: 214010 )
  21. Potassium dihydrogen phosphate (KH2PO4) (Wako Pure Chemical Industries, catalog number: 164-22635 )
  22. Dipotassium hydrogen phosphate (K2HPO4) (Wako Pure Chemical Industries, catalog number: 164-04295 )
  23. Ethylenediamine-N,N,N’,N’-tetraacetic acid disodium salt dihydrate (2NA) (EDTA) (Dojindo, catalog number: N001 )
  24. LB medium (see Recipes)
  25. TB medium (see Recipes)
  26. LB agar plate (see Recipes)
  27. Motility buffer (see Recipes)

Equipment

  1. pH meter (Beckman Coulter, model: Φ34 )
  2. Shaking incubator (30 °C, at 200 rpm) (TAITEC, model: BR-40LF )
  3. Spectrophotometer (able to measure OD600) (Shimadzu, model: UV-1800 )
  4. Centrifuge (able to hold 1.5 ml tubes, spin at 6,000 x g) (TOMY SEIKO, model: MX-305 )
  5. Single channel pipettes (1,000 µl, 100 µl) (PIPETMAN® Classic; Gilson, models: P1000 and P100 )
  6. Inverted fluorescence microscope (Olympus, model: IX71 ) (see Figure 1)
    1. 150x oil immersion objective lens (Olympus, model: UApo150XOTIRFM , NA1.45)
    2. Electron-multiplying charge-coupled device (EMCCD) camera (Hamamatsu Photonics, model: C9100-02 )
    3. Excitation filter (Omega Optical, model: 400AF30 )
    4. Excitation filter (Olympus, model: BP470-490 )
    5. Emission filter (Omega Optical, model: 520DF40 )
    6. High-speed wavelength switcher (Sutter Instrument, model: Lambda DG-4 )
    7. Dichroic mirror (Semrock, model: FF510-Di01-25x36 )

Software

  1. MetaMorph (Molecular Devices)
  2. ImageJ (National Institutes of Health, https://imagej.nih.gov/ij/)
  3. IGOR Pro (WaveMetrics)
  4. Microsoft Excel (Microsoft)

Procedure

Note: All procedures are carried out at room temperature unless otherwise specified.

  1. Fluorescence microscopy
    1. Pick a single colony of Salmonella cells expressing pHluorin(M153R)-FliG (YVM1004 and YVM1049 for local pH measurements and SJW1368 transformed with pYVM008 for in vivo calibration) from LB agar plate and inoculate it into a glass test tube with 5 ml LB and shaking at 150 rpm at 30 °C overnight. When SJW1368/pYVM008 cells are grown, ampicillin is added to a final concentration of 100 µg/ml.
    2. Transfer 50 µl of overnight culture to 5 ml of fresh TB and incubate at 30 °C with shaking at 150 rpm until the cell density reaches an OD600 of ca. 1.8.
      Note: It takes about 15 h.
    3. Transfer 500 µl of each culture to a 1.5 ml microcentrifuge tube and then collect the cells by centrifugation (6,000 x g, 2 min).
    4. Discard supernatant.
    5. Resuspend the cell pellet in 1.0 ml of motility buffer.
    6. Centrifuge at 6,000 x g for 2 min.
    7. Discard supernatant.
    8. Repeat steps A5-A7 twice.
    9. Resuspend the cells in 500 µl of fresh motility buffer.
    10. Make a tunnel slide by sandwiching double-sided tape between a 24 x 32 mm coverslip (bottom side) and an 18 x 18 mm coverslip (top side) (see Video 1).

      Video 1. Preparation for a tunnel slide (from Morimoto et al., 2017)

    11. Add 50 µl of the cell suspension to the tunnel slide and leave for 5 min to attach the cells to the coverslip surface.
    12. Wash out unbound cells by supplying 100 µl of the motility buffer using a pipette (P100). Absorb the excess amount of the buffer with a piece of a filter paper. (see Video 1)
    13. Put the sample on a custom-built inverted fluorescence microscope (Figure 1).


      Figure 1. Schematic of pH imaging system using ratiometric pHluorin(M153R). The pH imaging system was built on an inverted fluorescence microscope with a 150x oil immersion objective lens, 1.6x variable inserts and a C9100-02 EMCCD camera. The fluorescent spots of pHluorin(M153R)-FliG were excited by a xenon lamp with two excitation filters, 400AF30 (Omega Optical) for 410-nm excitation and BP470-490 (Olympus) for 470-nm excitation. A high-speed wavelength switcher was used to switch these two excitation filters. Fluorescence emission was passed through a dichroic mirror (DM) (FF510-Di01-25x36, Semrock) and an emission filter (EmF) (520DF40, Omega Optical). Each fluorescent image was captured by the EMCCD camera. The high-speed wavelength switcher and the EMCCD camera were controlled by the MetaMorph software. ND: neutral density filter.

    14. Select a 150x objective and insert a 1.6x variable lens.
      Note: High magnification is required to measure the local cytoplasmic pH in living bacterial cells (150x objective and 1.6x variable lens with C9100-02 EMCCD [cell size: 8 x 8 µm]: 33 nm/pixel).
    15. Switch between 410 and 470 nm excitation filters using a high-speed wavelength switcher.
    16. Capture each image of the pHluorin(M153R)-FliG fluorescent spots by the EMCCD camera with a 5 sec exposure time (Figure 2).
      Notes:
      1. The culture is diluted with motility buffer to adjust the number of cells to about 10 cells per 10 x 10 µm image field.
      2. Fluorescent spots are excited by a xenon lamp through an ND filter to avoid photobleaching. Time-lapse measurements are required to test the effect of photobleaching.


        Figure 2. Typical fluorescence image of Salmonella cells expressing pHluorin(M153R)-FliG. Fluorescence and bright field images of the Salmonella YVM1004 strain. The red square indicates a typical fluorescent spot of pHluorin(M153R)-FliG.

  2. In vivo calibration
    1. After step A8: Resuspend SJW1368 cells carrying pYVM008 in 500 µl of motility buffer containing 20 µM gramicidin and 20 mM potassium benzoate.
    2. Add 50 µl of the cell suspension to the tunnel slide and leave for 5 min.
    3. Wash out unbound cells by supplying 100 µl of motility buffer containing 20 µM gramicidin and 20 mM potassium benzoate. Absorb the excess amount of the buffer using a filter paper.
    4. Put the sample on the fluorescence microscope.
    5. Acquire ratiometric fluorescence images of the cells at external pH 6.0, 6.5, 7.0 or 7.5 in the presence of 20 µM gramicidin and 20 mM potassium benzoate. The tunnel slide and the cells are freshly prepared for each external pH measurement.

  3. In vitro calibration
    1. Suspend purified pHluorin(M153R)-FliG proteins in motility buffer with distinct pH values, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 or 8.5.
    2. Add 50 µl of the solution containing pHluorin(M153R)-FliG proteins to the tunnel slide and absorb the excess amount of the solution with a piece of filter paper.
    3. Acquire fluorescence images of the pHluorin(M153R)-FliG protein solution excited by 410 and 470 nm at each pH values.
      Note: When the protein concentration of pHluorin(M153R)-FliG is too high, its fluorescence intensity is considerably high, thereby saturating the sensor chip of the EMCCD camera. Therefore, it is necessary to adjust the protein concentration of pHluorin(M153R)-FliG to ca. ~100 µg/ml in order to make an in vitro calibration curve.

Data analysis

Analyze fluorescent images by image analysis software IGOR Pro or ImageJ.

  1. Calibration curve
    1. Define the total fluorescence intensity of the cell body determined by the image profile of the SJW1368 cells carrying pYVM008 (Figure 3).
      Note: Since the flhDC deletion strain produces no flagella, the pHluorin(M153R)-FliG probe freely diffuses in the cytoplasm.


      Figure 3. Typical image processes for determination of cytoplasmic bulk pH by pHluorin(M153R)-FliG. See text for details.

    2. Define the instrumental background intensity as the mean pixel intensity within a 50 x 50 pixel ROI (region of interest) of a nearby cell-less region.
    3. Define the autofluorescence intensity as the mean pixel intensity of 50 wild-type SJW1103 cells producing no fluorescent proteins.
    4. Subtract the instrumental background and the autofluorescence of the cells from the total fluorescence intensity of the fluorescent cell body to define the fluorescence intensity of freely diffusing pHluorin(M153R)-FliG within the cells at 410 and 470 nm excitation wavelengths.
    5. Calculate the fluorescence intensity ratio, R410/470, of the pHluorin(M153R)-FliG probe.
    6. Determine the cytoplasmic pH from the R410/470 of the pHluorin(M153R)-FliG that was obtained at each external pH in the presence of 20 µM gramicidin and 20 mM potassium benzoate, both of which equilibrate the pH inside and outside Salmonella cells, to make the in vivo calibration curve (Figure 4).
      Note: In vivo calibration clearly shows that pHluorin(M153R)-FliG works as a pH indicator in living cells.


      Figure 4. Calibration curve of pHluorin(M153R)-FliG in vivo and in vitro. Fluorescent images of purified pHluorin(M153R)-His protein solution were taken by the pH imaging system over a pH range from 5.5 to 8.5. The fluorescent intensity ratio 410/470 was calculated at each pH value (black, in vitro), and the data were fitted by a sigmoid function. Then, Salmonella SJW1368 cells harboring pYVM008 were observed by the same system in motility buffer at four distinct pH values, 6.0, 6.5, 7.0 and 7.5, in the presence of 20 µM gramicidin and 20 mM potassium benzoate, and the intracellular pH was measured (orange, in vivo). Vertical bars indicate standard deviations (Modified from Morimoto et al., 2016).

    7. Define the fluorescence intensity of the purified pHluorin(M153R)-FliG protein after subtraction of the instrumental background intensity as the mean pixel intensity within a 100 x 100 pixel ROI of an image of the solution without fluorescent proteins.
    8. Calculate R410/470 of the purified pHluorin(M153R)-FliG at each pH value.
    9. Plot the emission intensity ratios as a function of pH to make the in vitro calibration curve over a pH range from 5.5 to 8.5 (Figure 4).
    10. Fit the calibration data by a sigmoid curve function using the IGOR Pro software.



  2. Determination of local pH values
    1. Apply a rectangular mask for the fluorescent spots of the pHluorin(M153R)-FliG of 20 x 20 pixels to the ROI in a fluorescent image of the YVM1004 cells (Figure 2).
    2. Subtract the total background intensity including the instrumental background and the autofluorescence of the cell from each pixel value. The instrumental background intensity is defined as the mean pixel intensity within a 50 x 50 pixel ROI of a nearby cell-less region. The autofluorescence intensity of the cell is defined as the mean pixel intensity within the ROI of a non-fluorescent cell SJW1103.
    3. Define the fluorescence intensity of a single fluorescent spot of the pHluorin(M153R)-FliG probe as an integral fluorescent intensity value within the ROI.
    4. Calculate the ratio of the fluorescent intensity of the pHluorin(M153R)-FliG spots at 410 and 470 nm excitation wavelengths.
    5. Determine the local pH from the R410/470 value of each fluorescent spot using the in vitro calibration curve.
    6. Calculate the average of the local pH values and the standard error of the mean from the data of more than 100 fluorescent spots using Microsoft Excel (Microsoft) (Figure 5).


      Figure 5. Measurement of local pH using pHluorin(M153R)-FliG. The local pH around the flagellar type III protein export apparatus of the pH-fliG (YVM1004) and the ∆fliHI flhB(P28T) pH-fliG (YVM1049) cells was measured. More than 300 fluorescent spots were analyzed. Vertical bars indicate standard errors (Modified from Morimoto et al., 2016).

Notes

Salmonella cells expressing pHluorin(M153R)-FliG should be incubated with shaking for more than 5 h to be fully matured the fluorophores of pHluorin(M153R), because sufficient fluorescence intensity is required to detect the local pH.

Recipes

  1. LB medium (per liter)
    10 g Bacto tryptone
    5 g yeast extract
    5 g NaCl
  2. TB medium (per liter)
    10 g Bacto tryptone
    10 mM potassium phosphate, pH 7.0
  3. LB agar plate
    LB medium
    1.5% Bacto agar
  4. Motility buffer
    10 mM potassium phosphate
    0.1 mM EDTA
    Adjust to different pH values: 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 or 8.5

Acknowledgments

This protocol was modified from a previous work (Morimoto et al., 2016). This research was supported in part by JSPS KAKENHI Grant Numbers JP15K14498 and JP15H05593 to YVM, JP25000013 to KN and JP26293097 to TM, and MEXT KAKENHI Grant Numbers JP26115720 and JP15H01335 to YVM and JP24117004, JP25121718 and JP15H01640 to TM.

References

  1. Che, Y. S., Nakamura, S., Morimoto, Y. V., Kami-ike, N., Namba, K. and Minamino, T. (2014). Load-sensitive coupling of proton translocation and torque generation in the bacterial flagellar motor. Mol Microbiol 91(1): 175-184.
  2. Furukawa, A., Yoshikaie, K., Mori, T., Mori, H., Morimoto, Y. V., Sugano, Y., Iwaki, S., Minamino, T., Sugita, Y., Tanaka, Y. and Tsukazaki, T. (2017). Tunnel formation inferred from the I-form structures of the proton-driven protein secretion motor SecDF. Cell Rep 19(5): 895-901.
  3. Miesenböck, G., De Angelis, D. A. and Rothman, J. E. (1998). Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394(6689): 192-195.
  4. Minamino, T. (2014). Protein export through the bacterial flagellar type III export pathway. Biochim Biophys Acta 1843(8): 1642-1648.
  5. Minamino, T., Morimoto, Y. V., Hara, N., Aldridge, P. D. and Namba, K. (2016). The bacterial flagellar type III export gate complex is a dual fuel engine that can use both H+ and Na+ for flagellar protein export. PLoS Pathog 12(3): e1005495.
  6. Minamino, T., Morimoto, Y. V., Hara, N. and Namba, K. (2011). An energy transduction mechanism used in bacterial flagellar type III protein export. Nat Commun 2: 475.
  7. Minamino, T., Yamaguchi, S. and Macnab, R. M. (2000). Interaction between FliE and FlgB, a proximal rod component of the flagellar basal body of Salmonella. J Bacteriol 182(11): 3029-3036.
  8. Morimoto, Y. V., Che, Y. S., Minamino, T. and Namba, K. (2010). Proton-conductivity assay of plugged and unplugged MotA/B proton channel by cytoplasmic pHluorin expressed in Salmonella. FEBS Lett 584(6): 1268-1272.
  9. Morimoto, Y. V., Kami-ike, N., Miyata, T., Kawamoto, A., Kato, T., Namba, K. and Minamino, T. (2016). High-resolution pH imaging of living bacterial cells to detect local pH differences. mBio 7(6).
  10. Morimoto, Y. V., Kojima, S., Namba, K. and Minamino, T. (2011). M153R mutation in a pH-sensitive green fluorescent protein stabilizes its fusion proteins. PLoS One 6(5): e19598.
  11. Morimoto, Y. V., Namba, K. and Minamino, T. (2017). Bacterial intracellular sodium ion measurement using CoroNa Green. Bio Protoc 7(1) e2092.
  12. Ohnishi, K., Ohto, Y., Aizawa, S., Macnab, R. M. and Iino, T. (1994). FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium. J Bacteriol 176(8): 2272-2281.
  13. Yamaguchi, S., Fujita, H., Sugata, K., Taira, T. and Iino, T. (1984).Genetic analysis of H2, the structural gene for phase-2 flagellin in Salmonella. J Gen Microbiol 130(2): 255-265.

简介

细菌鞭毛III型蛋白质输出装置由跨膜出口门复合物和胞质ATP酶复合物组成。出口设备需要ATP水解和跨细胞质膜的质子动力来展开和转运鞭毛成分蛋白以构建细菌鞭毛(Minamino,2014)。出口设备是质子/蛋白质反向转运体,其将质子流与通过门络合物的蛋白质转运相结合(Minamino等,2011)。跨膜输出门蛋白FlhA与胞质ATP酶复合物一起作为能量转导(Minamino等,2016)。为了直接测量通过与鞭毛蛋白输出相结合的FlhA通道的质子流,我们开发了具有高空间和pH分辨率的体内pH成像系统(Morimoto等,2016)。在这里,我们描述了我们如何测量生活沙门氏菌细胞出口设备附近的局部pH(Morimoto et al。,2016)。我们的方法可以应用于广泛的活细胞。由于局部pH值是监测活细胞活性的最重要参数之一,因此我们的方案将广泛应用于生命科学的各个领域。
【背景】已经检测到跨膜质子通道复合物的质子通道活性是细菌细胞的细胞质pH降低(Morimoto等,2010; Che等,2014; Furukawa等,2017)。然而,为了详细测量活细胞中膜复合物的质子通道活性,需要精确测量局部细胞质pH。绿色荧光蛋白(GFP)的衍生物,pHluorin,激发波长为410和470 nm,发射于508 nm是测量活细胞胞质pH值的有用探针(Miesenböcket al。,1998)。该探针使得我们能够精确和定量地测量细胞内pH,因为两个激发波长R410 / 470的荧光强度比显示出显着的pH依赖性。 pHluorin可以遗传融合到靶蛋白上,以监测蛋白质周围的局部pH。然而,蛋白水解切割通常从其融合蛋白中去除pHluorin,不仅导致差的信噪比,而且导致数据的错误解释。我们发现,pH荧光素中的突变M153R显着稳定其融合产物,同时保持了荧光强度的410/470nm激发比的显着的pH依赖性(Morimoto等,2011)。因此,我们使用pHluorin(M153R)开发了一种以高度定量和精确的方式测量活细菌细胞中局部细胞质pH值的方法(Morimoto等,2016)。

关键字:细胞内pH, 局部pH值, 荧光显微镜检查, 细菌, 细菌鞭毛, 质子动力, III型蛋白质输出

材料和试剂

  1. 1.5 ml微量离心管
  2. 玻璃试管(IWAKI,目录号:A-18P)
  3. 双面胶带(NICHIBAN,目录号:NW-5)
  4. 24 x 32毫米盖玻片(厚度:0.12-0.17毫米)(松本玻璃,目录号:C024321)
  5. 18×18毫米盖玻片(厚度:0.12-0.17毫米)(松本玻璃,目录号:C018181)
  6. 移液器提示
  7. 滤纸(Toyo Roshi Kaisha,ADVANTEC,目录号:00011090)
  8. 沙门氏菌 YVM1004菌株(pH5.R)-fliG )(Morimoto等人,2011)
  9. 沙门氏菌 YVM1049菌株(ΔFiH-fliI flhB)(P28T)pHluorin(M153R)-fliG )(Morimoto等人, em>,2016)
  10. 沙门氏菌 SJW1103菌株(运动性和趋化性的野生型)(Yamaguchi等人,1984)
  11. 沙门氏菌 SJW1368菌株(Δ(cheW-flhD);鞭毛主操纵子突变体)(Ohnishi等人,1994)
  12. pYVM008(pTrc99A / pHluorin(M153R)-FliG)(Morimoto等人,2016)
  13. PBS中纯化的pHluorin(M153R)-FliG-His6蛋白质
    注意:通过镍 - 次氮基三乙酸(Ni-NTA)亲和层析从过表达pHluorin(M153R)-FliG-1的大肠杆菌BL21(DE3)细胞的可溶性级分纯化pHluorin(M153R)-FliG-His蛋白质,他由Minamino等人(2000)。
  14. 氨苄青霉素钠盐(和光纯药,目录号:014-23302)
  15. 格列美星(Thermo Fisher Scientific,目录号:G6888)
  16. 苯甲酸钾(和光纯药,目录号:164-19342)
  17. Bacto胰蛋白胨(BD,Bacto TM ,目录号:211705)
  18. 酵母提取物(BD,Bacto TM ,目录号:212750)
  19. 氯化钠(NaCl)(Wako Pure Chemical Industries,目录号:192-13925)
  20. Bacto琼脂(BD,Bacto TM ,目录号:214010)
  21. 磷酸二氢钾(KH 2 PO 4)(Wako Pure Chemical Industries,目录号:164-22635)
  22. 磷酸氢二钾(K 2 H 2 HPO 4)(和光纯药工业公司,目录号:164-04295)
  23. 乙二胺-N,N,N',N'-四乙酸二钠盐二水合物(2NA)(EDTA)(Dojindo,目录号:N001)
  24. LB培养基(见食谱)
  25. TB介质(见配方)
  26. LB琼脂平板(参见食谱)
  27. 运动缓冲(见配方)

设备

  1. pH计(Beckman Coulter,型号:Φ34)
  2. 摇动培养箱(30℃,200rpm)(TAITEC,型号:BR-40LF)
  3. 分光光度计(能够测量OD 600)(Shimadzu,型号:UV-1800)
  4. 离心机(能够容纳1.5ml管,以6,000xg旋转)(TOMY SEIKO,型号:MX-305)
  5. 单通道移液管(1000μl,100μl)(PIPETMAN Classic; Gilson,型号:P1000和P100)
  6. 倒置荧光显微镜(Olympus,型号:IX71)(见图1)
    1. 150x油浸物镜(Olympus,型号:UApo150XOTIRFM,NA1.45)
    2. 电子倍增电荷耦合器件(EMCCD)相机(Hamamatsu Photonics,型号:C9100-02)
    3. 激发滤波器(Omega Optical,型号:400AF30)
    4. 励磁过滤器(Olympus,型号:BP470-490)
    5. 发射滤光片(Omega Optical,型号:520DF40)
    6. 高速波长切换器(Sutter Instrument,型号:Lambda DG-4)
    7. 分色镜(Semrock,型号:FF510-Di01-25x36)

软件

  1. MetaMorph(Molecular Devices)
  2. ImageJ(National Institutes of Health, https://imagej.nih.gov/ij/
  3. IGOR Pro(WaveMetrics)
  4. Microsoft Excel(Microsoft)

程序

注意:除非另有说明,所有程序均在室温下进行。

  1. 荧光显微镜
    1. 从LB琼脂板上挑选表达pHluorin(M153R)-FliG(YVM1004和YVM1049用于局部pH测量和用pYVM008转化的SJW1368进行体内校准)的单个菌落细胞<并用5ml LB接种到玻璃试管中,在30℃下以150rpm摇动过夜。当生长SJW1368 / pYVM008细胞时,加入氨苄青霉素至终浓度为100μg/ ml。
    2. 将50μl过夜培养物转移到5ml新鲜的TB中,并在30℃下以150rpm摇动孵育直到细胞密度达到约600的OD 600。 1.8。
      注意:大约需要15小时。
    3. 转移500μl每种培养物至1.5 ml微量离心管,然后通过离心(6,000 x g,2分钟)收集细胞。
    4. 弃去上清液。
    5. 将细胞沉淀重悬于1.0ml运动缓冲液中
    6. 以6,000 x g离心2分钟。
    7. 弃去上清液。
    8. 重复步骤A5-A7两次。
    9. 将细胞重新悬浮在500μl新鲜运动缓冲液中。
    10. 通过将双面胶带夹在24 x 32毫米的盖玻片(底侧)和18 x 18毫米的盖玻片(上侧)之间进行隧道滑动(见视频1)。

      Video 1. Preparation for a tunnel slide (from Morimoto et al., 2017)

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    11. 将50μl细胞悬浮液加入隧道载玻片上,放置5分钟,将细胞连接到盖玻片表面。
    12. 通过使用移液管(P100)提供100μl动力缓冲液清洗未结合的细胞。用一张滤纸吸收多余的缓冲液。 (见视频1)
    13. 将样品放在定制的倒置荧光显微镜上(图1)

      图1.使用比例pHluorin(M153R)的pH成像系统的示意图。 pH成像系统建立在具有150x油浸物镜,1.6x可变插入物和C9100-02 EMCCD相机的倒置荧光显微镜上。通过具有两个激发滤光器的氙灯(用于410nm激发的400AF30(Omega Optical))和用于470nm激发的BP470-490(Olympus))激发pHluorin(M153R)-FliG的荧光斑点。使用高速波长切换器来切换这两个激励滤波器。荧光发射通过分色镜(DM)(FF510-Di01-25x36,Semrock)和发射滤光片(EmF)(520DF40,Omega Optical)。每个荧光图像被EMCCD相机捕获。高速波长切换器和EMCCD摄像机由MetaMorph软件控制。 ND:中性密度滤光片。

    14. 选择150x物镜并插入1.6x可变镜头。
      注意:需要高倍率来测量活细菌细胞中的局部细胞质pH(150x物镜和1.6x可变透镜,C9100-02CEMCD [单元尺寸:8×8μm]:33nm /像素)。 em>
    15. 使用高速波长切换器在410和470 nm的激发滤波器之间切换
    16. 通过EMCCD照相机以5秒的曝光时间捕获pHluorin(M153R)-FliG荧光斑点的每个图像(图2)。
      注意:
      1. 用动力缓冲液稀释培养物,以将细胞数量调整到每10×10 10个图像区域约10个细胞。
      2. 荧光斑被氙灯通过ND滤光器激发,以避免光漂白。需要延时测量来测试漂白效果。


        图2.表达pHluorin(M153R)-FliG的沙门菌细胞的典型荧光图像。 沙门氏菌 YVM1004菌株的荧光和亮场图像。红色方块表示pHluorin(M153R)-FliG的典型荧光斑点。

  2. 校准
    1. 步骤A8:将携带pYVM008的SJW1368细胞重悬于含有20μM短杆菌肽和20mM苯甲酸钾的500μl动力缓冲液中。
    2. 将50μl细胞悬浮液加入隧道载玻片,放置5 min
    3. 通过提供100μl含有20μM短杆菌肽和20mM苯甲酸钾的运动缓冲液来清洗未结合的细胞。使用滤纸吸收过量的缓冲液。
    4. 将样品放在荧光显微镜上
    5. 在20μM短杆菌肽和20mM苯甲酸钾的存在下,在外部pH 6.0,6.5,7.0或7.5的条件下获得细胞的比例荧光图像。每次外部pH测量时,隧道载玻片和细胞都是新鲜准备的。

  3. 校准
    1. 在不同pH值5.5,6.0,6.5,7.0,7.5,8.0或8.5的运动缓冲液中悬浮纯化的pHluorin(M153R)-FliG蛋白。
    2. 加入50μl含有pHluorin(M153R)-FliG蛋白的溶液到隧道载玻片上,并用一张滤纸吸收多余的溶液。
    3. 获得在每个pH值下由410和470nm激发的pHluorin(M153R)-FliG蛋白溶液的荧光图像。
      注意:当pH值(M153R)-FliG的蛋白质浓度太高时,其荧光强度相当高,从而使EMCCD相机的传感器芯片饱和。因此,有必要将pHluorin(M153R)-FliG的蛋白质浓度调节至约〜100μg/ ml,以便进行体外校准曲线。

数据分析

通过图像分析软件IGOR Pro或ImageJ分析荧光图像。

  1. 校正曲线
    1. 定义由携带pYVM008的SJW1368细胞的图像分布确定的细胞体的总荧光强度(图3)。
      注意:由于flhDC缺失菌株不产生鞭毛,所以pHluorin(M153R)-FliG探针在细胞质中自由扩散。


      图3.通过pHluorin(M153R)-FliG测定细胞质体积pH的典型图像过程。详见文本。

    2. 将工具背景强度定义为附近无细胞区域的50 x 50像素ROI(感兴趣区域)内的平均像素强度。
    3. 将自发荧光强度定义为不产生荧光蛋白的50种野生型SJW1103细胞的平均像素强度。
    4. 从荧光细胞体的总荧光强度减去细胞的仪器背景和自发荧光,以定义在410和470nm激发波长下自由扩散pHluorin(M153R)-FliG的荧光强度。
    5. 计算pH值荧光素(M153R)-FliG探针的荧光强度比(R 410/470)。
    6. 从20μM的短杆菌肽和20mM的苯甲酸钠存在下,在每个外部pH下得到的pHluorin(M153R)-FliG的R 410/470的R p值确定细胞质pH,两者平衡了内部和外部的沙门氏菌细胞内的pH,以使体内校准曲线(图4)。
      注意:体内校准清楚地表明,pHluorin(M153R)-FliG可作为活细胞中的pH指示剂。


      图4.体外的pHluorin(M153R)-FliG在体外的校准曲线。 通过pH成像系统在5.5至8.5的pH范围内摄取纯化的pHluorin(M153R)-His蛋白质溶液的荧光图像。在每个pH值(黑色,体外)下计算荧光强度比410/470,并且通过乙状结肠功能拟合数据。然后,在20μM短杆菌肽和20mM苯甲酸钾的存在下,在四种不同pH值,6.0,6.5,7.0和7.5的运动缓冲液中,通过相同的体系观察携带pYVM008的SJW1368细胞并测量细胞内pH(体内橙色)。垂直条表示标准偏差(从Morimoto等人修改,2016)。

    7. 在减去仪器背景强度后,定义纯化的pHluorin(M153R)-FliG蛋白的荧光强度,作为不含荧光蛋白的溶液图像的100×100像素ROI内的平均像素强度。
    8. 在每个pH值下计算纯化的pHluorin(M153R)-FliG的R 410/470。
    9. 绘制发射强度比值作为pH值的函数,以在5.5至8.5的pH范围内进行体外校准曲线(图4)。
    10. 使用IGOR Pro软件通过S形曲线功能调整校准数据。



  2. 局部pH值的测定
    1. 对于YVM1004细胞的荧光图像,将20×20像素的pHluorin(M153R)-FliG的荧光斑点应用于ROI的图案(图2)。
    2. 从每个像素值减去包括工具背景和单元格自动荧光的总背景强度。仪器背景强度定义为邻近无细胞区域的50 x 50像素ROI内的平均像素强度。细胞的自发荧光强度定义为非荧光细胞SJW1103的ROI内的平均像素强度。
    3. 定义pHluorin(M153R)-FliG探针的单个荧光斑点的荧光强度作为ROI内的积分荧光强度值。
    4. 计算在410和470nm激发波长的pHluorin(M153R)-FliG斑点的荧光强度的比例。
    5. 使用体外校准曲线确定每个荧光斑点的R 410/470值的局部pH值。
    6. 使用Microsoft Excel(Microsoft),从100多个荧光斑点的数据计算局部pH值的平均值和平均值的标准误差(图5)。


      图5.使用pHluorin(M153R)-FliG测量局部pH。 pH-fliG(YVM1004)的鞭毛III型蛋白质输出装置周围的局部pH值和pH值(P28T)的pH-测量fliG (YVM1049)细胞。分析了300多个荧光斑点。垂直条表示标准错误(从Morimoto等人修改,2016)。

笔记

由于需要足够的荧光强度以检测局部pH,所以表达pHluorin(M153R)-FliG的细胞应在振荡下孵育超过5小时才能使pHluorin(M153R)的荧光团完全成熟。

食谱

  1. LB培养基(每升)
    10克Bacto胰蛋白胨
    5克酵母提取物
    5克NaCl
  2. TB培养基(每升)
    10克Bacto胰蛋白胨
    10mM磷酸钾,pH7.0
  3. LB琼脂板
    LB培养基
    1.5%Bacto琼脂
  4. 运动缓冲区
    10 mM磷酸钾
    0.1 mM EDTA
    调整为不同的pH值:5.5,6.0,6.5,7.0,7.5,8.0或8.5

致谢

该协议是从以前的工作(Morimoto等人,2016)修改的。该研究部分由JSS KAKENHI Grant Numbers JP15K14498和JP15H05593给予YVM,JP25000013至KN和JP26293097至TM,以及MEXT KAKENHI Grant Numbers JP26115720和JP15H01335至YVM和JP24117004,JP25121718和JP15H01640至TM。

参考

  1. Che,Y. S.,Nakamura,S., Morimoto,Y.V.,Kami-ike,N.,Namba,K。和Minamino,T。(2014)。 质子易位的负载敏感耦合 和细菌鞭毛马达中的扭矩产生。 Mol Microbiol 91(1):175-184。
  2. 古河, A.,Yoshikaie,K.,Mori,T.,Mori,H.,Morimoto,Y.V.,Sugano,Y.,Iwaki,S., Minamino,T.,Sugita,Y.,Tanaka,Y。和Tsukazaki,T。(2017)。 从我推断的隧道形成 - 形成质子驱动的结构 蛋白质分泌马达SecDF。 Cell Rep 19(5):895-901。
  3. Miesenböck,G.,De Angelis,D. A.和 Rothman,J.E。(1998)。 可视化分泌和突触传播 与pH敏感的绿色荧光蛋白。自然 394(6689): 192-195。
  4. 南, (2014)。 蛋白质通过 细菌性鞭毛III型出口途径。 Biochim Biophys Acta 1843(8):1642-1648。
  5. 南, T.,Morimoto,Y.V.,Hara,N.,Aldridge,P.D。和Namba,K。(2016)。 fl fl fl 复合物是双燃料发动机,可以使用H + 和Na + 进行鞭毛蛋白质输出。 Pathog 12(3):e1005495。
  6. 南, T.,Morimoto,Y.V.,Hara,N。和Namba,K。(2011)。 使用的能量转换机制 细菌鞭毛III型蛋白质出口。 2:475.
  7. 南, T.,Yamaguchi,S.and Macnab,R.M。(2000)。 FliE与FlgB之间的相互作用 沙门氏菌的鞭毛基底体的杆部分。 Bacteriol 182(11):3029-3036。
  8. 森本, Y.V.,Che,Y.S.,Minamino,T.and Namba,K。(2010)。 插入和插入的质子电导率测定 通过沙门氏菌中表达的细胞质pHluorin来拔除MotA / B质子通道。 FEBS Lett 584(6): 1268-1272。
  9. 森本, Y.V.,Kami-ike,N.,Miyata,T.,Kawamoto,A.,Kato,T.,Namba,K。 Minamino,T。(2016)。 活细菌的高分辨率pH成像 细胞以检测局部pH差异。 7(6)。
  10. 森本, Y.V.,Kojima,S.,Namba,K.and Minamino,T。(2011)。 pH敏感绿色中的M153R突变 荧光蛋白稳定其融合蛋白。 6(5): e19598。
  11. Morimoto,Y.V.,Namba,K。和Minamino,T. (2017年)。 细菌细胞内 使用CoroNa Green进行钠离子测量。 7(1)e2092。
  12. 大西, K.,Ohto,Y.,Aizawa,S.,Macnab,R.M。和Iino,T。(1994)。 FlgD是需要的脚手架蛋白质 鞭毛钩组件在沙门氏菌 中。 176(8): 2272-2281。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Morimoto, Y. V., Kami-ike, N., Namba, K. and Minamino, T. (2017). Determination of Local pH Differences within Living Salmonella Cells by High-resolution pH Imaging Based on pH-sensitive GFP Derivative, pHluorin(M153R). Bio-protocol 7(17): e2529. DOI: 10.21769/BioProtoc.2529.
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