搜索

Characterising Maturation of GFP and mCherry of Genomically Integrated Fusions in Saccharomyces cerevisiae
鉴定酿酒酵母中GFP和mCherry融合蛋白的成熟特征   

评审
匿名评审
引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
eLIFE
Sep 2017

Abstract

Single-molecule fluorescence microscopy enables unrivaled sub-cellular quantitation of genomically encoded fusions of native proteins with fluorescent protein reporters. Fluorescent proteins must undergo in vivo maturation after expression before they become photoactive. Maturation effects must be quantified during single-molecule analysis. Here we present a method to characterise maturation of GFP and mCherry genetic protein fusions in budding yeast Saccharomyces cerevisiae.

Keywords: Single-molecule (单分子), Fluorescence (荧光), Fluorescent protein maturation (荧光蛋白成熟), Protein fusion (蛋白融合), GFP (GFP), mCherry (mCherry), Yeast (酵母)

Background

Single-molecule fluorescence microscopy enables sensitive quantification of molecular stoichiometry, mobility and copy number, not only on a cell-by-cell basis but also precisely to individual sub-cellular compartments (Leake, 2012; Wollman and Leake, 2015; Shashkova et al., 2017). The technique relies on endogenously expressed fluorescent protein fusions of the wild type protein of interest such that there is one-to-one labelling. However, all fluorescent proteins have an in vivo maturation time varying from a few minutes to several tens of minutes before entering a bright fluorescing state (Badrinarayanan et al., 2012). It is therefore of upmost importance to measure any maturation effects and quantify if there is any immature ‘dark fraction’ of labelled protein. These measurements are also particularly relevant to fluorescence recovery after photobleaching (FRAP). FRAP can be used to study molecular turnover in living cells (Beattie et al., 2017). FRAP is based on photobleaching of a cell region where a fluorescently labelled component is localized, followed by quantification of any fluorescence recovery in that region over time. The measured relation between the fluorescence intensity as a function of time following an initial photobleach can be used to determine molecular mobility and kinetics parameters, such as the rate of dissociation of a particular fluorescent component from a molecular complex (Leake et al., 2006). Therefore, any ‘new’ fluorescence coming from fluorescent protein maturation might affect this apparent result. We present here a protocol to characterise the maturation of Mig1-GFP and Nrd1-mCherry fusion proteins in living yeast Saccharomyces cerevisiae cells used in our single-molecule studies (Wollman et al., 2017).

We blocked protein translation in living cells by adding cycloheximide (Hartwell et al., 1970), and then measured any cellular fluorescence recovery after cells were completely photobleached by continuous illumination. Such fluorescence recovery is then used as a metric for newly matured GFP and/or mCherry in the cell. Our results are broadly consistent with in vivo maturation of GFP and mCherry reported previously (Badrinarayanan et al., 2012; Khmelinskii et al., 2012), but since maturation kinetics may be dependent on cell type and the specific extracellular microenvironment, it is important to quantify these maturation effects under the same experimental conditions used for the in vivo microscopy on the actual fusion strains of interest.

Materials and Reagents

  1. Sterile pipette tips, 1 ml, 200 µl, 10 µl (STARLAB, catalog numbers: S1111-6801 , S1111-0806 , S1111-3800 )
  2. 14 ml conical tubes (Corning, Falcon®, catalog number: 352059 )
  3. Petri dishes 92 mm diameter (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 172931 )
  4. Microscopy slides (Fisher Scientific, catalog number: FB58622 )
  5. Cover slips (Scientific Laboratory Supplies, catalog number: MIC3124 )
  6. Yeast S. cerevisiae YSH2348 with Mig1-GFP and Nrd1-mCherry genetically integrated protein fusions, MATa MIG1-GFP-HIS3 NRD1-mCherry-hphNT1 MET LYS (Hohmann lab, University of Gothenburg, Sweden)
  7. D(+)-Glucose (VWR, catalog number: 101176K )
  8. Bacto-yeast extract (BD, BactoTM, catalog number: 212750 )
  9. Peptone from meat (Merck, catalog number: 1072241000 )
  10. Agar-agar (Merck, catalog number: 1016141000 )
  11. MilliQ water
  12. Yeast nitrogen base without amino acids, without (NH4)2SO4 (Sigma-Aldrich, catalog number: Y1251 )
  13. Ammonium sulfate, (NH4)2SO4 (Merck, catalog number: 1012171000 )
  14. Complete supplement mixture (ForMedium, catalog number: DCS0019 )
  15. Cycloheximide (Sigma-Aldrich, catalog number: C7698 )
  16. EtOH (VWR, catalog number: VWRC20821.330 )
  17. Glucose 50% w/v (see Recipes)
  18. YPD agar with 4% glucose (see Recipes)
  19. YPD liquid medium with 4% glucose (see Recipes)
  20. YNB (Yeast Nitrogen Base) liquid medium without glucose (see Recipes)
  21. YNB (Yeast Nitrogen Base) liquid medium with 4% glucose (see Recipes)
  22. 100 mg/ml cycloheximide solution in EtOH (see Recipes)

Equipment

  1. Pipettes (STARLAB, model: ErgoOne® Single-Channel Pipette, 2-20 µl, 20-200 µl and 100-1,000 µl)
  2. pH-meter (Scientific & Chemical Supplies, catalog number: PHM975050 )
  3. Two timers (Fisher Scientific, catalog number: 15177414 )
  4. Autoclave (Getinge, model: 400/500LS-E Series Steam Sterilizers (533LS-E))
  5. Magnetic stirrer (Chemtech Scientific, model: C-MAG HS7 )
  6. 30 °C incubator (Eppendorf, New Brunswick ScientificTM, model: Innova® 4000 )
  7. Spectrophotometer (Biochrom, model: WPA S800 )
  8. Centrifuge (Eppendorf, model: 5810 R )
  9. Mercury-arc excitation fluorescence microscope Zeiss Axiovert 200M (Carl Zeiss, model: Axiovert 200 M ) with an AxioCamMR3 camera with separate filter sets: 38HE for GFP and 43HE for mCherry excitation; Plan-Apochromat 1.40-numerical-aperture oil immersion, 100x objective

Software

  1. AxioVision
  2. ImageJ 1.50g
  3. Excel
  4. MATLAB 2017a

Procedure

  1. Cell preparation
    1. Streak cells from a frozen stock, using a sterile pipette tip on a freshly-prepared YPD agar plate (see Recipes), and incubate at 30 °C for at least 24 h.
    2. Set an overnight culture in a 14 ml tube by inoculating 3 ml of YPD with cells grown on a YPD plate. Single colonies are not needed for genomically integrated strains. Incubate at 30 °C, 180 rpm.
    3. In the morning exchange the YPD medium (see Recipes) to YNB medium (see Recipes) supplemented with 4% glucose:
      1. Pellet the cells by centrifugation at 1,000 x g for 3 min, remove the supernatant.
      2. Resuspend the cells in 3 ml of YNB medium without any carbon source.
      3. Pellet the cells by centrifugation at 1,000 x g for 3 min, remove the supernatant.
      4. Suspend the cells in 3 ml of YNB supplemented with 4% glucose and incubate at 30 °C, 180 rpm, for ~4 h.
      5. Wash the culture by centrifugation (1,000 x g, 3 min) and re-suspend in 2 ml of YNB with 4% glucose. Incubate at 30 °C, 180 rpm, for about 10 min.
      6. Add 2 μl of 100 mg/ml cycloheximide solution (see Recipes) to the final concentration of 100 μg/ml. Incubate for 1 h at room temperature, without shaking, protect from light.
      7. Place 5 μl of the culture on a microscope slide and cover with a 22 x 22 mm coverslip. Avoid any air under the coverslip.

  2. Data acquisition
    1. Place the sample under the microscope, coverslip on the objective and find a region of interest containing 5-10 cells (100x magnification) which appear stationary and firmly anchored to the glass surface.
    2. Optimize exposure times for in vivo imaging of both GFP and mCherry fluorescent proteins to be able to detect a clear signal without saturating the detector. Under our microscope: GFP exposure time–22 sec, mCherry–7 sec.
    3. Find another region with 5-10 cells positioned far away from the previous one to avoid any potential bleaching from previous illumination exposure.
    4. Take a brightfield and a fluorescence image, by pressing the ‘snap’ button, with both channels using chosen exposure times, opening the mercury lamp shutter for only the length of exposure.
    5. Photobleach GFP or mCherry by continuous illumination of the appropriate wavelength until the region appears completely dark. Continue for 1 min longer. With our settings the total exposure time is: 3 min 40 sec for GFP and 4 min for mCherry. Immediately after, begin timing and acquire a picture of the bleached fluorescent protein with an appropriate channel and a brightfield image. This is denoted time point 0 min.
    6. Continue acquiring both fluorescent and brightfield pictures at the following time points after bleaching: 7.5, 15, 25, 30, 40, 60, 90 and 120 min.
    7. As simultaneous photobleaching of GFP and mCherry is not possible under this microscope, the time points were staggered for GFP and mCherry as listed in Table 1.

      Table 1. Order of photobleaching and data acquisition using two channels


  3. Data analysis
    1. Images are converted into open standard tiff files from zvi by AxioVision software.
    2. Further analysis is performed using ImageJ.
      1. Open the first unbleached brightfield image.
      2. By choosing an ‘oval’ selection tool, define a region of interest (ROI), an area around a cell as shown in Figure 1. It does not matter how much of non-cell area is included as every cell will be background-correct during the analysis.


        Figure 1. Selection of the region of interest for cell measurements. Scale bar = 20 µm.

      3. Open a fluorescence image of the same set, and define the same area of the same cell by simultaneously choosing ‘Shift’ and ‘E’ keys on the keyboard (Selection → Restore).
      4. From the menu bar select: Analyze → set measurements. Pick ‘area’ (represents a number of pixels, N) and ‘integrated density’ (sum intensity for the cell, Scell). Press ‘OK’.
      5. To obtain numeric values press ‘Ctrl’ + ‘M’ (Analyse → Measure). Record the result in Excel.
      6. Repeat throughout the entire data set for both channels keeping the same ROI.
      7. Repeat the entire procedure for all cells.
      8. Background correction: Choose random background areas around cells (Figure 2) and obtain numerical results for sum intensity (Sbg).


        Figure 2. Selection of the region of interest for the background measurements. Scale bar = 20 µm.

      9. Find the average (SAbg) and multiply by the number of pixels from cell (N) measurements. This is the intensity of the background represented within the cell area (Ibg).

      10. Subtraction of the average background sum intensity (Ibg) from the total intensity of the cell (Scell) represents Icell, the cellular fluorescence intensity with background correction.

      11. The average of fluorescence intensity of all cells analysed within the data set gives the final value of the fluorescence intensity (Ifinal) with appropriate estimation of SD and/or SE.

      12. Plot the final fluorescence intensity (Ifinal) vs. experimental time (Figures 3A and 3B) for fluorescently labelled cells and wild type autofluorescent cells. Any signal above autofluorescence is due to fluorescent protein maturation. For GFP (Figure 3A), no maturation was detected so it can be assumed that all of the fluorescent protein was mature in the cells and there is no ‘dark’ fraction. For mCherry (Figure 3B), some fluorescence recovery was measured. The following steps outline quantification of the maturation time and dark fraction.
      13. Subtract the autofluorescence from the mean fluorescent protein intensity at each time point (Figure 3C).
      14. Export the intensity and time values after the bleach by copying and pasting into two new variables in MATLAB, called x (for the time values ) and y (intensity values):
        1. Right click on the Workspace → New. Name it x or y. Press ‘Enter’ on the keyboard.
        2. Double click on this new variable opens a table in the Editor where values of time (for x) or intensity (for y) can be pasted.
      15. Open the curve fitting toolbox from the Apps menu.
      16. Select x for ‘X data’ and y for ‘Y data’.
      17. Choose custom equation and type:

        where, Ibleach is the remaining intensity after the bleach, Irec is the recovered intensity above Ibleach and tmat is the maturation time.
      18. If ‘Auto fit’ is ticked, fitting will be automatic.
      19. If the fit has not converged correctly, adjust the ‘Start point’ parameters in ‘Fit Options’ to reasonable estimates from the data i.e., y at x = 0 for Ibleach and y at x = end minus Ibleach for Irec.
      20. If the fit has converged record the fit and goodness of fit parameters from the ‘Results’ panel. For Figure 3C, Irec = 4.3 x 104 ± 3 x 104 counts, Ibleach = 5.2 x 104 ± 2.5 x 104 counts, tmat =17 ± 10 min with R2 = 0.7.
      21. To calculate the proportion dark, immature protein; divide Irec by the initial, autofluorescence corrected pre-bleach intensity. Here give ~5%.


        Figure 3. Characterisation of GFP and mCherry maturation times in vivo. GFP maturation within genomically integrated protein fusion (A). Maturation of a genomically integrated mCherry fusion (B) and its’ exponential recovery fit (C). Time normalized to bleach time at t = 0.

Data analysis

Data was analyzed as outlined in section C of the Procedure. Statistical methods are outlined in Wollman et al. (2017) but also briefly outlined here. In imaging experiments, each cell can be defined as a biological replicate sampled from the cell population. Sample sizes of ~10 cells were used to generate reasonable estimates of fluorescent protein maturation and are similar to previous studies (Badrinarayanan et al., 2012). Technical replicates are not possible with irreversible photobleaching however noise is characterized by the autofluorescent of wild type control cell measurements.

Notes

Autofluorescence is calculated as indicated in the protocol above but using a wild type yeast strain (i.e., without any fluorescent proteins present).

Recipes

  1. Glucose 50% w/v
    Weigh 500 g of glucose
    Bring up to 1 L with MilliQ water
    Dissolve by using magnetic stirrer with heating
    Autoclave for 20 min at 121 °C
  2. YPD agar with 4% glucose
    Mix yeast extract 5 g, Bacto-peptone (peptone from meat) 10 g and agar 10 g
    Bring up to 460 ml with MilliQ water
    Autoclave for 20 min at 121 °C
    Add 40 ml of glucose 50% w/v
    Cast plates: approximately 25 ml of the medium per plate
    Let them solidify, store upside down at 4 °C
  3. YPD liquid medium with 4% glucose
    Mix yeast extract 5 g and Bacto-peptone (peptone from meat) 10 g
    Bring up to 460 ml with MilliQ water
    Autoclave for 20 min at 121 °C
    Add 40 ml of glucose 50% w/v
  4. YNB (Yeast Nitrogen Base) liquid medium without glucose
    Mix yeast nitrogen base without amino acids, without (NH4)2SO4 1.7 g, complete supplement 0.79 g, (NH4)2SO4 5 g
    Dissolve in 900 ml of MilliQ water, adjust pH 5.8-6.0 using NaOH
    Bring up to 1,000 ml with MilliQ water
    Autoclave for 20 min at 121 °C
  5. YNB (Yeast Nitrogen Base) liquid medium with 4% glucose
    Mix yeast nitrogen base without amino acids, without (NH4)2SO4 1.7 g, complete supplement 0.79 g, (NH4)2SO4 5 g
    Dissolve in 900 ml of MilliQ water, set pH 5.8-6.0 using NaOH
    Bring up to 920 ml with MilliQ water
    Autoclave for 20 min at 121 °C
    Add 80 ml of glucose 50% w/v
  6. 100 mg/ml cycloheximide solution in EtOH
    Weigh 0.5 g of cycloheximide and dissolve in 5 ml of absolute EtOH
    Aliquot and store at -20 °C

Acknowledgments

This work was supported by the Biological Physical Sciences Institute, Royal Society, MRC (grant MR/K01580X/1), BBSRC (grant BB/N006453/1), the European Commission via Marie Curie-Network for Initial Training ISOLATE (Grant agreement No.: 289995), and the Royal Society Newton International Fellowship (NF160208). This protocol was adapted from Wollman et al. (2017).
Conflict of interest: Authors declare no conflict of interest.

References

  1. Badrinarayanan, A., Reyes-Lamothe, R., Uphoff, S., Leake, M. C. and Sherratt, D. J. (2012). In vivo architecture and action of bacterial structural maintenance of chromosome proteins. Science 338(6106): 528-531.
  2. Beattie, T. R., Kapadia, N., Nicolas, E., Uphoff, S., Wollman, A. J., Leake, M. C. and Reyes-Lamothe, R. (2017). Frequent exchange of the DNA polymerase during bacterial chromosome replication. Elife 6.
  3. Hartwell, L. H., Culotti, J. and Reid, B. (1970). Genetic control of the cell-division cycle in yeast. I. Detection of mutants. Proc Natl Acad Sci U S A 66(2): 352-359.
  4. Khmelinskii, A., Keller, P. J., Bartosik, A., Meurer, M., Barry, J. D., Mardin, B. R., Kaufmann, A., Trautmann, S., Wachsmuth, M., Pereira, G., Huber, W., Schiebel, E. and Knop, M. (2012). Tandem fluorescent protein timers for in vivo analysis of protein dynamics. Nat Biotechnol 30(7): 708-714.
  5. Leake, M. C. (2012). The physics of life: one molecule at a time. Philos Trans R Soc Lond B Biol Sci 368(1611): 20120248.
  6. Leake, M. C., Chandler, J. H., Wadhams, G. H., Bai, F., Berry, R. M. and Armitage, J. P. (2006). Stoichiometry and turnover in single, functioning membrane protein complexes. Nature 443(7109): 355-358.
  7. Shashkova, S., Wollman, A. J. M., Leake, M. C. and Hohmann, S. (2017). The yeast Mig1 transcriptional repressor is dephosphorylated by glucose-dependent and -independent mechanisms. FEMS Microbiol Lett 364(14).
  8. Wollman, A. J. and Leake, M. C. (2015). Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time. Faraday Discuss 184: 401-424.
  9. Wollman, A. J., Shashkova, S., Hedlund, E. G., Friemann, R., Hohmann, S. and Leake, M. C. (2017). Transcription factor clusters regulate genes in eukaryotic cells. Elife 6: e27451.

简介

单分子荧光显微镜技术使天然蛋白与荧光蛋白报告基因的基因组编码融合成为无与伦比的亚细胞定量。 荧光蛋白质在表达后必须进行体内成熟,然后它们变成光敏的。 成熟效应必须在单分子分析过程中进行量化。 在这里,我们提出一种方法来表征GFP和mCherry遗传蛋白融合在芽殖酵母酿酒酵母中的成熟。

【背景】单分子荧光显微技术能够灵敏定量分子化学计量,流动性和拷贝数,不仅在逐个细胞的基础上,而且精确到个别的亚细胞区室(Leake,2012; Wollman和Leake,2015; Shashkova, >等。,2017)。该技术依赖于感兴趣的野生型蛋白质的内源表达的荧光蛋白融合,从而存在一对一的标记。然而,所有荧光蛋白在进入明亮的荧光状态之前的体内成熟时间从几分钟到几十分钟不等(Badrinarayanan等人,2012)。因此,测量任何成熟效应和量化是否存在标记蛋白质的不成熟“暗部分”是最重要的。这些测量也与光漂白(FRAP)后的荧光恢复特别相关。 FRAP可用于研究活细胞中的分子转换(Beattie等人,2017)。 FRAP基于荧光标记组分定位的细胞区域的光漂白,随后定量该区域随时间的任何荧光恢复。可以使用在初始光漂白剂之后作为时间函数的荧光强度之间的测量关系来确定分子迁移率和动力学参数,例如特定荧光组分从分子复合物解离的速率(Leake等人, ,2006)。因此,来自荧光蛋白质成熟的任何“新”荧光都可能影响这种明显的结果。我们在此提出一种方案来表征Mig1-GFP和Nrd1-mCherry融合蛋白在用于我们的单分子研究的活酵母酿酒酵母细胞中的成熟(Wollman等人 >,2017)。

我们通过加入放线菌酮阻断活细胞中的蛋白质翻译(Hartwell等人,1970),然后在连续照射完全光漂白之后测量任何细胞荧光恢复。然后将这种荧光恢复用作细胞中新成熟的GFP和/或mCherry的度量。我们的结果与之前报道的GFP和mCherry(Badrinarayanan等人,2012; Khmelinskii等人,2012年)在体内成熟大体一致,2012 ),但是由于成熟动力学可能取决于细胞类型和特定的细胞外微环境,所以重要的是在用于体内显微镜观察的相同实验条件下对这些成熟效应进行定量利益。

关键字:单分子, 荧光, 荧光蛋白成熟, 蛋白融合, GFP, mCherry, 酵母

材料和试剂

  1. 无菌移液枪头,1毫升,200微升,10微升(STARLAB,目录号:S1111-6801,S1111-0806,S1111-3800)
  2. 14ml锥形管(Corning,Falcon ,目录号:352059)
  3. 培养皿直径92mm(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:172931)
  4. 显微镜幻灯片(Fisher Scientific,目录号:FB58622)
  5. 盖玻片(科学实验室用品,目录号:MIC3124)
  6. 酵母S。 YSH2348与Mig1-GFP和Nrd1-mCherry基因整合蛋白融合物,MATa MIG1-GFP-HIS3 NRD1-mCherry-hphNT1 MET LYS (Hohmann实验室,瑞典哥德堡大学)< br />
  7. D(+) - 葡萄糖(VWR,目录号:101176K)
  8. 细菌酵母提取物(BD,Bacto TM,目录号:212750)
  9. 肉类蛋白胨(默克,目录号:1072241000)
  10. 琼脂(Merck,目录号:1016141000)
  11. MilliQ水
  12. 不含(NH4)2SO4(Sigma-Aldrich,目录号:Y1251)的无氨基酸酵母氮碱
  13. 硫酸铵,(NH 4)2 SO 4(Merck,目录号:1012171000)
  14. 完全补充混合物(ForMedium,目录号:DCS0019)
  15. 环己酰亚胺(Sigma-Aldrich,目录号:C7698)
  16. EtOH(VWR,目录号:VWRC20821.330)
  17. 葡萄糖50%w / v(见食谱)
  18. YPD琼脂与4%的葡萄糖(见食谱)
  19. 含有4%葡萄糖的YPD液体培养基(见食谱)
  20. YNB(酵母氮基)无葡萄糖的液体培养基(见食谱)
  21. YNB(酵母氮基)液体培养基含4%葡萄糖(见食谱)
  22. 100毫克/毫升放线菌酮的乙醇溶液(见食谱)

设备

  1. 移液器(STARLAB,型号:ErgoOne®单通道移液器,2-20μl,20-200μl和100-1000μl)
  2. pH计(Scientific&amp; Chemical Supplies,目录号:PHM975050)
  3. 两个定时器(Fisher Scientific,目录号:15177414)
  4. 高压灭菌器(Getinge,型号:400 / 500LS-E系列蒸汽灭菌器(533LS-E))
  5. 磁力搅拌器(Chemtech Scientific,型号:C-MAG HS7)
  6. 30℃培养箱(Eppendorf,New Brunswick Scientific TM,型号:Innova 4000)。
  7. 分光光度计(Biochrom,型号:WPA S800)
  8. 离心机(Eppendorf,型号:5810 R)
  9. 带有AxioCamMR3相机的汞弧激发荧光显微镜Zeiss Axiovert 200M(Carl Zeiss,型号:Axiovert 200M),具有单独的滤波器组:38HE用于GFP和43HE用于mCherry激发; Plan-Apochromat 1.40数值孔径油浸式,100倍物镜

软件

  1. AxioVision
  2. ImageJ 1.50g
  3. Excel
  4. MATLAB 2017a

程序

  1. 细胞制备
    1. 使用新鲜制备的YPD琼脂平板(参见食谱)上的无菌移液器尖端从冷冻的原料中取出细胞,并在30°C孵育至少24小时。
    2. 在YPD平板上接种3ml YPD与在YPD平板上生长的细胞,在14ml试管中设置过夜培养物。基因组整合菌株不需要单个菌落。在30°C,180转/分下孵育。
    3. 在早上将YPD培养基(参见食谱)交换到补充有4%葡萄糖的YNB培养基(参见食谱)
      1. 通过在1000×g离心3分钟沉淀细胞,除去上清液。

      2. 在3毫升YNB培养基中重悬细胞,不含任何碳源。
      3. 通过在1000×g离心3分钟沉淀细胞,除去上清液。
      4. 将细胞悬浮于3ml补充有4%葡萄糖的YNB中,并在30℃,180rpm下孵育约4小时。
      5. 通过离心(1000×g,3分钟)洗涤培养物并重悬于含有4%葡萄糖的2ml YNB中。
        在30°C,180转/分下孵育10分钟
      6. 添加2微升的100毫克/毫升放线菌酮溶液(见食谱)到100微克/毫升的最终浓度。在室温下孵育1小时,不摇晃,避光。
      7. 将5μL的文化放在显微镜幻灯片上,并盖上一个22×22毫米的盖玻片。避免盖玻片下的空气。

  2. 数据采集
    1. 将样品置于显微镜下,在物镜上盖上盖子,找到一个包含5-10个细胞(100x放大倍数)的感兴趣区域,该区域显示为静止并牢固地固定在玻璃表面上。
    2. 优化GFP和mCherry荧光蛋白的体内成像的曝光时间,以便能够在不使检测器饱和的情况下检测到清晰的信号。在我们的显微镜下:GFP曝光时间22秒,mCherry-7秒
    3. 找到另一个5-10个细胞远离前一个区域的区域,以避免任何可能的漂白从以前的照明曝光。
    4. 通过按下“捕捉”按钮,使用选定的曝光时间,使用两个通道拍摄明视野和荧光图像,然后打开水银灯光闸,使其长时间曝光。
    5. Photobleach GFP或mCherry通过连续照射适当的波长,直到区域看起来完全黑暗。再继续1分钟。使用我们的设置,总曝光时间为:GFP 3分40秒,mCherry 4分。紧接着,开始计时并获得具有适当通道和明场图像的漂白荧光蛋白的图片。这是表示0分钟的时间点。
    6. 在漂白后的下列时间点继续采集荧光和明场照片:7.5,15,25,30,40,60,90和120分钟。
    7. 由于GFP和mCherry在这个显微镜下不可能同时发生漂白,所以GFP和mCherry的时间点如表1所示。

      表1.使用两个通道的光漂白和数据采集的顺序


  3. 数据分析

    1. 使用AxioVision软件将图像转换为zvi的开放标准tiff文件
    2. 进一步的分析是使用ImageJ进行的。
      1. 打开第一个未漂白的明场图像。
      2. 通过选择一个“椭圆形”选择工具,定义一个感兴趣区域(ROI),一个区域周围的区域,如图1所示。包括非细胞区域的多少并不重要,因为每个细胞都是背景正确的在分析过程中。


        图1.选择细胞测量的感兴趣区域。比例尺= 20微米。

      3. 打开同一组的荧光图像,并通过同时选择键盘上的“Shift”和“E”键(选择→恢复)来定义相同单元的相同区域。
      4. 从菜单栏中选择:分析→设置测量。选取“区域”(代表像素的数量,N )和“积分密度”(细胞的总和强度,S )。按“确定”。
      5. 要获得数值,请按'Ctrl'+'M'(分析→测量)。在Excel中记录结果。
      6. 两个渠道的整个数据集重复保持相同的投资回报率。
      7. 对所有细胞重复整个过程。
      8. 背景校正:选择细胞周围的随机背景区域(图2),并获得总和强度的数值结果(

        图2.选择背景测量的感兴趣区域比例尺= 20μm

      9. 找到平均值( S
      10. 平均背景总和强度( bg 代表细胞荧光强度与背景校正。 >
      11. 在数据集内分析的所有细胞的荧光强度的平均值给出了荧光强度的最终值( final ), SD和/或SE。

      12. 为荧光标记的细胞和野生型自体荧光细胞绘制最终的荧光强度(实验时间)(图3A和3B)与实验时间(图3A和3B)。自发荧光以上的任何信号都是由于荧光蛋白成熟。对于GFP(图3A),没有检测到成熟,因此可以认为所有荧光蛋白在细胞中都是成熟的并且没有“黑色”部分。对于mCherry(图3B),测量了一些荧光恢复。以下步骤概述了成熟时间和暗分数的量化。

      13. 在每个时间点从平均荧光蛋白强度中减去自发荧光(图3C)
      14. 通过在MATLAB中复制并粘贴到两个新变量(称为x(时间值)和y(强度值)),输出漂白后的强度和时间值:
        1. 右键单击工作区→新建。将其命名为x或y。按下键盘上的“Enter”。
        2. 双击这个新变量可以在编辑器中打开一个表格,其中可以粘贴时间(对于x)或强度(对于y)的值。

      15. 从“应用程序”菜单打开曲线拟合工具箱

      16. 选择x代表'X数据',y代表'Y数据'
      17. 选择自定义公式并键入:

        其中,漂白剂
      18. 如果勾选“自动适应”,则自动装配。
      19. 如果拟合没有正确收敛,则将“拟合选项”中的“起始点”参数调整为合理的估计值,即 ,x =漂白剂 I
      20. 如果拟合已经收敛,则从“结果”面板记录拟合参数的合适性和良好性。对于图3C, 计数,漂白 = 5.2×10 4 4±2.5×10 4 4 计数,
      21. 计算比例黑暗,不成熟的蛋白质;通过初始自发荧光校正的预漂白强度来分开

        图3. GFP和mCherry成熟时间体内特征。GFP在基因组整合蛋白融合(A)中成熟。基因组整合mCherry融合(B)的成熟及其指数恢复拟合(C)。时间归一化漂白时间在t = 0。

数据分析

按照程序的C部分所述分析数据。 Wollman等人(2017)概述了统计方法,但也在此简要概述。在成像实验中,每个细胞可以定义为从细胞群取样的生物学复制品。使用〜10个细胞的样本大小来产生对荧光蛋白质成熟的合理估计,并且与之前的研究相似(Badrinarayanan等人,2012)。技术上的重复不可能具有不可逆的光漂白,然而噪音的特征是野生型对照细胞测量的自发荧光。

笔记

如上述方案中所述计算自发荧光,但使用野生型酵母菌株(即,不存在任何荧光蛋白)。

食谱

  1. 葡萄糖50%w / v
    称重500克葡萄糖
    用MilliQ水带上1升
    用磁力搅拌器加热溶解

    在121°C高压灭菌20分钟
  2. YPD琼脂与4%葡萄糖
    混合酵母提取物5克,细菌蛋白胨(肉蛋白胨)10克和琼脂10克
    用MilliQ水带上460毫升

    在121°C高压灭菌20分钟 加入40毫升葡萄糖50%w / v
    铸盘:每盘大约25毫升的培养基
    让它们固化,在4℃下倒置。
  3. 含有4%葡萄糖的YPD液体培养基
    混合酵母提取物5克和细菌蛋白胨(肉胨)10克
    用MilliQ水带上460毫升

    在121°C高压灭菌20分钟 加入40毫升葡萄糖50%w / v
  4. YNB(酵母氮基)液体培养基无葡萄糖
    混合无氨基酸的酵母氮碱,无(NH4)2 SO4 1.7 g,完全补充0.79 g,(NH4) 4 <)2 <4> 5 溶解在900毫升MilliQ水中,用NaOH调节pH 5.8-6.0
    用MilliQ水带上1000毫升

    在121°C高压灭菌20分钟
  5. YNB(酵母氮基)液体培养基含4%葡萄糖
    混合无氨基酸的酵母氮碱,无(NH4)2 SO4 1.7 g,完全补充0.79 g,(NH4) 4 <)2 <4> 5 溶解在900毫升MilliQ水中,使用NaOH将pH设定为5.8-6.0 用MilliQ水带上920毫升

    在121°C高压灭菌20分钟 加入80毫升葡萄糖50%w / v
  6. 100mg / ml的放线菌酮在EtOH中的溶液 称取0.5克放线菌酮,并溶于5毫升无水乙醇中 分装和存储在-20°C

致谢

这项工作得到了生物物理科学研究所,皇家学会,MRC(授予MR / K01580X / 1),BBSRC(授予BB / N006453 / 1),欧盟委员会通过玛丽居里初步培训ISOLATE(拨款协议No :289995)和英国皇家学会牛顿国际奖学金(NF160208)。该协议改编自Wollman等人。 (2017)。
利益冲突:作者声明不存在利益冲突。

参考

  1. Badrinarayanan,A.,Reyes-Lamothe,R.,Uphoff,S.,Leake,M.C。和Sherratt,D.J。(2012)。 体内体细胞结构维持染色体蛋白质的结构和作用< /科学 338(6106):528-531。
  2. Beattie,T.R.,Kapadia,N.,Nicolas,E.,Uphoff,S.,Wollman,A.J.,Leake,M.C。和Reyes-Lamothe,R.(2017)。 细菌染色体复制过程中频繁交换DNA聚合酶 Elife < 6。
  3. Hartwell,L.H.,Culotti,J。和Reid,B。(1970)。 酵母细胞分裂周期的遗传控制。 I.检测突变体








    国外科学技术学院66(2):352-359。

  4. Khmelinskii,A.,Keller,PJ,Bartosik,A.,Meurer,M.,Barry,JD,Mardin,BR,Kaufmann,A.,Trautmann,S.,Wachsmuth,M.,Pereira,G.,Huber,W Schiebel,E。和Knop,M。(2012)。 串联荧光蛋白定时器体内蛋白质动力学分析 Nat Biotechnol 30(7):708-714。
  5. Leake,M.C。(2012)。 生命物理学:一次一个分子 Philos Trans R Soc Lond B Biol Sci 368(1611):20120248。
  6. Leake,M.C.,Chandler,J.H.,Wadhams,G.H.,Bai,F.,Berry,R.M。和Armitage,J.P.(2006)。 单一功能膜蛋白复合物的化学计量和转换率
    443(7109):355-358
  7. Shashkova,S.,Wollman,A.J.M。,Leake,M.C。和Hohmann,S。(2017)。 酵母Mig1转录阻遏物是通过葡萄糖依赖性和非依赖性机制去磷酸化的。 FEMS Microbiol Lett 364(14)。
  8. Wollman,A.J。和Leake,M.C。(2015)。 毫秒单分子定位显微镜结合卷积分析和自动图像分割来确定复杂结构中的蛋白质浓度 ,功能细胞,一次一个细胞。 法拉第讨论 184:401-424。
  9. Wollman,A.J.,Shashkova,S.,Hedlund,E.G.,Friemann,R.,Hohmann,S.and Leake,M.C。(2017)。 转录因子簇调节真核细胞中的基因 Elife 6:e27451。
  • English
  • 中文翻译
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 Shashkova et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Shashkova, S., Wollman, A. J., Hohmann, S. and Leake, M. (2018). Characterising Maturation of GFP and mCherry of Genomically Integrated Fusions in Saccharomyces cerevisiae. Bio-protocol 8(2): e2710. DOI: 10.21769/BioProtoc.2710.
  2. Wollman, A. J., Shashkova, S., Hedlund, E. G., Friemann, R., Hohmann, S. and Leake, M. C. (2017). Transcription factor clusters regulate genes in eukaryotic cells. Elife 6: e27451.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。