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Fluorescence Recovery After Photobleaching (FRAP) in the Fission Yeast Nucleus
裂殖酵母细胞核的荧光光漂白技术(FRAP)   

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Abstract

We use fluorescence recovery after photobleaching (FRAP) to calculate the diffusion coefficient of GFP in the nucleoplasm of fission yeast. The FRAP method can be generally used to measure the mobility of proteins inside the cell or its organelles.
In our experiment we only measured the diffusion of GFP inside the nucleoplasm of fission yeast mitotic cells. However, if GFP is fused to a protein, the mobility of the protein of interest can be calculated following the GFP signal in the bleached area. We did not, however, address this in our experiments; therefore other sources could be searched for this topic.
To compare FRAP and FLIP, both techniques can be used to measure protein mobility inside a cell. However, with FRAP, the diffusion of a protein is measured in the region of interest (ROI), to observe the recovering of fluorescence in this area. In FLIP, fluorescence recovery is measured in an area different from where the bleaching was done, to observe whether the tagged protein is able to move into that area, which would become darker, gaining the bleached proteins. The major difference here is that for FRAP a single bleaching event is sufficient, while FLIP requires a number of bleaching steps, in order to avoid reflux of fluorescent protein in the same region.
Technically FRAP in the nucleus und FRAP in the cytoplasm has no difference. However, we measured a difference between the diffusion coefficient inside the nucleus (D = 5.6 ± 2.8 μm2/s) and in the cytoplasm (D = 8.6 ± 2.2 μm2/s). This is due to the different compositions inside these compartments, consisting of differing amounts of proteins, DNA and RNA.

Materials and Reagents

  1. Fission yeast cells expressing GFP in the nucleus (e.g. PD31 from Kalinina et al., 2013)
  2. Lectin (Sigma-Aldrich, catalog number: L2380 )
  3. MgCl2.6H2O
  4. CaCl2.2H2O
  5. KCl
  6. Na2SO4
  7. Pantothenic acid
  8. Nicotinic acid
  9. Inositol
  10. Biotin
  11. Boric acid
  12. MnSO4
  13. ZnSO4.7H2O
  14. FeCl2.6H2O
  15. Molybdic acid
  16. KI
  17. CuSO4.5H2O
  18. Citric acid
  19. Edinburgh minimal medium (EMM) (see Recipes)

Equipment

  1. Glass bottom dish (35 mm dish with a coverslip number = 1.5 and thickness 0.16 – 0.19) (MatTek, catalog number: P35G-1.5-14-C ) covered with Lectin.Spinning disk confocal microscope with a scanner-based FRAP system
  2. We use the Andor Revolution Spinning Disc System (Andor Technology), consisting of a Yokogawa CSU-X1 spinning disk scan head (Yokogawa Electric), which is connected to an Olympus IX81 inverted microscope (OLYMPUS). The microscope is equipped with a Prior ProScanIII xy scanning stage (Prior Scientific, Rockland MA, USA) and an Olympus UPlanSApo 100x/1.4 NA oil objective (OLYMPUS). Excitation for acquisition and bleaching is done using a Sapphire 488 nm solid-state laser (50 mW; Coherent). Laser power is controlled using the acousto-optic tunable filter in the Andor Revolution laser combiner (ALC, Andor Technology). The microscope is equipped with an iXon EM + DU-897 BV back illuminated EMCCD camera, cooled to -80 °C, pre-amp gain 2.4, EM gain 300 (Andor Technology). The resulting xy-pixel size in the images is 129 nm.
    Note: Prior to the FRAP experiments, calibration has to be done following the FRAP calibration routine of Andor iQ software using the Andor FRAPPA calibration slide. Briefly, the software guides the user through a series of point-bleach steps during which one has to indicate the bleached point location in the image. This way the scanner bleach position is calibrated with the point position in the image.
  3. BL HC 525/30 (Semrock)
  4. Spectrometer

Software

  1. Andor iQ2 software (Andor Technology)
  2. Fiji (http://fiji.sc/Fiji)
  3. Matlab (MathWorks) software

Procedure

  1. A 5 ml pre-culture of cells is grown in EMM plus supplements over night. The next day, the cells are refreshed in a 20 ml culture until exponential growth, which is done by diluting 1 ml pre-culture with 19 ml fresh EMM (1:20). Cell number can be measured via optical density measurements on a spectrometer. To obtain cells in exponential growth, an OD600 = 0.1 – 0.2 should be achieved. This would approximate to a cell number between 2 x 106 cells/ml – 1 x 107 cells/ml. An OD600 = 1, is linear to the cell number.
  2. Imaging culture dishes with a glass-bottom are covered with 1 μl Lectin (2 mg/ml) and left to dry.
  3. 100 μl of cells in EMM from the culture are added to the dish and left to settle for 10 minutes.
  4. Cells, which were stuck to the dish via lectin, are washed once with 2 ml EMM to remove loose/un-attached cells, and 1.5 ml EMM is added.
  5. FRAP Experiments are performed at room temperature in three successive steps (a. b. c. see below) controlled by Andor iQ2 software. Ideally 30 – 50 cells are measured and analysed.
    1. Prebleach: after focusing the cell of interest, a time-lapse of 50 images of a single plane of the nucleus is acquired with 50 ms exposure time with a 488 nm laser, at 10-15% (details see above, emission filter is BL HC 525/30).
    2. Bleach: bleaching is performed on a 2 x 2 square pixel area (inside the nucleus, away from the nucleolus) with 50% of the 488 nm laser, with a dwell time of 1 ms and 2 repeats on each pixel (details above, emission filter is BL HC 525/30).
    3. Postbleach: following the bleaching, imaging is continued as before for 400 times with 50 ms exposure time of 10-15% of the 488 nm laser (emission filter is BL HC 525/30).
      Note: It is important to establish the appropriate laser exposure time and intensity before the FRAP experiment in a ‘not saturated fluorescence condition’ (Arbitrary units of fluorescence go from a range of 0 – 255. In order to have non- saturated conditions, the laser has to be set to an average saturation above 0 but below 255) to avoid excessive bleaching and allow fluorescence quantification for further analysis. This might mean that the image quality is rather poor.
  6. Analysis
    1. Briefly, a region of interest (ROI, see Figure 1) with a width of 5 pixels and a length roughly equal to the length of the nucleus, L, is drawn.


      Figure 1. FRAP experiments on GFP in the nucleus of fission yeast. From left to right: A scheme and 3 images of a cell expressing NLS-GFP (strain PD31, Kalinina et al., 2013) before photobleaching (-2.95 s), just after photobleaching (0 s), and the subsequent image (0.059 s). The cross in the middle panel marks the center of the bleached region (2 x 2 pixels). A region of interest (ROI, magenta rectangle) was drawn along the nucleus. Next to the images of the cell, a time-lapse sequence of the enlarged ROI in consecutive images shows the recovery of the GFP. The graph on the right shows the temporal decay of the first Fourier mode. Circles indicate data points, the solid line is a 3-parameter fit (C++) to the function A1(t) = A1(0) exp(-π2Dt/L2) + offset. Here, A1 is the amplitude of the first Fourier mode, t is time, D is the diffusion coefficient, and L is the length of the bleached area. Figure was taken and modified from Kalinina et al. 2013.

    2. The intensities inside the ROI on each image of the movie are summed along the short axis of the ROI.
    3. The resulting one-dimensional fluorescence intensity profiles, corresponding to consecutive time points, are used to calculate the temporal decay of the first Fourier mode.
    4. The diffusion coefficient D was calculated from the decay rate of the amplitude of the first Fourier mode A1(t), as described by Elowitz et al. 1999. For this particular example we get a diffusion coefficient D = 3.7 μm2/s.

Recipes

  1. Depending on the strain, the appropriate medium should be used. For imaging the transparent EMM is best (Forsburg and Rhind, 2006)
    For 1 liter solution, weigh in the following components:
    3 g/L   Potassium hydrogen phthallate 14.7 mM
    2.2 g/L
      Na2HPO4
    15.5 mM
    5 g/L
      NH4Cl
    93.5 mM
    20 g/L
      Glucose
    2% w/v
    20 ml/L
      Salts (see below, stock solutions)
    1 ml/L
      Vitamins (see below, stock solutions)
    0.1 ml/L
      Minerals (see below, stock solutions)
    225 mg/L
      Leucin (for PD31, which is auxotroph for Leucin)

    Salts: 50x Stock solution
    Amount
    Component
    Final concentration
    52.5 g/L
    MgCl2.6H2O
    0.26 M
    0.735 g/L
    CaCl2.2H2O
    4.99 mM
    50 g/L
    KCl
    0.67 M
    2 g/L
    Na2SO4
    14.1 mM
    Vitamins: 1,000x Stock
    1 g/L
    Pantothenic acid
    4.20 mM
    10 g/L
    Nicotinic acid
    81.2 mM
    10 g/L
    Inositol
    55.5 mM
    10 mg/L
    Biotin
    40.8 μM
    Minerals: 10,000x Stock
    5 g/L
    Boric acid
    80.9 mM
    4 g/L
    MnSO4
    23.7 mM
    4 g/L
    ZnSO4.7H2O
    13.9 mM
    2 g/L
    FeCl2.6H2O
    7.40 mM
    0.4 g/L
    Molybdic acid
    2.47 mM
    1 g/L
    KI
    6.02 mM
    0.4 g/L
    CuSO4.5H2O
    1.60 mM
    10 g/L
    Citric acid
    47.6 mM
    Note: Taken and modified from: http://www-bcf.usc.edu/~forsburg/media.html.

Acknowledgments

We thank E. Guarino and the Yeast Genetic Resource Center (YGRC, Japan) for strains and plasmids; the Light Microscopy Facility of MPI-CBG (Dresden, Germany) for discussions and advice; the German Research Foundation (DFG) and the Human Frontier Science Program for financial support. M.R.C. was supported by a Marie Curie Intra-European Fellowship. This protocol was adapted from Kalinina et al. (2013).

References

  1. Elowitz, M. B., Surette, M. G., Wolf, P. E., Stock, J. B. and Leibler, S. (1999). Protein mobility in the cytoplasm of Escherichia coli. J Bacteriol 181(1): 197-203.
  2. Forsburg, S. L. and Rhind, N. (2006). Basic methods for fission yeast. Yeast 23(3): 173-183.
  3. Kalinina, I., Nandi, A., Delivani, P., Chacon, M. R., Klemm, A. H., Ramunno-Johnson, D., Krull, A., Lindner, B., Pavin, N. and Tolic-Norrelykke, I. M. (2013). Pivoting of microtubules around the spindle pole accelerates kinetochore capture. Nat Cell Biol 15(1): 82-87. 

简介

我们使用光漂白后荧光恢复(FRAP)计算GFP在裂殖酵母的核质中的扩散系数。 FRAP方法通常可用于测量细胞或其细胞器内的蛋白质的移动性。
在我们的实验中,我们只测量GFP在分裂酵母有丝分裂细胞的核质内的扩散。然而,如果GFP融合到蛋白质,可以在漂白区域中的GFP信号之后计算感兴趣的蛋白质的移动性。然而,我们没有在我们的实验中解决这个问题;因此可以搜索其他来源的这个主题。
要比较FRAP和FLIP,这两种技术都可用于测量细胞内的蛋白质迁移率。然而,使用FRAP,在感兴趣的区域(ROI)中测量蛋白质的扩散,以观察在该区域中荧光的恢复。在FLIP中,在不同于进行漂白的区域测量荧光恢复,以观察标记的蛋白质是否能够移动到该区域,其将变得更暗,获得漂白的蛋白质。这里的主要区别在于,对于FRAP,单一漂白事件是足够的,而FLIP需要许多漂白步骤,以避免在同一区域的荧光蛋白的回流。
技术上FRAP在核和FRAP中细胞质没有区别。然而,我们测量了核内扩散系数(D = 5.6±2.8μm2/s/s)和细胞质中的扩散系数之间的差异(D = 8.6±2.2μm2//s)。这是由于这些隔室内不同的组成,由不同量的蛋白质,DNA和RNA组成。

材料和试剂

  1. 在细胞核中表达GFP的分裂酵母细胞(例如来自Kalinina等人的PD31等人,2013)。
  2. 凝胶(Sigma-Aldrich,目录号:L2380)
  3. MgCl 2 6H <2> O
  4. CaCl 2 2H O
  5. KCl
  6. Na 2 4
  7. 泛酸
  8. 烟酸
  9. 肌醇
  10. 生物素
  11. 硼酸
  12. MnSO 4
  13. ZnSO 4 。 7H O
  14. FeCl <2> 6H <2> O
  15. 钼酸
  16. KI
  17. CuSO 4 5H sub 2 O
  18. 柠檬酸
  19. 爱丁堡基本培养基(EMM)(参见食谱)

设备

  1. 覆盖有Lectin的玻璃底皿(盖玻片号= 1.5且厚度为0.16-0.19的35mm皿)(MatTek,目录号:P35G-1.5-14-C)。具有基于扫描仪的FRAP系统的透射盘共聚焦显微镜/>
  2. 我们使用安道尔革命旋转盘系统(安道科技),由横河CSU-X1旋转磁盘扫描头(横河电机)组成,连接到奥林巴斯IX81倒置显微镜(奥林巴斯)。显微镜配备有Prior ProScanIII xy扫描台(Prior Scientific,Rockland MA,USA)和Olympus UPlanSApo 100x/1.4 NA油物镜(OLYMPUS)。采集和漂白的激发使用Sapphire 488nm固态激光器(50mW;相干)进行。使用Andor Revolution激光组合器(ALC,Andor Technology)中的声光可调谐滤波器控制激光功率。显微镜配备有iXon EM + DU-897 BV背照式EMCCD照相机,冷却至-80℃,前置放大增益2.4,EM增益300(安道尔技术)。所得到的图像中的xy像素尺寸为129nm 注意:在FRAP实验之前,必须在使用Andor FRAPPA校准载片的Andor iQ软件的FRAP校准程序之后进行校准。简而言之,软件引导用户通过一系列点漂移步骤,在此期间必须指示图像中的漂白点位置。这样,扫描仪漂白位置用图像中的点位置校准。
  3. BL HC 525/30(Semrock)
  4. 光谱仪

软件

  1. Andor iQ2软件(Andor技术)
  2. 斐济( http://fiji.sc/Fiji
  3. Matlab(MathWorks)软件

程序

  1. 将5ml预培养的细胞在EMM +补充物中生长 晚。 第二天,将细胞在20ml培养物中更新直至 指数生长,其通过用19稀释1ml预培养物来进行 ml新鲜EMM(1:20)。 细胞数目可以通过光密度测量 在光谱仪上的测量。 为了获得指数生长的细胞, 应当实现OD <600> = 0.1-0.2。 这将近似于a 细胞数在2×10 6个细胞/ml-1×10 7个细胞/ml之间。 OD <600> = 1, 与单元格数量成线性关系。
  2. 带有玻璃底的成像培养皿用1μl凝集素(2mg/ml)覆盖,并使其干燥。
  3. 将100μl来自培养物的EMM中的细胞加入培养皿中并静置10分钟
  4. 通过凝集素粘附于培养皿的细胞用2洗涤一次 ml EMM以除去松散/未附着的细胞,并加入1.5ml EMM。
  5. FRAP实验在室温下连续进行三次 由Andor iQ2软件控制的步骤(a.b.c.见下文)。 理想情况下30 -   50细胞。
    1. Prebleach:在聚焦感兴趣的细胞后,使用488nm激光以50-15%的曝光时间以10-15%获得核的单个平面的50个图像的延时(细节参见上文,发射滤光片是BL HC 525/30)。
    2. 漂白:漂白在50%的488nm激光器的2×2正方形像素区域(在核内部,远离核仁)上进行,每个像素上的停留时间为1ms,并且2个重复(上面的细节, 发射滤光片为BL HC 525/30)。
    3. Postbleach:在漂白之后,如前所述继续成像400次,其中50nm曝光时间为488nm激光(发射滤光片为BL HC 525/30)的10-15%。
      注意:在"不饱和荧光条件"(任意荧光的范围从0到255的范围内)之前,在FRAP实验之前建立适当的激光曝光时间和强度是重要的。为了具有非饱和条件下,激光器必须设置为高于0但低于255的平均饱和度),以避免过度漂白并允许荧光定量用于进一步分析。这可能意味着图片质量相当差。
  6. 分析
    1. 简而言之,绘制具有5个像素的宽度和大致等于核的长度L的长度的感兴趣区域(ROI,参见图1)。


      图1.裂变酵母细胞核中GFP上的FRAP实验从左到右:表达NLS-GFP的细胞的方案和3个图像(菌株PD31,Kalinina等人, ,光漂白(-2.95s),光漂白后(0s)和随后的图像(0.059s)。中间面板中的十字标记了漂白区域的中心(2 x 2像素)。沿着核绘制感兴趣区域(ROI,品红色矩形)。旁边的 细胞的图像,连续图像中放大的ROI的延时序列显示GFP的恢复。右图显示了第一傅立叶模式的时间衰减。圆圈表示数据点,实线是函数 A 1 (t) = 的三参数拟合A 1 (0)exp(-π 2 D t/ )+ offset。这里, 1 是第一傅立叶模式的振幅, t 是时间, ,并且 L 是漂白区域的长度。从Kalinina等人采取和修改图。 。

    2. 沿着ROI的短轴对电影的每个图像上的ROI内部的强度求和。
    3. 得到的对应于连续时间点的一维荧光强度分布用于计算第一傅里叶模式的时间衰减。
    4. 从第一傅里叶模式的振幅的衰减速率计算扩散系数D,如Elowitz所描述的,其中Elowitz em>等。对于该特定示例,我们得到扩散系数 D =3.7μm 2 /s。

食谱

  1. 根据应变,应使用适当的介质。 对于成像透明EMM是最好的(Forsburg和Rhind,2006)
    对于1升溶液,称重以下组分:
    3  g/L Φ  Phthallate hydrogen potassium 14.7 mM
    2.2 g/L
       Na 2 HPO 4
    15.5 mM
    5 g/L
       NH 4 Cl
    93.5 mM
    20 g/L
      葡萄糖
    2%w/v
    20 ml/L
       盐(见下文,储备溶液)
    1 ml/L
       维生素(见下文,储备溶液)
    0.1 ml/L
       矿物(见下文,库存解决方案)
    225 mg/L
       Leucin(对于PD31,其是亮氨酸的营养缺陷型)

    盐:50x储液
    金额
    组件
    最终浓度
    52.5克/升
    MgCl 2 6H <2> O
    0.26 M
    0.735g/L
    CaCl 2 2H O
    4.99 mM
    50克/升
    KCl
    0.67 M
    2 g/L
    Na 2 4
    14.1 mM
    维生素:1,000x股
    1 g/L
    泛酸
    4.20 mM
    10 g/L
    烟酸
    81.2 mM
    10 g/L
    肌醇
    55.5 mM
    10 mg/L
    生物素
    40.8μM
    矿物:10,000x股
    5 g/L
    硼酸
    80.9 mM
    4 g/L
    MnSO 4
    23.7 mM
    4 g/L
    ZnSO 4 。 7H O
    13.9 mM
    2 g/L
    FeCl <2> 6H <2> O
    7.40 mM
    0.4 g/L
    钼酸
    2.47 mM
    1 g/L
    KI
    6.02 mM
    0.4 g/L
    CuSO 4 5H sub 2 O
    1.60 mM
    10 g/L
    柠檬酸
    47.6 mM
    注意:取自并修改自: http://www-bcf .usc.edu /〜forsburg/media.html

致谢

我们感谢E. Guarino和酵母基因资源中心(YGRC,日本)的菌株和质粒; MPI-CBG(德国德累斯顿)的光学显微设备进行讨论和建议; 德国研究基金会(DFG)和人类前沿科学计划提供财政支持。 M.R.C. 得到了玛丽·居里欧洲奖学金的支持。 该协议改编自Kalinina等人(2013)。

参考文献

  1. Elowitz,M.B.,Surette,M.G.,Wolf,P.E.,Stock,J.B.and Leibler,S。(1999)。 大肠杆菌细胞质中的蛋白质迁移。 em> J Bacteriol 181(1):197-203。
  2. Forsburg,S.L。和Rhind,N。(2006)。 裂殖酵母的基本方法 酵母 23(3 ):173-183。
  3. Kalinina,I.,Nandi,A.,Delivani,P.,Chacon,MR,Klemm,AH,Ramunno-Johnson,D.,Krull,A.,Lindner,B.,Pavin,N.and Tolic-Norrelykke,IM (2013年)。 微管在心轴极点周围的枢转加速了神经细胞的捕获。 15(1):82-87。 
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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: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Delivani, P., Chacón, M. R., Schroth-Diez, B. and Tolić-Nørrelykke, I. M. (2013). Fluorescence Recovery After Photobleaching (FRAP) in the Fission Yeast Nucleus. Bio-protocol 3(20): e941. DOI: 10.21769/BioProtoc.941.
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