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Subcellular Localization Experiments and FRET-FLIM Measurements in Plants
植物中的亚细胞定位实验和FRET-FLIM 测定   

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Abstract

Determining the localization of proteins within living cells may be very essential for understanding their biological function. Usually for analysis of subcellular localization, a construct encoding the translational fusion of a cDNA of interest with a fluorescent protein (FP) is engineered, transiently expressed in plant cells and examined with confocal microscopy.
In co-localization and interaction studies, two plasmids, each encoding one of the potential interacting/binding partners tagged with an appropriate pair of fluorescence proteins (for instance CFP/YFP) are co-expressed in plant cells. If proteins co-localize in certain cellular compartments it does not necessarily mean that they bind/interact to each other, therefore an additional technique should be applied for in vivo verification of putative interaction, e.g. Fluorescence Lifetime Imaging (FLIM) to detect Fluorescence Resonance Energy Transfer (FRET).
The protocol describes in detail the method that has been used to verify interaction between the bacterial effector HopQ1 and a 14-3-3a host protein and additionally to check the necessity of the central serine in the canonical 14-3-3 binding site within HopQ1 (Giska et al., 2013) for this association.

Materials and Reagents

  1. 3 – 4 weeks old Nicotiana benthamiana plants grown in a greenhouse
  2. Agrobacterium tumefaciens strain GV3101
  3. For co-localization and FRET-FLIM measurement selected pGWB (Gateway) binary vectors were used (Nakagawa et al., 2007):
    1. pGWB454 encoding Protein1 (here HopQ1-mRFP) -mRFP
    2. pGWB441 encoding Protein2 (here 14-3-3a-YFP) -YFP
    3. pGWB444 encoding unfused, free CFP
    4. pGWB441 encoding unfused, free YFP
    5. pGWB444 encoding CFP fused to YFP
    6. pGWB441 encoding Protein1- (here HopQ1-YFP) -YFP
    7. pGWB441 encoding Protein1a-YFP (e.g. mutated variant of Protein1)
    8. pGWB444 encoding Protein2 (here 14-3-3a-CFP) -CFP

Equipment

  1. For Microscopic evaluation
    Nikon Stereomicroscope SMZ 1500 with epi-fluorescence equipment (optional)
    Nikon Eclipse TE2000-E inverted C1 confocal laser scanning microscope, equipped with PlanApo 63x immersion oil objective, solid-state Coherent Sapphire 488-nm laser, Helium-Neon (HeNe) 543 nm laser, detector unit with three photomultiplier
    Useful link, http://www.microscopyu.com/
  2. For FRET-FLIM measurement
    FRET-FLIM experimental procedure is optimized for a Zeiss LSM 510 META confocal laser scanning microscope equipped with a FLIM module SPC730 (Becker and Hickl) for time correlated single photon counting (TCSPC). Single photons were detected with a photomultiplier MCP-PMT, R3809U-52, Hamamatsu Photonics. For excitation of photons, an ultrafast oscillating multiphoton excitation laser (titanium-sapphire, Chameleon, Coherent) was used (Chameleon XS, Coherent)
  3. Microscope slides (Gerhard Menzel GmbH, Menzel-GlaserTM, catalog number: AA00000112E )
  4. Microscope Cover Slips no. 1 (Gerhard Menzel GmbH, Menzel-GlaserTM, catalog number: BB024060A1 )

Software

  1. EZ C1 for Nikon C1 confocal – image acquisition
  2. EZ C1 Viewer for Nikon C1 confocal – image analysis
  3. LSM 510 for Zeiss Meta 510 Confocal – image acquisition
  4. Zeiss LSM Image Browser – image analysis
  5. SPC operation software for FLIM measurement (Becker & Hickl GmbH)
  6. SPC-Image 2.9.1 software (Becker & Hickl GmbH, http://www.becker-hickl.de/pdf/flim-zeiss-man37.pdf) – image analysis
  7. ImageJ freeware (http://rsbweb.nih.gov/ij/) – image analysis
  8. MS Excel

Procedure

  1. Experimental design to study subcellular localization of fusion proteins co-expressed in living plant cells
    1. Choosing a fluorescence protein
      There is a broad range of fluorescent protein variants used for the construction of fluorescence chimeric proteins that can be expressed in living cells (http://www.microscopyu.com/articles/livecellimaging/fpintro.html). Not all have been tested in plants. Recently a set of fluorescent protein Gateway entry vectors was generated by Mylle et al., 2013 that offers a valuable resource for plant cell biologists (http://gateway.psb.ugent.be). The Green Fluorescence Protein (GFP) and its color shifted genetic derivatives are most commonly used in fusion with proteins of interest to monitor their cellular localization. For more complex imaging experiments like co-localization studies of multiple fusion proteins, the selection of FPs must be based on the technical specifications of the confocal microscope that will be used. Instruments with simple optical setup can clearly distinguish between fluorescence proteins having none or minimal spectral overlap (for example CFP/GFP/RFP), combination of GFP and YFP should be avoided. Confocal microscopes enabling spectral imaging coupled to mathematically linear unmixing of the measured spectral profiles provide the ability to distinguish between large numbers of different fluorophores with partially overlapping spectra.
      In our lab for analysis of subcellular localization we usually make constructs encoding the protein of interest fused to GFP or YFP while red (e.g. mRFP, mCherry) and green fluorescence proteins are commonly used in combination for co-localization studies. The position of the fluorescence marker may affect the expression level of the gene construct. Therefore it is recommended to test the potential consequence of the N-terminal or C-terminal fusion of the FP to protein of interest on its expression level and localization pattern.
      Note: If the subcellular localization pattern of protein of interest with FP at N and C-terminus is similar we can assume that fusion with FP does not influence protein sorting and protein fusions reach their destinations in the cell. However, we observed that some protein fusions with FP at N-terminus may be completely retained in the endoplasmic reticulum while FP at C-terminus does not influence their subcellular targeting. The opposite situation is also possible.
    2. Choosing the transient gene expression system
      We perform different transient expression assays; each has its own advantages and disadvantages depending on research goals:
      1. Routinely infiltration of Nicotiana benthamiana leaves with Agrobacterium tumefaciens strain GV3101 carrying an appropriate construct is used [procedure described already by Xiyan Li (Li, 2011)]
        1. Advantages: Provides usually high transformation rates and allows the simultaneous expression of multiple proteins in single cells with levels sufficient for detection of fluorescence signals with confocal microscopy.
        2. Disadvantage: Limited applicability for other plants; Agrobacterium may also affect distribution of some proteins within plant cells, e.g. related to plant immunity.
      2. Biolistic bombardment of plant leaves with tungsten particles coated with plasmid DNA (Lichocka, 2014)
        1. Advantage: Can be applied for a wide range of plants for which agroinfiltration does not work efficiently; this method is fast, fluorescence of free GFP that is expressed under control of 35S promoter can be detected already after 6 h.
        2. Disadvantage: Requires expensive equipment, limited transformation efficiency.
      3. Transfection of protoplasts isolated from Arabidopsis thaliana leaves or seedlings
        1. Advantage: Enables better single cell imaging with high resolution.
        2. Disadvantage: Requires substantial expertise, limited applicability e.g. cell wall proteins cannot be analyzed, subcellular morphology might be altered compared to intact cells.
          Note: Only infiltration with Agrobacterium require cloning of expression cassette into binary plasmid, for biolistic bombardment and transfection of protoplast plasmids encoding the gene of interest under control of a plant promoter are sufficient.

  2. Microscopic evaluation
    1. Prior to confocal microscopy, plant samples can be screened under a fluorescence stereomicroscope to check if transient expression of the introduced gene construct was successful. Under low magnification, transfection rate and expression level can be estimated and an appropriate leaf peace with cells exhibiting strong fluorescence can also be cut out. Precise localization of the fusion protein within single plant cells must be done with confocal microscopy.
    2. Plant leaf fragments are placed on microscopic slides in a drop of water and covered with cover slips.
    3. For recording of images a PlanApo 63x immersion oil objective is usually used. GFP and YFP are excited with a Coherent Sapphire 488-nm laser; images of mRFP are obtained using 543 nm HeNe laser excitation. The fluorescence of GFP/YFP and mRFP is detected using the 515/30 and 605/75 band pass emission filter, respectively. The image must be optimized by adjusting the pinhole, detector gain, and laser power. If the detected fluorescence signal is low, pinhole diameter may be increased, however, this will lower optical resolution. If the pinhole is opened, more and more noise fluorescence is detected resulting in loss of confocal imaging. The laser power should be adjusted with care to avoid photodamage and photobleaching of the fluorophore. The general idea is to use the minimum amount of laser power to get sufficient signal at gain levels that does not result in too much background noise. To monitor the level of image saturation a special LUT (indicator box) should be checked which will display saturated pixels in a contrasting color. Only few pixels should be highlighted by showing the brightest signal ensuring that the full dynamic range from 0 to 255 is utilized in 8-bit color depth.
    4. To optimally separate the fluorescence of co-expressed fluorophores the imaging is performed in sequential mode. Images are collected in z- series with a step size of 0.5 μm. In our experiments the proteins of interest localize to cytoplasm and nucleus, therefore z-series included the epidermal cell volume including nucleus. For quantitative evaluation of fluorescence intensity within the cytoplasm and the nucleus, only certain type of cells are chosen for imaging. To eliminate the influence of the imaging depth on the fluorescence intensity only cells with nucleus located near the outer epidermal cell wall facing the coverslip are recorded. Usually plant cells with the nucleus located deeper or close to the transverse cell walls are omitted. The quality of images collected from deeper tissue layers is worse due to the scattering of laser light and definitely not appropriate for quantification and comparison of fluorescence intensity.
    5. It is recommended to compare single optical sections including nuclei with clearly visible nucleoli. Collected images can be processed using image analysis freeware ImageJ (http://rsbweb.nih.gov/ij/). ImageJ is a Java image processing program that can be supplemented by the user with a large number of plugins (available on ImageJ web site) that extend the capabilities of ImageJ and are useful in extracting quantitative data from confocal images.

  3. ImageJ for average fluorescence intensity measurement
    1. We applied ImageJ to measure average fluorescence intensities in cellular compartments for proteins that change their distribution in the presence of interacting partners. Sometimes it is difficult to distinguish by eye whether fluorescence intensity has changed in certain cellular compartment; therefore it is recommended to include quantitative evaluation of fluorescence images.
      Note: For quantitative evaluation and comparison of fluorescence intensities it is absolutely necessary to take images with the same hardware parameters, i.e. objective, laser power, pinhole opening, gain and offset values, zoom factor, otherwise comparison of relative image brightness is not really accurate. To minimize the influence of experimental condition on the fluorescence intensity in the plant samples it is recommended to prepare and analyze all experimental variants at the same time under the same conditions.
    2. Presented example illustrates (Figure 1) shift in the localization of 14-3-3a-YFP out of the nucleus and into the cytoplasm in the presence of the interacting partner, the bacterial effector HopQ1-mRFP. The observed phenomenon can be presented as the ratio of average fluorescence intensity in the cell nucleus versus average intensity in the cell cytoplasm (N/C ratio). Therefore average fluorescence intensity in specific cellular compartments must be quantified as follows:


      Figure 1. Transient expression of construct encoding 14-3-3a-YFP (A) and co-expression of 14 3-3a-YFP and HopQ1-mRFP (B&C) in N. benthamiana leaves. White arrowheads indicate cytoplasm, asterisks – nucleus, arrow – nucleoli. Scale bars represent 10 μm.

      1. For fluorescence quantification confocal images with zoomed nuclear region were chosen.
      2. Export images to ImageJ; open Plugins list and select ROI/ Multi Measure.
        Using drawing/selection tool draw small rectangular or circular region of interest (ROI) within the selected image. Add each ROI selection to the Multi Measure list; we usually select three different ROIs in each cellular compartment (i.e. nucleus and cytoplasm) and three to four in the background. To analyze ROIs of the same size one can move the ROI selection by dragging with the cursor to the next place on the image and add it to the Multi Measure list (Figure 2).
      3. From main menu select Analyze/Set Measurements and in the check box list select Area, Integrated Density and Mean Gray Value.
        Area: area of selection in square pixels
        Integrated Density: the product of Area and Mean Gray Value
        Mean Grey Value: the sum of the gray values of all the pixels in the selection divided by the number of pixels
      4. In Multi Measure window press Select All and Measure, the window Results appears with quantified data that can be exported to MS Excel (Figure 2).


        Figure 2. Multi ROIs fluorescence intensity measurement by ImageJ

      5. All selected ROIs can be drawn on image by clicking the Draw button in the Multi Measure window.
      6. Background correction of fluorescence intensity must be performed before calculation of fluorescence distribution ratio (Figure 3) for each ROI as follows:
      7. ROI1 = Integrated Density ROI1 – (Area ROI1 * average background Mean Grey Value)
      8. Background corrected N/C ratios are calculated from mean fluorescence intensity in nucleus and cytoplasm. For N/C ratios calculation, ca. 25 cells from each experimental combination should be analyzed. The absolute N/C values may vary somewhat between experiments depending on the imaging conditions and thus comparisons should be made for paired experiments.


        Figure 3. Background correction of fluorescence intensity within nucleus and cytoplasm and calculation of N/C ratio for 14-3-3 protein expressed alone or in HopQ1 presence

  4. FRET-FLIM for analysis of interaction between proteins fused to YFP and CFP, co-expressed in leaf epidermal cells of Nicotiana benthamiana
    1. Design of FRET-FLIM experiments
      1. For FRET-FLIM experiments each of the potentially interacting proteins must be fused to appropriate fluorophores displaying spectral properties that together constitute a "FRET pair". Here we describe a protocol for one of the commonly used FRET fluorophore pair: cyan fluorescence protein (CFP, donor) and yellow fluorescence protein (YFP, acceptor). There are other combinations of FP-tagged proteins that can be used for FRET-FLIM measurements, such as eGFP (Enhanced-Green Fluorescent Protein) as a donor and the mCherry (monomeric Cherry red fluorescent variant) as an acceptor (more http://www.microscopyu.com/articles/fluorescence/fret/fretintro.html). The available microscope setup usually determines the fluorophore pair that will be used.
      2. FRET is a process of radiantionless energy transfer from donor to acceptor fluorophore if they are in close vicinity (distance of < 10 nm). In FRET-FLIM imaging only the donor fluorophore lifetime is measured, here CFP. This parameter refers to the duration of excited state of the fluorophore and can be influenced by external factors such as pH or in our case presence of the acceptor. When FRET occurs, shortening of fluorescence lifetime of the donor can be measured.
      3. Plant cells transiently expressing the following combinations of fusion proteins are required:
        1. Unfused, free CFP alone (used for initial imaging calibration)
        2. Co-expressed unfused, free CFP and unfused, free YFP (negative control no.1)
        3. CFP fused to YFP (positive control, separated by 11 amino acids)
        4. Protein2-CFP alone
        5. Protein2-CFP and unfused, free YFP (negative control no.2)
        6. Protein2-CFP and Protein1-YFP and optionally
        7. Protein2-CFP and mutated variant of Protein1a-YFP
    2. Transient co-expression of gene constructs in plant cells
      1. Infiltrations of N. benthamiana with A. tumefaciens (GV3101) were performed as already described by Xiyan Li (Li, 2011).
        Notes:
        1. For infiltration chose older leaves (but avoid cotyledons) with flat blade, younger leaves are more folded that make them not convenient for microscopy because stretching on microscope slide is not possible.
        2. Avoid damaging of leaf tissue with syringe during infiltration; strong autofluorescence of dead cells may be confusing; you can make small 4-5 injuries of the lower (abaxial) epidermis with pipette tip (size up to 200 ul) around the blade edge and by pressing syringe at injury sites infiltrate the whole leaf, it markedly improves infiltration of leaf tissue.
      2. Prior to infiltration Agrobacterium cultures carrying plasmid encoding proteins fused to CFP and YFP are mixed in combinations in equal amounts to a final OD600 0.6-1.0.
      3. After three days leaves are examined under fluorescence stereomicroscope to check for plant cells expressing both CFP and YFP fusion proteins at acceptable levels. If expression level is very low, the number of detected photons during FLIM measurements might be not sufficient for lifetime calculations.
    3. FLIM measurement
      1. N. benthamiana leaf fragments are placed on slides in a drop of water and covered with coverslips.
      2. Measurements are performed on plant samples prepared as described in section "design of FRET-FLIM experiment" and confocal microscope settings are as described earlier by Kwaaitaal et al. (2010).
      3. For confocal imaging of plant cells co-expressed CFP and YFP fusion proteins, a 40x plan-apochromat water immersion objective is used. CFP and YFP are excited with the 458 nm and 514 nm laser line from the Agron-laser, respectively. Excitation of CFP for FLIM requires the ultrafast, multiphoton excitation Titanium:Sapphire laser tuned to a wavelength of 840 nm for 2-photon excitation of CFP, adjusted to 15-20% of transmission. Higher laser transmission can produce local heating which cause cell damage leading to autofluorescences and artificial photon emission.
      4. A selected area of interest is scanned continuously for 120 sec with a resolution of 128 x 128 pixels and 256 time channels. Usually this is sufficient to obtain a histogram of fluorescence decay, representing the number of photons recorded at each detection time in each pixel of the scanned ROI.
      5. The histograms are analyzed with the SPC-Image 2.9.1 software. The raw data are displayed as an intensity image (Figure 4, b + w image, left), from this a lifetime image is calculated (color coded image Figure 4 right), showing the distribution of lifetimes of the scanned ROI. In the images (Figure 4) sub-ROIs can be selected to display respective lifetime distributions. To calculate fluorescence lifetimes the decay curves in the individual pixels are fitted to an appropriate model. For estimations the brightest area on the intensity image must be selected. Typical models are either single exponential or multiexpotential decay curves. We assumed that the donor alone displays a monoexponential decay whereas a bi-expotential model adequately describes the FRET-situation. The  value is a measure for the fitting of data to the model, values of < 1.2 are regarded as good fit.


        Figure 4. The main panel of the SPCImage analysis software with displayed data from FLIM measurement collected for the positive control CFP-YFP protein fusion

      6. As Instrument Response Function (IRF), which is defined as the pulse shape of the FLIM system records for an infinitely short fluorescence lifetime, the value calculated by the SPCImage data analysis software was taken which in our experiments was sufficient for obtaining relevant lifetime data.
      7. The fit of fluorescence decay function can be improved by using introducing a binning factor and threshold level to exclude pixels with low photon number (more http://www.becker-hickl.de/pdf/flim-zeiss-man37.pdf). For calculation of mean lifetime of the individual cell the data are exported to Ms-Excel program.
    4. FRET efficiency calculation
      1. FLIM data should be recorded for a sufficient number of cells (>10) from each experimental combination to obtain statistically significant results. By comparing the average lifetime of the donor in the presence of an acceptor tDA τ with the average lifetime measured in absence of the acceptor tD, FRET efficiency can be calculated as follows:
        EFRET = 1- (tDA FRET/tD) * 100%
        Note: FLIM theory is hard-core physics, which is hard (or even not) to understand for biologists, we should be cautious with theoretical explanations and better refer to literature Becker & Hickl Manual (Becker, 2010) that is very useful in this regard.

Acknowledgments

This protocol is based on the procedure described by Giska et al. (2013).

References

  1. Becker W. (2010). The bh TCSPC Handbook. Fourth edition. Becker and Hickl GmbH. 
  2. Giska, F., Lichocka, M., Piechocki, M., Dadlez, M., Schmelzer, E., Hennig, J. and Krzymowska, M. (2013). Phosphorylation of HopQ1, a type III effector from Pseudomonas syringae, creates a binding site for host 14-3-3 proteins. Plant Physiol 161(4): 2049-2061.
  3. Kwaaitaal, M., Keinath, N. F., Pajonk, S., Biskup, C. and Panstruga, R. (2010). Combined bimolecular fluorescence complementation and Forster resonance energy transfer reveals ternary SNARE complex formation in living plant cells. Plant Physiol 152(3): 1135-1147.
  4. Lichocka, M. (2014). Biolistic bombardment for co-expression of proteins fused to YFP and mRFP in leaf epidermal cells of Phaseolus vulgaris 'red mexican'. Bio-Protocol 4(1): e1019.
  5. Li X. (2011). Infiltration of Benthamiana protocol for transient expression via Agrobacterium. Bio-Protocol 1(14): e95.
  6. Mylle, E., Codreanu, M. C., Boruc, J. and Russinova, E. (2013). Emission spectra profiling of fluorescent proteins in living plant cells. Plant Methods 9(1): 10.
  7. Nakagawa, T., Suzuki, T., Murata, S., Nakamura, S., Hino, T., Maeo, K., Tabata, R., Kawai, T., Tanaka, K., Niwa, Y., Watanabe, Y., Nakamura, K., Kimura, T. and Ishiguro, S. (2007). Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem 71(8): 2095-2100.

简介

确定蛋白质在活细胞内的定位可能是非常必要的了解其生物学功能。通常对于亚细胞定位的分析,编码感兴趣的cDNA与荧光蛋白(FP)的翻译融合的构建体被改造,在植物细胞中瞬时表达并用共聚焦显微镜检查。在共定位和相互作用研究,在植物细胞中共表达两种质粒,每种质粒编码用适当的荧光蛋白对(例如CFP/YFP)标记的一种潜在的相互作用/结合伴侣。如果蛋白质共定位在某些细胞区室中,其不一定意味着它们彼此结合/相互作用,因此应当应用另外的技术用于体内假定相互作用的验证,/em>荧光寿命成像(FLIM)以检测荧光共振能量转移(FRET)。该协议详细描述了用于验证细菌效应物HopQ1和14-3-3a宿主之间的相互作用的方法蛋白质,并且另外检查HopQ1中的规范14-3-3结合位点中的中心丝氨酸的必要性(Giska等人,2013)。

材料和试剂

  1. 3至4周龄的在温室中生长的烟草Nicosiana benthamiana 植物
  2. 根癌土壤杆菌菌株GV3101
  3. 对于共定位和FRET-FLIM测量,使用选择的pGWB(Gateway)二元载体(Nakagawa等人,2007):
    1. 编码Protein1(此处为HopQ1-mRFP)-mRFP的pGWB454
    2. 编码Protein2(这里是14-3-3a-YFP)-YFP的pGWB441
    3. pGWB444编码未融合,游离的CFP
    4. pGWB441编码未融合,游离YFP
    5. 编码与YFP融合的CFP的pGWB444
    6. 编码蛋白1(此处为HopQ1-YFP)-YFP的pGWB441
    7. 编码Protein1a-YFP(例如蛋白质1的突变变体)的pGWB441
    8. 编码Protein2(这里是14-3-3a-CFP)-CFP的pGWB444

设备

  1. 对于显微镜评价
    尼康立体显微镜SMZ 1500配备落射荧光设备(可选)
    Nikon Eclipse TE2000-E倒置C1共聚焦激光扫描显微镜,配有PlanApo 63x浸油物镜,固态相干蓝宝石488nm激光器,氦氖(HeNe)543nm激光器,带三个光电倍增管的检测器单元
    实用链接, http://www.microscopyu.com/
  2. 用于FRET-FLIM测量
    FRET-FLIM实验程序对于装备有用于时间相关单光子计数(TCSPC)的FLIM模块SPC730(Becker和Hickl)的Zeiss LSM 510 META共焦激光扫描显微镜进行优化。 单光子 用光电倍增器MCP-PMT,R3809U-52,Hamamatsu Photonics检测。 为了激发光子,使用超快振荡多光子激发激光器(钛 - 蓝宝石,Chameleon,Coherent)(Chameleon XS,Coherent)
  3. 显微镜载片(Gerhard Menzel GmbH,Menzel-Glaser TM,目录号:AA00000112E)
  4. 显微镜 1(Gerhard Menzel GmbH,Menzel-Glaser TM ,目录号:BB024060A1)

软件

  1. EZ C1适用于尼康C1共焦 - 图像采集
  2. EZ C1查看器用于尼康C1共焦 - 图像分析
  3. 用于Zeiss Meta 510共聚焦的LSM 510 - 图像采集
  4. Zeiss LSM图像浏览器 - 图像分析
  5. 用于FLIM测量的SPC操作软件(Becker& Hickl GmbH)
  6. SPC-Image 2.9.1软件(Becker& Hickl GmbH, http:///www.becker-hickl.de/pdf/flim-zeiss-man37.pdf ) - 图像分析
  7. ImageJ免费软件( http://rsbweb.nih.gov/ij/) - 图像分析
  8. MS Excel

程序

  1. 实验设计研究在活的植物细胞中共表达的融合蛋白的亚细胞定位
    1. 选择荧光蛋白
      存在广泛范围的荧光蛋白变体,其用于构建可在活细胞中表达的荧光嵌合蛋白 ( http://www.microscopyu.com/articles/livecellimaging/fpintro.html)。不是所有的都已经在植物中测试。最近,一组荧光蛋白Gateway条目载体由Mylle等人于2013年产生,其为植物细胞生物学家提供了有价值的资源( http://gateway.psb.ugent.be )。绿色荧光蛋白(GFP)及其变色的遗传衍生物最常用于与感兴趣的蛋白质融合以监测其细胞定位。对于更复杂的成像实验,如多个融合蛋白的共定位研究,FP的选择必须基于将使用的共聚焦显微镜的技术规格。具有简单光学设置的仪器可以清楚地区分不具有或具有最小光谱重叠的荧光蛋白(例如CFP/GFP/RFP),应避免GFP和YFP的组合。使得光谱成像耦合到测量的光谱轮廓的数学线性解混合的共焦显微镜提供区分具有部分重叠光谱的大量不同荧光团的能力。
      在我们的用于分析亚细胞定位的实验室中,通常使编码感兴趣的蛋白的构建体与GFP或YFP融合,而红色(例如mRFP,mCherry)和绿色荧光蛋白通常组合用于共定位学习。荧光标记物的位置可以影响基因构建体的表达水平。因此,建议测试FP的N-末端或C-末端融合蛋白对其表达水平和定位模式的潜在后果。
      注意:如果感兴趣的蛋白质在N和C末端的FP的亚细胞定位模式是相似的,我们可以假设与FP的融合不影响蛋白质分选和蛋白质融合到达其在细胞中的目的地。然而,我们观察到一些蛋白融合与FP在N端可能完全保留在内质网,而FP在C端不影响其亚细胞靶向。相反的情况也是可能的。
    2. 选择瞬时基因表达系统
      我们执行不同的瞬时表达测定;每个都有自己的优势和劣势,取决于研究目标:
      1. 使用携带合适构建体的根癌农杆菌菌株GV3101常规浸润本氏烟草叶[已经由Xiyan Li(Li,2011)描述的方法]
        1. 优点:通常提供高转化率,并允许在单个细胞中同时表达多种蛋白质,其水平足以通过共聚焦显微镜检测荧光信号。
        2. 缺点:对其他植物适用性有限;土壤杆菌也可以影响植物细胞内一些蛋白质的分布,例如。与植物免疫有关
      2. 用涂覆有质粒DNA的钨颗粒对植物叶子进行生物轰击(Lichocka,2014)
        1. 优点:可应用于农业生产过滤不能有效工作的各种植物; 这种方法是快速的,在35S启动子的控制下表达的游离GFP的荧光可以在6小时后被检测到
        2. 缺点:需要昂贵的设备,转换效率有限。
      3. 从拟南芥叶或幼苗中分离的原生质体的转染
        1. 优点:实现更高分辨率的单细胞成像
        2. 缺点:需要大量专业知识,有限的适用性,例如无法分析细胞壁蛋白,与完整细胞相比,亚细胞形态可能会改变。
          注意:只有用农杆菌的浸润需要将表达盒克隆到二元质粒中,用于生物弹射轰击和在植物启动子控制下转染编码目的基因的原生质体质粒是足够的。

  2. 显微评价
    1. 在共聚焦显微镜检查之前,可以在荧光立体显微镜下筛选植物样品,以检查引入的基因构建体的瞬时表达是否成功。在低放大倍数下,可以估计转染速率和表达水平,并且也可以切出表现出强荧光的细胞的适当叶平衡。融合蛋白在单个植物细胞内的精确定位必须用共聚焦显微镜进行。
    2. 将植物叶碎片置于一滴水中的显微镜载玻片上,并用盖玻片覆盖
    3. 对于图像的记录,通常使用PlanApo 63x浸油物镜。 GFP和YFP用Coherent Sapphire 488-nm激光激发;使用543nm HeNe激光激发获得mRFP的图像。使用515/30和605/75带通发射滤光片分别检测GFP/YFP和mRFP的荧光。必须通过调整针孔,检测器增益和激光功率来优化图像。如果检测到的荧光信号低,则针孔直径可能增加,然而,这将降低光学分辨率。如果针孔打开,则检测到越来越多的噪声荧光,导致共聚焦成像的损失。激光功率应该小心调整,以避免光致损伤和荧光团的光漂白。一般的想法是使用最小量的激光功率以在不导致太多背景噪声的增益水平下获得足够的信号。为了监视图像饱和度的水平,应当检查将以对比颜色显示饱和像素的特殊LUT(指示符框)。只有几个像素应通过显示最亮的信号来突出显示,确保从0到255的全动态范围用于8位颜色深度。
    4. 为了最佳地分离共表达荧光团的荧光,以顺序模式进行成像。图像以0.5μm的步长以z-系列收集。在我们的实验中,感兴趣的蛋白定位于细胞质和细胞核,因此z系列包括表皮细胞体积包括核。为了定量评估细胞质和细胞核内的荧光强度,仅选择某些类型的细胞用于成像。为了消除成像深度对荧光强度的影响,仅记录具有位于靠近面向盖玻片的外表皮细胞壁的细胞的细胞。通常植物细胞 其中核位于更靠近或靠近横向细胞壁的位置。从更深的组织层收集的图像的质量由于激光的散射而更差,并且绝对不适合于荧光强度的定量和比较。
    5. 建议比较单个光学切片,包括核和清晰可见的核仁。收集的图片可以使用图片分析免费软件ImageJ处理( http://rsbweb.nih.gov/ij/)。 ImageJ是一个Java图像处理程序,可以由用户使用大量插件(在ImageJ网站上提供)进行补充,这些插件扩展了ImageJ的功能,可用于从共焦图像中提取定量数据。

  3. ImageJ用于平均荧光强度测量
    1. 我们应用ImageJ来测量在相互作用的配偶体存在下改变其分布的蛋白质的细胞区室中的平均荧光强度。有时,难以通过眼睛区分某些细胞区室中荧光强度是否已经改变;因此建议包括荧光图像的定量评价 注意:对于荧光强度的定量评估和比较,绝对需要用相同的硬件参数拍摄图像,即物镜,激光功率,针孔开度,增益和偏移值,缩放因子,否则相对图像亮度的比较不是真的准确。为了最小化实验条件对植物样品中荧光强度的影响,建议准备和分析所有 实验变体同时在相同条件下。
    2. 所提出的实施例说明(图1)在相互作用配偶体,细菌效应物HopQ1-mRFP的存在下,14-3-3a-YFP定位于细胞核外并进入细胞质。观察到的现象可以表示为细胞核中的平均荧光强度与细胞质中的平均强度的比(N/C比)。因此,特定细胞区室中的平均荧光强度必须定量如下:


      图1.编码14-3-3a-YFP(A)的构建体的瞬时表达和14-3-3a-YFP和HopQ1-mRFP(B& C)在N中的共表达。本哈姆氏病叶。白色箭头表示细胞质,星号 - 细胞核,箭头 - 核仁。比例尺表示10μm。

      1. 对于荧光定量,选择具有缩放的核区域的共焦图像
      2. 将图像导出到ImageJ;打开插件列表并选择ROI /多测量 使用绘图/选择工具在所选图像中绘制小的矩形或圆形感兴趣区域(ROI)。将每个ROI选择添加到多测量列表;我们通常在每个细胞区室(即细胞核和细胞质)中选择三个不同的ROI,并在背景中选择三个到四个。为了分析相同大小的ROI,可以通过将光标拖动到图像上的下一个位置来移动ROI选择,并将其添加到多测量列表 (图2)。
      3. 从主菜单中选择分析/设置测量,并在复选框列表中选择面积,积分密度和平均灰度值。
        面积:方形像素的选择范围
        积分密度:区域和平均灰度值的乘积
        平均灰度值:选择中所有像素的灰度值之和除以像素数
      4. 在"多重测量"窗口中,按选择全部和测量,窗口结果将显示带有可导出到MS Excel(图2)的量化数据。


        图2. ImageJ
        的多个ROI的荧光强度测量
      5. 所有选定的ROI可以通过单击多个测量窗口中的绘制按钮在图像上绘制。
      6. 在计算每个ROI的荧光分布比(图3)之前,必须进行荧光强度的背景校正,如下所示:
      7. ROI1 =综合密度ROI1 - (面积ROI1 *平均背景平均灰度值)
      8. 背景校正的N/C比率是从细胞核和细胞质中的平均荧光强度计算的。对于N/C比率计算,应分析来自每个实验组合的25个细胞。根据成像条件,绝对N/C值可能在实验之间有些变化,因此应当对配对实验进行比较。


        图3.细胞核和细胞质内的荧光强度的背景校正和单独或在HopQ1存在下表达的14-3-3蛋白的N/C比的计算

  4. FRET-FLIM用于分析与YFP和CFP融合的蛋白质之间的相互作用,在本氏烟草的叶表皮细胞中共表达
    1. FRET-FLIM实验设计
      1. 对于FRET-FLIM实验,每个潜在相互作用的蛋白必须融合到合适的荧光团,显示一起构成"FRET对"的光谱性质。在这里我们描述一个常用的FRET荧光对之一的协议:青色荧光蛋白(CFP,捐助者)和黄色荧光蛋白(YFP,受体)。存在可用于FRET-FLIM测量的FP标记蛋白的其他组合,例如作为供体的eGFP(增强型绿色荧光蛋白)和作为受体的mCherry(单体樱桃红色荧光变体)(更多 http://www.microscopyu.com/articles/fluorescence/fret/fretintro.html )。可用的显微镜​​设置通常决定将使用的荧光团对。
      2. FRET是一种从供体到受体荧光团的无辐射能量转移的过程,如果它们在附近(距离<10nm)。在FRET-FLIM成像中,仅测量供体荧光团寿命,这里是CFP。该参数是指荧光团的激发态的持续时间,并且可以受外部因素如pH或在受体的存在情况下的影响。当FRET发生时,可以测量施主的荧光寿命的缩短
      3. 需要瞬时表达以下融合蛋白组合的植物细胞:
        1. 未熔化,单独的CFP(用于初始成像校准)
        2. 共表达的未融合的游离CFP和未融合的游离YFP(阴性对照1号)
        3. CFP与YFP(阳性对照,由11个氨基酸分开)融合
        4. 仅蛋白2-CFP
        5. 蛋白2-CFP和未融合的游离YFP(阴性对照2号)
        6. 蛋白2-CFP和蛋白1-YFP和任选地
        7. 蛋白2-CFP和蛋白1a-YFP的突变变体
    2. 基因构建体在植物细胞中的瞬时共表达
      1. 渗透。 本bent与 tumefaciens (GV3101),如Xiyan Li(Li,2011)所述。
        注意:
        1. 对于浸润,用平叶片选择较老的叶子(但避免子叶),较年轻的叶子更加折叠,使得它们不方便用于显微镜,因为在显微镜载玻片上拉伸是不可能的。
        2. 在注射过程中避免用叶子组织损伤; 死细胞的强自发荧光可能是混乱的; 你可以让小4-5受伤 (背部)表皮,在刀刃周围用移液管尖端(尺寸高达200μl),并且通过在损伤部位按压注射器渗透整个叶,这显着改善了叶组织的渗透。
      2. 在浸润之前,将携带有与CFP和YFP融合的编码蛋白质的质粒的土壤杆菌培养物以等量混合到最终的OD 600 0.6-1.0。
      3. 三天后,在荧光立体显微镜下检查叶片,以检查表达CFP和YFP融合蛋白的植物细胞在可接受的水平。如果表达水平非常低,在FLIM测量期间检测到的光子的数量可能不足以用于寿命计算。
    3. FLIM测量
      1. N。本生烟草叶碎片置于载玻片上的一滴水中,并盖上盖玻片
      2. 对如"FRET-FLIM实验设计"部分所述制备的植物样品进行测量,并且共聚焦显微镜设置如先前由Kwaaitaal等人所述(2010)。
      3. 对于植物细胞共表达的CFP和YFP融合蛋白的共聚焦成像,使用40x平面复消色差水浸物镜。 CFP和YFP分别用来自Agron激光器的458nm和514nm激光线激发。 FLIM的CFP激发需要调谐到840nm波长的超快多光子激发钛:蓝宝石激光用于CFP的双光子激发,调节到15-20%的透射率。较高的激光透射可以产生局部加热,导致细胞损伤,导致自发荧光和人工光子发射
      4. 所选的感兴趣区域连续扫描120秒,分辨率为128×128像素和256个时间通道。通常这足以获得荧光衰减的直方图,表示在扫描的ROI的每个像素中在每个检测时间记录的光子的数量。
      5. 使用SPC-Image 2.9.1软件分析直方图。原始数据显示为强度图像(图4,b + w图像,左),从中计算生命期图像(彩色编码图像右图),显示扫描的ROI的寿命分布。在图像(图4)中,可以选择子ROI以显示相应的寿命分布。为了计算荧光寿命,将各个像素中的衰减曲线拟合到适当的模型。对于估计,必须选择强度图像上的最亮区域。典型模型是单指数或多重电位衰减曲线。我们假设供体单独显示单指数衰减,而双指数模型充分描述了FRET情况。   用于将数据拟合到模型的度量, 1.2被认为是适合的。


        图4. SPCImage分析软件的主面板,显示来自FLIM测量的数据,收集阳性对照CFP-YFP蛋白融合

      6. 作为仪器响应函数(IRF),其定义为FLIM系统的脉冲形状记录无限短荧光寿命,采用通过SPCImage数据分析软件计算的值,在我们的实验中其足以获得相关的寿命数据。
      7. 可以通过使用合并因子和阈值水平来排除具有低光子数的像素来改善荧光衰减函数的拟合(更多http://www.becker-hickl.de/pdf/flim-zeiss-man37.pdf )。对于单个单元格的平均寿命的计算,数据导出到Ms-Excel程序
    4. FRET效率计算
      1. 应当记录来自每个实验组合的足够数量的细胞(> 10)的FLIM数据以获得统计学显着的结果。 通过比较在受体t DA DA存在下的给体的平均寿命与在没有受体t D的情况下测量的平均寿命,可以如下计算FRET效率:< br /> E FRET = 1-(t sub DA FRET /t )* 100%
        注意:FLIM理论是硬核物理学,对于生物学家来说很难(甚至不)理解,我们应该谨慎理论解释,更好地参考文献Becker& Hickl手册(Becker,2010),在这方面非常有用。

致谢

该协议基于Giska等人(2013)描述的程序。

参考文献

  1. Becker W.(2010)。 bh TCSPC手册。第四版。 Becker和Hickl GmbH。
  2. Giska,F.,Lichocka,M.,Piechocki,M.,Dadlez,M.,Schmelzer,E.,Hennig,J.and Krzymowska,M。(2013)。 HopQ1的磷酸化,一种来自丁香假单胞菌的III型效应物,创造了一种用于宿主14-3-3蛋白的结合位点。 植物生理学 161(4):2049-2061。
  3. Kwaaitaal,M.,Keinath,N.F.,Pajonk,S.,Biskup,C.and Panstruga,R。(2010)。 组合的双分子荧光互补和Forster共振能量转移显示活植物细胞中的三元SNARE复合物形成。 a> Plant Physiol 152(3):1135-1147。
  4. Lichocka,M。(2014)。 用于共表达与菜豆绿叶片表皮细胞中的YFP和mRFP融合的蛋白质的生物轰击em>'red mexican'。 生物协议 4(1):e1019。
  5. 李X(2011)。 通过土壤杆菌介导瞬时表达 。 生物协议 1(14):e95
  6. Mylle,E.,Codreanu,M.C.,Boruc,J.and Russinova,E。(2013)。 荧光蛋白在活植物细胞中的发射光谱分析植物方法 9(1):10.
  7. Nakagawa,T.,Suzuki,T.,Murata,S.,Nakamura,S.,Hino,T.,Maeo,K.,Tabata,R.,Kawai,T.,Tanaka,K.,Niwa, Watanabe,Y.,Nakamura,K.,Kimura,T。和Ishiguro,S。(2007)。 改进的Gateway二元载体:用于在植物的转基因分析中创建融合构建体的高性能载体。/a> Biosci Biotechnol Biochem 71(8):2095-2100
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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: © 2014 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. Lichocka, M. and Schmelzer, E. (2014). Subcellular Localization Experiments and FRET-FLIM Measurements in Plants. Bio-protocol 4(1): e1018. DOI: 10.21769/BioProtoc.1018.
  2. Giska, F., Lichocka, M., Piechocki, M., Dadlez, M., Schmelzer, E., Hennig, J. and Krzymowska, M. (2013). Phosphorylation of HopQ1, a type III effector from Pseudomonas syringae, creates a binding site for host 14-3-3 proteins. Plant Physiol 161(4): 2049-2061.
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