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Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)
采用荧光漂白恢复法(FRAP)评估蛋白质合成速率   

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Abstract

Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals.

Keywords: Caenorhabditis elegans(秀丽隐杆线虫), FRAP(荧光漂白恢复), Messenger RNA(信使RNA), Protein synthesis(蛋白质合成), Protein translation(蛋白质翻译)

Background

Proper regulation of protein synthesis is critical for cell homeostasis and growth. Deregulation of protein synthesis has been implicated in pathologies such as cancer and senescent decline (Bjornsti and Houghton, 2004; Syntichaki et al., 2007). Currently available biochemical methods for measuring general protein synthesis rate include metabolic labeling and polysomal profiling (Martin, 1998; Rennie et al., 1994). The applicability of these methodologies is limited due to poor intake and uncontrolled or unequal distribution of the label throughout the animal or tissue of interest. Also, these methods lack specificity and significant changes in specific cells or tissues of interest may be masked due to variability in intrinsic rates of translation of the bulk of the sample. In this protocol, we describe a method for monitoring protein synthesis rates in the nematode Caenorhabditis elegans, based on fluorescence recovery after photobleaching (FRAP). The experimental approach is based on the expression of fluorescent proteins, in cells and tissues of interest of transgenic animals. Fluorescence is then photobleached by irradiating cells, tissues or whole animals with a powerful light source. Recovery of fluorescence, indicative of new protein synthesis, is then monitored in cells or tissues of interest.

Materials and Reagents

  1. Greiner Petri dishes (60 x 15 mm) (Greiner Bio One, catalog number: 628161 )
  2. 35 mm plates (Corning, catalog number: 430165 )
  3. Microscope slides 75 x 25 x 1 mm (Marienfeld-Superior, catalog number: 10 006 12 )
  4. Microscope cover glass 18 x 18 mm (Marienfeld-Superior, catalog number: 01 010 30 )
  5. C. elegans strains (wild type [N2], ife-2[ok306], N2; Ex[pife-2GFP, pRF4], ife-2[ok306]; Ex[pife-2GFP, pRF4])
  6. Escherichia coli OP50 strain (obtained from the Caenorhabditis Genetics Center)
  7. Cycloheximide (Sigma-Aldrich, catalog number: C7698 )
    Note: Cycloheximide has significant, toxic side effects.
  8. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: 7778-77-0 )
  9. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: 7758-11-4 )
  10. Sodium chloride (NaCl) (EMD Millipore, catalog number: 106404 )
  11. Peptone (BD, Bacto, catalog number: 211677 )
  12. Streptomycin (Sigma-Aldrich, catalog number: S6501 )
  13. Agar (Sigma-Aldrich, catalog number: 05040 )
  14. Cholesterol stock solution (SERVA Electrophoresis, catalog number: 17101.01 )
  15. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  16. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  17. Nystatin stock solution (Sigma-Aldrich, catalog number: N3503 )
  18. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 7558-79-4 )
  19. Phosphate buffer (1 M; sterile, see Recipes)
  20. Nematode growth medium (NGM) agar plates (see Recipes)
  21. M9 buffer (see Recipes)

Equipment

  1. Dissecting stereomicroscope (Nikon, model: SMZ645 )
  2. UV crosslinker (VilberLourmat, model: BIO-LINK – BLX-E365 )
  3. Epifluorescence microscope (ZEISS, model: Axioskop 2 Plus )
  4. Standard equipment for preparing agar plates (autoclave, Petri dishes, etc.) (Sambrook and Russell, 2001)
  5. Standard equipment for maintaining worms (platinum wire pick, incubators, etc.)
    Note: For basic C. elegans culture, maintenance and manipulation techniques see refs (Epstein and Shakes, 1995; Lewis and Fleming, 1995; Hope, 1999; Strange, 2006). For information on C. elegans biology see refs (Wood, 1988; Epstein and Shakes, 1995; Riddle, 1997) and WormBook (http://www.wormbook.org/).

Software

  1. Camera control and imaging software (Carl Zeiss, Axio Vision 3.1 software)
  2. ImageJ image processing software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/) (Abràmoff et al., 2004)
  3. Microsoft Office 2011 Excel software package (Microsoft Corporation, Redmond, USA)
  4. Prism software package (GraphPad Software Inc., San Diego, USA)

Procedure

  1. Transgenic nematode generation and maintenance
    To implement the protocol described below, start by generating appropriate transgenic C. elegans strains expressing the fluorescent proteins of choice in the cells or tissues of interest, under the control of promoters active for the cell or tissues investigated. Both transcriptional and translational fusions can be used with this method. Various methods are available for introducing recombinant DNA into nematodes. A widely exercised technique is the microinjection of DNA into the gonadal syncytium of gravid adult hermaphrodite animals (Rieckher et al., 2009).
    1. Grow transgenic animals expressing the fluorophore in cells or tissues of interest on 60 mm plates seeded with OP50 (the E. coli strain used as C. elegans’ food source), at the standard temperature of 20 °C or other appropriate temperature (depends on genetic background and other experimental considerations such as the temperature sensitivity of animals examined).
    2. Unless an environmentally controlled chamber or room is available, the procedure of fluorescence photobleaching and recovery is performed at ambient temperature. Allow animals to equilibrate at this temperature for 2-3 h before photobleaching.

  2. Sample preparation, photobleaching and recovery
    1. Sample preparation
      The procedure can be performed either directly on a plate (step B1a) or on a coverslip (step B1b). When examining worms that express the fluorescent marker in individual cells, photographs of moving worms are hard to analyze. In this case follow step B1b, below. For worms expressing the fluorescent marker protein globally or in many tissues, the more convenient step B1a may be applicable. We generally avoid the use of anesthetics, especially the commonly used sodium azide, which blocks the mitochondrial respiratory chain, perturbs energy production and is likely to interfere with the fluorescence recovery process by hindering protein synthesis. To limit animal mobility (step B1a) we find particularly helpful the use of the dominant rol-6(su1006) allele as co-transformation marker (plasmid RF4), when constructing transgenic lines. This allele causes animals to roll instead of moving sinusoidally, which confines them in a relatively small area of the plate.
      1. Transfer single worms to fresh 35 mm plates, seeded with OP50 bacteria. A small bacterial spot in the center of the plate will make localization of the worm easier, while focusing the sample.
      2. Spot a drop of 15 μl M9 buffer on a microscope slide and place the worm on the drop with the help of an eyelash glued on a pick. Add a cover slip on the top of the drop. The weight of the cover slip is sufficient to keep the worm immobile during the procedure, without damaging it.
        Notes:
        1. M9 buffer instead of water ensures a favourable osmotic environment for the worm. Animals should not be allowed to dry out during photobleaching. Supply fresh M9 in the form of 5 μl drops applied to the side of the cover slip during lengthy photobleaching sessions. (CRITICAL STEP)
        2. Exercise caution when removing the cover slip to recover the animal. Accidentally pressing on the cover slip will crush the worms. (CRITICAL STEP)
    2. Photograph single animals before photobleaching
      Photograph single animals before photobleaching using a camera attached to the microscope (e.g., Axio Cam HR, Carl Zeiss). Images of fluorescent cells or tissues of interest are collected. Imaging parameters such as microscope and camera settings (lens and magnifier used, filters exposure time, resolution, etc.) should be documented.
    3. Perform photobleaching
      Use an epifluorescence, compound light microscope (e.g., Axioskop 2 Plus, Carl Zeiss) equipped with a high power light source (HBO 100; 100 Watt mercury arc lamp; Osram, Munich, Germany) and the appropriate excitation/emission filter sets to photobleach the animal. For the applications described here, 10 min of photobleaching reduce the initial emission intensity adequately (to within 30-50% of pre-bleach levels). The light intensity and the duration of the bleaching period are adjusted accordingly for the specific fluorophore, animal stage and cell or tissue under examination. Investigators should experimentally determine the appropriate duration of irradiation required to reach the extent of photobleaching, appropriate for different specimens (see Note 1).
      Notes:
      1. At least 20 individual animals should be processed for each experimental condition. The photobleaching period should be kept identical for all animals tested.
      2. CRITICAL STEP: Proper photobleaching conditions (light intensity, duration) should be set aiming to avoid injuring worms. The absolute level of fluorescence reduction by photobleaching is not important. We assess damage to worms by looking for apparent changes in behavior such as lethargy and movement defects or diminished responsiveness to touch, and for reduced fecundity in animals subjected to photobleaching. Animals showing signs of damage after photobleaching are excluded from further analysis.
    4. Fluorescence recovery
      Photograph each animal immediately after photobleaching. Collect several images of cells or tissues of interest. Move animals to fresh OP50-seeded NGM plates. Recover animals photobleached on a microscope slide by adding 100 μl of M9 at the edges of the cover slip and sliding off the cover slip. These worms are also returned to an OP50-seeded NGM plate for recovery. Recovery timing starts at this point.
      Note: All imaging parameters such as microscope and camera settings (lens and magnification used, filters exposure time, resolution, etc.) should be set as in step B2 above. (CRITICAL STEP)
    5. Photographing animals at defined time points after fluorescence recovery
      Follow fluorescence recovery by photographing animals at defined time points. We use 1 h intervals between successive photography sessions. A suitable time interval can be determined for each experimental application. Collect several images of cells or tissues of interest. As in step B4 above, it is critical that all imaging parameters (microscope and camera settings) are kept identical to those initially set in step B2 (see Note 2).
    6. Prepare a stock solution of cycloheximide by diluting in water to a concentration of 10 mg/ml. Keep refrigerated. Add cycloheximide on top of OP50-seeded, 35 mm NGM plates to 500 μg/ml final concentration in the agar volume and allow plates to dry.
      Notes:
      1. The antibiotic cycloheximide, a potent and specific inhibitor of mRNA translation can be used to discriminate between the contribution of new protein synthesis and protein diffusion in overall fluorescence recovery after photobleaching.
      2. Cycloheximide has significant, toxic side effects. Kill bacteria on plates before adding cycloheximide by exposing bacterial lawns on NGM plates to UV radiation. Irradiate at 254 nm for 10 min at 100 mJ/cm2 in a UV crosslinker (Garigan et al., 2002; Gems and Riddle, 2000) (such as the Stratalinker 2400; Statagene, La Jolla, USA). (CAUTION)
    7. Transfer animals onto cycloheximide-containing plates and incubate for 2 h, at the growth temperature. Prepare animals for photobleaching as in step B1a or B1b. Return worms in cycloheximide-containing plates during fluorescence recovery (see Note 3).

Data analysis

  1. Image acquisition
    Process images acquired in steps B2, B4 and B5 with ImageJ to determine the average and maximum pixel intensity for each image of fluorescent cell or tissue of interest in the collected photomicrographs. For each cell, tissue or animal, images should be acquired or converted to a pixel depth of 8 bit (256 shades of grey). Representative images of transgenic animals expressing pife-2GFP throughout somatic tissues in a wild type background or in IFE-2 deficient animals, in the presence or absence of cycloheximide, are shown in Figure 1.
  2. Fluorescence intensity quantification
    To analyze the area of interest manually, use the ‘freehand selection’ tool to enclose the fluorescent area. Select the ‘measurement’ command via the ‘analyze’ drop-down menu to perform pixel intensity analysis. On occasion (area continuity, high contrast ratios), selection of the fluorescent area can be done automatically. Select ‘adjust’ and then the ‘threshold’ command, within the ‘image’ drop-down menu of ImageJ. Adjust the threshold until the region of interest is marked. Within the ‘analyze’ drop-down menu, select the ‘analyze particles’ command. By selecting ‘outlines’ at the ‘show’ drop-down menu, check whether measurements correspond to the area of interest. Average and maximum pixel intensity values are collected for each transgenic line and grouped into ‘Pre-bleach’, ‘Bleach’ and ‘Recovery(n)’, where n is the time interval after photobleaching.
  3. Statistical analysis
    Statistical analysis of data is carried out using the Microsoft Office 2011 Excel software package (Microsoft Corporation, Redmond, USA) and the Prism software package (GraphPad Software Inc., San Diego, USA). The best-fit function that describes the recovery phase is generated by regression analysis (Figure 2). The slope of the best-fit lines provides quantification of the recovery rate. In the example shown, recovery is diminished in IFE-2-deficient animals or animals treated with cycloheximide.


    Figure 1. Photobleaching and recovery of fluorescence in vivo. Representative images of roller, transgenic animals expressing pife-2GFP throughout somatic tissues, in a wild type or IFE-2 deficient background before photobleaching, immediately following an 8 min whole-animal photobleaching session, and after a 5 h recovery period, in the absence (a) and presence (b) of cycloheximide (500 μg/ml). Scale bars = 100 μm.


    Figure 2. Regression analysis of fluorescence recovery in both wild type and IFE-2 deficient animals expressing pife-2GFP throughout somatic tissues. Best-fit lines are generated for average pixel intensity values obtained during the recovery phase for the indicated genetic backgrounds (a, wild type; b, ife-2[ok306]; black lines). The respective equations describing best-fit lines as well as R2 values for each line are also shown. Line slope corresponds to the first derivative of fluorescent change within a time unit (Δf/dt), which is a measure of the recovery rate. Cycloheximide treatment (CHX; 500 μg/ml) results in negligible recovery rate (blue lines).

Notes

  1. Inadequate photobleaching may be the result of insufficient duration of photobleaching and/or excitation light that is not concentrated on the tissue of interest. To overcome this problem, increase the duration of photobleaching, increase the light intensity or use an objective lens with higher magnification and numerical aperture.
  2. No significant recovery (step B5) may be an indication of specimen damage due to excessive photobleaching. It is also possible that the recovery kinetics are too slow for the chosen time interval. In addition, the promoter controlling the expression of the fluorophore may not be active during recovery. To address these issues, reduce photobleaching, allow longer recovery periods or switch promoter.
  3. Inadequate inhibition of recovery from photobleaching by cycloheximide may result from insufficient exposure to cycloheximide. Increase the pre-incubation time of the animals in the presence of cycloheximide.

Recipes

  1. Phosphate buffer (1 M)
    1. For 1 L, dissolve 102.2 g KH2PO4 and 57.06 g K2HPO4 in distilled water and fill up to 1 L. This is a 1 M solution, pH 6.0
    2. Autoclave at 121 °C for 15 min and store at room temperature
  2. Nematode growth medium (NGM) agar plates
    1. Mix 3 g NaCl, 2.5 g Bacto peptone, 0.2 g streptomycin, 17 g agar and add 900 ml distilled water. Autoclave at 121 °C for 15 min
    2. Let cool to 55-60 °C
    3. Add 1 ml cholesterol stock solution, 1 ml 1 M CaCl2, 1 ml 1 M MgSO4, 1 ml nystatin stock solution, 25 ml sterile 1 M phosphate buffer, pH 6.0, and distilled sterile water up to 1 L
    4. Pour about 8 ml medium per Petri dish and leave to solidify
    5. Store the plates at 4 °C until used
  3. M9 buffer
    1. Dissolve 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl in 1 L distilled water. Autoclave at 121 °C for 15 min
    2. Let cool and add 1 ml 1 M MgSO4 (sterile)
    3. Store M9 buffer at 4 °C

Acknowledgments

This work was funded by grants from the European Research Council (ERC), the European Commission 7th Framework Programme. The protocol has been adapted from Syntichaki et al. (2007), Nature 445, 922-926.

References

  1. Abràmoff, M. D., Magalhães, P. J. and Ram, S. J. (2004). Image processing with ImageJ. Biophotonics international 11(7): 36-42.
  2. Bjornsti, M. A. and Houghton, P. J. (2004). Lost in translation: dysregulation of cap-dependent translation and cancer. Cancer Cell 5(6): 519-523.
  3. Epstein, H. and Shakes, D. (1995). Caenorhabditis elegans: Modern biological analysis of an organism. Academic Press.
  4. Garigan, D., Hsu, A. L., Fraser, A. G., Kamath, R. S., Ahringer, J. and Kenyon, C. (2002). Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Genetics 161(3): 1101-1112.
  5. Gems, D. and Riddle, D. L. (2000). Genetic, behavioral and environmental determinants of male longevity in Caenorhabditis elegans. Genetics 154(4): 1597-1610.
  6. Hope, I. A. (1999). C. elegans: a practical approach. OUP Oxford.
  7. Lewis, J. A. and Fleming, J. T. (1995). Basic culture methods. Methods Cell Biol 48: 3-29.
  8. Martin, R. (1998). Protein synthesis : methods and protocols. Humana.
  9. Rennie, M. J., Smith, K. and Watt, P. W. (1994). Measurement of human tissue protein synthesis: an optimal approach. Am J Physiol 266(3 Pt 1): E298-307.
  10. Riddle, D. L., Blumenthal, T., Meyer, B. J. and Priess, J. R. (1997). C. elegans II. Cold Spring Harbor Laboratory.
  11. Rieckher, M., Kourtis, N., Pasparaki, A. and Tavernarakis, N. (2009). Transgenesis in Caenorhabditis elegans. Methods Mol Biol 561: 21-39.
  12. Sambrook, J. and Russell, D. W. (2001). Molecular cloning: a laboratory manual 3rd edition. Cold Spring Harbor Laboratory.
  13. Strange, K. (2006). C. elegans: methods and applications. Humana.
  14. Syntichaki, P., Troulinaki, K. and Tavernarakis, N. (2007). eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans. Nature 445(7130): 922-926.
  15. Wood, W. B. (1988). The nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory.

简介

目前可用的生物化学方法不能用于监测活体标本中特定细胞或组织中的蛋白质合成。在这里,我们描述了在活的秀丽隐杆线虫动物中监测具有本质上不同的翻译速率的单个细胞或组织中的蛋白质合成的非侵入性方法。

背景 蛋白质合成的适当调节对于细胞稳态和生长至关重要。蛋白质合成的放松规律已经涉及到诸如癌症和衰老衰退的病理学(Bjornsti和Houghton,2004; Syntichaki等人,2007)。目前可用的用于测量一般蛋白质合成速率的生物化学方法包括代谢标记和多糖成像(Martin,1998; Rennie等人,1994)。这些方法的适用性受限于标签在整个动物或感兴趣组织中的摄取不足和不受控制或不平等的分布。此外,这些方法缺乏特异性,由于大部分样品的固有翻译速率的变异性,可能会掩盖特定细胞或感兴趣的组织的显着变化。在本协议中,我们描述了一种基于光漂白(FRAP)后的荧光恢复来监测线虫秀丽隐杆线虫的蛋白质合成速率的方法。实验方法是基于转基因动物感兴趣的细胞和组织中荧光蛋白的表达。然后通过用强大的光源照射细胞,组织或整个动物来对荧光进行光漂白。然后在感兴趣的细胞或组织中监测指示新蛋白质合成的荧光的恢复。

关键字:秀丽隐杆线虫, 荧光漂白恢复, 信使RNA, 蛋白质合成, 蛋白质翻译

材料和试剂

  1. Greiner Petri菜(60 x 15毫米)(Greiner Bio One,目录号:628161)
  2. 35 mm板(Corning,目录号:430165)
  3. 显微镜滑动75 x 25 x 1 mm(Marienfeld-Superior,目录号:10 006 12)
  4. 显微镜盖玻璃18 x 18毫米(Marienfeld-Superior,目录号:01 010 30)
  5. C。 elegans 菌株(野生型[N2],ife-2 [ok306] ,N2; Ex [p ife-2 GFP ,pRF4], ife-2
  6. 大肠杆菌OP50菌株(从Caenorhabditis遗传学中心获得)
  7. 环己酰亚胺(Sigma-Aldrich,目录号:C7698)
    注意:环己酰亚胺具有显着的毒副作用。
  8. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:7778-77-0)
  9. 磷酸氢二钾(K 2 H 2 HPO 4)(Sigma-Aldrich,目录号:7758-11-4)
  10. 氯化钠(NaCl)(EMD Millipore,目录号:106404)
  11. 胨(BD,Bacto,目录号:211677)
  12. 链霉素(Sigma-Aldrich,目录号:S6501)
  13. 琼脂(Sigma-Aldrich,目录号:05040)
  14. 胆固醇储备溶液(SERVA Electrophoresis,目录号:17101.01)
  15. 氯化钙脱水(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C5080)
  16. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  17. 制霉菌素储备溶液(Sigma-Aldrich,目录号:N3503)
  18. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:7558-79-4)
  19. 磷酸盐缓冲液(1M;无菌,见食谱)
  20. 线虫生长培养基(NGM)琼脂平板(参见食谱)
  21. M9缓冲区(见配方)

设备

  1. 解剖立体显微镜(尼康,型号:SMZ645)
  2. 紫外线交联剂(VilberLourmat,型号:BIO-LINK-BLX-E365)
  3. 荧光显微镜(ZEISS,型号:Axioskop 2 Plus)
  4. 用于制备琼脂板(高压釜,培养皿,等等)的标准设备(Sambrook和Russell,2001)
  5. 用于维护蠕虫的标准设备(铂金丝选择,孵化器,等等。)
    注意:对于基本的线虫文化,维护和操纵技术参见参考文献(Epstein和Shakes,1995; Lewis和Fleming,1995; Hope,1999; Strange,2006)。关于秀丽隐杆线虫生物学的信息参见参考文献(Wood,1988; Epstein和Shakes,1995; Riddle,1997)和WormBook( http://www.wormbook.org/)。

软件

  1. 相机控制和成像软件(Carl Zeiss,Axio Vision 3.1软件)
  2. ImageJ图像处理软件(Rasband,WS,ImageJ,美国国立卫生研究院,Bethesda,Maryland,USA, http://rsb.info.nih.gov/ij/)(Abràmoff等人,2004)
  3. Microsoft Office 2011 Excel软件包(Microsoft Corporation,Redmond,USA)
  4. Prism软件包(GraphPad Software Inc.,San Diego,USA)

程序

  1. 转基因线虫生成和维护
    为了实现下述协议,首先生成适当的转基因C。在所研究的细胞或组织的启动子的控制下,在感兴趣的细胞或组织中表达选择的荧光蛋白的线虫菌株。转录和翻译融合都可以用于这种方法。各种方法可用于将重组DNA引入线虫。广泛采用的技术是将DNA显微注射到妊娠成年雌雄同体动物的性腺合胞体中(Rieckher等人,2009)。
    1. 在用OP50(用作线虫食品来源的大肠杆菌菌株)的60mm板上培养在感兴趣的细胞或组织中表达荧光团的转基因动物,在标准温度为20°C或其他合适的温度(取决于遗传背景和其他实验考虑因素,如所检测的动物的温度敏感性)。
    2. 除非有环境控制的室或房间可用,否则荧光漂白和回收的程序在环境温度下进行。允许动物在此温度下平衡2-3小时,然后再进行光漂白。

  2. 样品制备,光漂白和回收
    1. 样品制备
      该程序可以直接在板上(步骤B1a)或盖玻片(步骤B1b)进行。当检查在单个细胞中表达荧光标记的蠕虫时,移动蠕虫的照片很难分析。在这种情况下,请遵循下面的步骤B1b。对于在全局或许多组织中表达荧光标记蛋白的蠕虫,可以应用更方便的步骤B1a。我们通常避免使用麻醉剂,特别是通常使用的叠氮化钠,其阻断线粒体呼吸链,扰乱能量产生,并且可能通过阻碍蛋白质合成来干扰荧光恢复过程。为了限制动物的流动性(步骤B1a),我们发现在构建转基因品系时,使用优势的rol-6(su1006)等位基因作为共同转录标记(质粒RF4)。这种等位基因使动物滚动而不是正弦波移动,这将它们限制在板的相对较小的区域。
      1. 将单个蠕虫转移到新鲜的35mm板上,接种OP50细菌。在中心的一个小细菌斑点将使得蠕虫的定位更容易,同时聚焦样品。
      2. 在显微镜载玻片上点一滴15μlM9缓冲液,并将蚯蚓放在滴下,并用睫毛胶粘在拣选上。在水滴的顶部添加一个盖子。盖子滑块的重量足以在该过程中保持蜗杆不动,而不会损坏蜗杆。
        注意:
        1. M9缓冲液代替水确保了蠕虫的有利渗透环境。在漂白过程中不要让动物干涸。在长时间的漂白过程中,以5μl滴液的形式提供新鲜的M9,用于护盖的侧面。 (CRITICAL STEP)
        2. 在卸下盖板以恢复动物时,请务必小心。意外地压在盖板上将粉碎蠕虫。 (CRITICAL STEP)
    2. 照相漂白前照片单一动物
      使用连接到显微镜的照相机(例如Axio Cam HR,Carl Zeiss)在照相漂白之前拍摄单个动物。收集感兴趣的荧光细胞或组织的图像。应记录成像参数,如显微镜和相机设置(使用的镜头和放大镜,过滤曝光时间,分辨率,等)。
    3. 执行光漂白
      使用装备有高功率光源(HBO 100; 100瓦汞弧灯;欧司朗,德国慕尼黑)的表面荧光,复合光学显微镜(例如,Axioskop 2 Plus,Carl Zeiss)和适当的激发/发射过滤器套件可以对动物进行漂白。对于本文所述的应用,10分钟的漂白漂白可以充分降低初始发射强度(达到漂白前水平的30-50%)。对于特定的荧光团,动物阶段和被检查的细胞或组织,相应地调整漂白期的光强度和持续时间。研究人员应通过实验来确定达到光漂白程度所需的适当照射持续时间,适用于不同的标本(见注1)。
      注意:

      1. 每个实验条件应至少处理20只个体动物。对于所有测试的动物,漂白期应保持相同。
      2. 关键步骤:应设置适当的漂白条件(光强度,持续时间),以避免伤害蠕虫。通过光漂白的荧光还原的绝对水平并不重要。我们通过寻找行为的明显变化来评估对蠕虫的损害,例如嗜睡和运动缺陷或对触摸的反应性降低,以及减少经过光漂白的动物的繁殖力。光漂白后出现损伤迹象的动物将不再进一步分析。
    4. 荧光恢复
      光漂白后立即拍摄每只动物。收集感兴趣的细胞或组织的几个图像。将动物移动到新鲜的OP50种子NGM板上。通过在盖板的边缘处加入100μl的M9,在显微镜载玻片上将漂白动物回收,并滑出盖板。这些蠕虫也返回到OP50种子的NGM板上进行恢复。恢复时间从此开始。
      注意:所有成像参数如显微镜和相机设置(使用镜头和倍率,过滤器曝光时间,分辨率等)应按照上述步骤B2进行设置。 (CRITICAL STEP)
    5. 在荧光恢复后定义的时间点拍摄动物
      通过在规定的时间点拍摄动物来追踪荧光恢复。我们在连续拍摄会议之间使用1小时的间隔。可以为每个实验应用确定合适的时间间隔。收集感兴趣的细胞或组织的几个图像。如上述步骤B4所示,至关重要的是,所有成像参数(显微镜和照相机设置)与步骤B2中最初设置的成像参数保持一致(见注2)。
    6. 通过在水中稀释至10mg/ml的浓度来制备放线菌酮的储备溶液。保持冷藏。在OP50接种的35mm NGM平板的顶部加入放线菌酮至琼脂体积的最终浓度为500μg/ml,并使板干燥。
      注意:
























      1. 环己酰胺具有显着的毒副作用。通过将NGM平板上的细菌草坪暴露于紫外线辐射之前,在放入放线菌酮后,将细菌杀死。在UV交联剂(Garigan等人,2002; Gems和Riddle,2000)(例如Stratalinker 2400; Statagene,La Jolla,Inc。)中,在254nm处以100mJ/cm 2在254nm下辐射10分钟美国)。 (小心)
    7. 在含放线菌酮的板上转移动物,并在生长温度下孵育2小时。准备如步骤B1a或B1b中的光漂白动物。在荧光恢复期间回收含放线菌酮的板中的蠕虫(见注3)

数据分析

  1. 图像采集
    使用ImageJ处理在步骤B2,B4和B5中获取的图像,以确定所收集的显微照片中感兴趣的荧光细胞或组织的每个图像的平均和最大像素强度。对于每个细胞,组织或动物,应该获取图像或将其转换为8位(256灰度)的像素深度。在野生型背景或IFE-2缺陷型动物中,在存在或不存在放线菌酮的情况下,在整个体细胞组织中表达p ife-2 GFP的转基因动物的代表性图像是如图1所示
  2. 荧光强度量化
    要手动分析感兴趣区域,请使用"手写选择"工具包围荧光区域。通过"分析"下拉菜单选择"测量"命令来执行像素强度分析。有时(区域连续性,高对比度),可以自动选择荧光区域。在ImageJ的"image"下拉菜单中选择"调整",然后选择"阈值"命令。调整阈值,直到感兴趣的区域被标记为止。在"分析"下拉菜单中,选择"分析粒子"命令。通过在"显示"下拉菜单中选择"轮廓",检查测量是否对应于感兴趣的区域。收集每个转基因品系的平均和最大像素强度值,并分组为"漂白前","漂白"和"恢复(n)",其中n是光漂白后的时间间隔。
  3. 统计分析
    使用Microsoft Office 2011 Excel软件包(Microsoft Corporation,Redmond,USA)和Prism软件包(GraphPad Software Inc.,San Diego,USA)进行数据的统计分析。描述恢复阶段的最佳拟合函数是通过回归分析产生的(图2)。最佳拟合线的斜率提供了回收率的量化。在所示的例子中,IFE-2缺乏的动物或用放线菌酮治疗的动物的恢复减少。


    图1.体内荧光的漂白和回收。。表达p 在光动力漂白之前的野生型或IFE-2缺陷背景中,在8分钟全动物光漂白期之后,以及在5小时恢复期之后,在不存在(a)和(b)放线菌酮(500μg/ml)。比例尺=100μm

    图2.在整个体细胞组织中表达p ife-2 GFP的野生型和IFE-2缺陷型动物中的荧光回收的回归分析针对所指示的遗传背景(a,野生型; b, )在恢复阶段期间获得的平均像素强度值生成 - 线)。还示出了描述每行的最佳拟合线以及R 2 值的各个等式。线斜率对应于时间单位内的荧光变化的一阶导数(Δf/dt),其为回收率的量度。环己酰亚胺处理(CHX;500μg/ml)导致回收率可忽略不计(蓝线)。

笔记

  1. 光漂白不足可能是光漂白和/或不集中在感兴趣组织上的激发光持续时间不足的结果。为了克服这个问题,增加光漂白的持续时间,提高光强或者使用更高倍率和数值孔径的物镜。
  2. 没有明显的恢复(步骤B5)可能是由于过度光漂白引起的样品损伤的指示。对于所选择的时间间隔,恢复动力学也可能太慢。此外,控制荧光团表达的启动子在恢复期间可能不活跃。为了解决这些问题,减少光漂白,允许更长的恢复期或切换启动器。
  3. 由放线菌酮对光漂白的回收抑制不足可能是由于曝露不到放线菌酮而导致的。增加放线菌酮存在下动物的预孵育时间。

食谱

  1. 磷酸盐缓冲液(1M)
    1. 对于1L,在蒸馏水中溶解102.2g KH 2 PO 4和57.06g K 2 HPO 4,填充1 L.这是一个1M溶液,pH 6.0
    2. 在121℃高压灭菌15分钟,并在室温下储存
  2. 线虫生长培养基(NGM)琼脂板
    1. 混合3g NaCl,2.5g细菌蛋白胨,0.2g链霉素,17g琼脂并加入900ml蒸馏水。高压灭菌在121°C 15分钟
    2. 让凉爽至55-60°C
    3. 加入1ml胆固醇储备溶液,1ml 1M CaCl 2,1ml 1M MgSO 4,1ml制霉菌素储备溶液,25ml无菌1M磷酸盐缓冲液,pH 6.0,蒸馏无菌水至1升
    4. 倾倒约8毫升培养基每培养皿,并离开巩固
    5. 将板保存在4°C直到使用
  3. M9缓冲区
    1. 将3g KH 2 PO 4,6g Na 2 HPO 4,5L NaCl的1L蒸馏水溶解水。高压灭菌在121°C 15分钟
    2. 冷却并加入1ml 1M MgSO 4(无菌)
    3. 在4°C存放M9缓冲液

致谢

这项工作是由欧洲研究委员会(ERC),欧洲委员会第七届支持框架计划的资助提供的。该协议已经由Syntichaki等人改编。 (2007),Nature 445,922-926。

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引用:Kourtis, N. and Tavernarakis, N. (2017). Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP). Bio-protocol 7(5): e2156. DOI: 10.21769/BioProtoc.2156.
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