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Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells
在活细胞中使用HaloTag配体标记HaloTag融合蛋白   

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eLIFE
18-Oct 2016

Abstract

HaloTag has been widely used to label proteins in vitro and in vivo (Los et al., 2008). In this protocol, we describe labelling HaloTag-Cbx fusion proteins by HaloTag ligands for live-cell single-molecule imaging (Zhen et al., 2016).

Keywords: HaloTag (HaloTag), Live-cell single-molecule imaging (活细胞单分子成像), Polycomb (多梳), Cbx (Cbx), Epigenetics (表观遗传学), Janelia FluorTM dye (Janelia FluorTM染料)

Background

Molecular processes of living organisms are intrinsically dynamics. Direct observation of the molecular processes in living cells is critical for quantitatively understanding of how biological systems function. Recent advances in fluorescence microscopy and fluorescent labelling enable to visualize trajectories of individually single molecules in living cells, providing insights about dynamic interactions and assemblies of biological molecules (Kusumi et al., 2014; Liu et al., 2015; Tatavosian et al., 2015; Cuvier and Fierz, 2017). Specific labelling of biomolecules with fluorophores is the key for fluorescence single-molecule imaging. HaloTag is self-labeling tag proteins that can be coupled to synthetic dyes in living cells (Los et al., 2008). The reaction occurs rapidly in living cells and the formed covalent bond is specific and irreversible. This technique has been utilized to study the genetic information flow in vivo, and to measure the kinetic of gene regulation in living mammalian cells (Liu et al., 2015; Zheng and Lavis, 2017). Janelia FluorTM dyes, such as Janelia FluorTM 549 (JF549), are bright and photostable fluorescent HaloTag ligands (Grimm et al., 2015). This protocol describes how to label HaloTag-Cbx proteins with JF549 for live-cell single-molecule imaging, which was developed in the recent publication (Zhen et al., 2016).

Materials and Reagents

  1. Pipette tips (BioExpress, catalog number: P-1236-200)
    Manufacturer: Biotix, catalog number: P-1236-200CS .
  2. 35 mm glass bottom dish made in the laboratory (see Video 1 for making glass-bottom dishes)

    Video 1. Making glass-bottom dishes. The video elaborates how to make glass-bottom dishes for live-cell single-molecule imaging.

  3. Cell lines used in protocol: mouse embryonic stem cells and HEK293T cells
  4. Janelia Fluor 549 dye (JF549) provided by Dr. Luke D. Lavis (Janelia Research Campus, Howard Hughes Medical Institute)
  5. Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  6. Phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537-500ML )
  7. Gelatin (Sigma-Aldrich, catalog number: G1890-100G )
  8. DMEM (Sigma-Aldrich, catalog number: D5796 )
  9. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F0926 )
  10. Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  11. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  12. β-Mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 21985023 )
  13. Non-essential amino acids (Thermo Fisher Scientific, catalog number: 1114050 )
  14. Leukemia inhibitor factor (LIF, made in the laboratory)
  15. FluoroBrite DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: A1896701 )
  16. ES cell medium (see Recipes)
  17. Live-cell imaging medium (see Recipes)

Equipment

  1. Single channel pipette (BioExpress, Kaysville, USA)
  2. Heater controller (Warner Instruments, catalog number: TC-324 )
  3. Microscope (Manual Microscopy) (ZEISS, model: Axio Observer D1 )
  4. Alpha Plan-Apochromatic 100/1.46 NA Oil-immersion Objective (ZEISS, Germany)
  5. Evolve 512 x 512 EMCCD camera (Photometrics, Tucson, USA)
  6. Solid state laser (Intelligent Imaging Innovations, 3i LaserStack with Fiber)

Software

  1. Slidebook 6.0 software (Intelligent Imaging Innovations, Denver, Colorado)
  2. MATLAB R2015a (8.5.0.197613) (MathWorks, Natick, USA)
  3. U-track 2.0 (Danusar Lab, UT South Western Medical Center, Dallas, USA)

Procedure

  1. Trypsinize 70-90% confluent cells (mouse embryonic stem cells or HEK293 cells) of 100 mm plate stably expressing HaloTag-Cbx proteins (we recommend 0.6 ml of 0.05% trypsin-EDTA [1x] for 60 mm plate and 1.5 ml for 100 mm plate).
  2. Seed 20% of cells to 35 mm glass-bottom dish coated by Gelatin overnight (Note 1) (see Video 2 for gelatinization).

    Video 2. Gelatinization of glass-bottom dishes. The video describes how to gelatinize glass-bottom dishes before seeding cells.

  3. Following by overnight culture, the final confluency of the cells before was between 80-90%. Several concentrations (5 nM, 15 nM, and 30 nM) of JF549 are used to incubate with cells for 15 min at 37 °C in 5% CO2 (Notes 2 and 3) (see Video 3 for adding the JF549 dye).

    Video 3. Adding JF549 dyes to cells


  4. Gently wash cells with ES medium (see Recipes) once and incubate in ES medium for 30 min at 37 °C and 5% CO2 (see Video 4 for washing cells).

    Video 4. Washing cells with ES cell medium

  5. Replace ES medium with live-cell imaging medium (see Recipes) (see Video 5 for adding live-cell imaging medium).

    Video 5. Replacing ES cell medium with live-cell imaging medium

  6. Maintain 37 °C conditions during imaging by using a heater controller. Each plate should be imaged for the maximum of 1.5 h after placing on the microscope (see Video 6 for placing dishes on objective).

    Video 6. Placing dishes on objective

  7. The number of individual fluorescent spot per nucleus should be between 10-50 spots, controlled by adjusting the JF549 dye concentrations (Notes 4 and 5) (see Video 7 for single-molecule imaging).

    Video 7. Single-molecule imaging. The video describes how to image individual HaloTag-Cbx proteins within living cells.

  8. Movies are then uploaded to u-track 2.0. Each cell is cropped from the larger movie. Cropped movies are processed (Note 6).

Data analysis

  1. Our data were analyzed using MATLAB with u-track 2.0 plug-in, detail guide can be found at http://www.utsouthwestern.edu/labs/danuser/software/ (the software and pdf file guide are included in the download).
  2. Representative images and movies can be found in Zhen et al., 2016.

Notes

  1. Imaging dishes should be gelatinized overnight.
  2. We recommend using pipettes, not suction, when removing medium from imaging dishes.
  3. When add medium to the cells in imaging dishes, we recommend tilting plate at an angle, slowly add medium to the lower edge and let the medium reach the cells slowly.
  4. The TIRF angle used is unique for individual cells and should be adjusted to achieve the best possible movies.
  5. To obtain representative movies, the focus of the movie should be at the middle layer of the nucleus. This can be recognized by the deeper black area (this can be achieved by adjusting the angle). Bright field can be used to check the nucleus area.
  6. In u-track 2.0, multiple movies can be loaded and processed. After being processed, each movie should be checked to ensure that cells should be no drift and rotation.

Recipes

  1. ES cell medium
    DMEM
    15% FBS
    2 mM glutamine
    100 U/ml penicillin-streptomycin
    0.1 mM β-mercaptoethanol
    1,000 U/ml LIF
    0.1 mM non-essential amino acids
  2. Live-cell imaging medium
    FluoroBrite DMEM supplemented with 15% FBS
    2 mM glutamine
    100 U/ml penicillin-streptomycin
    0.1  mM β-mercaptoethanol
    1,000 U/ml LIF
    0.1 mM non-essential amino acids

Acknowledgments

This work was supported, in whole or in part, by the National Cancer Institute of the National Institutes of Health under Award Number R03CA191443 (to XR). This work was also supported by grants from the CU-Denver Office Research Service (to XR) and the American Cancer Society Grant IRG 57-001-53 subaward (to XR). This protocol was originally developed in Zhen et al., 2016.

References

  1. Cuvier, O. and Fierz, B. (2017). Dynamic chromatin technologies: from individual molecules to epigenomic regulation in cells. Nat Rev Genet.
  2. Grimm, J. B., English, B. P., Chen, J., Slaughter, J. P., Zhang, Z., Revyakin, A., Patel, R., Macklin, J. J., Normanno, D., Singer, R. H., Lionnet, T. and Lavis, L. D. (2015). A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat Methods 12(3): 244-250, 243 p following 250.
  3. Kusumi, A., Tsunoyama, T. A., Hirosawa, K. M., Kasai, R. S. and Fujiwara, T. K. (2014). Tracking single molecules at work in living cells. Nat Chem Biol 10(7): 524-532.
  4. Liu, Z., Lavis, L. D. and Betzig, E. (2015). Imaging live-cell dynamics and structure at the single-molecule level. Mol Cell 58(4): 644-659.
  5. Los, G. V., Encell, L. P., McDougall, M. G., Hartzell, D. D., Karassina, N., Zimprich, C., Wood, M. G., Learish, R., Ohana, R. F., Urh, M., Simpson, D., Mendez, J., Zimmerman, K., Otto, P., Vidugiris, G., Zhu, J., Darzins, A., Klaubert, D. H., Bulleit, R. F. and Wood, K. V. (2008). HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3(6): 373-382.
  6. Tatavosian, R., Zhen, C. Y. and Ren, X. J. (2015). Single-molecule fluorescence microscopy methods in chromatin biology. Acs Sym Ser 1215:129-136.
  7. Zhen, C. Y., Tatavosian, R., Huynh, T. N., Duc, H. N., Das, R., Kokotovic, M., Grimm, J. B., Lavis, L. D., Lee, J., Mejia, F. J., Li, Y., Yao, T. and Ren, X. (2016). Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin. Elife 5.
  8. Zheng, Q. and Lavis, L. D. (2017). Development of photostable fluorophores for molecular imaging. Curr Opin Chem Biol 39: 32-38.

简介

HaloTag已广泛用于体外和体内标记蛋白质(Los et al。,2008)。 在本协议中,我们描述了通过HaloTag配体标记HaloTag-Cbx融合蛋白的活细胞单分子成像(Zhen等,2016)。
【背景】生物体的分子过程本质上是动态的。直接观察活细胞中的分子过程对于定量地了解生物系统的功能至关重要。荧光显微镜和荧光标记的最新进展使得能够可视化活细胞中单个单个分子的轨迹,提供有关动态相互作用和生物分子装配的见解(Kusumi等,2014; Liu等,2015; Tatavosian等, 2015; Cuvier和Fierz,2017)。用荧光团对生物分子进行特异性标记是荧光单分子成像的关键。 HaloTag是可以与活细胞中合成染料偶联的自标记标签蛋白(Los et al。,2008)。反应在活细胞中迅速发生,形成的共价键是特异性的和不可逆的。该技术已被用于研究体内遗传信息流,并测定活体哺乳动物细胞中基因调控的动力学(Liu et al。,2015; Zheng and Lavis,2017)。 Janelia FluorTM染料,如Janelia FluorTM 549(JF549),是明亮和光稳定的荧光HaloTag配体(Grimm等,2015)。该协议描述了如何使用JF549标记HaloTag-Cbx蛋白质用于活细胞单分子成像,这是在最近的出版物(Zhen等,2016)中开发的。

关键字:HaloTag, 活细胞单分子成像, 多梳, Cbx, 表观遗传学, Janelia FluorTM染料

材料和试剂

  1. 移液器提示(BioExpress,目录号:P-1236-200)
    制造商:Biotix,目录号:P-1236-200CS。
  2. 实验室制造的35毫米玻璃底盘(参见视频1制作玻璃底盘)

    Video 1. Making glass-bottom dishes. The video elaborates how to make glass-bottom dishes for live-cell single-molecule imaging.

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

    Get Adobe Flash Player


  3. 方案中使用的细胞系:小鼠胚胎干细胞和HEK293T细胞
  4. 由Luke D. Lavis博士(Janelia Research Campus,Howard Hughes Medical Institute)提供的Janelia Fluor 549染料(JF <549
  5. 胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
  6. 磷酸缓冲盐水(PBS)(Sigma-Aldrich,目录号:D8537-500ML)
  7. 明胶(Sigma-Aldrich,目录号:G1890-100G)
  8. DMEM(Sigma-Aldrich,目录号:D5796)
  9. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F0926)
  10. 谷氨酰胺(Thermo Fisher Scientific,Gibco TM ,目录号:25030081)
  11. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  12. β-巯基乙醇(Thermo Fisher Scientific,Gibco TM,目录号:21985023)
  13. 非必需氨基酸(Thermo Fisher Scientific,目录号:1114050)
  14. 白血病抑制因子(LIF,实验室制作)
  15. FluoroBrite DMEM(Thermo Fisher Scientific,Gibco TM,目录号:A1896701)
  16. ES细胞培养基(见食谱)
  17. 活细胞成像介质(参见食谱)

设备

  1. 单通道移液器(BioExpress,Kaysville,USA)
  2. 加热器控制器(Warner Instruments,目录号:TC-324)
  3. 显微镜(手动显微镜)(ZEISS,型号:Axio Observer D1)
  4. Alpha Plan-Apochromatic 100 / 1.46 NA浸油目标(ZEISS,德国)
  5. 演进512 x 512 EMCCD相机(Photometrics,美国图森)
  6. 固态激光(智能成像创新,3i LaserStack与光纤)

软件

  1. Slidebook 6.0软件(智能影像创新,丹佛,科罗拉多州)
  2. MATLAB R2015a(8.5.0.197613)(MathWorks,Natlilck,USA)
  3. U-track 2.0(Danusar Lab,UT South Western Medical Center,Dallas,USA)

程序

  1. 将稳定表达HaloTag-Cbx蛋白质的100mm板的70-90%汇合细胞(小鼠胚胎干细胞或HEK293细胞)胰蛋白酶化(我们推荐将0.6ml 0.05%胰蛋白酶-EDTA [1x]用于60mm板,1.5ml用于100mm 盘子)。
  2. 种子20%细胞至明胶包被的35毫米玻璃底盘过夜(注1)(见视频2进行凝胶化)。

    Video 2. Gelatinization of glass-bottom dishes. The video describes how to gelatinize glass-bottom dishes before seeding cells.

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

    Get Adobe Flash Player


  3. 随后通过过夜培养,细胞的最终融合率在80-90%之间。 使用几种浓度(5nM,15nM和30nM)的JF 549 ,在37℃,5%CO 2(注释)中与细胞孵育15分钟 2和3)(参见视频3添加JF <549 染料)。

    Video 3. Adding JF549 dyes to cells

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

    Get Adobe Flash Player



  4. 用ES培养基轻轻洗涤细胞(参见食谱)一次,并在ES培养基中于37℃和5%CO 2孵育30分钟(参见视频4用于洗涤细胞)。

    Video 4. Washing cells with ES cell medium

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

    Get Adobe Flash Player


  5. 用活细胞成像介质代替ES培养基(参见食谱)(参见视频5添加活细胞成像培养基)。

    Video 5. Replacing ES cell medium with live-cell imaging medium

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

    Get Adobe Flash Player


  6. 使用加热器控制器在成像期间保持37°C的条件。 放置在显微镜上后,每个板片最多可成像1.5小时(见视频6,用于放置目标物)。

    Video 6. Placing dishes on objective

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

    Get Adobe Flash Player


  7. 通过调整JF 549 染料浓度(注4和5)(参见视频7进行单分子成像),每个细胞核上的个体荧光斑数应在10-50个点之间。/>
    Video 7. Single-molecule imaging. The video describes how to image individual HaloTag-Cbx proteins within living cells.

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

    Get Adobe Flash Player


  8. 电影然后上传到u-track 2.0。每个单元格都是从较大的电影中剪掉。裁剪的动画被处理(注6)。

数据分析

  1. 我们的数据使用MATLAB与u-track 2.0插件进行分析,详细指南可以在 http://www.utsouthwestern.edu/labs/danuser/software/ (软件和pdf文件指南包含在下载中)。
  2. 代表性的图像和电影可以在贞氏等等。中找到,

笔记

  1. 成像菜肴应胶化过夜。
  2. 从成像菜肴中取出培养基时,建议使用移液器,不要吸。
  3. 当在成像盘中添加细胞时,建议倾斜平板,慢慢地向下边缘添加介质,让培养基缓慢到达细胞。
  4. 所使用的TIRF角度对于单个单元格是独一无二的,应该进行调整以实现最佳的电影。
  5. 要获得代表性的电影,电影的焦点应该在核心的中间层。这可以被更深的黑色区域识别(这可以通过调整角度来实现)。光场可用于检查核区。
  6. 在u-track 2.0中,可以加载和处理多个动画。处理后,应检查每部电影,以确保细胞不应漂移和旋转。

食谱

  1. ES细胞培养基
    DMEM
    15%FBS
    2 mM谷氨酰胺
    100 U / ml青霉素 - 链霉素
    0.1mMβ-巯基乙醇
    1,000 U / ml LIF
    0.1mM非必需氨基酸
  2. 活细胞成像介质
    FluoroBrite DMEM补充了15%FBS
    2 mM谷氨酰胺
    100 U / ml青霉素 - 链霉素
    0.1mMβ-巯基乙醇
    1,000 U / ml LIF
    0.1mM非必需氨基酸

致谢

这项工作全部或部分由美国国家卫生研究院国家癌症研究所根据获奖号R03CA191443(至XR)进行了支持。这项工作也得到了丹佛办事处研究处(XR)和美国癌症协会授予IRG 57-001-53(向XR)的赠款的支持。这个协议最初是在臻等人,2016年开发的。

参考

  1. Cuvier,O.和Fierz,B.(2017)。动态染色质技术:从个体分子到细胞中的表观遗传调控。 Nat Rev Genet 。
  2. Grimm,JB,英语,BP,Chen,J.,Slaughter,JP,Zhang,Z.,Revyakin,A.,Patel,R.,Macklin,JJ,Normanno,D.,Singer,RH,Lionnet, Lavis,LD(2015)。一般的改进方法荧光团用于活细胞和单分子显微镜。 Nat方法:12(3):244-250,243 p在250之后。
  3. Kusumi,A.,Tsunoyama,TA,Hirosawa,KM,Kasai,RS和Fujiwara,TK(2014)。&lt; a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih。 gov / pubmed / 24937070"target ="_ blank">跟踪活细胞工作中的单分子。 Nat Chem Biol 10(7):524-532。
  4. Liu,Z.,Lavis,LD和Betzig,E.(2015)。在单分子水平上成像活细胞动力学和结构。分子细胞 58(4):644-659。
  5. Los,GV,Encell,LP,McDougall,MG,Hartzell,DD,Karassina,N.,Zimprich,C.,Wood,MG,Learish,R.,Ohana,RF,Urh,M.,Simpson,D.,Mendez ,J.,Zimmerman,K.,Otto,P.,Vidugiris,G.,Zhu,J.,Darzins,A.,Klaubert,DH,Bulleit,RF and Wood,KV(2008)。&lt; a class = "ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/18533659"target ="_ blank"> HaloTag:一种用于细胞成像和蛋白质分析的新型蛋白质标记技术。 ACS Chem Biol 3(6):373-382。
  6. Tatavosian,R.,Zhen,CY and Ren,XJ(2015)。&nbsp; 染色质生物学中的单分子荧光显微镜方法。 Acs Sym Ser 1215:129-136。
  7. Zhen,CY,Tatavosian,R.,Huynh,TN,Duc,HN,Das,R.,Kokotovic,M.,Grimm,JB,Lavis,LD,Lee,J.,Mejia,FJ,Li,Y.,Yao ,T.和Ren,X.(2016)。 活细胞单分子跟踪揭示了H3K27me3和DNA靶标polycomb Cbx7-PRC1与染色质的共识。 Elife 5.
  8. Zheng,Q. and Lavis,LD(2017)。&nbsp; 开发用于分子成像的光稳定荧光团。 Curr Opin Chem Biol 39:32-38。
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Copyright Duc and Ren . 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. Duc, H. N. and Ren, X. (2017). Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells. Bio-protocol 7(17): e2526. DOI: 10.21769/BioProtoc.2526.
  2. Zhen, C. Y., Tatavosian, R., Huynh, T. N., Duc, H. N., Das, R., Kokotovic, M., Grimm, J. B., Lavis, L. D., Lee, J., Mejia, F. J., Li, Y., Yao, T. and Ren, X. (2016). Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin. Elife 5.
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