3D-SIM结构照明超分辨率显微镜实现蛋白质在植物亚细胞器内的定位

doi:10.11983/CBB15038

植物学报 ›› 2015, Vol. 50 ›› Issue (4): 495-503.DOI: 10.11983/CBB15038

3D-SIM结构照明超分辨率显微镜实现蛋白质在植物亚细胞器内的定位

刘玥¹, 尹悦佳², 梁重阳³, 黄殿帅³, 王阳⁴, 刘艳芝², 窦瑶², 冯树丹^4,^*(), 郝东云^1,^2,^*()

¹吉林农业大学生命科学学院, 长春 130118
²吉林省农业科学院农业生物技术研究所, 长春 130033
³吉林大学再生医学科学研究所, 长春 130021
⁴哈尔滨师范大学生命科学学院, 哈尔滨 150080

收稿日期:2015-02-16 接受日期:2015-03-30 出版日期:2015-07-01 发布日期:2015-05-07
通讯作者: 冯树丹,郝东云
作者简介:
? 共同第一作者
基金资助:
国家自然科学基金青年基金(No.31200611)和国家自然科学基金(No.31170731)

Using 3D-SIM Structure Illumination Microscope to Localize Proteins in Plant Subcellular Compartments

Yue Liu¹, Yuejia Yin², Chongyang Liang³, Dianshuai Huang³, Yang Wang⁴, Yanzhi Liu², Yao Dou², Shudan Feng^{4, *}, Dongyun Hao^{1, 2, *}

¹College of Life Sciences, Jilin Agricultural University, Changchun 130118, China
²Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun 130033, China
³Institute of Frontier Medical Sciences, Jilin University, Changchun 130021, China
⁴College of Life Sciences, Harbin Normal University, Harbin 150080, China

Received:2015-02-16 Accepted:2015-03-30 Online:2015-07-01 Published:2015-05-07
Contact: Feng Shudan,Hao Dongyun
About author:
? These authors contributed equally to this paper

摘要/Abstract

摘要： 基因表达产物蛋白质的亚细胞定位是解析基因生物学功能的重要证据之一。近年来出现的超分辨率光学成像技术已成功应用于人类和动物细胞中, 预示着显微成像技术继激光共聚焦技术后的又一重要进步。由于植物细胞的特殊性和成像技术的研发取向, 超分辨率光学成像技术在植物细胞蛋白质亚细胞定位的应用尚未见报道。该研究利用DeltaVision OMX显微镜技术, 克服了叶绿体基粒中叶绿素自发荧光与融合蛋白荧光不易区分的缺陷, 解决了受分辨率局限无法将植物细胞中蛋白质在亚细胞器内可视化精确定位的技术难题, 成功地将植物蔗糖合成酶ZmSUS-SH1定位在烟草表皮细胞叶绿体基粒周围。该研究同时建立了一套基于撕片制片法的简便OMX显微镜制片方法, 并针对OMX显微成像技术在植物细胞中蛋白质亚细胞定位的应用进行了讨论。

Abstract: The information on protein subcellular localization is important to elucidate protein function, and imaging technology is one of the important approaches to visualize protein localization. However, conventional microscopy techniques can barely resolve details of subcellular structures mainly because of the auto-fluorescence interference with their limited imaging resolution. In recent years, super-resolution optical imaging technologies have been successfully used in human and animal cell research for their 8-fold higher spacial resolution over laser confocal microscopy. Application of these technologies in plant cells has not been reported, probably due to the peculiarity of plant cells. Here, we report the successful use of DeltaVision OMX microscope technology for visualizing Zea mays sucrose synthase 1 (ZmSUS-SH1) around chloroplast grana in transgenic tobacco epidermal cells. OMX microscopy can overcome the cellular chlorophyll fluorescence interference in imaging fluorescent fusion proteins. We also developed an optimized sample preparation protocol for using DeltaVision OMX microscope technology with plant materials.

刘玥, 尹悦佳, 梁重阳, 黄殿帅, 王阳, 刘艳芝, 窦瑶, 冯树丹, 郝东云. 3D-SIM结构照明超分辨率显微镜实现蛋白质在植物亚细胞器内的定位. 植物学报, 2015, 50(4): 495-503.

Yue Liu, Yuejia Yin, Chongyang Liang, Dianshuai Huang, Yang Wang, Yanzhi Liu, Yao Dou, Shudan Feng, Dongyun Hao. Using 3D-SIM Structure Illumination Microscope to Localize Proteins in Plant Subcellular Compartments. Chinese Bulletin of Botany, 2015, 50(4): 495-503.

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB15038

https://www.chinbullbotany.com/CN/Y2015/V50/I4/495

图/表 5

表1 引物序列

Table 1 Primer sequences

Gene names	Application	Amplification length (bp)		Sequence (5′-3′)
Zea mays sucrose synthase (ZmSH1)	PCR	1 900	SH1-L1 SH1-R1	ATGCCTCCTTTCCTCGTCCT CTCACGTACTTCCAGAACCCG
Green fluorescence protein (GFP)	PCR	487	GFP-L2 GFP-R	TTCGGTTATGGTGTTCAATGCCTGTTACAAACTCAAGAAGGACCA

图1 融合蛋白基因表达载体的构建和外源基因的表达 (A) 融合蛋白基因表达架构示意图; (B) 转融合蛋白基因烟草的PCR鉴定, 用ZmSH1引物(B1)和GFP引物(B2)进行PCR扩增的产物的琼脂糖凝胶电泳图谱; (C) 融合基因在烟草中表达的RT-PCR验证。M: Marker; T: 转基因植株; Wt: 野生型植株

Figure 1 Construction and validation of fusion reporters (A) Schematic represenntation of the fusion protein vector; (B) Electrophoretic assay of PCR products from the transgenic tobacco leaves with ZmSH1 (B1) and GFP (B2) primers respectively; (C) RT-PCR validation of the fusion protein gene expression in the transgenic tobacco. M: Molecular weight size marker; T: Transgenic tobacco; Wt: The wild-type tobacco as a control

图2 撕片标本制作流程 (A) 选取成熟烟草叶片; (B) 在载玻片中央滴一滴水; (C) 用镊子由外向内撕取表皮, 将标本在水中展开; (D) 切去多余部分; (E) 沿一侧盖上盖玻片; (F) 拇指按压除去气泡; (G) 指甲油封片。Bar=1.5 cm

Figure 2 Flow chart of specimen preparation (A) Select mature tobacco leaves; (B) Place a drop of water onto a microscope slide; (C) Tear the leaf epidermis with a forceps in an orientation from the leaf edge inwards; (D) Take the thinnest part as the specimen by cutting off the excessive tissue; (E) Place the coverslip onto the specimen carefully to avoid trapping air; (F) Press the coverslip gently with the thumb to remove air bubbles; (G) Seal the coverslip with nail varnish. Bar=1.5 cm

图3 烟草叶片表皮细胞激光共聚焦成像采用DeltaVision Olympus激光扫描共聚焦显微镜488 nm激光, 100X油镜, 距离物镜8 582-8 594 μm处观察得到。(A1), (B1), (C1), (D1) 未转化烟草成像; (A2), (B2), (C2), (D2) 瞬时转化融合蛋白标本成像; (A3), (B3), (C3), (D3) 稳定转化融合蛋白标本成像; (E) 瞬时转化GFP标本成像。图A2和A3为激发光下融合蛋白的荧光成像; 图B1-B3为激发光下叶绿体自发荧光成像; 图C1-C3为激发光下绿色荧光蛋白和叶绿体自发荧光; 图D1-D3为可见光下表皮细胞成像。Bar=20 μm

Figure 3 Confocal imaging of tobacco epidermal cells Images were taken by DeltaVision Olympus scanning confocal microscope with the lens to sample distance between 8 582 and 8 594 μm upon excitation with a 488 nm laser. (A1), (B1), (C1), (D1) Images of non-transgenic tobacco epidermal cells; (A2), (B2), (C2), (D2) Images of the ZmSH1-GFP transiently transformed tobacco epidermal cells; (A3), (B3), (C3), (D3) Images of the stably transformed tobacco epidermal cells; (E) Image of GFP transiently transformed tobacco epidermal cells. Figures A2 and A3 are the fusion protein images taken upon excitation; Figures B1-B3 are chlorophyll autofluorescence taken upon excitation; Figures C1-C3 are the overlapping images of the fusion protein fluorescence and the chlorophyll autofluorescence taken upon excitation; Figures D1-D3 are the tobacco epidermal cell images taken under the visible light. Bar=20 μm

图4 转融合蛋白基因烟草表皮细胞标本超分辨率显微镜成像采用DeltaVision OMX超分辨率显微镜, 在488 nm激发光下, 经软件将荧光信号重构得到的图像。(A1), (A2) 不同角度成像的融合蛋白; (B1), (B2) 叶绿素自发荧光; (C1)-(C6) 相对应两者图像的叠加图像; (D1)-(D3) 上述叠加图像的横切图像; (E) 上述叠加图像的纵切图像。

Figure 4 The super-resolution images of ZmSUS-SH1-GFP fusion protein in transgenic tobacco epidermal cells Images were taken by DeltaVision OMX Super-resolution microscopes upon excitation with a 488 nm laser. (A1), (A2) Images of different viewing angles of the fusion protein fluorescence; (B1), (B2) Chlorophyll autofluorescence; (C1)-(C6) The corres- ponding overlapping images of the above two channels; (D1)-(D3) The latitudinal section of the images C; (E) The longitudinal one.

参考文献 19

1	龚燕华, 彭小忠 (2010). 蛋白质相互作用及亚细胞定位原理与技术. 北京: 中国协和医科大学出版社. pp. 210-234.
2	李楠, 王黎明, 杨军 (1996). 激光共聚焦显微镜的原理和应用. 军医进修学院学报 17, 232-234.
3	邢浩然, 刘丽娟, 刘国振 (2006). 植物蛋白质的亚细胞定位研究进展. 华北农学报 21(增刊), 1-6.
4	赵启韬, 苗俊英 (2003). 激光共聚焦显微镜在生物医学研究中的应用. 北京生物医学工程 22, 52-54.
5	Brown ACN, Oddos S, Dobbie IM, Alakoskela JM, Parton RM, Eissmann P, Neil MAA, Dunsby C, French PMW, Davis I, Davis DM (2011). Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super resolution microscopy.PLoS Biol 9, 1-18.
6	Coltharp C, Xiao J (2012). Superresolution microscopy for microbiology.Cell Microbiol 14, 1808-1818.
7	Dickerson D (2010). A study of chromatin dynamics during transcription by fluorescence light microscopy. Doctoral Thesis. Dundee: University of Dundee.
8	Dobbie IM, King E, Parton RM, Carlton PM, Sedat JW, Swedlow JR, Davis I (2011). OMX: a new platform for multimodal, multichannel wide-field imaging. In: Goldmam RD, ed. Live Cell Imaging, 2nd edn. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. pp. 898-909.
9	Gustafsson MGL (2008). Super-resolution light microscopy goes live.Nat Methods 5, 385-387.
10	Gustafsson MGL, Shao L, Carlton PM, Wang CJR, Golubovskaya IN, Cande WZ, Agard DA, Sedat JW (2008). Three-dimensional resolution doubling in wide- field fluorescence microscopy by structured illumination.Biophys J 94, 4957-4970.
11	Masters BR (2010). The development of fluorescence microscopy. eLS 1-9, doi: 10.1002/9780470015902. a0022- 093.
12	Minden JS, Agard DA, Sedat JW, Alberts BM (1989). Direct cell lineage analysis in Drosophila melanogaster by time-lapse, three-dimensional optical microscopy of living embryos.J Cell Biol 109, 505-516.
13	Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P, Burke B, Cardoso MC, Agard DA, Gustafsson MGL, Leonhardt H, Sedat JW (2008). Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.Science 320, 1332-1336.
14	Seabold GK, Wang PY, Petralia RS, Chang K, Zhou A, McDermott MI, Wang YX, Milgram SL, Wenthold RJ (2012). Dileucine and PDZ-binding motifs mediate synaptic adhesion-like molecule 1 (SALM1) trafficking in hippocampal neurons.J Biol Chem 287, 4470-4484.
15	Swedlow JR (2012). Innovation in biological microscopy: current status and future directions.BioEssays 34, 333-340.
16	Tanos BE, Yang HJ, Soni R, Wang WJ, Macaluso FP, Asara JM, Tsou MFB (2013). Centriole distal appendages promote membrane docking, leading to cilia initiation.Genes Dev 27, 163-168.
17	van de Corput MPC, de Boer E, Knoch TA, van Cappellen WA, Quintanilla A, Ferrand L, Grosveld FG (2012). Super-resolution imaging reveals three-dimensional folding dynamics of the β-globin locus upon gene activation.J Cell Sci 125, 4630-4639.
18	Weil TT, Xanthakis D, Parton R, Dobbie I, Rabouille C, Gavis ER, Davis I (2010). Distinguishing direct from indirect roles for bicoid mRNA localization factors.Deve- lopment 137, 169-176.
19	Zheng CY, Wang YX, Kachar B, Petralia RS (2011). Differential localization of SAP102 and PSD-95 is revealed in hippocampal spines using super-resolution light microscopy.Commun Integr Biol 4, 104-105.

3D-SIM结构照明超分辨率显微镜实现蛋白质在植物亚细胞器内的定位

Using 3D-SIM Structure Illumination Microscope to Localize Proteins in Plant Subcellular Compartments

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 19

相关文章 0

编辑推荐

Metrics

本文评价