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

doi:10.11983/CBB15038

Abstract

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.

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[J]. Chinese Bulletin of Botany, 2015, 50(4): 495-503.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB15038

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

Figures/Tables 5

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

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

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

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

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.

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