植物中的亚细胞定位实验和FRET-FLIM 测定

Malgorzata  Lichocka; Elmon  Schmelzer

doi:10.21769/BioProtoc.1018

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Peer-reviewed

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

ML Malgorzata Lichocka email

ES Elmon Schmelzer

发布: 2014年01月05日第4卷第1期 DOI: 10.21769/BioProtoc.1018 浏览次数: 17625

评审: Anonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

Apr 2013

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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

3 – 4 weeks old Nicotiana benthamiana plants grown in a greenhouse
Agrobacterium tumefaciens strain GV3101
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

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/
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)
Microscope slides (Gerhard Menzel GmbH, Menzel-Glaser^TM, catalog number: AA00000112E )
Microscope Cover Slips no. 1 (Gerhard Menzel GmbH, Menzel-Glaser^TM, catalog number: BB024060A1 )

Software

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

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

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.
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.