Abstract
Plasmodesmata (PD) are intercellular communication channels that form long, membrane-lined cylinders across cellular junctions. A fluorescent-tagging approach is most commonly used for an initial assessment to address whether a protein of interest may localize or associate with PD domain. However, owing to the dimension of PD being at nanoscale, PD-associated fluorescent signals are detected only as small spots scattered at the cell periphery, hence requiring additional confirmatory evidence. Immunogold labeling provides such information, but suitable antibodies are not always available and morphological preservation is often compromised with this approach. Here we describe an alternative approach using a correlative light and electron microscopy (CLEM) technique, which combines fluorescent imaging and transmission electron microscopy. By employing this method, a clear correlation between fluorescent speckles and the presence of individual or clusters of PD is achieved.
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References
Ding B, Turgeon R, Parthasarathy MV (1992) Substructure of freeze-substituted plasmodesmata. Protoplasma 169:28–41
Ding B, Haudenshield JS, Hull RJ, Wolf S, Beachy RN, Lucas WJ (1992) Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants. Plant Cell 4:915–928
Lee JY, Wang X, Cui W, Sager R, Modla S, Czymmek K et al (2011) A plasmodesmata-localized protein mediates crosstalk between cell-to-cell communication and innate immunity in arabidopsis. Plant Cell 23:3353–3373
Simpson C, Thomas C, Findlay K, Bayer E, Maule AJ (2009) An arabidopsis gpi-anchor plasmodesmal neck protein with callose binding activity and potential to regulate cell-to-cell trafficking. Plant Cell 21:581–594
Blackman LM, Harper JDI, Overall RL (1999) Localization of a centrin-like protein to higher plant plasmodesmata. Eur J Cell Biol 78:297–304
Tian Q, Olsen L, Sun B, Lid SE, Brown RC, Lemmon BE et al (2007) Subcellular localization and functional domain studies of defective kernel1 in maize and arabidopsis suggest a model for aleurone cell fate specification involving crinkly4 and supernumerary aleurone layer1. Plant Cell 19:3127–3145
van Rijnsoever C, Oorschot V, Klumperman J (2008) Correlative light-electron microscopy (clem) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nat Methods 5:973–980
Giepmans BN, Deerinck TJ, Smarr BL, Jones YZ, Ellisman MH (2005) Correlated light and electron microscopic imaging of multiple endogenous proteins using quantum dots. Nat Methods 2:743–749
Polishchuk RS, Polishchuk EV, Marra P, Alberti S, Buccione R, Luini A et al (2000) Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane. J Cell Biol 148:45–58
Robinson JM, Vandre DD (1997) Efficient immunocytochemical labeling of leukocyte microtubules with fluoronanogold: an important tool for correlative microscopy. J Histochem Cytochem 45:631–642
Deerinck TJ, Martone ME, Lev-Ram V, Green DP, Tsien RY, Spector DL et al (1994) Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy. J Cell Biol 126:901–910
Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, Davidson MW et al (2011) A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 9:e1001041
Martell JD, Deerinck TJ, Sancak Y, Poulos TL, Mootha VK, Sosinsky GE et al (2012) Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol 30:1143–1148
Wei D, Jacobs S, Modla S, Zhang S, Young CL, Cirino R et al (2012) High-resolution three-dimensional reconstruction of a whole yeast cell using focused-ion beam scanning electron microscopy. Biotechniques 53:41–48
Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2:e329
Micheva KD, Smith SJ (2007) Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55:25–36
Modla S, Mendonca J, Czymmek KJ, Akins RE (2010) Identification of neuromuscular junctions by correlative confocal and transmission election microscopy. J Neurosci Methods 191:158–165
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 1:473–497
Fiala JC (2005) Reconstruct: a free editor for serial section microscopy. J Microsc 218:52–61
Hanson HH, Reilly JE, Lee R, Janssen WG, Phillips GR (2010) Streamlined embedding of cell monolayers on gridded glass-bottom imaging dishes for correlative light and electron microscopy. Microsc Microanal 16:747–754
Rowley JC, Moran DT (1975) A simple procedure for mounting wrinkle-free sections on formvar-coated slot grids. Ultramicroscopy 1:151–155
Acknowledgements
The research pertinent to the development of this protocol was supported by grants provided by the National Science Foundation (IOB 0954931) and partially by the grants from the National Center for Research Resources (5P30RR031160-03) and the National Institute of General Medical Sciences (8 P30 GM103519-03) from the National Institutes of Health to J.-Y. L.
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Modla, S., Caplan, J.L., Czymmek, K.J., Lee, JY. (2015). Localization of Fluorescently Tagged Protein to Plasmodesmata by Correlative Light and Electron Microscopy. In: Heinlein, M. (eds) Plasmodesmata. Methods in Molecular Biology, vol 1217. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1523-1_8
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DOI: https://doi.org/10.1007/978-1-4939-1523-1_8
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