Abstract
Studies of protein subcellular localization and dynamics are helpful in understanding the cellular functions of proteins in an organism. In the past decade, the use of green fluorescent protein (GFP) as a fusion tag has dramatically extended our knowledge in this field. Transient expression and stable transformation of GFP-tagged proteins have been wildly used to study protein localization in vivo in different systems. Although GFP-based tags provide a fast and convenient way to characterize protein properties in living cells, several reports have demonstrated that GFP fusions might not accurately reflect the localization of the native protein as GFP tags may alter the protein properties. To facilitate proper usage of GFP tags in plant cell biology study, we describe detailed protocols to identify possible inhibitory effects of fluorescent tags on protein subcellular localization and to determine if a fluorescently tagged protein is localized to the correct subcellular compartment. Using Arabidopsis Endomembrane protein 12 (EMP12) as an example, we first show the possible inhibitory effect of GFP tags on proper protein localization and then describe the immunofluorescence labeling method to verify the correct localization of GFP fusion proteins. Next, a method is presented using the ImageJ program with the Pearson–Spearman correlation (PSC) colocalization plug-in for statistical quantification of colocalization ratios of two fluorophores. Finally we provide a detailed method for protein dynamics studies using spinning disk confocal microscopy in Arabidopsis cells.
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Acknowledgements
We are grateful to the current and previous members of Prof Jiang’s Laboratory for their contributions in setting up and improving the above methods. This work was supported by grants from the Research Grants Council of Hong Kong (CUHK466011, 465112,466613, CUHK2/CRF/11G, C4011-14R, and AoE/M-05/12), NSFC/RGC (N_CUHK406/12), the National Natural Science Foundation of China (31270226 and 31470294), the Chinese Academy of Sciences-Croucher Funding Scheme for Joint Laboratories, and Shenzhen Peacock Project (KQTD201101) (to L.J.).
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Cui, Y., Gao, C., Zhao, Q., Jiang, L. (2016). Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells. In: Schwartzbach, S., Skalli, O., Schikorski, T. (eds) High-Resolution Imaging of Cellular Proteins. Methods in Molecular Biology, vol 1474. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6352-2_7
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DOI: https://doi.org/10.1007/978-1-4939-6352-2_7
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