title: Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells.
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.
authors: Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. cuiyongwhu@gmail.com.
authors: Yong
authors: Y
authors: Cui
authors: Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
authors: Caiji
authors: C
authors: Gao
authors: Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
authors: Qiong
authors: Q
authors: Zhao
authors: Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. ljiang@cuhk.edu.hk.|CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, 518057, China. ljiang@cuhk.edu.hk.
authors: Liwen
authors: L
authors: Jiang
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques; D005455:Fluorescent Antibody Technique / Q000379:methods*; D015870:Gene Expression; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D059828:Plant Cells / Q000378:metabolism / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D020691:rab GTP-Binding Proteins; D000097573:Red Fluorescent Protein; C081080:Rha1 protein, Arabidopsis; C054562:yellow fluorescent protein, Bacteria
keywords: Colocalization analysis; GFP-tagged proteins; Immunofluorescence labeling; Protein dynamics; Spinning disk confocal microscopy; Subcellular localization