Abstract
To perform quantitative live cell imaging, investigators require fluorescent reporters that accurately report protein localization and levels, while minimally perturbing the cell. Yet, within the biochemically distinct environments of cellular organelles, popular fluorescent proteins (FPs), including EGFP, can be unreliable for quantitative imaging, resulting in the underestimation of protein levels and incorrect localization. Specifically, within the secretory pathway, significant populations of FPs misfold and fail to fluoresce due to non-native disulphide bond formation. Furthermore, transmembrane FP-fusion constructs can disrupt organelle architecture due to oligomerizing tendencies of numerous common FPs. Here, we describe a powerful set of bright and inert FPs optimized for use in multiple cellular compartments, especially oxidizing environments and biological membranes. Also, we provide new insights into the use of red FPs in the secretory pathway. Our monomeric 'oxFPs' finally resolve long-standing, underappreciated and important problems of cell biology and should be useful for a number of applications.
Highlights
To perform quantitative live cell imaging, investigators require fluorescent reporters that accurately report protein localization and levels, while minimally perturbing the cell
We established that the cysteines in superfolder GFP (sfGFP) are resistant to disulphide bond formation in oxidizing environments[15]
We found that EBFP2 and mTagBFP possess strong propensities to oligomerize and form Organized Smooth Endoplasmic Reticulum (OSER) whorl structures (Table 3) (Supplementary Fig. 4c)
Summary
To perform quantitative live cell imaging, investigators require fluorescent reporters that accurately report protein localization and levels, while minimally perturbing the cell. Within the secretory pathway, significant populations of FPs misfold and fail to fluoresce due to non-native disulphide bond formation. A secretory pathway targeted cytoplasmic protein that contains cysteines is at risk for the formation of disulphide bonds that do not occur in the cytoplasm. Our study of optimizing FPs for the eukaryotic secretory pathway allowed us to determine the consequences of (1) the oxidizing environment, (2) non-native posttranslational modifications and (3) intracellular trafficking on FP folding and behaviour. When using FPs within the secretory pathway, it is preferable to select FPs that naturally lack cysteine residues, such as the family of mFruit FPs13 or are resistant to disulphide bond formation, such as superfolder GFP (sfGFP)[14,15]. Some RFPs, like mNeptune, develop significant pools of dead-end products of chromophore formation that happen to fluoresce green[28], a highly undesirable characteristic for imaging multiple FPs
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