Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway

Costantini, Lindsey M.; Subach, Fedor V.; Jaureguiberry‐Bravo, Matias; Verkhusha, Vladislav V.; Snapp, Erik L.

doi:10.1016/j.bbrc.2012.12.028

Cited by 19 publications

(28 citation statements)

References 23 publications

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“…Second, many FPs contain consensus sequences (N-X-S/T, where X is any amino acid except proline) for N-linked sugar additions. These sugars increase an FP's size and can influence its diffusive behavior (Costantini, Subach, Jaureguiberry-Bravo, Verkhusha, & Snapp, 2013) and cause a fusion protein to interact with the lectin chaperones calnexin and calreticulin. Third, FPs have a characteristic p K a or pH at which the fluorescence intensity of the FP decreases by half and eventually becomes dark as the pH decreases further.…”

Section: 2 Methodsmentioning

confidence: 99%

“…The best behaved FPs we have identified are superfolder GFP (sfGFP) and its derivatives (Aronson et al, 2011; Pedelacq, Cabantous, Tran, Terwilliger, & Waldo, 2006). A blue FP suitable for the secretory pathway has been described (Costantini et al, 2013) and can be used in conjunction with sfGFP without significant crosstalk for most microscope filter sets. FPs with cysteines or N-linked glycosylation sites can be used on the cytoplasmic face of the GC, as long as the FPs are robustly monomeric.…”

Section: 2 Methodsmentioning

confidence: 99%

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Photobleaching Methods to Study Golgi Complex Dynamics in Living Cells

Snapp

2013

Methods in Cell Biology

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The Golgi complex (GC) is a highly dynamic organelle that constantly receives and exports proteins and lipids from both the endoplasmic reticulum and the plasma membrane. While protein trafficking can be monitored with traditional biochemical methods, these approaches average the behaviors of millions of cells, provide modest temporal information and no spatial information. Photobleaching methods enable investigators to monitor protein trafficking in single cells or even single GC stacks with subsecond precision. Furthermore, photobleaching can be exploited to monitor the behaviors of resident GC proteins to provide insight into mechanisms of retention and trafficking. In this chapter, general photobleaching approaches with laser scanning confocal microscopes are described. Importantly, the problems associated with many fluorescent proteins (FPs) and their uses in the secretory pathway are discussed and appropriate choices are suggested. For example, Enhanced Green Fluorescent Protein (EGFP) and most red FPs are extremely problematic. Finally, options for data analyses are described.

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Section: 2 Methodsmentioning

confidence: 99%

Section: 2 Methodsmentioning

confidence: 99%

Photobleaching Methods to Study Golgi Complex Dynamics in Living Cells

Snapp

2013

Methods in Cell Biology

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“…Several popular FPs, including EGFP, have a propensity to oligomerize [49, 50] and proteins advertised as “monomeric,” may still oligomerize in cells [47]. This is especially true when the FPs are attached to integral membrane proteins, which confines the FPs to a two-dimensional plane and increases the effective concentration leading to a higher probability of oligomerizing.…”

Section: Approaches For Imaging Er Stress and Upr Activity In Livinmentioning

confidence: 99%

“…At this time, we recommend superfolder GFP [56, 57], secBFP2 [47], and FusionRed [58] for protein fusions. For transcriptional reporters, mNeonGreen matures rapidly and produces an intense signal [59] and TagRFP has similar properties for a red reporter [60].…”

Section: Approaches For Imaging Er Stress and Upr Activity In Livinmentioning

confidence: 99%

Approaches to imaging unfolded secretory protein stress in living cells

Lajoie

Fazio

Snapp

2014

Endoplasmic Reticulum Stress in Diseases

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The endoplasmic reticulum (ER) is the point of entry of proteins into the secretory pathway. Nascent peptides interact with the ER quality control machinery that ensures correct folding of the nascent proteins. Failure to properly fold proteins can lead to loss of protein function and cytotoxic aggregation of misfolded proteins that can lead to cell death. To cope with increases in the ER unfolded secretory protein burden, cells have evolved the Unfolded Protein Response (UPR). The UPR is the primary signaling pathway that monitors the state of the ER folding environment. When the unfolded protein burden overwhelms the capacity of the ER quality control machinery, a state termed ER stress, sensor proteins detect accumulation of misfolded peptides and trigger the UPR transcriptional response. The UPR, which is conserved from yeast to mammals, consists of an ensemble of complex signaling pathways that aims at adapting the ER to the new misfolded protein load. To determine how different factors impact the ER folding environment, various tools and assays have been developed. In this review, we discuss recent advances in live cell imaging reporters and model systems that enable researchers to monitor changes in the unfolded secretory protein burden and activation of the UPR and its associated signaling pathways.

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“…9–11 Similarly, the N-linked glycosylation machinery in the ER can append large sugar groups to asparagines that are followed by a consensus glycosylation sequence (N-X-S/T). 12, 13 We and others have previously shown such modifications can generate misfolded populations of dark FPs 9, 10 , enlarge their size 13 , and incorrectly localize fusion proteins. 11 To avoid such problems, we have identified and optimized multiple monomeric FPs for use in oxidizing environments including the ER, the eukaryotic secretory pathway, the mitochondrial inner membrane space, the chloroplast inner membrane space, and the periplasm of gram-negative bacteria.…”

mentioning

confidence: 97%