Bright monomeric red fluorescent protein with an extended fluorescence lifetime

Merzlyak, Ekaterina M.; Goedhart, Joachim; Shcherbo, Dmitry; Bulina, Mariya E; Shcheglov, Aleksandr S; Fradkov, Arkady F.; Gaintzeva, Anna; Lukyanov, Konstantin A.; Lukyanov, Sergey; Gadella, Theodorus W. J.; Chudakov, Dmitriy M.

doi:10.1038/nmeth1062

Cited by 590 publications

(533 citation statements)

References 16 publications

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“…A survey of Sun2 immunoreactivity in cells containing LULL1-redistributed TorA-mGFP indicated that changes in Sun2 were widespread; 60% of these cells lacked a clearly delineated nuclear rim of Sun2 compared with only 4% of untransfected cells (n Ͼ 110 for each). To rule out the alternative possibility that redistributed TorA masked the lumenal Sun2 antibody epitope (Hodzic et al, 2004), we made a tetracycline-inducible LULL1-myc cell line that also constitutively expressed Sun2-GFP and assessed the effect of introducing TorA fused to TagRFP (Merzlyak et al, 2007). Direct examination of protein fluorescence showed that Sun2-GFP was displaced by TorA-TagRFP ( Figure 6D), paralleling the effect of TorA on endogenous Sun2.…”

Section: Redistributed Tora Displaces a Subset Of Linc Complexmentioning

confidence: 99%

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LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by theDYT1Dystonia Mutation

Heyden

Naismith

Snapp

et al. 2009

MBoC

115

162

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TorsinA (TorA) is an AAA؉ ATPase in the endoplasmic reticulum (ER) lumen that is mutated in early onset DYT1 dystonia. TorA is an essential protein in mice and is thought to function in the nuclear envelope (NE) despite localizing throughout the ER. Here, we report that transient interaction of TorA with the ER membrane protein LULL1 targets TorA to the NE. FRAP and Blue Native PAGE indicate that TorA is a stable, slowly diffusing oligomer in either the absence or presence of LULL1. Increasing LULL1 expression redistributes both wild-type and disease-mutant TorA to the NE, while decreasing LULL1 with shRNAs eliminates intrinsic enrichment of disease-mutant TorA in the NE. When concentrated in the NE, TorA displaces the nuclear membrane proteins Sun2, nesprin-2G, and nesprin-3 while leaving nuclear pores and Sun1 unchanged. Wild-type TorA also induces changes in NE membrane structure. Because SUN proteins interact with nesprins to connect nucleus and cytoskeleton, these effects suggest a new role for TorA in modulating complexes that traverse the NE. Importantly, once concentrated in the NE, disease-mutant TorA displaces Sun2 with reduced efficiency and does not change NE membrane structure. Together, our data suggest that LULL1 regulates the distribution and activity of TorA within the ER and NE lumen and reveal functional defects in the mutant protein responsible for DYT1 dystonia.

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Section: Redistributed Tora Displaces a Subset Of Linc Complexmentioning

confidence: 99%

“…TorA-TagRFP was made by transferring TorA mutants excised with XhoI and EcoRI from EGFP-N1 into a RFP-N1 vector containing TagRFP (Merzlyak et al, 2007). The TorA ⌬26-43 deletion was created by QuikChange mutagenesis (Stratagene, La Jolla, CA).…”

Section: Plasmidsmentioning

confidence: 99%

LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by theDYT1Dystonia Mutation

Heyden

Naismith

Snapp

et al. 2009

MBoC

115

162

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“…3 and Supplementary movies [1][2][3][4][5][6][7][8]. Owing to the lack of a statistical methodology to make direct comparisons of fluorescent protein performance in fusions, the practice is utilized only to provide one or several representative images that demonstrate the ability of the reporter to properly perform in each of the constructs.…”

Section: Performance Of Fusionred In Fusionsmentioning

confidence: 99%

“…Over the past decade, a number of red dimeric and tetrameric fluorescent proteins have been subjected to artificial monomerization, which in most cases has resulted in a loss of brightness, a slower maturation rate and reduced fluorescence stability. Engineered red monomers, such as mCherry and other mFruit proteins 3 , along with mKO 4 , TagRFP 5 and mKate2 (refs 6,7) are suitable fusion partners for many proteins of interest. However, these proteins usually do not perform as well as Aequorea victoria GFP derivatives in fusions, and often present issues related to abnormal subcellular protein localization, aggregation, or toxic effects 8 .…”

mentioning

confidence: 99%

A monomeric red fluorescent protein with low cytotoxicity

et al. 2012

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Multicolour labelling with fluorescent proteins is frequently used to differentially highlight specific structures in living systems. Labelling with fusion proteins is particularly demanding and is still problematic with the currently available palette of fluorescent proteins that emit in the red range due to unsuitable subcellular localization, protein-induced toxicity and low levels of labelling efficiency. Here we report a new monomeric red fluorescent protein, called FusionRed, which demonstrates both high efficiency in fusions and low toxicity in living cells and tissues.

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“…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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Bright monomeric red fluorescent protein with an extended fluorescence lifetime

Cited by 590 publications

References 16 publications

LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by theDYT1Dystonia Mutation

LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by theDYT1Dystonia Mutation

A monomeric red fluorescent protein with low cytotoxicity

Approaches to imaging unfolded secretory protein stress in living cells

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