A monomeric red fluorescent protein with low cytotoxicity

Shemiakina, Irina I.; Ermakova, Galina V.; Cranfill, Paula J.; Baird, Michelle A.; Evans, R. A.; Souslova, Ekaterina A.; Staroverov, Dmitry B; Gorokhovatsky, Andrey Yu.; Putintseva, Ekaterina V.; Gorodnicheva, Tatiana; Chepurnykh, T. V.; Strukova, Lydia A.; Lukyanov, Sergey; Zaraisky, Andrey G.; Davidson, Michael W.; Chudakov, Dmitriy M.; Shcherbo, Dmitry

doi:10.1038/ncomms2208

Cited by 187 publications

(193 citation statements)

References 23 publications

(27 reference statements)

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“…17 Another member of the eqFP578 variant family is FusionRed (λ ex ¼ 580 nm and λ em ¼ 608 nm), which exhibits decreased cytotoxicity when expressed in mammalian cells. 37 The eqFP611 lineage has also yielded mRuby (λ ex ¼ 558 nm and λ em ¼ 605 nm) and the brighter mRuby2 variant (λ ex ¼ 559 nm and λ em ¼ 600 nm), which exhibit a relatively LSS (∼50 nm) between the excitation and emission maxima. 35,36 Although a growing number of engineered standard, far, and LSS RFPs have an "m" (as an abbreviation for monomeric) in front of their name, many do not behave as monomers when expressed in cells.…”

Section: Standard Rfpsmentioning

confidence: 99%

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Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

2015

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Section: Standard Rfpsmentioning

confidence: 99%

“…49 In some cases, the protein may form dimers or higher order oligomers, which can lead to aggregation and/or cytotoxicity. 37 Yet other RFPs, many of which are unambiguously monomeric, can form bright puncta in certain cells types due to accumulation in lysosomes or autophagosomes.…”

Section: Standard Rfpsmentioning

confidence: 99%

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

2015

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“…Fluorescent protein fusions have been used extensively as markers for ChR expression (42). To quantify ChR expression, we fused the red fluorescent protein, mKate2.5 (mKate) (43), to the C termini of the ChRs. To quantify membrane insertion and plasma membrane localization, we used the SpyTag/SpyCatcher labeling method (44).…”

Section: Resultsmentioning

confidence: 99%

Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins

Bedbrook

Rice

Yang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Integral membrane proteins (MPs) are key engineering targets due to their critical roles in regulating cell function. In engineering MPs, it can be extremely challenging to retain membrane localization capability while changing other desired properties. We have used structure-guided SCHEMA recombination to create a large set of functionally diverse chimeras from three sequence-diverse channelrhodopsins (ChRs). We chose 218 ChR chimeras from two SCHEMA libraries and assayed them for expression and plasma membrane localization in human embryonic kidney cells. The majority of the chimeras express, with 89% of the tested chimeras outperforming the lowest-expressing parent; 12% of the tested chimeras express at even higher levels than any of the parents. A significant fraction (23%) also localize to the membrane better than the lowest-performing parent ChR. Most (93%) of these welllocalizing chimeras are also functional light-gated channels. Many chimeras have stronger light-activated inward currents than the three parents, and some have unique off-kinetics and spectral properties relative to the parents. An effective method for generating protein sequence and functional diversity, SCHEMA recombination can be used to gain insights into sequence-function relationships in MPs.membrane proteins | channelrhodopsin | structure-guided recombination | chimeragenesis

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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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A monomeric red fluorescent protein with low cytotoxicity

Cited by 187 publications

References 23 publications

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins

Approaches to imaging unfolded secretory protein stress in living cells

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