pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

Shen, Yi; Rosendale, Morgane; Campbell, Robert E.; Perrais, David

doi:10.1083/jcb.201404107

Cited by 213 publications

(231 citation statements)

References 39 publications

(76 reference statements)

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“…More importantly, it provides an orthogonal handle for scaffolding protein oligomerization 3 and biochemical isolation of native protein complexes 11 . For the sfCherry2 1-10/11 system, the fact that we have only observed lysosome puncta when labeling ER proteins suggests that this problem could be potentially resolved by increasing its pKa with rational designs 32 . Moreover, our engineering platform can be easily adapted to generating other red split FPs based on novel bright FPs such as TagRFP-T 34 , mRuby3 35 , or mScarlet 36 .…”

Section: Discussionmentioning

confidence: 95%

“…These split constructs have allowed us to obtain two-color or super-resolution images of endogenous proteins and have revealed ER tubules with greatly reduced abundance of the translocon component Sec61B. Our platform can be easily extended to the engineering of other self-complmenting split FPs with distinct colors (e.g., mTurquoise2, mTagBFP2) 28,29 , good photoactivation performance (e.g., mMaple3, PATagRFP) 30,31 , or new functionalities (e.g., pH sensitivity) 32 . We note that for non-selfcomplementing split FPs, which are used to detect protein-protein interactions in bimolecular fluorescence complementation (BiFC) assays 33 , a different engineering platform is needed to ensure minimum affinity between the two FP fragments by themselves.…”

Section: Discussionmentioning

confidence: 99%

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Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Feng

Sekine

Pessino

et al. 2017

Preprint

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Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact.To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen2 1-10/11 that improves the ratio of complemented signal to the background of FP 1-10 -expressing cells compared to the commonly used split GFP 1-10/11 ; as well as a 10-fold brighter red-colored split-sfCherry2 1-10/11 . Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry2 11 and GFP 11 , revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes. 5,6 , identification of cell contacts and synapses 7,8 , as well as scaffolding protein assembly 3, 9, 10 . Recently, they have also enabled the generation of large-scale human cell line libraries with fluorescently tagged endogenous proteins through CRISPR/Cas9-based gene editing 11 .So far, the most commonly used self-complementing split FP was GFP 1-10D7/11M3 OPT (which we refers to as GFP 1-10/11 ), engineered from super-folder GFP (sfGFP) 12 . With the splitting point between the tenth and eleventh β-strands, the resulting GFP 11 fragment is a 16-amino acid (a.a.) short peptide. The corresponding GFP 1-10 fragment remains almost non-fluorescent until complementation, making GFP 1-10/11 well suited for protein labeling by fusing GFP 11 to the target protein and over-expressing GFP 1-10 in the corresponding subcellular compartments. However, there lacks a second, orthogonal split FP system with comparable complementation performance for multicolor imaging and multiplexed scaffolding of protein assembly. Previously, a sfCherry 1-10/11 system 3 was derived from super-folder Cherry, an mCherry variant optimized for folding efficiency 13 . However, its overall fluorescent brightness is substantially weaker than an intact sfCherry fusion, potentially due to its limited complementation efficiency 3 . Although two-color imaging with sfCherry 1-10/11 and GFP 1-10/11 has been done using tandem sfCherry 11 to amplify the sfCherry signal for over-expressed targets, it is still too dim to detect most endogenous proteins.In this paper, we report a screening strategy for the direct engineering of self-complementing split FPs. Using this strategy, we have generated a yellow-green-colored mNeonGreen2 1-10/11 (mNG2) that has an improved ratio of complemented signal to the background of FP 1-10 -expressing cells as compared to GFP 1-10/11 , as well as a red-colored sfCherry2 1-...

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Feng

Sekine

Pessino

et al. 2017

Preprint

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“…According to the reported photophysical properties of pHuji (Table 1) [13], it has no cross-color interference with EGFP and is hopeful for dualcolor chemical reactivation imaging. But whether these properties of pHuji remain in hydrophobic resin is unknown.…”

Section: Resultsmentioning

confidence: 99%

“…The fluorescent protein labeling technique including GFP and its variants was proved to be compatible with resin embedding in our previous report [3]. To find a pH sensitive red fluorescent protein for simultaneous two-color imaging, among the pH-sensitive red fluorescent proteins derived not from GFP, we selected pHuji [13], a bright red fluorescent protein developed in 2014 that can provide in situ pHdependent changes in fluorescence intensity. We found pHuji remained pH-sensitive in resin and could be quenched and reactivated simultaneously with EGFP or EYFP.…”

Section: Discussionmentioning

confidence: 99%

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Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP

Guo

Liu

et al. 2017

Biomed. Opt. Express

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Abstract:The pH-sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EGFP or EYFP is good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is an urgent need. Here a pH-sensitive red fluorescent protein, pHuji, is selected and verified to remain pH-sensitive in HM20 resin. We observe 183% fluorescence intensity of pHuji in resin-embeded mouse brain and 29.08-fold fluorescence intensity of reactivated pHuji compared to the quenched state. pHuji and EGFP can be quenched and chemically reactivated simultaneously in resin, thus enabling simultaneous two-color micro-optical sectioning tomography of resin-embedded mouse brain. This method may greatly facilitate the visualization of neuronal morphology and neural circuits to promote understanding of the structure and function of the brain.

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Early Endosomes Act as Local Exocytosis Hubs to Repair Endothelial Membrane Damage

et al. 2023

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The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio‐temporal membrane dynamics at the wound site and the source of the membrane required for wound repair are poorly understood. Here, it is shown that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live‐cell imaging and correlative light and electron microscopy, it is demonstrated that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal soluble N‐ethylmaleimide sensitive factor attachment protein receptor (SNARE) VAMP2, which is crucial for efficient membrane repair. Thus, the newly identified Ca2+‐evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells.

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pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

Abstract: A new pH-sensitive red fluorescent protein called pHuji, in combination with green fluorescent superecliptic pHluorin, allows two-color detection of endocytic events in live cells.

Cited by 213 publications

References 39 publications

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP

Early Endosomes Act as Local Exocytosis Hubs to Repair Endothelial Membrane Damage

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