Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport

Johnson, Danielle; Ai, Hui‐wang; Wong, Pierre; Young, James D.; Campbell, Robert E.; Casey, Joseph R.

doi:10.1074/jbc.m109.019042

Cited by 64 publications

(72 citation statements)

References 59 publications

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“…89 The majority of intrinsic single FP-based biosensors are green or yellow fluorescent, and only a few intrinsic red single FP-based biosensors have been described. Examples include mNectarine, which was applied for detection of nucleoside transport, 90 and pHTomato, which was used to report synaptic neurotransmitter release at nerve terminals. 57 An excitation ratiometric pH biosensor, pHRed, was engineered from LSS RFP mKeima and used to image energy-dependent changes of cytosolic and mitochondrial pH.…”

Section: Rfp Biosensors Based On a Single Fpmentioning

confidence: 99%

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

2015

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Section: Rfp Biosensors Based On a Single Fpmentioning

confidence: 99%

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

2015

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“…With few exceptions, the fluorescence of FPs is pH dependent and a number of variants that change fluorescence intensity at near-physiological pH values have been reported. 6 Some FPs also exhibit an intrinsic sensitivity towards halide ions and have been used to image physiologically-relevant changes in chloride ion concentrations. 7 The creation of single FP-based biosensors that specifically respond to environmental variables other than pH or halide ions has required some relatively sophisticated engineering of avGFP chimeras.…”

Section: Introductionmentioning

confidence: 99%

Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet

2010

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Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein-based biosensors for Ca 21 and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)-based biosensors by providing a means for varying the critical dipole-dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th b-strand of mCherry. These new variants may ultimately prove useful for the creation of single FP-based Ca 21 biosensors.

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“…To this end, we engineered a genetically encoded melanosome-localized pH sensor (MELOPS) capable of detecting pH changes in the melanosome lumen of living cells. MELOPS was created by inserting the coding sequence of the pH sensor protein Nectarine (25) into the first luminal loop of the melanosome protein OCA2, the product of the gene mutated in oculocutaneous albinism type 2 (Fig. S5).…”

Section: Tpc2 Regulates Melanosome Luminal Phmentioning

confidence: 99%

TPC2 controls pigmentation by regulating melanosome pH and size

Ambrosio

Boyle

Aradi

et al. 2016

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

109

103

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Melanin is responsible for pigmentation of skin and hair and is synthesized in a specialized organelle, the melanosome, in melanocytes. A genome-wide association study revealed that the two pore segment channel 2 (TPCN2) gene is strongly linked to pigmentation variations. TPCN2 encodes the two-pore channel 2 (TPC2) protein, a cation channel. Nevertheless, how TPC2 regulates pigmentation remains unknown. Here, we show that TPC2 is expressed in melanocytes and localizes to the melanosome-limiting membrane and, to a lesser extent, to endolysosomal compartments by confocal fluorescence and immunogold electron microscopy. Immunomagnetic isolation of TPC2-containing organelles confirmed its coresidence with melanosomal markers. TPCN2 knockout by means of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 gene editing elicited a dramatic increase in pigment content in MNT-1 melanocytic cells. This effect was rescued by transient expression of TPC2-GFP. Consistently, siRNA-mediated knockdown of TPC2 also caused a substantial increase in melanin content in both MNT-1 cells and primary human melanocytes. Using a newly developed genetically encoded pH sensor targeted to melanosomes, we determined that the melanosome lumen in TPC2-KO MNT-1 cells and primary melanocytes subjected to TPC2 knockdown is less acidic than in control cells. Fluorescence and electron microscopy analysis revealed that TPC2-KO MNT-1 cells have significantly larger melanosomes than control cells, but the number of organelles is unchanged. TPC2 likely regulates melanosomes pH and size by mediating Ca 2+ release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca 2+ sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size.melanosome | pigmentation | two-pore channel 2 | organelle pH | membrane traffic

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Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport

Cited by 64 publications

References 59 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

Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet

TPC2 controls pigmentation by regulating melanosome pH and size

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