Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Tuesday Speaker Abstracts

Acid Resistant Monomeric GFP for Quantitative Single-Cell Analyses Takeharu Nagai . Osaka University, Osaka, Japan.

Fluorescent proteins (FPs) cloned from diverse luminous organisms have made a great contribution as fusion tags and units of FRET-based indicators for visualizing biological phenomena. However, most FPs lose their fluorescence in an acidic environment due to their high pH sensitivity (pKa ≈ 6.0), and this has hampered their application to reliable imaging in compartments such as late-endosomes, secretory granules, lysosomes and vacuoles (pH ≈ 4.5- 5.5). Up to date, some acid resistant FPs have been developed, including mTurquoise2 (cyan FP, pKa = 3.1) and TagRFP (red FP, pKa = 3.8), but there is little report of acid resistant green FPs [1]. Therefore, it has been restricted to perform multiple color imaging by FPs to track different proteins simultaneously, and to develop FRET-based indicators for observing ion dynamics. Here, we report an acid resistant monomeric GFP, Gamillus derived from flower hat jellyfish, Olindias formosa living in the Sea of Japan, and engineered by directed evolution. Compared with the most conventional GFP, EGFP (pKa = 6.0), Gamillus has much higher pH stability (pKa = 3.5). Gamillus also showed 1.9-folds higher brightness, 1.7-folds faster chromophore maturation speed and similar photo-stability compared with EGFP. Gamillus expressed in HeLa cells was properly localized in various cellular organelles including lysosomes. We applied Gamillus to develop a FRET-based ratiometric pH indicator by tandem fusion of acid resistant Gamillus with a pH-sensitive RFP, tdTomato. We also have determined a three dimensional structure of Gamillus at 1.7 Å resolution by X-ray crystallography, and been analyzing the structural characteristics. In near future, application of Gamillus to multi-color imaging and development of FRET-based indicators will accelerate revelation of mystery hiding in acidic organelles.

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