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.