Novel application of Macrolampis sp2 firefly luciferase for intracellular pH-biosensing in mammalian cells

Author(s)

de Mello Gabriel, Gabriele Veronica ^[1] ; Yasuno, Rie ^[2] ; Mitani, Yasuo ^[3] ; Ohmiya, Yoshihiro ^[2] ; Viviani, Vadim Ravara ^{[4, 1]}

Affiliation

^[1] Fed Univ Sao Carlos UFSCar, Grad Sch Biotechnol & Environm Monitoring, Campus Sorocaba, Sorocaba, SP - Brazil

^[2] Natl Inst Adv Ind Sci & Technol, Biomed Res Inst, Tsukuba, Ibaraki - Japan

^[3] Natl Inst Adv Ind Sci & Technol, Bioprod Res Inst, Sapporo, Hokkaido - Japan

^[4] Fed Univ Sao Carlos UFSCar, Grad Sch Evolut Genet & Mol Biol, Sao Carlos, SP - Brazil

• Document type: Journal article
• Source: PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES; v. 18, n. 5, p. 1212-1217, MAY 1 2019.
• Web of Science Citations: 3

Abstract

Bioluminescence is widely used in biosensors. Firefly luciferase-based bioluminescent sensors are among the most popular ones. Firefly luciferases are pH-sensitive, displaying a large red shift at acidic pH, a property that has been considered undesirable for most applications. Currently, biosensors that can detect intracellular pH are in demand, and some fluorescent biosensors are available. However, pH sensors using bioluminescence have not been used yet. Thus, we decided to harness a firefly luciferase to measure the intracellular pH in mammalian cells. For this purpose, we engineered the luciferase derived from Macrolampis sp2 firefly to localize it on the cytosol or nucleus, in order to observe pH variation in these compartments during biological activities. We first calibrated the emission ratios (R = I-green/I-red) at different pH values. As expected, we observed a red shift of light emission under acidic conditions when the cells were subjected to different pH conditions in the presence of the K+/H+ ionophore, nigericin. Based on these results, we concluded that this firefly luciferase can be used as a diagnostic tool for measuring the intracellular pH variation in pathogenic cells or in cells during apoptosis. This is the first example of real time-monitoring of pH change using color tuning luciferase. (AU)

• FAPESP's process: 10/05426-8 - Arthropod bioluminescence: biological diversity in Brazilian biomes, biochemical origin, structural/functional evolution of luciferases, molecular differentiation of lanterns, biotechnological, environmental and educational applications
• Grantee: Vadim Viviani
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 14/04477-9 - Developing of bioluminescent ratiometric biosensors for pH and divalent metals based on the engineering of the pH-sensing moiety of firefly luciferases
• Grantee: Gabriele Verônica de Mello Gabriel
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 16/15946-5 - Expression of Macrolampis sp2 firefly luciferase in mammalian cells for intracelular pH-biosnsing
• Grantee: Gabriele Verônica de Mello Gabriel
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• FAPESP's process: 11/23961-0 - pH-sensitive luciferases: structural origin of pH-sensitivity, and suitability of use in cell biosensors for pH and heavy metals
• Grantee: Vadim Viviani
• Support Opportunities: Regular Research Grants
• FAPESP's process: 15/22603-4 - Humanization and expression of Macrolampis sp2 firefly luciferase for intracelular pH-biosensing
• Grantee: Gabriele Verônica de Mello Gabriel
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate