Abstract
Photoconvertible fluorescence proteins are potential tools for investigating dynamic processes in living cells. Monomeric Eos-fluorescent protein (mEosFP) was used as an autofluorescent tag to monitor aquaporin-2 (AQP2) trafficking in mpkCCD cells grown on semipermeable filter. The emission of mEosFP can be switched from green to red upon UV irradiation. mEos2 (an mEOSFP variant) was fused to the amino-terminus of AQP2. mpkCCD was transfected with the chimeric construct and cultured without vasopressin to diminish endogenous AQP2 expression. Apical osmotic water permeability (Pf) of mpkCCD was measured with calcein fluorescence. Trafficking of mEOSFP-AQP2 was monitored using two-photon confocal microscopy. Expression of mEOSFP-AQP2 conferred Pf to the transfected cells as indicated by cell swelling when exposed to apical hypotonic solution. Pf of transfected cells was further augmented by 10 μM forskolin. No corresponding increase in cell volume and Pf were found in non-transfected cells. Forskolin treatment was also accompanied with apical targeting of mEOSFP-AQP2 in live cells, which was confirmed with immunohistochemistry. Rapid local photoconversion from green mEOSFP-AQP2 to red mEOSFP-AQP2 was successfully induced with a 405 nm diode laser. These results provide proof-of-concept to monitor the trafficking of two populations of AQP2-containing vesicles in live cells.
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