A novel analytical method for in vivo phosphate tracking
Research output: Contribution to journal › Journal article › Research › peer-review
Hong Gu, Sylvie Lalonde, Sakiko Okumoto, Loren L. Looger, Anne Marie Scharff-Poulsen, Arthur R. Grossman, Jens Kossmann, Iver Jakobsen, Wolf B. Frommer
Genetically-encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (Pi) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate-binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows Pi-dependent increases in FRET efficiency. FLIPPi affinity mutants cover Pi changes over eight orders of magnitude. COS-7 cells co-expressing a low-affinity FLIPPi and a Na+/Pi co-transporter exhibited FRET changes when perfused with 100µM Pi, demonstrating concentrative Pi uptake by PiT2. FLIPPi sensors are suitable for real-time monitoring of Pi metabolism in living cells, providing a new tool for fluxomics, analysis of pathophysiology or changes of Pi during cell migration.
|Number of pages||9|
|Publication status||Published - 2006|
FLIPPi, fluorescent indicator protein for inorganic phosphate, FRET, fluorescence resonance energy transfer, FP, fluorescent protein
- LIFE - Fluorescence energy transfer, Phosphate starvation, Biosensor, Synechococcus