Poster: Emerging Technologies

Abs # 872: FLIP nanosensors for dynamic imaging of metabolites in living cells

Presenter:	Frommer, Wolf B., frommer@zmbp.uni-tuebingen.de
Authors	Frommer, Wolf B. (A) (B)  Fehr, Marcus  (A) (B)  Lager, Ida  (A)   Lalonde, Sylvie  (A)
Affiliations:	(A): ZMBP University Tübingen (B): Carnegie Institution of Washington, Stanford
Web Site:	http://www-ciwdpb.stanford.edu/

Static analysis of metabolite composition in organs, tissues and cellular compartments involves cell disruption. Most techniques cannot measure metabolite changes in real-time, nor account for likely variations in local metabolite concentration at the cellular level. The methods have low resolution and are prone to artifacts, e.g. contamination by other cell types or subcellular compartments. A set of novel nanosensors was developed that allows imaging of various sugars dynamically with subcellular resolution. Using the bacterial periplasmic maltose and glucose binding proteins, FLIP nanosensors were constructed that transform their substrate-induced hinge-bend movement into altered fluorescence resonance energy transfer (FRET) between two fluorescent proteins. To obtain broader detection ranges, affinity mutants were generated. Optical measurements confirm that the fusions do not influence binding properties. To test the system for detecting maltose in complex solutes, a German beer was shown to contain 28 mM maltosides. The nanosensors were introduced into various cell lines and into transgenic organisms and used to determine cytosolic concentrations of maltosides and glucose, and the effect of inhibitors on homeostasis. The results show that periplasmic binding proteins, which comprise more than 50 proteins covering a wide spectrum of substrates are suitable for FRET-based in vitro, ex vivo and in vivo detection, providing numerous scientific, medical and environmental applications.