Affinity Series of Genetically Encoded Förster Resonance Energy-Transfer Sensors for Sucrose

Abstract

Genetically encoded fluorescent sugar sensors are valuable tools for the discovery of transporters and for quantitative monitoring of sugar steady-state levels in intact tissues. Genetically encoded Forster resonance energy-transfer sensors for glucose have been designed and optimized extensively, and a full series of affinity mutants is available for in vivo studies. However, to date, only a single improved sucrose sensor FLIPsuc-90μΔ1 with K m for sucrose of ∼90 μM was available. This sucrose sensor was engineered on the basis of an Agrobacterium tumefaciens sugar-binding protein. Here, we took a two-step approach to first improve the dynamic range of the FLIPsuc sensor and then expand the detection range from micro-to millimolar sucrose concentrations by mutating a key residue in the binding site. The resulting series of sucrose sensors may enable investigation of sucrose transporter candidates and comprehensive in vivo analyses of sucrose concentration in plants. Since FLIPsuc-90μ also detects trehalose in animal cells, the new series of sensors will likely be suitable for investigating trehalose transport and monitor trehalose steady-state levels in vivo.

OsSWEET11b, a potential sixth leaf blight susceptibility gene involved in sugar transport‐dependent male fertility

“…Transport assays were performed in 96‐well plates (Chen et al ., 2010). HEK293T cells were cotransfected with a plasmid carrying the sucrose sensor FLIPsuc90µ‐sCsA (Sadoine et al ., 2021) and a plasmid carrying a candidate transporter gene (100 ng) using Lipofectamine2000 (Invitrogen). For FRET imaging, culture media in each well were replaced with 100 µl Hanks Balanced Saline Salt (HBSS) buffer followed by the addition of 100 µl HBSS buffer containing 25 mM sucrose.…”

Imaging Approaches for the Study of Metabolism in Real Time Using Genetically Encoded Reporters

“…The sensor exhibits significant response (up to 200% increase in fluorescence intensity) and flexible applicability, even allowing intravital imaging. Following similar strategies, sensors for mono (ribose) or di-saccharides (sucrose), have been developed and applied in non-mammalian systems or even in small animals such as Drosophila and C. elegans ( Lager et al, 2006 ; Sadoine et al, 2020 ). Along with the above, a FLIM-based sensor has been reported ( Diaz-Garcia et al, 2017 ; Diaz-Garcia et al, 2019 ) yielding a maximum lifetime change in the range of 0.38 ns, yet as with every FLIM measurement, special equipment is needed and read outs are not straightforward.…”

Fluorescence-based sensing of the bioenergetic and physicochemical status of the cell

“…Finally, a donor and an acceptor can be connected through a linker containing a sensing domain for specific metabolite, ion or physical factor such as temperature or excluded volume. A change in conformation in this linker will generally result in a change in the positions of the two fluorescent probes relative to one another, bringing them closer together or further apart, depending on the sensor's design (Imamura et al, 2009;Sadoine et al, 2021).…”

“…Binding affinities of some sensors, such as ion probes, are often different when measured in vitro or in vivo. Substrate-binding proteins associated with ATP-binding cassette importers and other types of transporters and ion channels (Scheepers, Nijeholt, & Poolman, 2016) have been a popular source of proteins to which a fluorescence donor and acceptor can be engineered, either by gene fusion with FPs (Isoda et al, 2021;Okumoto, Jones, & Frommer, 2012;Sadoine et al, 2021) or via chemical modification of strategically engineered Cys pairs (de Boer et al, 2019;Gouridis et al, 2015). These proteins are readily engineered to obtain sensors in the appropriate affinity range(s), but other ligand-specific proteins have also been used to design ratiometric or FRET-based sensors (vide infra).…”

“…Subsequently, FPs have been used to develop a great variety of sensors (Berg, Hung, & Yellen, 2009;Miyawaki et al, 1997;Nadler, Morgan, Flamholz, Kortright, & Savage, 2016) to study both molecular and global changes in the tagged macromolecule or structure of the cell. The availability of FPs emitting at different wavelengths allowed the application of the F€ orster Resonance Energy Transfer (FRET) mechanism to create sensors (Calamera et al, 2019;Miyawaki et al, 1997;Otten et al, 2019;Sadoine, Reger, Wong, & Frommer, 2021) and to determine interaction between proteins or protein domains (Ivanusic, Eschricht, & Denner, 2014;Kaufmann et al, 2020). Circularly permuted fluorescent proteins (cpFPs) variants (Topell, Hennecke, & Glockshuber, 1999) have been developed to create sensors (Kostyuk, Demidovich, Kotova, Belousov, & Bilan, 2019) to detect variations in the concentration of specific molecules.…”