Robust Red FRET Sensors Using Self-Associating Fluorescent Domains

Abstract

Elucidation of subcellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here, we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dynamic range of protease sensors up to 10-fold and was essential to generate functional red variants of CFP-YFP-based Zn(2+) sensors. The large dynamic range afforded by the new red FRET pair allowed simultaneous use of differently colored Zn(2+) FRET sensors to image Zn(2+) over a broad concentration range in the same cellular compartment.

Genetically-encoded FRET-based sensors for monitoring Zn2+ in living cells

“…36,37 In order to simultaneously monitor Zn 2+ in different cellular compartments in the same cell or study the relation between Zn 2+ concentration and intracellular signal transduction, sensor variants are required that can be used together with CFP-YFP based sensors. 21 Recently red-shifted variants of eCALWY were obtained by replacing Cerulean and Citrine by mOrange and mCherry, respectively. To obtain sensors with a sufficient FRET response, hydrophobic mutations needed to be introduced at the surface of both fluorescent domains to promote association of mOrange and mCherry in the Zn (Table 1).…”

Intracellular zinc in insulin secretion and action: a determinant of diabetes risk?

“…The reasons for these variations are unclear and may involve differences in intracellular pH on which the probes are steeply dependent. Red-shifted variant have been created for Zap and eCALWY sensors (32,34) . Hybrid probes include both genetically-encoded and small molecular elements.…”

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

“…78,79 The orange-red FRET pair was recently improved by developing self-associating variants of mOrange and mCherry by reversion of the hydrophobic dimeric interface breaking mutations. 80,81 LSS mOrange and mKate2 are yet another orange-red FRET pair that has been simultaneously imaged with a CFP-YFP FRET pair using a single laser excitation wavelength. 47 Generally speaking, the single most important advantage of FRET-based biosensors is that they provide ratiometric fluorescent changes that can typically be calibrated, making this class of indicators most appropriate for quantitative imaging.…”