Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins

Ast, Cindy; J, Foret; Oltrogge, Luke M.; Michele, Roberto De; Kleist, Thomas; Ho, Cheng-Hsun; Frommer, Wolf B.

doi:10.1038/s41467-017-00400-2

Cited by 90 publications

(87 citation statements)

References 59 publications

(87 reference statements)

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“…Different approaches have been used to overcome this obstacle. For example, a second FP can be fused to the one of the probe termini or inserted into cpFP as an internal control [70,71,72,73]. The basic principles of GEFIs are presented in Figure 1.…”

Section: Circularly Permuted Afps As Reporter Units For Gefismentioning

confidence: 99%

Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification

Kostyuk

Demidovich

Kotova

et al. 2019

IJMS

106

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Genetically encoded biosensors based on fluorescent proteins (FPs) are a reliable tool for studying the various biological processes in living systems. The circular permutation of single FPs led to the development of an extensive class of biosensors that allow the monitoring of many intracellular events. In circularly permuted FPs (cpFPs), the original N- and C-termini are fused using a peptide linker, while new termini are formed near the chromophore. Such a structure imparts greater mobility to the FP than that of the native variant, allowing greater lability of the spectral characteristics. One of the common principles of creating genetically encoded biosensors is based on the integration of a cpFP into a flexible region of a sensory domain or between two interacting domains, which are selected according to certain characteristics. Conformational rearrangements of the sensory domain associated with ligand interaction or changes in the cellular parameter are transferred to the cpFP, changing the chromophore environment. In this review, we highlight the basic principles of such sensors, the history of their creation, and a complete classification of the available biosensors.

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Section: Circularly Permuted Afps As Reporter Units For Gefismentioning

confidence: 99%

Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification

Kostyuk

Demidovich

Kotova

et al. 2019

IJMS

106

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“…Here we used an optogenetic biosensing approach, based on a Förster Resonance Energy Transfer (FRET) biosensor for GA for high-resolution quantification of spatiotemporal GA distribution. Small molecule FRET biosensors that directly interact with and report on ligand concentration have been used in living plants to generate high-resolution measurements of calcium [20,21], sugars [22,23], and the plant hormone abscisic acid [24,25]. In these synthetic fusion proteins, binding of the ligand to a sensory domain induces structural rearrangements that alter the amount of energy transfer between a 5 donor fluorescent protein moiety and an acceptor fluorescent protein moiety.…”

Section: Introductionmentioning

confidence: 99%

In vivo gibberellin gradients visualized in rapidly elongating tissues

et al. 2017

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The phytohormone gibberellin (GA) is a key regulator of plant growth and development. Although the upstream regulation and downstream responses to GA vary across cells and tissues, developmental stages and environmental conditions, the spatiotemporal distribution of GA in vivo remains unclear. Using a combinatorial screen in yeast, we engineered an optogenetic biosensor, GIBBERELLIN PERCEPTION SENSOR 1 (GPS1), that senses nanomolar levels of bioactive GAs. Arabidopsis thaliana plants expressing a nuclear localized GPS1 report on GAs at the cellular level. GA gradients were correlated with gradients of cell length in rapidly elongating roots and dark-grown hypocotyls. In roots, accumulation of exogenously applied GA also correlated with cell length, intimating that a root GA gradient can be established independently of GA biosynthesis. In hypocotyls, GA levels were reduced in a phytochrome interacting factor (pif) quadruple mutant in the dark and increased in a phytochrome double mutant in the light, indicating that PIFs elevate GA in the dark and that phytochrome inhibition of PIFs could lower GA in the light. As GA signalling directs hypocotyl elongation largely through promoting PIF activity, PIF promotion of GA accumulation represents a positive feedback loop within the molecular framework driving rapid hypocotyl growth.

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“…Analysis of glutamate dynamics are thus best performed with apoplasmic glutamate sensors. To further improve sensor performance, Matryoshka-type indicators, along with affinity mutants to control for artifacts, should be employed, as they are ratiometric, have an increased dynamic range and are less affected by changes in pH (Ast et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Sponging of glutamate at the outer plasma membrane surface reveals roles for glutamate in development

Castro-Rodríguez

Kleist

Gappel

et al. 2020

Preprint

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Plants use electrical and chemical signals for systemic communication. Herbivory, for instance, appears to trigger local apoplasmic glutamate accumulation, systemic electrical signals and calcium waves that travel to report insect damage to neighboring leaves and initiate defense. To monitor extra-and intracellular glutamate concentrations in plants, we generated lines expressing genetically encoded fluorescent glutamate sensors. In contrast to cytosolically localized sensors, extracellularly displayed variants inhibited plant growth and proper development. Phenotypic analyses of high-affinity display sensor lines revealed that root meristem development, particularly the quiescent center (QC), the number of lateral roots, vegetative growth and flower architecture were affected. Notably, the severity of the phenotypes was proportional to the affinity of the displayed glutamate sensors, intimating that their ability to bind extracellular glutamate caused the observed defects. Congruously, root growth defects were suppressed by supplementing culture media with low levels of glutamate. Overall, our data indicate sensor noxiousness was dependent on its membrane-tethering and likely caused by sequestration of extracellular glutamate, specifically at the cell surface, thereby either disrupting the supply of glutamate to meristematic cells and/or by impairing local glutamatergic signaling during development.

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Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins

Cited by 90 publications

References 59 publications

Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification

Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification

In vivo gibberellin gradients visualized in rapidly elongating tissues

Sponging of glutamate at the outer plasma membrane surface reveals roles for glutamate in development

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