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Review
, 41 (9), 809-817

Synergistic Ensemble of Optogenetic Actuators and Dynamic Indicators in Cell Biology

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Review

Synergistic Ensemble of Optogenetic Actuators and Dynamic Indicators in Cell Biology

Jihoon Kim et al. Mol Cells.

Abstract

Discovery of the naturally evolved fluorescent proteins and their genetically engineered biosensors have enormously contributed to current bioimaging techniques. These reporters to trace dynamic changes of intracellular protein activities have continuously transformed according to the various demands in biological studies. Along with that, light-inducible optogenetic technologies have offered scientists to perturb, control and analyze the function of intracellular machineries in spatiotemporal manner. In this review, we present an overview of the molecular strategies that have been exploited for producing genetically encoded protein reporters and various optogenetic modules. Finally, in particular, we discuss the current efforts for combined use of these reporters and optogenetic modules as a powerful tactic for the control and imaging of signaling events in cells and tissues.

Keywords: biosensor; optogenetics.

Figures

Fig. 1
Fig. 1. Strategies of engineering FRET-based biosensors
Schematics for the design of FRET-based dynamic biosensors as in (A) an intermolecular type or (B) an intramolecular type. (C) Strategy of linker design by varying size and sequences of amino acids. (D) Selection of sensing module topology for optimal efficiency of FRET. (E) Modification of fluorophore orientation to ensure sufficient energy transfer between a fluorescence pair of donor and acceptor.
Fig. 2
Fig. 2. Versatile utilization of CRY2-based optogenetic signaling actuators
(A) Schematic depicts modes of CRY2 interaction induced by blue light stimulation. (B, C) Schematic of Opto-TrkB and OptoFGFR1 activation by light-inducible CRY2 homo-interaction upon blue light stimulation. (D) Sequestration of CRY2-conjugated proteins by light-inducible trapping in clusters (LARIAT). (E) Schematic of intracellular membrane aggregation (IM-LARIAT) triggered by the light-induced binding of CRY2- and CIB1-Rab.
Fig. 3
Fig. 3. Simultaneous control and visualization of intracellular calcium signaling using a red-shifted reporter
(A) Schematic of optoSTIM1activation by blue light-inducible PHR homo-oligomerization. A red-shifted calcium reporter, R-GECO can be simultaneously visualized under the precise control of spatiotemporal OptoSTIM1 activation. (B) Pseudocolored fluorescence images of a red-shifted genetically encoded calcium indicator (R-GECO1). Three HeLa cells (R1, R2, and R3) co-expressing OptoSTIM1 and R-GECO1 were sequentially stimulated with blue light at different time points. White dotted lines indicate cell boundaries. Scale bar, 20 μm. (C) Pseudocolor image of R-GECO1PM fluorescence in a HeLa cell co-expressing OptoSTIM1. White circles designate three subcellular regions (R1, R2, and R3) sequentially stimulated with blue light. Scale bar, 20 μm. Graph represents changes in fold-changes of R-GECO1PM according to time at three subcellular regions. Red, green and blue arrows indicate time points of light stimulation in R1, R2 and R3, respectively.

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