Recent advances in the use of genetically encodable optical tools to elicit and monitor signaling events

Abstract

Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.

Keywords: Biosensors; Optogenetic tools; Signaling.

Figure 1.

Basic biosensor designs. A. Translocation-based biosensors. Phosphoinositide (PI) sensors utilize lipid-selective binding domains that translocate to or from the membrane to report the production and degradation of specific PIs, such as PIP₃ (a). Kinase translocation reporters similarly undergo translocation into or out of the nucleus depending on the phosphorylation of kinase-specific substrate sequences within the reporter (b). SPARK, a phase separation-based kinase reporter, visualizes dynamics of kinase activity with phase separation (c). B. Single FP-based sensor. The insertion of a conformational switch into a single FP can be used to alter chromophore behavior in response to a signaling event. C. FRET-based sensor. Changes in the proximity or orientation between a pair of FPs caused by the sensing unit upon detection of a signaling event results in a change in FRET.

Figure 2.

Recent advances in biosensors. A. Super-resolution compatible biosensors. Bimolecular fluorescence complementation (BiFC) using photoactivatable FPs combined with STORM/PALM imaging enables super-resolution PPI observation (a). TagRFP-T fluorescence shows increasing fluctuations as the proximity between Dronpa and TagRFP-T is closed by the sensing unit. This allows the use of pcSOFI, a super-resolution technique, to observe signaling activity at subdiffraction spatial resolution (b). B. Biochemical activity integrators. CaMPARI is a GCaMP-based Ca²⁺ sensor that features a green-to-red photoconvertible FP. The sensor undergoes irreversible green-to-red photoconversion only when exposed to both violet light and Ca²⁺. Thus, the red color persists even when Ca²⁺ levels drop, allowing post hoc analysis of signaling (a). When FLARE/Cal-Light is exposed to Ca²⁺ and blue light, a transcription factor that turns on the reporter translocates into the nucleus. Unlike CaMPARI, this system can be used with a variety of readouts (b). C. Semi-genetically encoded biosensors. SplitFAST is suitable for showing dynamic PPIs because of its reversible interaction. Conventional BiFC cannot report PPI dynamics due to the irreversible nature of FP complementation, whereas splitFAST makes this possible (a). New synthetic dyes with improved brightness and stability help to overcome the limitations of the low delivery efficiency of synthetic dyes. Voltron is based on the voltage sensor Ace2N and new bright synthetic dyes such as JF₅₂₅. This drastically improved in vivo voltage imaging in animals (b). D. Bimodal sensors. hyBRET sensors combine a FRET sensor with a bioluminescent protein (RLuc). In the absence of the luciferase substrate, the signaling activity can be measured in FRET mode, while addition of substrate allows monitoring in BRET mode (a). GLICO combines GCaMP with splitNLuc. In the presence of Ca²⁺, the GLICO sensing unit undergoes a conformational change that modulates cpGFP fluorescence and leads to splitNLuc complementation. Therefore, Ca²⁺ levels can be monitored via GFP emission caused by either external illumination (fluorescence mode) or luciferin addition to induce BRET between NLuc and cpGFP (bioluminescence mode) (b). PA: photoactivatable, hn: light, POI: protein of interest.

Figure 3.

Components and designs of optogenetic tools. A. Membrane-spanning actuators. Rhodopsin can act as a membrane-spanning actuator in the form of a light-driven ion channel, an ion pump, or a GPCR. The covalently bound chromophore of rhodopsin can trigger a conformational change in rhodopsin upon light illumination. B. Light-induced PPIs. Light-dependent modulation of protein translocation and/or signaling perturbation utilizes light-induced PPIs between photoreceptors and their effectors. Representative light-induced PPIs are shown in the table. C. Light-mediated second messenger control. In bPAC, adenylyl cyclase bound to the BLUF domain is activated by light to produce cAMP. A guanylyl cyclase-containing rhodopsin from Blastocladiella emersonii (BeCyclOp) similarly produces cGMP when activated by light. The red-light-activated phosphodiesterase (LAPD) degrades cGMP and cAMP. D. Engineered control of enzyme activity. The Jα helix of the AsLOV domain is unfolded by light, allowing enzyme “caging” and “uncaging” (a). Photodissociable dimeric Dronpa (pdDronpa1) can form dimers upon 500 nm light and dissociate upon 400nm light. This feature also allows reversible control of protein cagin/uncaging by light (b). hn: light, POI: protein of interest; DD: dimerization domain, AC: adenylyl cyclase, GC: guanylyl cyclase, PDE: phosphodiesterase

Figure 4.

Recent advances in optogenetic tools. A. Photocleavable protein and linker. The excitation of PhoCl by 400 nm light leads to its photoconversion and backbone cleavage. Proteins conjugated to PhoCl will thus translocate upon PhoCl cleavage (a). MeNV-HaXS is a photocleavable chemical dimerization system. HaloTagged and SNAP-tagged proteins that are tethered by this linker can be dissociated using 360 nm light irradiation (b). B. Optogenetic control with UAAs. Substitution of functionally critical amino acids with photocaged UAAs can be used to inhibit a target protein until light irradiation restores protein function (a). UAAs carrying BzF can induce photocrosslinking upon UV stimulation. The use of photocrosslinking UAAs can enable light-mediated inhibition of protein function (b). Azobenzene moieties can undergo reversible cis/trans photoisomerization in response to blue and UV light. Incorporating UAAs containing azobenzene moieties allows reversible cis/trans photoisomerization of an amino acid side chain, enabling reversible control of protein activity (c). C. Chemo-optogenetic control. Luminopsin combines channelrhodopsin with a bioluminescent protein. Bioluminescence from the luciferase can induce conformational changes in channelrhodopsin without the need for external illumination (a). Similarly, a luminescence-activated adenylyl cyclase also combines a bioluminescent protein (NLuc) with bPAC (b).