Genetically encoded molecular probes to visualize and perturb signaling dynamics in living biological systems

Abstract

In this Commentary, we discuss two sets of genetically encoded molecular tools that have significantly enhanced our ability to observe and manipulate complex biochemical processes in their native context and that have been essential in deepening our molecular understanding of how intracellular signaling networks function. In particular, genetically encoded biosensors are widely used to directly visualize signaling events in living cells, and we highlight several examples of basic biosensor designs that have enabled researchers to capture the spatial and temporal dynamics of numerous signaling molecules, including second messengers and signaling enzymes, with remarkable detail. Similarly, we discuss a number of genetically encoded biochemical perturbation techniques that are being used to manipulate the activity of various signaling molecules with far greater spatial and temporal selectivity than can be achieved using standard pharmacological or genetic techniques, focusing specifically on examples of chemically driven and light-inducible perturbation strategies. We then describe recent efforts to combine these diverse and powerful molecular tools into a unified platform that can be used to elucidate the molecular details of biological processes that may potentially extend well beyond the realm of signal transduction.

Keywords: Biosensors; FRET; Live cell imaging; Signaling; Targeted perturbations.

Fig. 1.

Different types of FP-based biosensor. (A) The fusion of an FP such as GFP (green) to a specific binding domain (gray) can be used to report on the production of certain signaling molecules (blue circle), for example through the redistribution of the probe – and thus fluorescence – from the cytosol (left) to the plasma membrane (right). (B) The Ca²⁺ sensor GCaMP consists of a molecular switch that contains calmodulin (CaM) and M13 inserted into a circularly permuted GFP (green), in which the native N- and C-termini of GFP are linked together, and new termini are generated from within the core β-barrel structure of GFP; the addition of Ca²⁺ causes CaM to bind to M13, which leads to increased GFP fluorescence. (C) FRET-based reporter for kinase activity. A kinase-specific substrate peptide and a phosphoamino-acid-binding domain (PAABD) are sandwiched between two FPs that can undergo FRET (e.g. CFP and YFP). Phosphorylation (P) of the substrate through the cognate kinase induces binding of the substrate peptide by the PAABD, resulting in a conformational rearrangement that produces a change in FRET.

Fig. 2.

Examples of chemically inducible perturbation tools. (A) A chemically inducible dimerization system utilizing a pair of rapamycin-binding domains. One binding domain, derived from the FKBP-rapamycin binding (FRB) protein, is tethered to the plasma membrane and the other, derived from the FK506-binding protein (FKBP), is fused to an enzyme effector such as the GTPase Rac in the cytosol. Rapamycin treatment induces the translocation of the enzyme into the vicinity of its substrate, thereby promoting activity. (B) A kinase-inducible bimolecular switch. Here, a membrane-tethered, kinase-specific substrate peptide is shown, together with a phosphoamino-acid-binding domain (PAABD) that is coupled to an enzyme effector in the cytosol. Phosphorylation (P) of the substrate peptide reversibly recruits the PAABD–enzyme fusion protein to the membrane, where the enzyme is active. (C) Spatiotemporal manipulation of cAMP using soluble adenylyl cyclase (sAC). sAC is exclusively stimulated by bicarbonate (HCO₃^–) and can be expressed in different subcellular compartments to selectively produce cAMP in different regions of the cell, for instance in the cytosol or nucleus.

Fig. 3.

Examples of optically inducible perturbation tools. (A) The light-induced ion channel channel rhodopsin. The associated retinal moiety photoisomerizes upon illumination with blue light, which promotes channel opening and subsequent ion influx. (B) Photoactivatable Rac. A constitutively active Rac isoform is fused to the light–oxygen–voltage (LOV) domain and Jα helix from Avena sativa phototropin 1, which results in an intramolecular complex that inhibits Rac activity. Upon illumination with blue light, a reversible conformational change disrupts the Jα helix and induces Rac activity. (C) Light-inducible control of protein-protein interactions using phytochrome B (PhyB) and a phytochrome-interacting factor (PIF). Phycocyanobilin (PCB) is covalently bound to the PhyB protein, which is tethered to the plasma membrane. Upon illumination with red light, PCB undergoes a photoisomerization reaction that results in the binding of PhyB to the PIF. This can be used to control the translocation of a PIF-tethered effector enzyme from the cytosol to the plasma membrane. In this example, a guanine nucleotide exchange factor for a Rho-family GTPase (Rho-GEF) is tethered to PIF and used to activate a Rho-family GTPase (Rho) in the plasma membrane. (D) Optical control of enzyme activity with a photoswitchable FP. A weakly oligomerizing Dronpa mutant (Dronpa145N; green) is tethered to the N- and C-termini of an effector enzyme; in the fluorescent state (Dronpa ON), two of the fusion proteins associate to form a Dronpa145N tetramer, thereby blocking enzyme activity. Switching Dronpa145N into the non-fluorescent state (Dronpa OFF) by using blue light causes dissociation of the tetramer and releases the active enzyme.