Live Imaging with Genetically Encoded Physiologic Sensors and Optogenetic Tools

Abstract

Barrier tissues such as the epidermis employ complex signal transduction systems to execute morphogenetic programs and to rapidly respond to environmental cues to promote homeostasis. Recent advances in live-imaging techniques and tools allow precise spatial and temporal monitoring and manipulation of intracellular signaling cascades. Leveraging the chemistry of naturally occurring light-sensitive proteins, genetically encoded fluorescent biosensors have emerged as robust tools for visualizing dynamic signaling events. In contrast, optogenetic protein constructs permit laser-mediated control of signal receptors and effectors within live cells, organoids, and even model organisms. In this paper, we review the basic principles underlying novel biosensors and optogenetic tools and highlight how recent studies in cutaneous biology have leveraged these imaging strategies to illuminate the spatiotemporal signals regulating epidermal development, barrier formation, and tissue homeostasis.

Figure 1.. Common biosensor design strategies.

a) Translocation-based biosensors contain dynamic sensor domains that change subcellular localization (i.e., from the cytoplasm to the nucleus) in response to various cellular signals, allowing fluorescent visualization of signal-induced changes in protein localization. b) Split fluorescence protein (FP)-based biosensors employ self-assembling FP fragments that are linked to protein(s) of interest i.e., dimeric binding partners. Signal-induced changes in the sensor domain unit bring the two FP fragments together resulting in fluorescence reconstitution. c) Circularly permuted FPs are sandwiched between sensing units composed of two or more interacting proteins. Switch-like conformational changes in the sensing unit trigger closure of the cpFP resulting in enhanced fluorescence intensity. d) pH-sensitive FPs, which, for example, become quenched in acidic environments, can be fused to organelle membrane-localization signals or proteins of interest such as endosomal or autophagosomal makers enabling the detection of pH dynamics within specific subcellular compartments. e) Fluorescence resonance energy transfer (FRET)-based biosensors rely on the distance-dependent transfer of energy between two compatible fluorophores (i.e., CFP and YFP) such that the emission spectrum of the donor FP overlaps with the excitation spectrum of the acceptor FP. Conformational changes in the sensing unit or protein-protein interactions bring the two FPs in close proximity, allowing energy transfer between the donor and acceptor fluorophores and an increase in FRET intensity.

Figure 2.. GCaMP biosensor design for calcium sensing and applications in keratinocyte biology.

a) Schematic representation of the GCaMP structure where a central circularly permuted enhanced GFP (cp-eGFP) is sandwiched between the M13 fragment of myosin light chain kinase (M13) at the N-terminus and Calmodulin (CaM) at the C-terminus. Ca²⁺-dependent binding of CaM to M13 triggers conformational rearrangement of cp-eGFP and fluorescence reconstitution. b) Keratinocytes are chemically coupled through the assembly of intercellular gap junctions (connexons), which enable the propagation of Ca²⁺ waves between neighboring cells within local signaling networks, which can be visualized in real-time using GCaMP sensors. c) Representative time-lapse fluorescence images of primary normal human epidermal keratinocytes (NHEKs) expressing GCaMP6 following treatment with the SERCA inhibitor, thapsigargin (at t=0s), which induces rapid translocation of calcium from the ER into the cytosol. Scale bar = 10 μm.

Figure 3.. pH- and REDOX-sensitive fluorescent biosensors.

a) Intensiometric pH sensors employ a single pH-sensitive FP which, for example, display reduced fluorescence intensity upon acidification. Ratiometric pH sensors contain two or more tandem FPs with unique spectral properties and pH sensitivities. Upon acidification, the pH-sensitive fluorophore is quenched, while the pH-insensitive fluorophore serves as a stable reference point enabling more robust quantification of pH changes. b) pH-responsive biosensors can be coupled to endogenous proteins to enable compartment-specific detection of pH changes. Fusion of a ratiometric pH sensor to the lysosome-associated membrane protein 1 (LAMP1) allows quantification of lysosomal acidification/maturation. c) Schematic representation of the dual-functional fluorescent biosensor, pHaROS, comprising a REDOX-sensitive iLOV domain and a pH-sensitive mBeRFP. d) Green-light illumination of the photosensitizing fluorescent protein, KillerRed, triggers rapid photobleaching and generation of reactive oxygen species including singlet oxygen and superoxide. KillerRed can be fused to endogenous proteins or organelle-localization signals to allow compartment-specific oxidative damage induced by laser illumination.

Figure 4.. Optogenetic manipulation of intracellular calcium dynamics.

a) Optogenetic dimerization systems employ photosensitive proteins capable of undergoing light-induced homo- or hetero-dimerization to facilitate clustering of endogenous proteins of interest. b) Light stimulation of cytochrome photoreceptor 2 (Cry2) initiates homo-oligomerization of activated proteins, allowing for precision control of oligomerization-dependent cellular processes. c) Schematic representation of OptoSTIM1-mediated endogenous CRAC/Orai1 Ca2+ channel activation. Blue light illumination triggers Cry2 mediated oligomerization of the STIM1 cytosolic domain and CRAC channel opening. d) Schematic representation of a light-operated Orai1 channel (LOCa) engineered via the introduction of a LOV2 domain within a cytoplasmic loop of constitutively active Orai1. Blue light-induced conformational changes within the LOV2 domain trigger Orai1 channel opening and Ca²⁺ influx.