Investigating neuronal function with optically controllable proteins

Abstract

In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain.

Keywords: development; optobiology; optogenetics; signal transduction; transcription.

FIGURE 1

Uses of opsins. (A) Opto-α₁-AR consists of bovine visual opsin with the intracellular loops of G_q-coupled human α_1a-adrenergic receptor. Opto-β₂-AR consists of bovine visual opsin with the intracellular loops of G_s-coupled hamster β₂-adrenergic receptor. When excited with blue light, opto-α₁-AR and opto-β₂-AR activate production of IP₃ and cAMP, increasing and decreasing neuronal firing in vivo, respectively. (B) Opto-MOR consists of rat visual opsin with the intracellular loops of the mu-opioid receptor. Optical stimulation results in inhibition of adenylate cyclase via G_i/o, activation of ERK via β-arrestin and G_i/o, and activation of GIRK via Gβγ. (C) Photoactivatable serotonin receptors can be produced by conjugating light-sensitive vertebrate opsins (here, denoted vOpsins) at various excitation wavelengths with the C terminal portion of a specific serotonin receptor subtype, which mediates proper localization within the cell via sorting proteins. Excitation with 400–600 nm light triggers activation of GIRK via Gβγ, decreasing neuronal firing.

FIGURE 2

Blue light using flavin adenine dinucleotide (BLUF) domain-regulated adenylate cyclases. (A) The euPACα polypeptide is composed of two BLUF and two catalytic domains in the order BLUF1, C1, BLUF2, C2, and likely dimerizes or tetramerizes when expressed heterologously. The C1 and C2 catalytic domains associate to form the adenylate cyclase active site. BLUF domains N-terminal to each catalytic domain enhance catalysis in response to light. (B) The bPAC is composed of a single BLUF and a single catalytic domain, and likely dimerizes when expressed, so that an adenylate cyclase active site forms at the interface of the catalytic domains. The BLUF domain enhances catalysis in response to light.

FIGURE 3

Uses of LOV domains. (A) Photoactivation of PA-Rac1 by blue light has been shown to prevent cocaine-induced increase in spine elongation and, separately, to increase synaptic currents. (B) Blue light-mediated LOV2-PDZ interaction can recruit motor proteins for locomotion of targeted organelles. PDZ-kinesin would produce anterograde movement of the organelle, while PDZ-dynein would produce retrograde movement of the organelle. (C) Lumitoxins are fusions of a channel-blocking peptide toxin, a flexible linker, the LOV2 domain, and a transmembrane helix. When excited by blue light, unfolding of the Jα helix is believed to lengthen the linkage to the membrane, decreasing the local concentration of the toxin near the ion channels. (D) The LightON transcription system fuses a truncated Gal4 DNA-binding domain (DBD) to the Vivid (VVD) LOV domain to produce a construct that homodimerizes upon excitation with blue light and binds to Gal4’s cognate DNA, activating downstream transcription. TAD is a transcription-activating domain. (E) A similar homodimerizing optobiological tool for transcription control. Bacterial transcription factor EL222’s light-sensitive LOV domain and helix-turn-helix (HTH) DBD are conjugated to a nuclear localizing sequence and VP16 transcription activating domain (TAD). The compound protein homodimerizes upon excitation with blue light to bind cognate C120 DNA sequences and activate downstream transcription. (F) Interaction between FKF1 and GIGANTEA (GI) domains can produce blue light-activatable transcription employing the VP16 TAD and the Gal4 DBD. (G) Proteins fused to miniSOG, a singlet oxygen-generating mutant of LOV2, can be destroyed by chromophore-assisted light inactivation (CALI). In the case of a VAMP2-miniSOG fusion, light results in blockade of synaptic transmission.

FIGURE 4

Uses of CRY2 domains. For all panels, CRY2 and CIB1 are the full-length domains. PHR is the photolyase homology region of CRY2 (a truncated CRY2) and CIBN is the N terminal portion of CIB1 (a truncated CIB1). (A) Light-sensitive receptor tyrosine kinases. Upon excitation with blue light, PHR homodimerizes to activate downstream components in the Trk signaling pathway. (B) Heterodimerization between PHR and CIBN used to produce light-activated Raf1 for optogenetic control of the Raf1/MEK/ERK pathway in PC12 cells. Membrane localization of Raf1 activated downstream kinases and eventually stimulated neurite growth in PC12 cells. (C) Light-activated phosphatidylinositol 3-kinase (PI3K). CIBN is membrane-localized and PHR is fused to the inter-SH2 domain of p85b, the regulatory subunit of PI3K kinase (iSH). The catalytic p110 component of PI3K was supplied endogenously by the cell expressing these two constructs. Blue light excitation recruited the PHR-iSH-p110 complex to the membrane, where it would produce PIP₃ from PIP₂. (D) Light-induced heterodimerization between CRY2 and CIBN can assemble a functional protein if an appropriate half of the protein is fused to each of CRY2 and CIBN. Fusing half of cre recombinase to CRY2 and CIBN produces light-dependent loxP site recombination. (E) The light-inducible transcriptional effector (LITE) system allows optical control of transcription or chromatin structure. Transcription activator-like effectors (TALEs) serve as modular DNA-binding domains. TALE fused to PHR comprises one component of LITE. An effector domain (TAD or HED, a histone effector domain) fused to CIB1 comprises the other component of LITE.

FIGURE 5

UVR8 in regulation of protein export. The Ultraviolet Response 8 (UVR8) plant photoreceptor forms homodimers in the dark and dissociates into monomers upon excitation with UV-B light. A protein secretion control system conjugates tandem UVR8 tags to a protein of interest (POI). As a result, these conjugates form aggregates in the ER and remain stationary until dissociated by UV-B light.

FIGURE 6

Recent uses of LOV and CRY2 domains with potential neuroscience applications. (A) Motor protein direction can be modified by changing the lever arm length using a LOV2 domain. An artificial lever arm was made by fusing two α-actinin structural elements (arm and extension) to LOV2, then this was attached to a motor protein’s catalytic domain (motor body). Change in lever arm geometry upon illumination resulted in a change in motor direction. (B) Light-induced LOV2 conformational change was also used to disrupt the folding of two fragments of calmodulin, allowing light-induced release of calcium. (C) The LARIAT method enables temporary inactivation of a POI by sequestration. LARIAT consists of CRY2 conjugated to a POI or an antibody fragment recognizing the POI (Ab) and CIB1 conjugated to a multimerizing protein (M). Blue light excitation causes the POI to aggregate to the multimer clusters formed by the CIB1-M construct.

FIGURE 7

Other photosensory modules with potential benefits for neurobiological applications. (A) Phytochrome – PIF based protein membrane translocation. Phytochrome (PhyB) is localized at plasma membrane by fusing to a membrane-trafficking peptide, and a POI is in fusion with the PIF domain. Red light-mediated PhyB-PIF interaction drives the POI to the plasma membrane, initiating downstream reactions. (B) Dronpa system for optogenetic control of protein function through steric blockade. Dronpa(K145N) forms tetramers. A POI is flanked by Dronpa on either terminus. When Dronpa is in multimeric form, the protein is caged and unable to perform its normal functions. Exposure to cyan light causes Dronpa to monomerize, uncaging the protein. A subsequent exposure to violet light will cause Dronpa to multimerize, again caging the protein.