Curated Optogenetic Publication Database

Abstract: The regulation of gene expression is crucial in diverse areas of biological science, engineering, and medicine. A genetically encoded system based on the RNA binding domain of the Pumilio and FBF (PUF) proteins was developed for the bidirectional regulation (i.e., either upregulation or downregulation) of the translation of a target mRNA. PUF domains serve as designable scaffolds for the recognition of specific RNA elements and the specificity can be easily altered to target any 8-nucleotide RNA sequence. The expression of a reporter could be varied by over 17-fold when using PUF-based activators and repressors. The specificity of the method was established by using wild-type and mutant PUF domains. Furthermore, this method could be used to activate the translation of target mRNA downstream of PUF binding sites in a light-dependent manner. Such specific bidirectional control of mRNA translation could be particularly useful in the fields of synthetic biology, developmental biology, and metabolic engineering.

Abstract: It has been proposed that differential activation kinetics allows cells to use a common set of signaling pathways to specify distinct cellular outcomes. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) induce different activation kinetics of the Raf/MEK/ERK signaling pathway and result in differentiation and proliferation, respectively. However, a direct and quantitative linkage between the temporal profile of Raf/MEK/ERK activation and the cellular outputs has not been established due to a lack of means to precisely perturb its signaling kinetics. Here, we construct a light-gated protein-protein interaction system to regulate the activation pattern of the Raf/MEK/ERK signaling pathway. Light-induced activation of the Raf/MEK/ERK cascade leads to significant neurite outgrowth in rat PC12 pheochromocytoma cell lines in the absence of growth factors. Compared with NGF stimulation, light stimulation induces longer but fewer neurites. Intermittent on/off illumination reveals that cells achieve maximum neurite outgrowth if the off-time duration per cycle is shorter than 45 min. Overall, light-mediated kinetic control enables precise dissection of the temporal dimension within the intracellular signal transduction network.

Abstract: Perturbations in the concentration of a specific protein are often used to study and control biological networks. The ability to "dial-in" and programmatically control the concentration of a desired protein in cultures of cells would be transformative for applications in research and biotechnology. We developed a culturing apparatus and feedback control scheme which, in combination with an optogenetic system, allows us to generate defined perturbations in the intracellular concentration of a specific protein in microbial cell culture. As light can be easily added and removed, we can control protein concentration in culture more dynamically than would be possible with long-lived chemical inducers. Control of protein concentration is achieved by sampling individual cells from the culture apparatus, imaging and quantifying protein concentration, and adjusting the inducing light appropriately. The culturing apparatus can be operated as a chemostat, allowing us to precisely control microbial growth and providing cell material for downstream assays. We illustrate the potential for this technology by generating fixed and time-varying concentrations of a specific protein in continuous steady-state cultures of the model organism Saccharomyces cerevisiae. We anticipate that this technology will allow for quantitative studies of biological networks as well as external tuning of synthetic gene circuits and bioprocesses.

Abstract: The extracellular signal-regulated kinase (ERK) plays a central role in the signaling cascades of cell growth. Here, we show that stochastic ERK activity pulses regulate cell proliferation rates in a cell density-dependent manner. A fluorescence resonance energy transfer (FRET) biosensor revealed that stochastic ERK activity pulses fired spontaneously or propagated from adjacent cells. Frequency, but not amplitude, of ERK activity pulses exhibited a bell-shaped response to the cell density and correlated with cell proliferation rates. Consistently, synthetic ERK activity pulses generated by a light-switchable CRaf protein accelerated cell proliferation. A mathematical model clarified that 80% and 20% of ERK activity pulses are generated by the noise and cell-to-cell propagation, respectively. Finally, RNA sequencing analysis of cells subjected to the synthetic ERK activity pulses suggested the involvement of serum responsive factor (SRF) transcription factors in the gene expression driven by the ERK activity pulses.

Abstract: A genetically encoded optogenetic system was constructed that activates mRNA translation in mammalian cells in response to light. Blue light induces the reconstitution of an RNA binding domain and a translation initiation domain, thereby activating target mRNA translation downstream of the binding sites.

Abstract: The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.

Abstract: Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is highly regulated in a spatiotemporal manner and plays multiple roles in individual cells. However, the local dynamics and primary functions of PIP3 in developing neurons remain unclear because of a lack of techniques for manipulating PIP3 spatiotemporally. We addressed this issue by combining optogenetic control and observation of endogenous PIP3 signaling. Endogenous PIP3 was abundant in actin-rich structures such as growth cones and "waves", and PIP3-rich plasma membranes moved actively within growth cones. To study the role of PIP3 in developing neurons, we developed a PI3K photoswitch that can induce production of PIP3 at specific locations upon blue light exposure. We succeeded in producing PIP3 locally in mouse hippocampal neurons. Local PIP3 elevation at neurite tips did not induce neurite elongation, but it was sufficient to induce the formation of filopodia and lamellipodia. Interestingly, ectopic PIP3 elevation alone activated membranes to form actin-based structures whose behavior was similar to that of growth-cone-like "waves". We also found that endocytosis regulates effective PIP3 concentration at plasma membranes. These results revealed the local dynamics and primary functions of PIP3, providing fundamental information about PIP3 signaling in neurons.

Abstract: We describe LOLLIbow, a Brainbow-based live imaging system with applications in developmental biology and neurobiology. The development of an animal, including the environmentally sensitive adaptation of its brain, is thought to proceed through continual orchestration among diverse cell types as they divide, migrate, transform and interact with one another within the body. To facilitate direct visualization of such dynamic morphogenesis by individual cells in vivo, we have modified the original Brainbow for Drosophila in which live imaging is practical during much of its development. Our system offers permanent fluorescent labels that reveal fine morphological details of individual cells without requiring dissection or fixation of the samples. It also features a non-invasive means to control the timing of stochastic tricolor cell labeling with a light pulse. We demonstrate applicability of the new system in a variety of settings that could benefit from direct imaging of the developing multicellular organism with single-cell resolution.

Abstract: Light inducible protein-protein interactions are powerful tools to manipulate biological processes. Genetically encoded light-gated proteins for controlling precise cellular behavior are a new and promising technology, called optogenetics. Here we exploited the blue light-induced transcription system in yeast and zebrafish, based on the blue light dependent interaction between two plant proteins, blue light photoreceptor Cryptochrome 2 (CRY2) and the bHLH transcription factor CIB1 (CRY-interacting bHLH 1). We demonstrate the utility of this system by inducing rapid transcription suppression and activation in zebrafish.

Abstract: A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light/cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems.

Abstract: Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly, locally, and reversibly. The inositol 5-phosphatase domain of OCRL (5-ptase(OCRL)), which acts on PI(4,5)P(2) and PI(3,4,5)P(3), was fused to the photolyase homology region domain of CRY2, and the CRY2-binding domain, CIBN, was fused to plasma membrane-targeting motifs. Blue-light illumination (458-488 nm) of mammalian cells expressing these constructs resulted in nearly instantaneous recruitment of 5-ptase(OCRL) to the plasma membrane, where it caused rapid (within seconds) and reversible (within minutes) dephosphorylation of its targets as revealed by diverse cellular assays: dissociation of PI(4,5)P(2) and PI(3,4,5)P(3) biosensors, disappearance of endocytic clathrin-coated pits, nearly complete inhibition of KCNQ2/3 channel currents, and loss of membrane ruffling. Focal illumination resulted in local and transient 5-ptase(OCRL) recruitment and PI(4,5)P(2) dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P(2) biosensor and membrane ruffling at the opposite side of the cells. Using the same approach for the recruitment of PI3K, local PI(3,4,5)P(3) synthesis and membrane ruffling could be induced, with corresponding loss of ruffling distally to the illuminated region. This technique provides a powerful tool for dissecting with high spatial-temporal kinetics the cellular functions of various PIs and reversibly controlling the functions of downstream effectors of these signaling lipids.

Abstract: Dimerizers allowing inducible control of protein-protein interactions are powerful tools for manipulating biological processes. Here we describe genetically encoded light-inducible protein-interaction modules based on Arabidopsis thaliana cryptochrome 2 and CIB1 that require no exogenous ligands and dimerize on blue-light exposure with subsecond time resolution and subcellular spatial resolution. We demonstrate the utility of this system by inducing protein translocation, transcription and Cre recombinase-mediated DNA recombination using light.