Showing 1 - 15 of 15 results
An Optogenetic Method to Study Signal Transduction in Intestinal StemCell Homeostasis.
Homeostasis in adult organs involves replacement of cells from a stem cell pool maintained in specialized niches regulated by extracellular signals. This cell-to-cell communication employs signal transduction pathways allowing cells to respond with a variety of behaviors. To study these cellular behaviors, signaling must be perturbed within tissues in precise patterns, a technique recently made possible by the development of optogenetic tools. We developed tools to study signal transduction in vivo in an adult fly midgut stem cell model where signaling was regulated by the application of light. Activation was achieved by clustering of membrane receptors EGFR and Toll, while inactivation was achieved by clustering the downstream activators ERK/Rolled and NFκB/Dorsal in the cytoplasm, preventing nuclear translocation and transcriptional activation. We show that both pathways contribute to stem and transit amplifying cell numbers and affect the lifespan of adult flies. We further present new approaches to overcome overexpression phenotypes and novel methods for the integration of optogenetics into the already-established genetic toolkit of Drosophila.
Optogenetic Control for Investigating Subcellular Localization of Fyn Kinase Activity in Single Live Cells.
Previous studies with various Src family kinase biosensors showed that the nuclear kinase activities are much suppressed compared to those in the cytosol, suggesting that these kinases are regulated differently in the nucleus and in the cytosol. In this study, using Fyn as an example, we first engineered a Fyn biosensor with a light-inducible nuclear localization signal (LINuS) to demonstrate that the Fyn kinase activity is significantly lower in the nucleus than in the cytosol. To understand how different equilibrium states between Fyn and the corresponding phosphatases are maintained in the cytosol and nucleus, we further engineered a Fyn kinase domain with LINuS. The results revealed that the Fyn kinase can be actively transported into the nucleus upon light activation and upregulate the biosensor signals in the nucleus. Our results suggest that there is limited transport or diffusion of Fyn kinase between the cytosol and nucleus in the cells, which is important for the maintenance of different equilibrium states of Fyn in situ.
Yeast Two Hybrid Screening of Photo-Switchable Protein-Protein Interaction Libraries.
Although widely used in the detection and characterization of protein-protein interactions, Y2H screening has been under-used for the engineering of new optogenetic tools or the improvement of existing tools. Here we explore the feasibility of using Y2H selection and screening to evaluate libraries of photoswitchable protein-protein interactions. We targeted the interaction between circularly permuted photoactive yellow protein (cPYP) and its binding partner BoPD (binder of PYP dark state) by mutating a set of four surface residues of cPYP that contribute to the binding interface. A library of ~10,000 variants was expressed in yeast together with BoPD in a Y2H format. An initial selection for the cPYP/BoPD interaction was performed using a range of concentrations of the cPYP chromophore. As expected, the majority (>90% of cPYP variants no longer bound to BoPD). Replica plating was the used to evaluate the photoswitchability of the surviving clones. Photoswitchable cPYP variants with BoPD affinities equal to, or higher than, native cPYP were recovered in addition to variants with altered photocycles and binders that interacted with BoPD as apo-proteins. Y2H results reflected protein-protein interaction affinity, expression, photoswitchability and chromophore uptake, and correlated well with results obtained both in vitro and in mammalian cells. Thus, by systematic variation of selection parameters, Y2H screens can be effectively used to generate new optogenetic tools for controlling protein-protein interactions for use in diverse settings.
An optogenetic tool for induced protein stabilization based on the Phaeodactylum tricornutum aureochrome 1a LOV domain.
Control of cellular events by optogenetic tools is a powerful approach to manipulate cellular functions in a minimally invasive manner. A common problem posed by the application of optogenetic tools is to tune the activity range to be physiologically relevant. Here, we characterized a photoreceptor of the light-oxygen-voltage domain family of Phaeodactylum tricornutum aureochrome 1a (AuLOV) as a tool for increasing protein stability under blue light conditions in budding yeast. Structural studies of AuLOVwt, the variants AuLOVM254 and AuLOVW349 revealed alternative dimer association modes for the dark state, which differ from previously reported AuLOV dark state structures. Rational design of AuLOV-dimer interface mutations resulted in an optimized optogenetic tool that we fused to the photoactivatable adenylyl cyclase from Beggiatoa sp.. This synergistic light-regulation approach using two photoreceptors resulted in an optimized, photoactivatable adenylyl cyclase with a cyclic AMP production activity that matches the physiological range of Saccharomyces cerevisiae. Overall, we enlarged the optogenetic toolbox for yeast and demonstrated the importance of fine-tuning the optogenetic tool activity for successful application in cells.
Optogenetic modulation of TrkB signaling in the mouse brain.
Optogenetic activation of receptors has advantages compared with chemical or ligand treatment because of its high spatial and temporal precision. Especially in the brain, the use of a genetically encoded light-tunable receptor is superior to direct infusion or systemic drug treatment. We applied light activatable TrkB receptor in mouse brain with reduced basal activity by incorporating Cry2PHR mutant, Opto-cytTrkB(E281A). Upon AAV mediated gene delivery, this form was expressed at sufficient levels in the mouse hippocampus (HPC) and medial entorhinal cortex (MEC) retaining normal canonical signal transduction by blue light stimulus, even by delivery of non-invasive LED light on the mouse head. Within target cells, where its expression was driven by a cell type-specific promoter, Opto-cytTrkB(E281A)-mediated TrkB signaling could be controlled by adjusting light-stimulation conditions. We further demonstrated that Opto-cytTrkB(E281A) could locally induce TrkB signaling in axon terminals in the MEC-HPC. In summary, Opto-cytTrkB(E281A) will be useful for elucidating time- and region-specific roles of TrkB signaling ranging from cellular function to neural circuit mechanisms.
A Computational Protocol for Regulating Protein Binding Reactions with a Light-Sensitive Protein Dimer.
Light-sensitive proteins can be used to perturb signaling networks in living cells and animals with high spatiotemporal resolution. We recently engineered a protein heterodimer that dissociates when irradiated with blue light and demonstrated that by fusing each half of the dimer to termini of a protein that it is possible to selectively block binding surfaces on the protein when in the dark. On activation with light, the dimer dissociates and exposes the binding surface, allowing the protein to bind its partner. Critical to the success of this system, called Z-lock, is that the linkers connecting the dimer components to the termini are engineered so that the dimer forms over the appropriate binding surface. Here, we develop and test a protocol in the Rosetta molecular modeling program for designing linkers for Z-lock. We show that the protocol can predict the most effective linker sets for three different light-sensitive switches, including a newly designed switch that binds the Rho-family GTPase Cdc42 on stimulation with blue light. This protocol represents a generalized computational approach to placing a wide variety of proteins under optogenetic control with Z-lock.
Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein-Protein Interactions.
Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein-protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.
Optogenetic Navigation of Routes Leading to Protein Amyloidogenesis in Bacteria.
Modulation of liquid-liquid and liquid-hydrogel phase transitions is central to avoid the cytotoxic aggregation of proteins in eukaryotic cells, but knowledge on its relevance in bacteria is limited. Here the power of optogenetics to engineer proteins as light-responsive switches has been used to control the balance between solubility and aggregation for LOV2-WH1, a chimera between the plant blue light-responsive domain LOV2 and the bacterial prion-like protein RepA-WH1. These proteins were first linked by fusing, as a continuous α-helix, the C-terminal photo-transducer Jα helix in LOV2 with the N-terminal domain-closure α1 helix in RepA-WH1, and then improved for light-responsiveness by including mutations in the Jα moiety. In the darkness and in a crowded solution in vitro, LOV2-WH1 nucleates the irreversible assembly of amyloid fibers into a hydrogel. However, under blue light illumination LOV2-WH1 assembles as soluble oligomers. When expressed in Escherichia coli, LOV2-WH1 forms in the darkness large intracellular amyloid inclusions compatible with bacterial proliferation. Strikingly, under blue light LOV2-WH1 aggregates decrease in size while they become detrimental for bacterial growth. LOV2-WH1 optogenetics governs the assembly of mutually exclusive inert amyloid fibers or cytotoxic oligomers, thus enabling the navigation of the conformational landscape of protein amyloidogenesis to generate potential photo-activated anti-bacterial devices (optobiotics).
Applications of optobiology in intact cells and multi-cellular organisms.
Temporal kinetics and spatial coordination of signal transduction in cells are vital for cell fate determination. Tools that allow for precise modulation of spatiotemporal regulation of intracellular signaling in intact cells and multicellular organisms remain limited. The emerging optobiological approaches use light to control protein-protein interaction in live cells and multicellular organisms. Optobiology empowers light-mediated control of diverse cellular and organismal functions such as neuronal activity, intracellular signaling, gene expression, cell proliferation, differentiation, migration, and apoptosis. In this review, we highlight recent developments in optobiology, focusing on new features of second-generation optobiological tools. We cover applications of optobiological approaches in the study of cellular and organismal functions, discuss current challenges, and present our outlook. Taking advantage of the high spatial and temporal resolution of light control, optobiology promises to provide new insights into the coordination of signaling circuits in intact cells and multicellular organisms.
Photoactivation Mechanism of a Bacterial Light-Regulated Adenylyl Cyclase.
Light-regulated enzymes enable organisms to quickly respond to changing light conditions. We characterize a photoactivatable adenylyl cyclase (AC) from Beggiatoa sp. (bPAC) that translates a blue light signal into the production of the second messenger cyclic AMP. bPAC contains a BLUF photoreceptor domain that senses blue light using a flavin chromophore, linked to an AC domain. We present a dark state crystal structure of bPAC that closely resembles the recently published structure of the homologous OaPAC from Oscillatoria acuminata. To elucidate the structural mechanism of light-dependent AC activation by the BLUF domain, we determined the crystal structures of illuminated bPAC and of a pseudo-lit state variant. We use hydrogen-deuterium exchange measurements of secondary structure dynamics and hypothesis-driven point mutations to trace the activation pathway from the chromophore in the BLUF domain to the active site of the cyclase. The structural changes are relayed from the residues interacting with the excited chromophore through a conserved kink of the BLUF β-sheet to a tongue-like extrusion of the AC domain that regulates active site opening and repositions catalytic residues. Our findings not only show the specific molecular pathway of photoactivation in BLUF-regulated ACs but also have implications for the general understanding of signaling in BLUF domains and of the activation of ACs.
The amino-terminal helix modulates light-activated conformational changes in AsLOV2.
The mechanism of light-triggered conformational change and signaling in light-oxygen-voltage (LOV) domains remains elusive in spite of extensive investigation and their use in optogenetic studies. The LOV2 domain of Avenasativa phototropin 1 (AsLOV2), a member of the Per-Arnt-Sim (PAS) family, contains a flavin mononucleotide chromophore that forms a covalent bond with a cysteine upon illumination. This event leads to the release of the carboxy-terminal Jα helix, the biological output signal. Using mutational analysis, circular dichroism, and NMR, we find that the largely ignored amino-terminal helix is a control element in AsLOV2's light-activated conformational change. We further identify a direct amino-to-carboxy-terminal "input-output" signaling pathway. These findings provide a framework to rationalize the LOV domain architecture, as well as the signaling mechanisms in both isolated and tandem arrangements of PAS domains. This knowledge can be applied in engineering LOV-based photoswitches, opening up new design strategies and improving existing ones.
From dusk till dawn: one-plasmid systems for light-regulated gene expression.
Signaling photoreceptors mediate diverse organismal adaptations in response to light. As light-gated protein switches, signaling photoreceptors provide the basis for optogenetics, a term that refers to the control of organismal physiology and behavior by light. We establish as novel optogenetic tools the plasmids pDusk and pDawn, which employ blue-light photoreceptors to confer light-repressed or light-induced gene expression in Escherichia coli with up to 460-fold induction upon illumination. Key features of these systems are low background activity, high dynamic range, spatial control on the 20-μm scale, independence from exogenous factors, and ease of use. In optogenetic experiments, pDusk and pDawn can be used to specifically perturb individual nodes of signaling networks and interrogate their role. On the preparative scale, pDawn can induce by light the production of recombinant proteins and thus represents a cost-effective and readily automated alternative to conventional induction systems.
Multichromatic control of gene expression in Escherichia coli.
Light is a powerful tool for manipulating living cells because it can be applied with high resolution across space and over time. We previously constructed a red light-sensitive Escherichia coli transcription system based on a chimera between the red/far-red switchable cyanobacterial phytochrome Cph1 and the E. coli EnvZ/OmpR two-component signaling pathway. Here, we report the development of a green light-inducible transcription system in E. coli based on a recently discovered green/red photoswitchable two-component system from cyanobacteria. We demonstrate that the transcriptional output is proportional to the intensity of green light applied and that the green sensor is orthogonal to the red sensor at intensities of 532-nm light less than 0.01 W/m(2). Expression of both sensors in a single cell allows two-color optical control of transcription both in batch culture and in patterns across a lawn of engineered cells. Because each sensor functions as a photoreversible switch, this system should allow the spatial and temporal control of the expression of multiple genes through different combinations of light wavelengths. This feature aids precision single-cell and population-level studies in systems and synthetic biology.
Design and signaling mechanism of light-regulated histidine kinases.
Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen-sensitive, light-inert histidine kinase by replacing its chemosensor domain by a light-oxygen-voltage photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by approximately 1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo. Signals are transmitted from the light-oxygen-voltage sensor domain to the histidine kinase domain via a 40 degrees -60 degrees rotational movement within an alpha-helical coiled-coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by alpha-helices and to chemo- and photosensors. Conserved sequence motifs guide the rational design of light-regulated variants of histidine kinases and other proteins.
Structural basis for light-dependent signaling in the dimeric LOV domain of the photosensor YtvA.
The photosensor YtvA binds flavin mononucleotide and regulates the general stress reaction in Bacillus subtilis in response to blue light illumination. It belongs to the family of light-oxygen-voltage (LOV) proteins that were first described in plant phototropins and form a subgroup of the Per-Arnt-Sim (PAS) superfamily. Here, we report the three-dimensional structure of the LOV domain of YtvA in its dark and light states. The protein assumes the global fold common to all PAS domains and dimerizes via a hydrophobic interface. Directly C-terminal to the core of the LOV domain, an alpha-helix extends into the solvent. Light absorption causes formation of a covalent bond between a conserved cysteine residue and atom C(4a) of the FMN ring, which triggers rearrangements throughout the LOV domain. Concomitantly, in the dark and light structures, the two subunits of the dimeric protein rotate relative to each other by 5 degrees . This small quaternary structural change is presumably a component of the mechanism by which the activity of YtvA is regulated in response to light. In terms of both structure and signaling mechanism, YtvA differs from plant phototropins and more closely resembles prokaryotic heme-binding PAS domains.