Showing 1 - 25 of 32 results
Optogenetics in the hot seat.
Optogenetic and thermogenetic tools have been limited to applications for single-state control of cellular processes. A single-component optogenetic tool was found to act as both a temperature sensor and a photoreceptor, enabling multi-state control of developmental signaling.
Temperature-responsive optogenetic probes of cell signaling.
We describe single-component optogenetic probes whose activation dynamics depend on both light and temperature. We used the BcLOV4 photoreceptor to stimulate Ras and phosphatidyl inositol-3-kinase signaling in mammalian cells, allowing activation over a large dynamic range with low basal levels. Surprisingly, we found that BcLOV4 membrane translocation dynamics could be tuned by both light and temperature such that membrane localization spontaneously decayed at elevated temperatures despite constant illumination. Quantitative modeling predicted BcLOV4 activation dynamics across a range of light and temperature inputs and thus provides an experimental roadmap for BcLOV4-based probes. BcLOV4 drove strong and stable signal activation in both zebrafish and fly cells, and thermal inactivation provided a means to multiplex distinct blue-light sensitive tools in individual mammalian cells. BcLOV4 is thus a versatile photosensor with unique light and temperature sensitivity that enables straightforward generation of broadly applicable optogenetic tools.
Designer membraneless organelles sequester native factors for control of cell behavior.
Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron-size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to 90% of targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.
Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering.
Plant-based photosensors, such as the light-oxygen-voltage sensing domain 2 (LOV2) from oat phototropin 1, can be modularly wired into cell signaling networks to remotely control protein activity and physiological processes. However, the applicability of LOV2 is hampered by the limited choice of available caging surfaces and its preference to accommodate the effector domains downstream of the C-terminal Jα helix. Here, we engineered a set of LOV2 circular permutants (cpLOV2) with additional caging capabilities, thereby expanding the repertoire of genetically encoded photoswitches to accelerate the design of optogenetic devices. We demonstrate the use of cpLOV2-based optogenetic tools to reversibly gate ion channels, antagonize CRISPR-Cas9-mediated genome engineering, control protein subcellular localization, reprogram transcriptional outputs, elicit cell suicide and generate photoactivatable chimeric antigen receptor T cells for inducible tumor cell killing. Our approach is widely applicable for engineering other photoreceptors to meet the growing need of optogenetic tools tailored for biomedical and biotechnological applications.
Engineering AraC to make it responsive to light instead of arabinose.
The L-arabinose-responsive AraC and its cognate PBAD promoter underlie one of the most often used chemically inducible prokaryotic gene expression systems in microbiology and synthetic biology. Here, we change the sensing capability of AraC from L-arabinose to blue light, making its dimerization and the resulting PBAD activation light-inducible. We engineer an entire family of blue light-inducible AraC dimers in Escherichia coli (BLADE) to control gene expression in space and time. We show that BLADE can be used with pre-existing L-arabinose-responsive plasmids and strains, enabling optogenetic experiments without the need to clone. Furthermore, we apply BLADE to control, with light, the catabolism of L-arabinose, thus externally steering bacterial growth with a simple transformation step. Our work establishes BLADE as a highly practical and effective optogenetic tool with plug-and-play functionality-features that we hope will accelerate the broader adoption of optogenetics and the realization of its vast potential in microbiology, synthetic biology and biotechnology.
Living materials fabricated via gradient mineralization of light-inducible biofilms.
Living organisms have evolved sophisticated cell-mediated biomineralization mechanisms to build structurally ordered, environmentally adaptive composite materials. Despite advances in biomimetic mineralization research, it remains difficult to produce mineralized composites that integrate the structural features and 'living' attributes of their natural counterparts. Here, inspired by natural graded materials, we developed living patterned and gradient composites by coupling light-inducible bacterial biofilm formation with biomimetic hydroxyapatite (HA) mineralization. We showed that both the location and the degree of mineralization could be regulated by tailoring functional biofilm growth with spatial and biomass density control. The cells in the composites remained viable and could sense and respond to environmental signals. Additionally, the composites exhibited a maximum 15-fold increase in Young's modulus after mineralization and could be applied to repair damage in a spatially controlled manner. Beyond insights into the mechanism of formation of natural graded composites, our study provides a viable means of fabricating living composites with dynamic responsiveness and environmental adaptability.
Optogenetic control of the lac operon for bacterial chemical and protein production.
Control of the lac operon with isopropyl β-D-1-thiogalactopyranoside (IPTG) has been used to regulate gene expression in Escherichia coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities, such as easy tunability, reversibility, dynamic induction strength and spatial control, that are difficult to obtain with chemical inducers. We have developed a series of circuits for optogenetic regulation of the lac operon, which we call OptoLAC, to control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least two-litre bioreactors. Furthermore, OptoLAC circuits enable control of recombinant protein production, reaching yields comparable to IPTG induction but with easier tunability of expression. OptoLAC circuits are potentially useful to confer light control over other cell functions originally designed to be IPTG-inducible.
m6A-binding YTHDF proteins promote stress granule formation.
Diverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are important for SG formation. Super-resolution imaging further reveals that YTHDF proteins appear to be in a super-saturated state, forming clusters that often reside in the periphery of or at the junctions between SG core clusters, and potentially promote SG formation by reducing the activation energy barrier and critical size for SG condensate formation. Our results suggest a new function of the m6A-binding YTHDF proteins in regulating SG formation.
Optogenetic control of cofilin and αTAT in living cells using Z-lock.
Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.
A blue light receptor that mediates RNA binding and translational regulation.
Sensory photoreceptor proteins underpin light-dependent adaptations in nature and enable the optogenetic control of organismal behavior and physiology. We identified the bacterial light-oxygen-voltage (LOV) photoreceptor PAL that sequence-specifically binds short RNA stem loops with around 20 nM affinity in blue light and weaker than 1 µM in darkness. A crystal structure rationalizes the unusual receptor architecture of PAL with C-terminal LOV photosensor and N-terminal effector units. The light-activated PAL-RNA interaction can be harnessed to regulate gene expression at the RNA level as a function of light in both bacteria and mammalian cells. The present results elucidate a new signal-transduction paradigm in LOV receptors and conjoin RNA biology with optogenetic regulation, thereby paving the way toward hitherto inaccessible optoribogenetic modalities.
A split CRISPR-Cpf1 platform for inducible genome editing and gene activation.
The CRISPR-Cpf1 endonuclease has recently been demonstrated as a powerful tool to manipulate targeted gene sequences. Here, we performed an extensive screening of split Cpf1 fragments and identified a pair that, combined with inducible dimerization domains, enables chemical- and light-inducible genome editing in human cells. We also identified another split Cpf1 pair that is spontaneously activated. The newly generated amino and carboxyl termini of the spontaneously activated split Cpf1 can be repurposed as de novo fusion sites of artificial effector domains. Based on this finding, we generated an improved split dCpf1 activator, which has the potential to activate endogenous genes more efficiently than a previously established dCas9 activator. Finally, we showed that the split dCpf1 activator can efficiently activate target genes in mice. These results demonstrate that the present split Cpf1 provides an efficient and sophisticated genome manipulation in the fields of basic research and biotechnological applications.
Rewiring bacterial two-component systems by modular DNA-binding domain swapping.
Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways and valuable sensors for synthetic biology. However, most TCSs remain uncharacterized or difficult to harness for applications. Major challenges are that many TCS output promoters are unknown, subject to cross-regulation, or silent in heterologous hosts. Here, we demonstrate that the two largest families of response regulator DNA-binding domains can be interchanged with remarkable flexibility, enabling the corresponding TCSs to be rewired to synthetic output promoters. We exploit this plasticity to eliminate cross-regulation, un-silence a gram-negative TCS in a gram-positive host, and engineer a system with over 1,300-fold activation. Finally, we apply DNA-binding domain swapping to screen uncharacterized Shewanella oneidensis TCSs in Escherichia coli, leading to the discovery of a previously uncharacterized pH sensor. This work should accelerate fundamental TCS studies and enable the engineering of a large family of genetically encoded sensors with diverse applications.
Light-based control of metabolic flux through assembly of synthetic organelles.
To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.
Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET.
Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Förster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.
Engineering RGB color vision into Escherichia coli.
Optogenetic tools use colored light to rapidly control gene expression in space and time. We designed a genetically encoded system that gives Escherichia coli the ability to distinguish between red, green, and blue (RGB) light and respond by changing gene expression. We use this system to produce 'color photographs' on bacterial culture plates by controlling pigment production and to redirect metabolic flux by expressing CRISPRi guide RNAs.
Optogenetics: Switching with red and blue.
Abstract not available.
Illuminating developmental biology through photochemistry.
Developmental biology has been continually shaped by technological advances, evolving from a descriptive science into one immersed in molecular and cellular mechanisms. Most recently, genome sequencing and 'omics' profiling have provided developmental biologists with a wealth of genetic and biochemical information; however, fully translating this knowledge into functional understanding will require new experimental capabilities. Photoactivatable probes have emerged as particularly valuable tools for investigating developmental mechanisms, as they can enable rapid, specific manipulations of DNA, RNA, proteins, and cells with spatiotemporal precision. In this Perspective, we describe optochemical and optogenetic systems that have been applied in multicellular organisms, insights gained through the use of these probes, and their current limitations. We also suggest how chemical biologists can expand the reach of photoactivatable technologies and bring new depth to our understanding of organismal development.
Near-infrared optogenetic pair for protein regulation and spectral multiplexing.
Multifunctional optogenetic systems are in high demand for use in basic and biomedical research. Near-infrared-light-inducible binding of bacterial phytochrome BphP1 to its natural PpsR2 partner is beneficial for simultaneous use with blue-light-activatable tools. However, applications of the BphP1-PpsR2 pair are limited by the large size, multidomain structure and oligomeric behavior of PpsR2. Here, we engineered a single-domain BphP1 binding partner, Q-PAS1, which is three-fold smaller and lacks oligomerization. We exploited a helix-PAS fold of Q-PAS1 to develop several near-infrared-light-controllable transcription regulation systems, enabling either 40-fold activation or inhibition. The light-induced BphP1-Q-PAS1 interaction allowed modification of the chromatin epigenetic state. Multiplexing the BphP1-Q-PAS1 pair with a blue-light-activatable LOV-domain-based system demonstrated their negligible spectral crosstalk. By integrating the Q-PAS1 and LOV domains in a single optogenetic tool, we achieved tridirectional protein targeting, independently controlled by near-infrared and blue light, thus demonstrating the superiority of Q-PAS1 for spectral multiplexing and engineering of multicomponent systems.
Evolution of a split RNA polymerase as a versatile biosensor platform.
Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies are often limited by a low signal-to-noise ratio, the extensive optimization required for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting activity-responsive RNAP (AR) system to create biosensors that can be activated by light and small molecules, demonstrating the 'plug-and-play' nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.
A photoactivatable Cre-loxP recombination system for optogenetic genome engineering.
Genome engineering techniques represented by the Cre-loxP recombination system have been used extensively for biomedical research. However, powerful and useful techniques for genome engineering that have high spatiotemporal precision remain elusive. Here we develop a highly efficient photoactivatable Cre recombinase (PA-Cre) to optogenetically control genome engineering in vivo. PA-Cre is based on the reassembly of split Cre fragments by light-inducible dimerization of the Magnet system. PA-Cre enables sharp induction (up to 320-fold) of DNA recombination and is efficiently activated even by low-intensity illumination (∼0.04 W m(-2)) or short periods of pulsed illumination (∼30 s). We demonstrate that PA-Cre allows for efficient DNA recombination in an internal organ of living mice through noninvasive external illumination using a LED light source. The present PA-Cre provides a powerful tool to greatly facilitate optogenetic genome engineering in vivo.
Light-induced nuclear export reveals rapid dynamics of epigenetic modifications.
We engineered a photoactivatable system for rapidly and reversibly exporting proteins from the nucleus by embedding a nuclear export signal in the LOV2 domain from phototropin 1. Fusing the chromatin modifier Bre1 to the photoswitch, we achieved light-dependent control of histone H2B monoubiquitylation in yeast, revealing fast turnover of the ubiquitin mark. Moreover, this inducible system allowed us to dynamically monitor the status of epigenetic modifications dependent on H2B ubiquitylation.
Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase.
Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use in controlling protein-protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2-CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2-CIB system by identifying versions of CRY2-CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates an α13-α14 turn motif within plant CRYs whose perturbation alters signaling-state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second-generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range, allowing robust recombination following exposure to a single, brief pulse of light.
Optogenetic oligomerization of Rab GTPases regulates intracellular membrane trafficking.
Intracellular membrane trafficking, which is involved in diverse cellular processes, is dynamic and difficult to study in a spatiotemporal manner. Here we report an optogenetic strategy, termed light-activated reversible inhibition by assembled trap of intracellular membranes (IM-LARIAT), that uses various Rab GTPases combined with blue-light-induced hetero-interaction between cryptochrome 2 and CIB1. In this system, illumination induces a rapid and reversible intracellular membrane aggregation that disrupts the dynamics and functions of the targeted membrane. We applied IM-LARIAT to specifically perturb several Rab-mediated trafficking processes, including receptor transport, protein sorting and secretion, and signaling initiated from endosomes. We finally used this tool to reveal different functions of local Rab5-mediated and Rab11-mediated membrane trafficking in growth cones and soma of young hippocampal neurons. Our results show that IM-LARIAT is a versatile tool that can be used to dissect spatiotemporal functions of intracellular membranes in diverse systems.
Frequency and amplitude control of cortical oscillations by phosphoinositide waves.
Rhythmicity is prevalent in the cortical dynamics of diverse single and multicellular systems. Current models of cortical oscillations focus primarily on cytoskeleton-based feedbacks, but information on signals upstream of the actin cytoskeleton is limited. In addition, inhibitory mechanisms--especially local inhibitory mechanisms, which ensure proper spatial and kinetic controls of activation--are not well understood. Here, we identified two phosphoinositide phosphatases, synaptojanin 2 and SHIP1, that function in periodic traveling waves of rat basophilic leukemia (RBL) mast cells. The local, phase-shifted activation of lipid phosphatases generates sequential waves of phosphoinositides. By acutely perturbing phosphoinositide composition using optogenetic methods, we showed that pulses of PtdIns(4,5)P2 regulate the amplitude of cyclic membrane waves while PtdIns(3,4)P2 sets the frequency. Collectively, these data suggest that the spatiotemporal dynamics of lipid metabolism have a key role in governing cortical oscillations and reveal how phosphatidylinositol 3-kinases (PI3K) activity could be frequency-encoded by a phosphatase-dependent inhibitory reaction.
Light-assisted small-molecule screening against protein kinases.
High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.