Curated Optogenetic Publication Database

Abstract: Collective cell migration is emerging as a major driver of embryonic development, organogenesis, tissue homeostasis, and tumor dissemination. In contrast to individually migrating cells, collectively migrating cells maintain cell-cell adhesions and coordinate direction-sensing as they move. While non-muscle myosin II has been studied extensively in the context of cells migrating individually in vitro, its roles in cells migrating collectively in three-dimensional, native environments are not fully understood. Here we use genetics, Airyscan microscopy, live imaging, optogenetics, and Förster resonance energy transfer to probe the localization, dynamics, and functions of myosin II in migrating border cells of the Drosophila ovary. We find that myosin accumulates transiently at the base of protrusions, where it functions to retract them. E-cadherin and myosin co-localize at border cell-border cell contacts and cooperate to transmit directional information. A phosphomimetic form of myosin is sufficient to convert border cells to a round morphology and blebbing migration mode. Together these studies demonstrate that distinct and dynamic pools of myosin II regulate protrusion dynamics within and between collectively migrating cells and suggest a new model for the role of protrusions in collective direction sensing in vivo. Movie S1 Movie S1 Live imaging of border cell specification and delamination from anterior epithelium From Figure 1D-I. Slbo promoter driving Lifeact-GFP (green) marks border cells, Upd-Gal4, UAS-DsRed.nls (red) mark polar cell nuclei. Hoechst 33342 (blue) marks DNA. Time resolution is 4 min. Movie S2 Movie S2 Representative Z-projected and registered live imaging of Sqh-mCherry accumulating in cortical junctions (flashing arrows) during border cell migration. From Figure 3J-K. Time resolution is 25 sec. Movie S3 Movie S3 Representative Z-projected and registered live imaging of E-cad-GFP during border cell migration. From Figure 3M-N. Time resolution is 60 sec. Movie S4 Movie S4 Representative Z-projection of control flpout cells from hs-Flp;, Slbo>Lifeact-GFP; AyGal4, UAS-RFP. Clonal cells are marked by magenta nuclei (nls-RFP). Time resolution is 2.5 min. From Supp. Figure 3 A-D. Movie S5 Movie S5 Representative Z-projection of Sqh-RNAi flpout cells from hs-Flp;, Slbo>Lifeact-GFP; AyGal4, UAS-RFP, UAS-sqh-RNAi. Clonal cells are marked by magenta nuclei (nls-RFP). Time resolution is 2.5 min. From Supp. Figure 3 E-H. Movie S6 Movie S6 Representative Z-projected c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-white RNAi. Time resolution is 2 min. From Supp. Figure 4 A-D. Movie S7 Movie S7 Representative Z-projected c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-sqh-RNAi showing frequent side protrusions. Time resolution is 2 min. From Supp. Figure 4 E-H. White arrows indicate ectopic side and rear protrusions. Movie S8 Movie S8 Representative Z-projected c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-sqh-RNAi showing long lived side protrusions. Time resolution is 2 min. From Supp. Figure 4 I-L. Movie S9 Movie S9 Representative Z-projected live imaging of c306-Gal4 driving UAS-white-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during periods of protrusive and round migration phases. From Figure 6A-D. 25 min corresponds to 6A and B and 1hr:25 min corresponds to 6C and D. Time resolution is 2.5 min. Movie S10 Movie S10 Sqh-mCherry (magenta) channel from Supplementary Movie 9. From Figure 6A-D. 25 min corresponds to 6A and B and 1hr:25 min corresponds to 6C and D. Time resolution is 2.5 min. Movie S11 Movie S11 Representative Z-projected live imaging of c306-Gal4 driving UAS-Ecad-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during a protrusive phase of migration. From Figure 6E-F. Time resolution is 2.5 min. Movie S12 Movie S12 Sqh-mCherry (magenta) channel from Supplementary Movie 11. From Figure 6E-F. Time resolution is 2.5 min. Movie S13 Movie S13 Representative Z-projected live imaging of c306-Gal4 driving UAS-Ecad-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during a rounded phase of migration. From Figure 6G-H. Time resolution is 2.5 min. Movie S14 Movie S14 Sqh-mCherry (magenta) channel from Supplementary Movie 13. From Figure 6G-H. Time resolution is 2.5 min. Movie S15 Movie S15 Example segmentation analysis from a representative Z-projected time lapse of a cluster expressing c306-Gal4 driving UAS-white-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during migration. Time lapse analyzed in Imaris by 1. segmentation of the cluster using Lifeact-GFP, 2. Rendering of Sqh-mCherry by masking the inside of the Life-act surface, 3. performing a distance transformation using the masked Sqh-mCherry that is color coded for distance from membrane (dark colors are short distances and bright/white colors are more distant), 4. combining the distance transformation with the Sqh-mCherry mask to only include the cortical 2 μm of the original Sqh-mCherry signal for quantification in Figure 6I. Movie S16 Movie S16 Representative Z-projected time lapse of Lifeact-GFP and Sqh-mCherry expressing clusters used for quantification of Figure 7B-C during protrusion/retractions cycles. Time resolution is 2 min. Movie S17 Movie S17 Sqh-mCherry channel from Supplementary movie 16. Time resolution is 2 min. Movie S18 Movie S18 Representative Z-projections of Lifeact-GFP (green) in c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-Sqh-E20E21 migrating border cells clusters that split. Time resolution is 2 min. Movie S19 Movie S19 Representative Z-projections of Lifeact-GFP (green) in c306-Gal4; tub-GAL80ts driving UAS-LifeactGFP and UAS-Sqh-E20E21 migrating border cells clusters during protrusive phase. Time resolution is 2 min. Movie S20 Movie S20 Representative Z-projection of Lifeact-GFP (green) in c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-Sqh-E20E21 border cells cluster at the oocyte border during a blebbing phase. Time resolution is 2 min. Movie S21 Movie S21 Representative Z-projection of control cluster expressing slbo-Gal4; UAS-PLCδ1-PH-GFP. Time resolution is 2 min. Movie S22 Movie S22 Representative Z-projection of cluster expressing slbo-Gal4; UAS-PLCδ1-PH-GFP, UAS-Rho1V14. Blebs are marked by white arrows. Time resolution is 2 min.

Abstract: Background Photocontrol of protein activity has become a helpful strategy for regulating biological pathways. Herein, a method for the precise and reversible photocontrol of oxidase activity was developed by using the conformational change of the AsLOV2 domain. Results The AsLOV2 domain was inserted into the nonconserved sites exposed on the surface of the AdhP protein, and the alov9 fusion was successfully screened for subsequent optical experiments under the assumption that neither of these actions affected the original activity of AdhP protein. The activity of alov9 was noticeably inhibited when the fusion was exposed to 470 nm blue light and recovered within 30 min. As a result, we could precisely and reversibly photocontrol alov9 activity through the optimization of several parameters, including cofactor concentration, light intensity, and illumination time. Conclusions An efficient method was developed for the photoinhibition of enzymatic activity based on the insertion of the light-sensitive AsLOV2 domain, providing new ideas for photocontrolling metabolic pathways without carriers in the future.

Abstract: Inactivation of the endosomal sorting complex required for transport (ESCRT) machinery has been reported to cause autophagic defects, but the exact functions of ESCRT proteins in macroautophagy/autophagy remain incompletely understood. Using live-cell fluorescence microscopy we found that the filament-forming ESCRT-III subunit CHMP4B was recruited transiently to nascent autophagosomes during starvation-induced autophagy and mitophagy, with residence times of about 1 and 2 min, respectively. Correlative light microscopy and electron tomography revealed CHMP4B recruitment at a late step in mitophagosome formation. The autophagosomal dwell time of CHMP4B was strongly increased by depletion of the regulatory ESCRT-III subunit CHMP2A. Using a novel optogenetic closure assay we observed that depletion of CHMP2A inhibited phagophore sealing during mitophagy. Consistent with this, depletion of CHMP2A and other ESCRT-III subunits inhibited both PRKN/PARKIN-dependent and -independent mitophagy. We conclude that the ESCRT machinery mediates phagophore closure, and that this is essential for mitophagic flux. Abbreviations: BSA: bovine serum albumin; CHMP: chromatin-modifying protein; CLEM: correlative light and electron microscopy; EGFP: enhanced green fluorescent protein; ESCRT: endosomal sorting complex required for transport; HEPES: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; HRP: horseradish peroxidase; ILV: intralumenal vesicle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; LOV2: light oxygen voltage 2; MLS: mitochondrial localization sequence; MT-CO2: mitochondrially encoded cytochrome c oxidase II; O+A: oligomycin and antimycin A; PBS: phosphate-buffered saline; PIPES: piperazine-N,N-bis(2-ethanesulfonic acid); PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RAB: RAS-related in brain; SD: standard deviation; SEM: standard error of the mean; TOMM20: TOMM20: translocase of outer mitochondrial membrane 20; VCL: vinculin; VPS4: vacuolar protein sorting protein 4; Zdk1: Zdark 1; TUBG: Tubulin gamma chain.

Abstract: Abstract not available.

Abstract: Targeted cell ablation is a powerful approach for studying the role of specific cell populations in a variety of organotypic functions, including cell differentiation, and organ generation and regeneration. Emerging tools for permanently or conditionally ablating targeted cell populations and transiently inhibiting neuronal activities exhibit a diversity of application and utility. Each tool has distinct features, and none can be universally applied to study different cell types in various tissue compartments. Although these tools have been developed for over 30 years, they require additional improvement. Currently, there is no consensus on how to select the tools to answer the specific scientific questions of interest. Selecting the appropriate cell ablation technique to study the function of a targeted cell population is less straightforward than selecting the method to study a gene's functions. In this review, we discuss the features of the various tools for targeted cell ablation and provide recommendations for optimal application of specific approaches.

Abstract: Because small-molecule activators of adenylyl cyclases (AC) affect ACs cell-wide, it is challenging to explore the signaling consequences of AC activity emanating from specific intracellular compartments. We explored this issue using a series of engineered, optogenetic, spatially restricted, photoactivable adenylyl cyclases (PACs) positioned at the plasma membrane (PM), the outer mitochondrial membrane (OMM), and the nucleus (Nu). The biochemical consequences of brief photostimulation of PAC is primarily limited to the intracellular site occupied by the PAC. By contrast, sustained photostimulation results in distal cAMP signaling. Prolonged cAMP generation at the OMM profoundly stimulates nuclear protein kinase (PKA) activity. We have found that phosphodiesterases 3 (OMM and PM) and 4 (PM) modulate proximal (local) cAMP-triggered activity, whereas phosphodiesterase 4 regulates distal cAMP activity as well as the migration of PKA's catalytic subunit into the nucleus.

Abstract: Decorating GUVs, used as minimal synthetic cell models, with photoswitchable proteins allows controlling the adhesion between them and their assembly into multicellular structures with light. Thereby, the chemical communication between a sender and a receiver GUV, which strongly depends on their spatial proximity, can also be photoregulated.

Abstract: cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli.

Abstract: Light-inducible approaches provide means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Recent developments of optogenetic and chemo-optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. The new expansions of the toolbox facilitate control of signal transduction, genome editing, 'painting' patterns of active molecules onto cellular membranes and light-induced cell cycle control. A combination of light- and chemically induced dimerization approaches has also seen interesting progress. Here we provide an overview of the optogenetic systems and the emerging chemo-optogenetic systems, and discuss recent applications in tackling complex biological problems.

Abstract: A common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.

Abstract: We developed VIEW-MOD (Versatile Illumination Engine with a Modular Optical Design): a compact, multi-modality microscope, which accommodates multiple illumination schemes including variable angle total internal reflection, point scanning and vertical/horizontal light sheet. This system allows combining and flexibly switching between different illuminations and imaging modes by employing three electrically tunable lenses and two fast-steering mirrors. This versatile optics design provides control of 6 degrees of freedom of the illumination source (3 translation, 2 tilt, and beam shape) plus the axial position of the imaging plane. We also developed standalone software with an easy-to-use GUI to calibrate and control the microscope. We demonstrate the applications of this system and software in biosensor imaging, optogenetics and fast 3D volume imaging. This system is ready to fit into complex imaging circumstances requiring precise control of illumination and detection paths, and has a broad scope of usability for a myriad of biological applications.

Abstract: Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been developed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback except for in silico control. Here, we engineer optogenetic gene circuits into mammalian cells to achieve noise-reduction for precise gene expression control by genetic, in vitro negative feedback. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-inhibiting peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide a range of nearly 4-fold gene expression control and up to 5-fold noise reduction from existing optogenetic systems. Moreover, we use the LITer gene circuit architecture to control gene expression of the cancer oncogene KRAS(G12V) and study its downstream effects through phospho-ERK levels and cellular proliferation. Overall, these novel LITer optogenetic platforms should enable precise spatiotemporal perturbations for studying multicellular phenotypes in developmental biology, oncology and other biomedical fields of research.

Abstract: The DNA damage response (DDR) generates transient repair compartments to concentrate repair proteins and activate signaling factors. The physicochemical properties of these spatially confined compartments and their function remain poorly understood. Here, we establish, based on live cell microscopy and CRISPR/Cas9-mediated endogenous protein tagging, that 53BP1-marked repair compartments are dynamic, show droplet-like behavior, and undergo frequent fusion and fission events. 53BP1 assembly, but not the upstream accumulation of γH2AX and MDC1, is highly sensitive to changes in osmotic pressure, temperature, salt concentration and to disruption of hydrophobic interactions. Phase separation of 53BP1 is substantiated by optoDroplet experiments, which further allowed dissection of the 53BP1 sequence elements that cooperate for light-induced clustering. Moreover, we found the tumor suppressor protein p53 to be enriched within 53BP1 optoDroplets, and conditions that disrupt 53BP1 phase separation impair 53BP1-dependent induction of p53 and diminish p53 target gene expression. We thus suggest that 53BP1 phase separation integrates localized DNA damage recognition and repair factor assembly with global p53-dependent gene activation and cell fate decisions.

Abstract: Contraction of cortical actomyosin networks driven by myosin activation controls cell shape changes and tissue morphogenesis during animal development. In vitro studies suggest that contractility also depends on the geometrical organization of actin filaments. Here we analyze the function of actomyosin network topology in vivo using optogenetic stimulation of myosin-II in Drosophila embryos. We show that early during cellularization, hexagonally arrayed actomyosin fibers are resilient to myosin-II activation. Actomyosin fibers then acquire a ring-like conformation and become contractile and sensitive to myosin-II. This transition is controlled by Bottleneck, a Drosophila unique protein expressed for only a short time during early cellularization, which we show regulates actin bundling. In addition, it requires two opposing actin cross-linkers, Filamin and Fimbrin. Filamin acts synergistically with Bottleneck to facilitate hexagonal patterning, while Fimbrin controls remodeling of the hexagonal network into contractile rings. Thus, actin cross-linking regulates the spatio-temporal organization of actomyosin contraction in vivo, which is critical for tissue morphogenesis.

Abstract: Inside the female genital tract, mammalian sperm undergo a maturation process called capacitation, which primes the sperm to navigate across the oviduct and fertilize the egg. Sperm capacitation and motility are controlled by 3',5'-cyclic adenosine monophosphate (cAMP). Here, we show that optogenetics, the control of cellular signaling by genetically encoded light-activated proteins, allows to manipulate cAMP dynamics in sperm flagella and, thereby, sperm capacitation and motility by light. To this end, we used sperm that express the light-activated phosphodiesterase LAPD or the photo-activated adenylate cyclase bPAC. The control of cAMP by LAPD or bPAC combined with pharmacological interventions provides spatiotemporal precision and allows to probe the physiological function of cAMP compartmentalization in mammalian sperm.

Abstract: Mammalian genomes are folded into tens of thousands of long-range looping interactions. The cause-and-effect relationship between looping and genome function is poorly understood, and the extent to which loops are dynamic on short time scales remains an unanswered question. Here, we engineer a new class of synthetic architectural proteins for directed rearrangement of the three-dimensional genome using blue light. We target our light-activated-dynamic-looping (LADL) system to two genomic anchors with CRISPR guide RNAs and induce their spatial colocalization via light-induced heterodimerization of cryptochrome 2 and a dCas9-CIBN fusion protein. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Using single-molecule RNA-FISH, we demonstrate that de novo formation of the Zfp462-SE loop correlates with a modest increase in Zfp462 expression. LADL facilitates colocalization of genomic loci without exogenous chemical cofactors and will enable future efforts to engineer reversible and oscillatory loops on short time scales.

Abstract: Subcellular localization of signal molecules is a hallmark in organizing the signaling network. OpEn-Tag is a modular optogenetic endomembrane targeting toolbox that allows alteration of the localization and therefore the activity of signaling processes with the spatiotemporal resolution of optogenetics. OpEn-Tag is a two-component system employing (1) a variety of targeting peptides fused to and thereby dictating the localization of mCherry-labeled cryptochrome 2 binding protein CIBN toward distinct endomembranes and (2) the cytosolic, fluorescence-labeled blue light photoreceptor cryptochrome 2 as a customizable building block that can be fused to proteins of interest. The combination of OpEn-Tag with growth factor stimulation or the use of two membrane anchor sequences allows investigation of multilayered signal transduction processes as demonstrated here for the protein kinase AKT.

Abstract: Optogenetic probes can be powerful tools for dissecting complexity in cell biology, but there is a lack of instrumentation to exploit their potential for automated, high-information-content experiments. This protocol describes the construction and use of the optoPlate-96, a platform for high-throughput three-color optogenetics experiments that allows simultaneous manipulation of common red- and blue-light-sensitive optogenetic probes. The optoPlate-96 enables illumination of individual wells in 96-well microwell plates or in groups of wells in 384-well plates. Its design ensures that there will be no cross-illumination between microwells in 96-well plates, and an active cooling system minimizes sample heating during light-intensive experiments. This protocol details the steps to assemble, test, and use the optoPlate-96. The device can be fully assembled without specialized equipment beyond a 3D printer and a laser cutter, starting from open-source design files and commercially available components. We then describe how to perform a typical optogenetics experiment using the optoPlate-96 to stimulate adherent mammalian cells. Although optoPlate-96 experiments are compatible with any plate-based readout, we describe analysis using quantitative single-cell immunofluorescence. This workflow thus allows complex optogenetics experiments (independent control of stimulation colors, intensity, dynamics, and time points) with high-dimensional outputs at single-cell resolution. Starting from 3D-printed and laser-cut components, assembly and testing of the optoPlate-96 can be accomplished in 3-4 h, at a cost of ~$600. A full optoPlate-96 experiment with immunofluorescence analysis can be performed within ~24 h, but this estimate is variable depending on the cell type and experimental parameters.

Abstract: Light-sensing protein domains that link an exogenous light signal to the activity of an enzyme have attracted much notice for engineering new regulatory mechanisms into proteins and for studying the dynamic behavior of intracellular reactions as well as reaction cascades. Light-oxygen-voltage (LOV) photoreceptors are blue light-sensing modules that have been intensely characterized for this purpose and linked to several proteins of interest. For successful application of these tools it is crucial to identify appropriate fusion strategies for combining sensor and enzyme domains that sustain activity and light-induced responsivity. Terminal fusion of LOV domains is the natural strategy; however, this is not transferrable to T7 RNA polymerase since both of its termini are involved in catalysis. We show here that it is possible to covalently insert LOV domains into the polymerase protein while preserving its activity and generating new light-responsive allosteric coupling.

Abstract: In order to study the role of signaling proteins, such as kinases and GTPases, in brain functions it is necessary to control their activity at the appropriate spatiotemporal resolution and to examine the cellular and behavioral effects of such changes in activity. Reduced spatiotemporal resolution in the regulation of these proteins activity will impede the ability to understand the proteins normal functions as longer modification of their activity in non-normal locations could lead to effects different from their natural functions. To control intracellular signaling proteins at the highest temporal resolution recent innovative optogenetic approaches were developed to allow the control of photoactivable signaling proteins activity by light. These photoactivatable proteins can be activated in selected cell population in brain and in specific subcellular compartments. Minimal-invasive tools are being developed to photoactivate these proteins for study and therapy. Together these techniques afford an unprecedented spatiotemporal control of signaling proteins activity to unveil the function of brain proteins with high accuracy in behaving animals. As dysfunctional signaling proteins are involved in brain diseases, the optogenetic technique has also the potential to be used as a tool to treat brain diseases.

Abstract: We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce ∼100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.

Abstract: 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.

Abstract: Mononegaviruses are promising tools as oncolytic vectors and transgene delivery vectors for gene therapy and regenerative medicine. By using the Magnet proteins, which reversibly heterodimerize upon blue light illumination, photocontrollable mononegaviruses (measles and rabies viruses) were generated. The Magnet proteins were inserted into the flexible domain of viral polymerase, and viruses showed strong replication and oncolytic activities only when the viral polymerases were activated by blue light illumination.

Abstract: Living cells can impart materials with advanced functions, such as sense-and-respond, chemical production, toxin remediation, energy generation and storage, self-destruction, and self-healing. Here, an approach is presented to use light to pattern Escherichia coli onto diverse materials by controlling the expression of curli fibers that anchor the formation of a biofilm. Different colors of light are used to express variants of the structural protein CsgA fused to different peptide tags. By projecting color images onto the material containing bacteria, this system can be used to pattern the growth of composite materials, including layers of protein and gold nanoparticles. This is used to pattern cells onto materials used for 3D printing, plastics (polystyrene), and textiles (cotton). Further, the adhered cells are demonstrated to respond to sensory information, including small molecules (IPTG and DAPG) and light from light-emitting diodes. This work advances the capacity to engineer responsive living materials in which cells provide diverse functionality.

Abstract: Membrane contact sites (MCSs) are specialized subcellular compartments formed by closely apposed membranes from two organelles. The intermembrane gap is separated by a distance ranging from 10 to 35 nm. MCSs are typically maintained through dynamic protein-protein and protein-lipid interactions. These intermembrane contact sites constitute important intracellular signalling hotspots to mediate a plethora of cellular processes, including calcium homeostasis, lipid metabolism, membrane biogenesis and organelle remodelling. In recent years, a series of genetically encoded probes and chemogenetic or optogenetic actuators have been invented to aid the visualization and interrogation of MCSs in both fixed and living cells. These molecular tools have greatly accelerated the pace of mechanistic dissection of membrane contact sites at the molecular level. In this review, we present an overview on the latest progress in this endeavour, and provide a general guide to the selection of methods and molecular tools for probing interorganellar membrane contact sites.