Curated Optogenetic Publication Database

Abstract: Cells under high confinement form highly polarized hydrostatic pressure-driven, stable leader blebs that enable efficient migration in low adhesion, environments. Here we investigated the basis of the polarized bleb morphology of metastatic melanoma cells migrating in non-adhesive confinement. Using high-resolution time-lapse imaging and specific molecular perturbations, we found that EGF signaling via PI3K stabilizes and maintains a polarized leader bleb. Protein activity biosensors revealed a unique EGFR/PI3K activity gradient decreasing from rear-to-front, promoting PIP3 and Rac1-GTP accumulation at the bleb rear, with its antagonists PIP2 and RhoA-GTP concentrated at the bleb tip, opposite to the front-to-rear organization of these signaling modules in integrin-mediated mesenchymal migration. Optogenetic experiments showed that disrupting this gradient caused bleb retraction, underscoring the role of this signaling gradient in bleb stability. Mathematical modeling and experiments identified a mechanism where, as the bleb initiates, CD44 and ERM proteins restrict EGFR mobility in a membrane-apposed cortical actin meshwork in the bleb rear, establishing a rear-to-front EGFR-PI3K-Rac activity gradient. Thus, our study reveals the biophysical and molecular underpinnings of cell polarity in bleb-based migration of metastatic cells in non-adhesive confinement, and underscores how alternative spatial arrangements of migration signaling modules can mediate different migration modes according to the local microenvironment.

Abstract: Collective cell migration and tissue morphogenesis play a variety of important roles in the development of many species. Tissue morphogenesis often generates mechanical forces that alter cell shapes and arrangements, resembling collective cell migration-like behaviors. Genetic methods have been widely used to study collective cell migration and its like behavior, advancing our understanding of these processes during development. However, a growing body of research shows that collective cell migration during development is not a simple behavior but is often combined with other cellular and tissue processes. In addition, different surrounding environments can also influence migrating cells, further complicating collective cell migration during development. Due to the complexity of developmental processes and tissues, traditional genetic approaches often encounter challenges and limitations. Thus, some methods with spatiotemporal control become urgent in dissecting collective cell migration and tissue morphogenesis during development. Optogenetics is a method that combines optics and genetics, providing a perfect strategy for spatiotemporally controlling corresponding protein activity in subcellular, cellular or tissue levels. In this review, we introduce the basic mechanisms underlying different optogenetic tools. Then, we demonstrate how optogenetic methods have been applied in vivo to dissect collective cell migration and tissue morphogenesis during development. Additionally, we describe some promising optogenetic approaches for advancing this field. Together, this review will guide and facilitate future studies of collective cell migration in vivo and tissue morphogenesis by optogenetics.

Abstract: The endoplasmic reticulum (ER) links to multiple organelles through membrane contact sites (MCS), which play critical roles in signal transduction, cell homeostasis and stress response. However, studying the behaviour and functions of MCS in plants is still challenging, partially due to the lack of site-specific markers. Here, we used an optogenetic reporter, LiMETER (Light-inducible Membrane-Tethered cortical ER), to study the structure and dynamics of ER-PM contact sites (EPCS) in plants. Upon blue light activation, LiMETER is recruited to the EPCS rapidly, while this process is reversible when blue light is turned off. Compared with other EPCS reporters, LiMETER specifically and reversibly labels the contact sites, causing little side-effects on the ER structure and plant development. With its help, we re-examined the formation of ER-PM connections induced by cell-intrinsic factors or extracellular stimuli. We found that EPCSs are preferably localised at ER tubules and the edge of ER cisternae, and their number increased significantly under abiotic stress conditions. The abundance of ER and PM interaction is also developmental dependent, suggesting a direct link between ER-PM interaction, ER function and cell homeostasis. Taken together, we showed that LiMETER is an improved marker for functional and microscopical studies of ER-PM interaction, demonstrating the effectiveness of optogenetic tools in future research.

Abstract: Research for biopharmaceutical production processes with mammalian cells steadily aims to enhance the cell-specific productivity as a means for optimizing total productivities of bioreactors. Whereas current technologies such as pH, temperature, and osmolality shift require modifications of the cultivation medium, the use of optogenetic switches in recombinant producer cells might be a promising contact-free alternative. However, the proper application of optogenetically engineered cells requires a detailed understanding of basic cellular responses of cells that do not yet contain the optogenetic switches. The knowhow of ideal light exposure to enable the optimum use of related approaches is missing so far. Consequently, the current study set out to find optimum conditions for IgG1 producing Chinese hamster ovary (CHO) cells which were exposed to blue LED light. Growth characteristics, cell-specific productivity using enzyme-linked immunosorbent assay, as well as cell cycle distribution using flow cytometry were analyzed. Whereas too harsh light exposure causes detrimental growth effects that could be compensated with antioxidants, a surprising boost of cell-specific productivity by 57% occurred at optimum high light doses. The increase coincided with an increased number of cells in the G1 phase of the cell cycle after 72 h of illumination. The results present a promising new approach to boost biopharmaceutical productivity of mammalian cells simply by proper light exposure without any further optogenetic engineering. KEY POINTS: • Blue LED light hinders growth in CHO DP-12 cells • Antioxidants protect to a certain degree from blue light effects • Illumination with blue LED light raises cell-specific productivity.

Abstract: Microtubule acetylation is implicated in regulating cell motility, yet its physiological role in directional migration and the underlying molecular mechanisms have remained unclear. This knowledge gap has persisted primarily due to a lack of tools capable of rapidly manipulating microtubule acetylation in actively migrating cells. To overcome this limitation and elucidate the causal relationship between microtubule acetylation and cell migration, we developed a novel optogenetic actuator, optoTAT, which enables precise and rapid induction of microtubule acetylation within minutes in live cells. Using optoTAT, we observed striking and rapid responses at both molecular and cellular level. First, microtubule acetylation triggers release of the RhoA activator GEF-H1 from sequestration on microtubules. This release subsequently enhances actomyosin contractility and drives focal adhesion maturation. These subcellular processes collectively promote sustained directional cell migration. Our findings position GEF-H1 as a critical molecular responder to microtubule acetylation in the regulation of directed cell migration, revealing a dynamic crosstalk between the actin and microtubule cytoskeletal networks.

Abstract: Morphogen gradients convey essential spatial information during tissue patterning. While both concentration and timing of morphogen exposure are crucial, how cells interpret these graded inputs remains challenging to address. We employed an optogenetic system to acutely and reversibly modulate the nuclear concentration of the morphogen Dorsal (DL), homologue of NF-κB, which orchestrates dorso-ventral patterning in the Drosophila embryo. By controlling DL nuclear concentration while simultaneously recording target gene outputs in real time, we identified a critical window for DL action that is required to instruct patterning, and characterized the resulting effect on spatio-temporal transcription of target genes in terms of timing, coordination, and bursting. We found that a transient decrease in nuclear DL levels at nuclear cycle 13 leads to reduced expression of the mesoderm-associated gene snail (sna) and partial derepression of the neurogenic ectoderm-associated target short gastrulation (sog) in ventral regions. Surprisingly, the mispatterning elicited by this transient change in DL is detectable at the level of single cell transcriptional bursting kinetics, specifically affecting long inter-burst durations. Our approach of using temporally-resolved and reversible modulation of a morphogen in vivo, combined with mathematical modeling, establishes a framework for understanding the stimulus-response relationships that govern embryonic patterning.

Abstract: CRISPR-Cas technologies have revolutionized life sciences by enabling programmable genome editing across diverse organisms. Achieving dynamic and precise control over CRISPR-Cas activity with exogenous triggers, such as light or chemical ligands, remains an important need. Existing tools for CRISPR-Cas control are often limited to specific Cas orthologs or selected applications, restricting their versatility. Anti-CRISPR (Acr) proteins, natural inhibitors of CRISPR-Cas systems, provide a flexible regulatory layer but are constitutively active in their native forms. In this study, we built on our previously reported concept for optogenetic CRISPR-Cas control with engineered, light-switchable anti-CRISPR proteins and expanded it from ortholog-specific Acrs towards AcrIIA5 and AcrVA1, broad-spectrum inhibitors of CRISPR-Cas9 and -Cas12a, respectively. We then conceived and implemented a novel, chemogenetic anti-CRISPR platform based on engineered, circularly permuted ligand receptor domains of human origin, that together respond to six different, clinically-relevant drugs. The resulting toolbox achieves both optogenetic and chemogenetic control of genome editing in human cells with a wide range of CRISPR-Cas effectors, including type II-A and -C CRISPR-Cas9s, and -Cas12a. In sum, this work establishes a versatile platform for multidimensional control of CRISPR-Cas systems, with immediate applications in basic research and biotechnology and potential for therapeutic use in the future.

Abstract: Light-Oxygen-Voltage (LOV) domains are the protein-based light switches used in nature to trigger and regulate various processes. They allow light signals to be converted into metabolic signaling cascades. Various LOV-domain proteins have been characterized in the last few decades and have been used to develop light-sensitive tools in cell biology research. LOV-based applications exploit the light-driven regulation of effector elements to activate signaling pathways, activate genes, or locate proteins within cells. A relatively new application of an engineered small LOV-domain protein called miniSOG (mini singlet oxygen generator) is based on the light-induced formation of reactive oxygen species (ROS). The first miniSOG was engineered from a LOV domain from Arabidopsis thaliana. This engineered 14 kDa light-responsive flavin-containing protein can be exploited as protein tag for the light-triggered localized production of ROS. Such tunable ROS production by miniSOG or similarly redesigned LOV-domains can be of use in studies focused on subcellular phenomena but may also allow new light-fueled catalytic processes. This review provides an overview of the discovery of LOV domains and their development into tools for cell biology. It also highlights recent advancements in engineering LOV domains for various biotechnological applications and cell biology studies.

Abstract: Studies utilizing electron microscopy and live fluorescence microscopy have significantly enhanced our understanding of the molecular mechanisms that regulate junctional dynamics during homeostasis, development and disease. To fully grasp the enormous complexity of cell-cell adhesions, it is crucial to study the nanoscale architectures of tight junctions, adherens junctions and desmosomes. It is important to integrate these junctional architectures with the membrane morphology and cellular topography in which the junctions are embedded. In this Review, we explore new insights from studies using super-resolution and volume electron microscopy into the nanoscale organization of these junctional complexes as well as the roles of the junction-associated cytoskeleton, neighboring organelles and the plasma membrane. Furthermore, we provide an overview of junction- and cytoskeletal-related biosensors and optogenetic probes that have contributed to these advances and discuss how these microscopy tools enhance our understanding of junctional dynamics across cellular environments.

Abstract: Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can repress or activate specific genes, how these functions are implemented at the molecular level has remained elusive, particularly in the endogenous context of developing animals. Here, we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible (~1 min) and memoryless. Furthermore, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.

Abstract: Chimeric antigen receptor T cell therapies have achieved great success in eradicating some liquid tumors, whereas the preclinical results in treating solid tumors have proven less decisive. One of the principal challenges in solid tumor treatment is the physical barrier composed of a dense extracellular matrix, which prevents immune cells from penetrating the tissue to attack intratumoral cancer cells. Here, we improve immune cell infiltration into solid tumors by manipulating septin-7 functions in cells. Using protein allosteric design, we reprogram the three-dimensional structure of septin-7 and insert a blue light-responsive light-oxygen-voltage-sensing domain 2 (LOV2), creating a light-controllable septin-7-LOV2 hybrid protein. Blue light inhibits septin-7 function in live cells, inducing extended cell protrusions and cell polarization, enhancing cell transmigration efficiency through confining spaces. We genetically edited human natural killer cell line (NK92) and mouse primary CD8+ T-cells expressing the engineered protein, and we demonstrated improved penetration and cytotoxicity against various tumor spheroid models. Our proposed strategy to enhance immune cell infiltration is compatible with other methodologies and therefore, could be used in combination to further improve cell-based immunotherapies against solid tumors.

Abstract: Lamellipodia are sheet-like protrusions essential for migration and endocytosis, yet the ultrastructure of the actin cytoskeleton during lamellipodia formation remains underexplored. Here, we combined the optogenetic tool PA-Rac1 with cryo-ET to enable ultrastructural analysis of newly formed lamellipodia. We successfully visualized lamellipodia at various extension stages, representing phases of their formation. In minor extensions, several unbundled actin filaments formed “Minor protrusions” at the leading edge. For moderately extended lamellipodia, cross-linked actin filaments formed small filopodia-like structures, termed “mini filopodia.” In fully extended lamellipodia, filopodia matured at multiple points, and cross-linked actin filaments running nearly parallel to the leading edge increased throughout the lamellipodia. These observations suggest that actin polymerization begins in specific plasma membrane regions, forming mini filopodia that either mature into full filopodia or detach from the leading edge to form parallel filaments. This actin turnover likely drives lamellipodial protrusion, providing new insights into actin dynamics and cell migration.

Abstract: Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway.

Abstract: The human nucleoporin RanBP2/Nup358 interacts with SUMO1-modified RanGAP1 and the SUMO E2 Ubc9 at the nuclear pore complex (NPC) to promote export and disassembly of exportin Crm1/Ran(GTP)/cargo complexes. In mitosis, RanBP2/SUMO1-RanGAP1/Ubc9 remains intact after NPC disassembly and is recruited to kinetochores and mitotic spindles by Crm1 where it contributes to mitotic progression. Interestingly, RanBP2 binds SUMO1-RanGAP1/Ubc9 with motifs that also catalyze SUMO E3 ligase activity. Here, we resolve cryo-EM structures of a RanBP2 C-terminal fragment bound to Crm1, SUMO1-RanGAP1/Ubc9, and two molecules of Ran(GTP), one bound to Crm1 and the other bound to RanGAP1 and RanBP2. These structures reveal several unanticipated interactions with Crm1 including a nuclear export signal (NES) for RanGAP1, the deletion of which mislocalizes RanGAP1 and the Ran GTPase in cells. Our structural and biochemical results support models in which RanBP2 E3 ligase activity is dependent on Crm1, the RanGAP1 NES and Ran GTPase cycling.

Abstract: Many bacteria swim by the rotation of the bacterial flagellar motor (BFM). The BFM is powered by proton translocation across the inner membrane through the hetero-heptameric MotA5MotB2 protein complex. Two periplasmic domains of MotB are critical in activating BFM rotation: (1) the peptidoglycan binding (PGB) domain that anchors MotB in the peptidoglycan layer and (2) the plug domain that modulates the proton flow. Existing cytoplasmic fluorescent probes have been shown to negatively affect motor rotation and switching. Here we inserted a fluorescent probe in the periplasm near the plug of MotB in an attempt to circumvent issues with cytoplasmic probes and for possible use in observing the mechanism of plug-based regulation of proton flow. We inserted green fluorescent protein (GFP) and iLOV, a fluorescent version of the light-oxygen-voltage (LOV) domain, in four periplasmic locations in MotB. Insertions near the plug retained motility but showed limited fluorescence for both fluorophores. Additional short, flexible glycine-serine (GS) linkers improved motility but did not improve brightness. Further optimization is necessary to improve the fluorescence of these periplasmic probes.

Abstract: The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.

Abstract: Biochemical reaction networks adapt to environmental conditions by sensing chemical or physical stimuli and using tightly controlled mechanisms. While most signals come from molecules, many cells can also sense and respond to light. Among the biomolecular structures that enable light sensing, we selected a light-oxygen-voltage (LOV) domain in a previous study that tested the engineering of novel regulatory mechanisms into a nucleic acid polymerase. In this follow-up study, we studied the activities of previously selected variants in kinetic detail, and we generated additional LOV-polymerase fusion variants based on further insertion criteria. Our results provide mechanistic insights into how LOV domain insertion influences polymerase activity in a light-responsive manner: All active and photoresponsive enzyme variants studied by us to date were partially inhibited (i.e., "turned off") after irradiation with blue light at 470 nm, which can be explained by specific obstructions of the polymerase entry or exit structures (substrate entry channels or product exit channels, or both). Although the effects observed are moderate, we anticipate further engineering strategies that could be used to improve the extent of switchability and possibly to develop a "turn-on mode" insertion.

Abstract: In recent decades, the field of synthetic biology has witnessed remarkable progress, driving advances in both research and practical applications. One pivotal area of development involves the design of transgene switches capable of precisely regulating specified outputs and controlling cell behaviors in response to physical cues, which encompass light, magnetic fields, temperature, mechanical forces, ultrasound, and electricity. In this review, we delve into the cutting-edge progress made in the field of physically controlled protein expression in engineered mammalian cells, exploring the diverse genetic tools and synthetic strategies available for engineering targeting cells to sense these physical cues and generate the desired outputs accordingly. We discuss the precision and efficiency limitations inherent in these tools, while also highlighting their immense potential for therapeutic applications.

Abstract: Disintegration of organelle membranes induces various cellular responses and has pathological consequences, including autoinflammatory diseases and neurodegeneration. Establishing methods to induce membrane rupture of organelles of interest is essential to analyze the downstream effects of membrane rupture; however, the spatiotemporal induction of rupture of specific membranes remains challenging. Here, we develop a series of optogenetic tools to induce organelle membrane rupture by using engineered Bcl-2-associated X protein (BAX), whose primary function is to form membrane pores in the outer mitochondrial membrane (OMM) during apoptosis. When BAX is forced to target mitochondria, lysosomes, or the endoplasmic reticulum (ER) by replacing its C-terminal transmembrane domain (TMD) with organelle-targeting sequences, the BAX mutants rupture their target membranes. To regulate the activity of organelle-targeted BAX, the photosensitive light-oxygen-voltage-sensing 2 (LOV2) domain is fused to the N-terminus of BAX. The resulting LOV2–BAX fusion protein exhibits blue light–dependent membrane-rupture activity on various organelles, including mitochondria, the ER, and lysosomes. Thus, LOV2–BAX enables spatiotemporal induction of membrane rupture across a broad range of organelles, expanding research opportunities on the consequences of organelle membrane disruption.

Abstract: Alcohol use disorder (AUD) is a complex condition, and it remains unclear which specific neuronal substrates mediate alcohol-seeking and -taking behaviors. Engram cells and their related ensembles, which encode learning and memory, may play a role in this process. We aimed to assess the precise neural substrates underlying alcohol-seeking and -taking behaviors and determine how they may affect one another.

Abstract: Here we present the Multisite Assembly of Gateway Induced Clones (MAGIC) system, which harnesses site-specific recombination-based cloning via Gateway technology for rapid, modular assembly of between 1 and 3 “Entry” vector components, all into a fourth, standard high copy “Destination” plasmid backbone. The MAGIC toolkit spans a range of in vitro and in vivo uses, from directing tunable gene expression, to driving simultaneous expression of microRNAs and fluorescent reporters, to enabling site-specific recombinase-dependent gene expression. All MAGIC system components are directly compatible with existing multisite gateway Tol2 systems currently used in zebrafish, as well as existing eukaryotic cell culture expression Destination plasmids, and available mammalian lentiviral and adenoviral Destination vectors, allowing rapid cross-species experimentation. Moreover, herein we describe novel vectors with flanking piggyBac transposon elements for stable genomic integration in vitro or in vivo when used with piggyBac transposase. Collectively, the MAGIC system facilitates transgenesis in cultured mammalian cells, electroporated mouse and chick embryos, as well as in injected zebrafish embryos, enabling the rapid generation of innovative DNA constructs for biological research due to a shared, common plasmid platform.

Abstract: Optogenetics' advancement has made light induction attractive for controlling biological processes due to its advantages of fine-tunability, reversibility, and low toxicity. The lactose operon induction system, commonly used in Escherichia coli, relies on the binding of lactose or isopropyl β-d-1-thiogalactopyranoside (IPTG) to the lactose repressor protein LacI, playing a pivotal role in controlling the lactose operon. Here, we harnessed the light-responsive light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1 as a tool for light control and engineered LacI into two light-responsive variants, OptoLacIL and OptoLacID. These variants exhibit direct responsiveness to light and darkness, respectively, eliminating the need for IPTG. Building upon OptoLacI, we constructed two light-controlled E. coli gene expression systems, OptoE.coliLight system and OptoE.coliDark system. These systems enable bifunctional gene expression regulation in E. coli through light manipulation and show superior controllability compared to IPTG-induced systems. We applied the OptoE.coliDark system to protein production and metabolic flux control. Protein production levels are comparable to those induced by IPTG. Notably, the titers of dark-induced production of 1,3-propanediol (1,3-PDO) and ergothioneine exceeded 110% and 60% of those induced by IPTG, respectively. The development of OptoLacI will contribute to the advancement of the field of optogenetic protein engineering, holding substantial potential applications across various fields.

Abstract: Diabetes mellitus (DM) is a common chronic disease affecting humans globally. It is characterized by abnormally elevated blood glucose levels due to the failure of insulin production or reduction of insulin sensitivity and functionality. Insulin and glucagon-like peptide (GLP)-1 replenishment or improvement of insulin resistance are the two major strategies to treat diabetes. Recently, optogenetics that uses genetically encoded light-sensitive proteins to precisely control cell functions has been regarded as a novel therapeutic strategy for diabetes. Here, we summarize the latest development of optogenetics and its integration with synthetic biology approaches to produce light-responsive cells for insulin/GLP-1 production, amelioration of insulin resistance and neuromodulation of insulin secretion. In addition, we introduce the development of cell encapsulation and delivery methods and smart bioelectronic devices for the in vivo application of optogenetics-based cell therapy in diabetes. The remaining challenges for optogenetics-based cell therapy in the clinical translational study are also discussed.

Abstract: Hydrophilic and biocompatible hydrogels are widely applied as ideal scaffolds in tissue engineering. The "smart" gelation material can alter its structural, physiochemical, and functional features in answer to various endo/exogenous stimuli to better biomimic the endogenous extracellular matrix for the engineering of cells and tissues. Light irradiation owns a high spatial-temporal resolution, complete biorthogonal reactivity, and fine-tunability and can thus induce physiochemical reactions within the matrix of photoresponsive hydrogels with good precision, efficiency, and safety. Both gel structure (e.g., geometry, porosity, and dimension) and performance (like conductivity and thermogenic or mechanical properties) can hence be programmed on-demand to yield the biochemical and biophysical signals regulating the morphology, growth, motility, and phenotype of engineered cells and tissues. Here we summarize the strategies and mechanisms for encoding light-reactivity into a hydrogel and demonstrate how fantastically such responsive gels change their structure and properties with light irradiation as desired and thus improve their applications in tissue engineering including cargo delivery, dynamic three-dimensional cell culture, and tissue repair and regeneration, aiming to provide a basis for more and better translation of photoresponsive hydrogels in the clinic.

Abstract: The cell-cell adhesion molecule E-cadherin has been intensively studied due to its prevalence in tissue function and its spatiotemporal regulation during epithelial-to-mesenchymal cell transition. Nonetheless, regulating and studying the dynamics of it has proven challenging. We developed a photoswitchable version of E-cadherin, named opto-E-cadherin, which can be toggled OFF with blue light illumination and back ON in the dark. Herein, we describe easy-to-use methods to test and characterise opto-E- cadherin cell clones for downstream experiments. Key features • This protocol describes how to implement optogenetic cell-cell adhesion molecules effectively (described here on the basis of opto-E-cadherin), while highlighting possible pitfalls. • Utilises equipment commonly found in most laboratories with high ease of use. • Phenotype screening is easy and done within a few hours (comparison of cell clusters in the dark vs. blue light in an aggregation assay). • Three different functionality assay systems are described. • After the cell line is established, all experiments can be performed within three days.