Curated Optogenetic Publication Database

Abstract: Upconversion-mediated optogenetics is an emerging powerful technique to remotely control and manipulate the deep-tissue protein functions and signaling pathway activation. This technique uses lanthanide upconversion nanoparticles (UCNPs) as light transducers and through near-infrared light to indirectly activate the traditional optogenetic proteins. With the merits of high spatiotemporal resolution and minimal invasiveness, this technique enables cell-type specific manipulation of cellular activities in deep tissues as well as in living animals. In this review, we introduce the latest development of optogenetic modules and UCNPs, with emphasis on the integration of UCNPs with cellular optogenetics and their biomedical applications on the control of neural/brain activity, cancer therapy and cardiac optogenetics in vivo. Furthermore, we analyze the current developed strategies to optimize and advance the upconversion-mediated optogenetics and discuss the remaining challenges of its further applications in biomedical study and clinical translational research. STATEMENT OF SIGNIFICANCE: Optogenetics harnesses photoactivatable proteins to optically stimulate and control intracellular activities. UCNPs-mediated NIR-activatable optogenetics uses lanthanide upconversion nanoparticles (UCNPs) as light transducers and utilizes near-infrared (NIR) light to indirectly activate the traditional optogenetic proteins. The integration of UCNPs with cellular optogenetics has showed great promise in biomedical applications in regulating neural/brain activity, cancer therapy and cardiac optogenetics in vivo. The evolution and optimization of functional UCNPs and the discovery and engineering of novel optogenetic modules would both contribute to the advance of such unique hybrid technology, which may lead to discoveries in biomedical research and provide new treatments for human diseases.

Abstract: Genetically encoded tools for the regulation of endogenous molecules (RNA, DNA elements and protein) are needed to study and control biological processes with minimal interference caused by protein overexpression and overactivation of signaling pathways. Here we focus on light-controlled optogenetic tools (OTs) that allow spatiotemporally precise regulation of gene expression and protein function. To control endogenous molecules, OTs combine light-sensing modules from natural photoreceptors with specific protein or nucleic acid binders. We discuss OT designs and group OTs according to the principles of their regulation. We outline characteristics of OT performance, discuss considerations for their use in vivo and review available OTs and their applications in cells and in vivo. Finally, we provide a brief outlook on the development of OTs.

Abstract: Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely, the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in S. cerevisiae to perturb both intercellular and interspecies interactions. IMPORTANCE Recent advances in microbial ecology have highlighted the importance of intercellular interactions in controlling the development, composition, and resilience of microbial communities. In order to better understand the role of these interactions in governing community development, it is critical to be able to alter them in a controlled manner. Optogenetically controlled interactions offer advantages over static perturbations or chemically controlled interactions, as light can be manipulated in space and time and does not require the addition of nutrients or antibiotics. Here, we report a system for rapidly achieving light control of a gene of interest in the important model organism Saccharomyces cerevisiae and demonstrate that by controlling expression of the enzyme invertase, we can control cooperative interactions. This approach will be useful for understanding intercellular and interspecies interactions in natural and synthetic microbial consortia containing S. cerevisiae and serves as a proof of principle for implementing this approach in other consortia.

Abstract: Biological information can be encoded within the dynamics of signaling components, which has been implicated in a broad range of physiological processes including stress response, oncogenesis, and stem cell differentiation. To study the complexity of information transfer across the eukaryotic promoter, we screened 119 dynamic conditions-modulating the pulse frequency, amplitude, and pulse width of light-regulating the binding of an epigenome editor to a fluorescent reporter. This system revealed tunable gene expression and filtering behaviors and provided a quantification of the limit to the amount of information that can be reliably transferred across a single promoter as ∼1.7 bits. Using a library of over 100 orthogonal chromatin regulators, we further determined that chromatin state could be used to tune mutual information and expression levels, as well as completely alter the input-output transfer function of the promoter. This system unlocks the information-rich content of eukaryotic gene regulation.

Abstract: Cytokinesis is required to cleave the daughter cells at the end of mitosis and relies on the spatiotemporal control of RhoA GTPase. Cytokinesis failure can lead to changes in cell fate or aneuploidy, which can be detrimental during development and/or can lead to cancer. However, our knowledge of the pathways that regulate RhoA during cytokinesis is limited, and the role of other Rho family GTPases is not clear. This is largely because the study of Rho GTPases presents unique challenges using traditional cell biological and biochemical methods, and they have pleiotropic functions making genetic studies difficult to interpret. The recent generation of optogenetic tools and biosensors that control and detect active Rho has overcome some of these challenges and is helping to elucidate the role of RhoA in cytokinesis. However, improvements are needed to reveal the role of other Rho GTPases in cytokinesis, and to identify the molecular mechanisms that control Rho activity. This review examines some of the outstanding questions in cytokinesis, and explores tools for the imaging and control of Rho GTPases.

Abstract: Migratory cells navigate through crowded 3D microenvironments in vivo. Amoeboid cells, such as immune cells and some cancer cells, are thought to do so by deforming their bodies to squeeze through tight spaces.1 Yet large populations of nearly spherical amoeboid cells migrate2–4 in microenvironments too dense5,6 to move through without extensive shape deformations. How they do so is unknown. We used high-resolution light-sheet microscopy to visualize metastatic melanoma cells in dense environments, finding that cells maintain a round morphology as they migrate and create a path through which to move via bleb-driven mechanical degradation and subsequent macropinocytosis of extracellular matrix components. Proteolytic degradation of the extracellular matrix via matrix metalloproteinases is not required. Membrane blebs are short-lived relative to the timescale of migration, and thus persistence in their polarization is critical for productive ablation of the extracellular matrix. Interactions between small but long-lived cortical adhesions and collagen at the cell front induce PI-3 Kinase signaling that drive bleb enlargement via branched actin polymerization. Large blebs in turn abrade collagen, creating a feedback between extracellular matrix structure, cell morphology, and cell polarization that results in both path generation and persistent cell movement.

Abstract: The last stage of cell division involves two daughter cells remaining interconnected by a cytokinetic bridge that is cleaved in a process called abscission. During pre-abscission, we identified that the centrosome moves in a Rab11-dependent manner towards the cytokinetic bridge in human cells grown in culture and in an in vivo vertebrate model, Danio rerio (zebrafish). Rab11-endosomes are dynamically organized in a Rab11-GTP dependent manner at the centrosome during pre-abscission and this organization is required for the centrosome protein, pericentrin, to be enriched at the centrosome. Using zebrafish embryos, we found that reduction in pericentrin expression or optogenetically disrupting Rab11-endosome function inhibited centrosome movement towards the cytokinetic bridge and abscission resulting in daughter cells prone to being binucleated and/or having supernumerary centrosomes. These studies suggest that Rab11-endosomes contribute to centrosome function during pre-abscission by regulating pericentrin organization resulting in appropriate centrosome movement towards the cytokinetic bridge and subsequent abscission.

Abstract: The ability to stimulate mammalian cells with light, brought along by optogenetic control, has significantly broadened our understanding of electrically excitable tissues. Backed by advanced (bio)materials, it has recently paved the way towards novel biosensing concepts supporting bio-analytics applications transversal to the main biomedical stream. The advancements concerning enabling biomaterials and related novel biosensing concepts involving optogenetics are reviewed with particular focus on the use of engineered cells for cell-based sensing platforms and the available toolbox (from mere actuators and reporters to novel multifunctional opto-chemogenetic tools) for optogenetic-enabled real-time cellular diagnostics and biosensor development. The key advantages of these modified cell-based biosensors concern both significantly faster (minutes instead of hours) and higher sensitivity detection of low concentrations of bioactive/toxic analytes (below the threshold concentrations in classical cellular sensors) as well as improved standardization as warranted by unified analytic platforms. These novel multimodal functional electro-optical label-free assays are reviewed among the key elements for optogenetic-based biosensing standardization. This focused review is a potential guide for materials researchers interested in biosensing based on light-responsive biomaterials and related analytic tools.

Abstract: Lipids are highly dynamic molecules that, due to their hydrophobicity, are spatially confined to membrane environments. From these locations, certain privileged lipids serve as signaling molecules. For understanding the biological functions of subcellular pools of signaling lipids, induced proximity tools have been invaluable. These methods involve controlled heterodimerization, by either small-molecule or light triggers, of functional proteins. In the arena of lipid signaling, induced proximity tools can recruit lipid-metabolizing enzymes to manipulate lipid signaling and create artificial tethers between organelle membranes to control lipid trafficking pathways at membrane contact sites. Here, we review recent advances in methodology development and biological application of chemical-induced and light-induced proximity tools for manipulating lipid metabolism, trafficking, and signaling.

Abstract: Distinct patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar-polarized pattern of myosin II localization regulated by Rho1 signaling during Drosophila body axis elongation is thought to drive cell behaviors that contribute to convergent extension. However, it is not well understood how specific aspects of a myosin pattern influence the multiple cell behaviors, including cell intercalation, cell shape changes, and apical cell area fluctuations, that simultaneously occur during morphogenesis. Here, we developed two optogenetic tools, optoGEF and optoGAP, to activate or deactivate Rho1 signaling, respectively. We used these tools to manipulate myosin patterns at the apical side of the germband epithelium during Drosophila axis elongation and analyzed the effects on contractile cell behaviors. We show that uniform activation or inactivation of Rho1 signaling across the apical surface of the germband is sufficient to disrupt the planar-polarized pattern of myosin at cell junctions on the timescale of 3-5 min, leading to distinct changes in junctional and medial myosin patterns in optoGEF and optoGAP embryos. These two perturbations to Rho1 activity both disrupt axis elongation and cell intercalation but have distinct effects on cell area fluctuations and cell packings that are linked with changes in the medial and junctional myosin pools. These studies demonstrate that acute optogenetic perturbations to Rho1 activity are sufficient to rapidly override the endogenous planar-polarized myosin pattern in the germband during axis elongation. Moreover, our results reveal that the levels of Rho1 activity and the balance between medial and junctional myosin play key roles not only in organizing the cell rearrangements that are known to directly contribute to axis elongation but also in regulating cell area fluctuations and cell packings, which have been proposed to be important factors influencing the mechanics of tissue deformation and flow.

Abstract: The field of optogenetics is rapidly growing in relevance and number of developed tools. Amongst other things, the optogenetic repertoire includes light-responsive ion channels and methods for gene regulation. This review will be confined to the optogenetic control of gene expression in mammalian cells as suitable models for clinical applications. Here optogenetic gene regulation might offer an excellent method for spatially and timely regulated gene and protein expression in cell therapeutic approaches. Well-known systems for gene regulation, such as the LOV-, CRY2/CIB-, PhyB/PIF-systems, as well as other, in mammalian cells not yet fully established systems will be described. Advantages and disadvantages with regard to clinical applications are outlined in detail. Among the many unanswered questions concerning the application of optogenetics, we discuss items such as the use of exogenous chromophores and their effects on the biology of the cells and methods for a gentle, but effective gene transfection method for optogenetic tools for in vivo applications. This article is protected by copyright. All rights reserved.

Abstract: Many organs and tissues have an intrinsic ability to regenerate from a dedicated, tissue-specific stem cell pool. As organisms age, the process of self-regulation or homeostasis begins to slow down with fewer stem cells available for tissue repair. Tissues become more fragile and organs less efficient. This slowdown of homeostatic processes leads to the development of cellular and neurodegenerative diseases. In this review, we highlight the recent use and future potential of optogenetic approaches to study homeostasis. Optogenetics uses photosensitive molecules and genetic engineering to modulate cellular activity in vivo, allowing precise experiments with spatiotemporal control. We look at applications of this technology for understanding the mechanisms governing homeostasis and degeneration as applied to widely used model organisms, such as Drosophila melanogaster, where other common tools are less effective or unavailable.

Abstract: Cell–cell adhesions are fundamental in regulating multicellular behavior and lie at the center of many biological processes from embryoid development to cancer development. Therefore, controlling cell–cell adhesions is fundamental to gaining insight into these phenomena and gaining tools that would help in the bioartificial construction of tissues. For addressing biological questions as well as bottom-up tissue engineering the challenge is to have multiple cell types self-assemble in parallel and organize in a desired pattern from a mixture of different cell types. Ideally, different cell types should be triggered to self-assemble with different stimuli without interfering with the other and different types of cells should sort out in a multicellular mixture into separate clusters. In this chapter, we will summarize the developments in photoregulation cell–cell adhesions using non-neuronal optogenetics. Among the concepts, we will cover is the control of homophylic and heterophilic cell–cell adhesions, the independent control of two different types with blue or red light and the self-sorting of cells into distinct structures and the importance of cell–cell adhesion dynamics. These tools will give an overview of how the spatiotemporal regulation of cell–cell adhesion gives insight into their role and how tissues can be assembled from cells as the basic building block.

Abstract: Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the relation between mitochondrial morphologies and functions are unclear due to the lack of methods to precisely exert mechano-stimulation on and deform mitochondria inside live cells. Here, we present an optogenetic approach that uses light to induce deformation of mitochondria by recruiting molecular motors to the outer mitochondrial membrane via light-activated protein-protein hetero-dimerization. Mechanical forces generated by motor proteins distort the outer membrane, during which the inner mitochondrial membrane can also be deformed. Moreover, this optical method can achieve subcellular spatial precision and be combined with different optical dimerizers and molecular motors. This method presents a mitochondria-specific mechano-stimulator for studying mitochondria mechanobiology and the interplay between mitochondria shapes and functions.

Abstract: Cell death events continuously challenge epithelial barrier function yet are crucial to eliminate old or critically damaged cells. How such apoptotic events are spatio-temporally organized to maintain epithelial homeostasis remains unclear. We observe waves of extracellular-signal-regulated kinase (ERK) and AKT serine/threonine kinase (Akt) activity pulses that originate from apoptotic cells and propagate radially to healthy surrounding cells. This requires epidermal growth factor receptor (EGFR) and matrix metalloproteinase (MMP) signaling. At the single-cell level, ERK/Akt waves act as spatial survival signals that locally protect cells in the vicinity of the epithelial injury from apoptosis for a period of 3-4 h. At the cell population level, ERK/Akt waves maintain epithelial homeostasis (EH) in response to mild or intense environmental insults. Disruption of this spatial signaling system results in the inability of a model epithelial tissue to ensure barrier function in response to environmental insults.

Abstract: Ischemia-reperfusion injury is a major cause of acute kidney injury. Recent studies on the pathophysiology of ischemia-reperfusion-induced acute kidney injury showed that immunologic responses significantly affect kidney ischemia-reperfusion injury and repair. Nuclear factor (NF)-ĸB signaling, which controls cytokine production and cell survival, is significantly involved in ischemia-reperfusion-induced acute kidney injury, and its inhibition can ameliorate ischemic acute kidney injury. Using EXPLOR, a novel, optogenetically engineered exosome technology, we successfully delivered the exosomal super-repressor inhibitor of NF-ĸB (Exo-srIĸB) into B6 wild type mice before/after kidney ischemia-reperfusion surgery, and compared outcomes with those of a control exosome (Exo-Naïve)-injected group. Exo-srIĸB treatment resulted in lower levels of serum blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin in post-ischemic mice than in the Exo-Naïve treatment group. Systemic delivery of Exo-srIĸB decreased NF-ĸB activity in post-ischemic kidneys and reduced apoptosis. Post-ischemic kidneys showed decreased gene expression of pro-inflammatory cytokines and adhesion molecules with Exo-srIĸB treatment as compared with the control. Intravital imaging confirmed the uptake of exosomes in neutrophils and macrophages. Exo-srIĸB treatment also significantly affected post-ischemic kidney immune cell populations, lowering neutrophil, monocyte/macrophage, and T cell frequencies than those in the control. Thus, modulation of NF-ĸB signaling through exosomal delivery can be used as a novel therapeutic method for ischemia-reperfusion-induced acute kidney injury.

Abstract: Materials in nature have fascinating properties that serve as a continuous source of inspiration for materials scientists. Accordingly, bio-mimetic and bio-inspired approaches have yielded remarkable structural and functional materials for a plethora of applications. Despite these advances, many properties of natural materials remain challenging or yet impossible to incorporate into synthetic materials. Natural materials are produced by living cells, which sense and process environmental cues and conditions by means of signaling and genetic programs, thereby controlling the biosynthesis, remodeling, functionalization, or degradation of the natural material. In this context, synthetic biology offers unique opportunities in materials sciences by providing direct access to the rational engineering of how a cell senses and processes environmental information and translates them into the properties and functions of materials. Here, we identify and review two main directions by which synthetic biology can be harnessed to provide new impulses for the biologization of the materials sciences: first, the engineering of cells to produce precursors for the subsequent synthesis of materials. This includes materials that are otherwise produced from petrochemical resources, but also materials where the bio-produced substances contribute unique properties and functions not existing in traditional materials. Second, engineered living materials that are formed or assembled by cells or in which cells contribute specific functions while remaining an integral part of the living composite material. We finally provide a perspective of future scientific directions of this promising area of research and discuss science policy that would be required to support research and development in this field.

Abstract: Optogenetics uses light-inducible protein-protein interactions to precisely control the timing, localization, and intensity of signaling activity. The precise spatial and temporal resolution of this emerging technology has proven extremely attractive to the study of embryonic development, a program faithfully replicated to form the same organism from a single cell. We have previously performed a comparative study for optogenetic activation of receptor tyrosine kinases, where we found that the cytoplasm-to-membrane translocation-based optogenetic systems outperform the membrane-anchored dimerization systems in activating the receptor tyrosine kinase signaling in live Xenopus embryos. Here, we determine if this engineering strategy can be generalized to other signaling pathways involving membrane-bound receptors. As a proof of concept, we demonstrate that the cytoplasm-to-membrane translocation of the low-density lipoprotein receptor-related protein-6 (LRP6), a membrane-bound coreceptor for the canonical Wnt pathway, triggers Wnt activity. Optogenetic activation of LRP6 leads to axis duplication in developing Xenopus embryos, indicating that the cytoplasm-to-membrane translocation of the membrane-bound receptor could be a generalizable strategy for the construction of optogenetic systems.

Abstract: Hundreds of genes interact with the yeast nuclear pore complex (NPC), localizing at the nuclear periphery and clustering with co-regulated genes. Dynamic tracking of peripheral genes shows that they cycle on and off the NPC and that interaction with the NPC slows their sub-diffusive movement. Furthermore, NPC-dependent inter-chromosomal clustering leads to coordinated movement of pairs of loci separated by hundreds of nanometers. We developed fractional Brownian motion simulations for chromosomal loci in the nucleoplasm and interacting with NPCs. These simulations predict the rate and nature of random sub-diffusion during repositioning from nucleoplasm to periphery and match measurements from two different experimental models, arguing that recruitment to the nuclear periphery is due to random sub-diffusion and transient capture by NPCs. Finally, the simulations do not lead to inter-chromosomal clustering or coordinated movement, suggesting that interaction with the NPC is necessary, but not sufficient, to cause clustering.

Abstract: Biological signals are sensed by their respective receptors and are transduced and processed by a sophisticated intracellular signaling network leading to a signal-specific cellular response. Thereby, the response to the signal depends on the strength, the frequency, and the duration of the stimulus as well as on the subcellular signal progression. Optogenetic tools are based on genetically encoded light-sensing proteins facilitating the precise spatiotemporal control of signal transduction pathways and cell fate decisions in the absence of natural ligands. In this review, we provide an overview of optogenetic approaches connecting light-regulated protein-protein interaction or caging/uncaging events with steering the function of signaling proteins. We briefly discuss the most common optogenetic switches and their mode of action. The main part deals with the engineering and application of optogenetic tools for the control of transmembrane receptors including receptor tyrosine kinases, the T cell receptor and integrins, and their effector proteins. We also address the hallmarks of optogenetics, the spatial and temporal control of signaling events.

Abstract: Abstract not available.

Abstract: Transient receptor potential canonical type 5 (TRPC5) ion channels are expressed in the brain and kidney, and have been identified as promising therapeutic targets whose selective inhibition can protect against diseases driven by a leaky kidney filter, such as Focal Segmental Glomerular Sclerosis (FSGS). TRPC5 channels are activated by elevated levels of extracellular Ca2+or lanthanide ions, but also by G protein (Gq/11) stimulation. Phosphatidylinositol bisphosphate (PIP2) hydrolysis by phospholipase C (PLC) enzymes leads to protein kinase C (PKC)-mediated phosphorylation of TRPC5 channels and their subsequent desensitization. However, the roles of PIP2 in activation and maintenance of TRPC5 channel activity via its hydrolysis product diacyl glycerol (DAG), as well as the mechanism of desensitization of TRPC5 activity by DAG-stimulated PKC activity remain unclear. Here, we designed experiments to distinguish between the processes underlying channel activation and inhibition. Using whole-cell patch clamp, we employed an optogenetic tool to dephosphorylate PIP2 and assess channel-PIP2 interactions influenced by activators, such as DAG, or inhibitors, such as PKC phosphorylation. Using total internal reflection microscopy, we assessed channel cell surface density. We show that PIP2 controls both the PKC-mediated inhibition as well as the DAG- and lanthanide-mediated activation of TRPC5 currents via control of gating rather than channel cell surface density. These mechanistic insights promise to aid in the development of more selective and precise inhibitors to block TRPC5 channel activity, and to illuminate new opportunities for targeted therapies for a group of chronic kidney diseases for which there is currently a great unmet need.

Abstract: Interaction of talin with the cytoplasmic tails of integrin β triggers integrin activation, leading to an increase of integrin affinity/avidity for extracellular ligands. In talin knockout mice, loss of talin interaction with platelet integrin αIIbβ3 causes a severe hemostatic defect, and loss of talin interaction with endothelial cell integrin αVβ3 affects angiogenesis. In normal cells, talin is auto-inhibited and localized in the cytoplasm. Here we employed an optogenetic platform to assess whether recruitment of full-length talin to the plasma membrane was sufficient to induce integrin activation. A dimerization module (CRY2 fused to the N-terminus of talin; CIBN-CAAX) responsive to 450 nm (blue) light was inserted into CHO cells and endothelial cells also expressing αIIbβ3 or αVβ3, respectively. Thus, exposure of the cells to blue light caused a rapid and reversible recruitment of CRY2-talin to the CIBN-CAAX-decorated plasma membrane. This resulted in β3 integrin activation in both cell types, as well as increasing migration of the endothelial cells. However, membrane recruitment of talin was not sufficient for integrin activation, as membrane-associated Rap1-GTP was also required. Moreover, talin mutations that interfered with its direct binding to Rap1 abrogated β3 integrin activation. Altogether, these results define a role for the plasma membrane recruitment of talin in β3 integrin activation, and they suggest a nuanced sequence of events thereafter involving Rap1-GTP.

Abstract: Dynamic membraneless compartments formed by protein condensates have multifunctional roles in cellular biology. Tools that inducibly trigger condensate formation have been useful for exploring their cellular function, however, there are few tools that provide inducible control over condensate disruption. To address this need we developed DisCo (Disassembly of Condensates), which relies on the use of chemical dimerizers to inducibly recruit a ligand to the condensate-forming protein, triggering condensate dissociation. We demonstrate use of DisCo to disrupt condensates of FUS, associated with amyotrophic lateral sclerosis, and to prevent formation of polyglutamine-containing huntingtin condensates, associated with Huntington's disease. In addition, we combined DisCo with a tool to induce condensates with light, CRY2olig, achieving bidirectional control of condensate formation and disassembly using orthogonal inputs of light and rapamycin. Our results demonstrate a method to manipulate condensate states that will have broad utility, enabling better understanding of the biological role of condensates in health and disease.

Abstract: Cells receive enormous amounts of information from their environment. How they act on this information-by migrating, expressing genes, or relaying signals to other cells-comprises much of the regulatory and self-organizational complexity found across biology. The "parts list" involved in cell signaling is generally well established, but how do these parts work together to decode signals and produce appropriate responses? This fundamental question is increasingly being addressed with optogenetic tools: light-sensitive proteins that enable biologists to manipulate the interaction, localization, and activity state of proteins with high spatial and temporal precision. In this review, we summarize how optogenetics is being used in the pursuit of an answer to this question, outlining the current suite of optogenetic tools available to the researcher and calling attention to studies that increase our understanding of and improve our ability to engineer biology. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.