Curated Optogenetic Publication Database

Abstract: Fundamental cellular properties are determined by the repertoire and abundance of proteins displayed on the cell surface. As such, the trafficking mechanisms for establishing and maintaining the surface proteome must be tightly regulated for cells to respond appropriately to extracellular cues, yet plastic enough to adapt to ever-changing environments. Not only are the identity and abundance of surface proteins critical, but in many cases, their regulated spatial positioning within surface nanodomains can greatly impact their function. In the context of neuronal cell biology, surface levels and positioning of ion channels and neurotransmitter receptors play essential roles in establishing important properties, including cellular excitability and synaptic strength. Here we review our current understanding of the trafficking pathways that control the abundance and localization of proteins important for synaptic function and plasticity, as well as recent technological advances that are allowing the field to investigate protein trafficking with increasing spatiotemporal precision.

Abstract: End-binding proteins (EBs) are adaptors that recruit functionally diverse microtubule plus-end-tracking proteins (+TIPs) to growing microtubule plus ends. To test with high spatial and temporal accuracy how, when and where +TIP complexes contribute to dynamic cell biology, we developed a photo-inactivated EB1 variant (π-EB1) by inserting a blue-light-sensitive protein–protein interaction module between the microtubule-binding and +TIP-binding domains of EB1. π-EB1 replaces endogenous EB1 function in the absence of blue light. By contrast, blue-light-mediated π-EB1 photodissociation results in rapid +TIP complex disassembly, and acutely and reversibly attenuates microtubule growth independent of microtubule end association of the microtubule polymerase CKAP5 (also known as ch-TOG and XMAP215). Local π-EB1 photodissociation allows subcellular control of microtubule dynamics at the second and micrometre scale, and elicits aversive turning of migrating cancer cells. Importantly, light-mediated domain splitting can serve as a template to optically control other intracellular protein activities.

Abstract: We developed a novel optogenetic tool, SxIP-improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein-dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes.

Abstract: Cells receive diverse signaling cues from their environment that trigger cascades of biochemical reactions in a dynamic manner. Single-cell imaging technologies have revealed that not only molecular species but also dynamic patterns of signaling inputs determine the fates of signal-receiving cells; however it has been challenging to elucidate how such dynamic information is delivered and decoded in complex networks of inter-cellular and inter-molecular interactions. The recent development of optogenetic technology with photo-sensitive proteins has changed this situation; the combination of microscopy and optogenetics provides fruitful insights into the mechanism of dynamic information processing at the single-cell level. Here, we review recent efforts to visualize the flows of dynamic patterns in signaling pathways, which utilize methods integrating single-cell imaging and optogenetics.

Abstract: Rac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble because of the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs and possibly GAPs, downstream effectors, and other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms.

Abstract: Mammalian fertilization relies on sperm finding the egg and penetrating the egg vestments. All steps in a sperm's lifetime crucially rely on changes in the second messenger cAMP (cyclic adenosine monophosphate). In recent years, it has become clear that signal transduction in sperm is not a continuum, but rather organized in subcellular domains, e.g. the sperm head and the sperm flagellum, with the latter being further separated into the midpiece, principal piece, and endpiece. To understand the underlying signaling pathways controlling sperm function in more detail, experimental approaches are needed that allow to study sperm signaling with spatial and temporal precision. Here, we will give a comprehensive overview on cAMP signaling in mammalian sperm, describing the molecular players involved in these pathways and the sperm functions that are controlled by cAMP. Furthermore, we will highlight recent advances in analyzing and manipulating sperm signaling with spatio-temporal precision using light.

Abstract: Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed blue light-induced chromatin recruitment (BLInCR) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a cluster of reporter genes in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ∼2 min after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models of transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.This article has an associated First Person interview with the first author of the paper.

Abstract: The biophysical framework of collective cell migration has been extensively investigated in recent years; however, it remains elusive how chemical inputs from neighboring cells are integrated to coordinate the collective movement. Here, we provide evidence that propagation waves of extracellular signal-related kinase (ERK) mitogen-activated protein kinase activation determine the direction of the collective cell migration. A wound-healing assay of Mardin-Darby canine kidney (MDCK) epithelial cells revealed two distinct types of ERK activation wave, a "tidal wave" from the wound, and a self-organized "spontaneous wave" in regions distant from the wound. In both cases, MDCK cells collectively migrated against the direction of the ERK activation wave. The inhibition of ERK activation propagation suppressed collective cell migration. An ERK activation wave spatiotemporally controlled actomyosin contraction and cell density. Furthermore, an optogenetic ERK activation wave reproduced the collective cell migration. These data provide new mechanistic insight into how cells sense the direction of collective cell migration.

Abstract: Although the cAMP-dependent protein kinase (PKA) is ubiquitously expressed, it is sequestered at specific subcellular locations throughout the cell, thereby resulting in compartmentalized cellular signaling that triggers site-specific behavioral phenotypes. We developed a three-step engineering strategy to construct an optogenetic PKA (optoPKA) and demonstrated that, upon illumination, optoPKA migrates to specified intracellular sites. Furthermore, we designed intracellular spatially segregated reporters of PKA activity and confirmed that optoPKA phosphorylates these reporters in a light-dependent fashion. Finally, proteomics experiments reveal that light activation of optoPKA results in the phosphorylation of known endogenous PKA substrates as well as potential novel substrates.

Abstract: Focal adhesion kinase (FAK) integrates signaling from integrins, growth factor receptors and mechanical stress to control cell adhesion, motility, survival and proliferation. Here, we developed a single-component, photo-activatable FAK, termed optoFAK, by using blue light-induced oligomerization of cryptochrome 2 (CRY2) to activate FAK-CRY2 fusion proteins. OptoFAK functions uncoupled from physiological stimuli and activates downstream signaling rapidly and reversibly upon blue light exposure. OptoFAK stimulates SRC creating a positive feedback loop on FAK activation, facilitating phosphorylation of paxillin and p130Cas in adherent cells. In detached cells or in mechanically stressed adherent cells, optoFAK is autophosphorylated upon exposure to blue light, however, downstream signaling is hampered indicating that the accessibility to these substrates is disturbed. OptoFAK may prove to be a useful tool to study the biological function of FAK in growth factor and integrin signaling, tension-mediated focal adhesion maturation or anoikis and could additionally serve as test system for kinase inhibitors.

Abstract: αVβ3 is reported to promote angiogenesis in some model systems but not in others. Here we used optogenetics to study effects of αVβ3 interaction with the intracellular adapter, kindlin-2, on endothelial cell functions potentially relevant to angiogenesis. Since interaction of kindlin-2 with αVβ3 requires the C-terminal three residues of the β3 cytoplasmic tail (Arg-Gly-Thr; RGT), optogenetic probes LOVpep and ePDZ1 were fused to β3ΔRGT-GFP and mCherry-kindlin2, respectively, and expressed in β3-null microvascular endothelial cells. Exposure of the cells to 450 nm (blue) light caused rapid and specific interaction of kindlin-2 with αVβ3 as assessed by immunofluorescence and TIRF microscopy, and it led to increased endothelial cell migration, podosome formation and angiogenic sprouting. Analyses of kindlin-2 mutants indicated that interaction of kindlin-2 with other kindlin-2 binding partners, including c-Src, actin, integrin-linked kinase and phosphoinositides, were also likely necessary for these endothelial cell responses. Thus, kindlin-2 promotes αVβ3-dependent angiogenic functions of endothelial cells through its simultaneous interactions with β3 and several other binding partners. Optogenetic approaches should find further use in clarifying spatiotemporal aspects of vascular cell biology.

Abstract: Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene’s transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.

Abstract: Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) signaling is transient and spatially confined in live cells. How this pattern of signaling regulates transmitter release and hormone secretion has not been addressed. We devised an optogenetic approach to control PI(4,5)P2 levels in time and space in insulin-secreting cells. Combining this approach with total internal reflection fluorescence microscopy, we examined individual vesicle-trafficking steps. Unlike long-term PI(4,5)P2 perturbations, rapid and cell-wide PI(4,5)P2 reduction in the plasma membrane (PM) strongly inhibits secretion and intracellular Ca(2+) concentration ([Ca(2+)]i) responses, but not sytaxin1a clustering. Interestingly, local PI(4,5)P2 reduction selectively at vesicle docking sites causes remarkable vesicle undocking from the PM without affecting [Ca(2+)]i. These results highlight a key role of local PI(4,5)P2 in vesicle tethering and docking, coordinated with its role in priming and fusion. Thus, different spatiotemporal PI(4,5)P2 signaling regulates distinct steps of vesicle trafficking, and vesicle docking may be a key target of local PI(4,5)P2 signaling in vivo.

Abstract: Rho GTPase family members are known regulators of directed migration and therefore play key roles in processes including development, immune response and cancer metastasis. However, their individual contributions to these processes are complex. Here, we regulate the activity of two family members, Rac and Cdc42, by optogenetically recruiting specific GEF DH/PH domains to defined regions on the cell membrane. We find that the localized activation of both GTPases produce lamellipodia in cells plated on a fibronectin substrate. Using a novel optotaxis assay, we show that biased activation can drive directional migration. Interestingly, in the absence of exogenous fibronectin, Rac activation is insufficient to produce stable lamellipodia or directional migration while Cdc42 activation is sufficient. We find that a remarkably small amount of fibronectin (<10 puncta per protrusion) is necessary to support stable GTPase-driven lamellipodia. Cdc42 bypasses the need for exogenous fibronectin by stimulating cellular fibronectin deposition under the newly formed lamellipodia.

Abstract: Sensory systems use adaptation to measure changes in signaling inputs rather than absolute levels of signaling inputs. Adaptation enables eukaryotic cells to directionally migrate over a large dynamic range of chemoattractant. Because of complex feedback interactions and redundancy, it has been difficult to define the portion or portions of eukaryotic chemotactic signaling networks that generate adaptation and identify the regulators of this process. In this study, we use a combination of optogenetic intracellular inputs, CRISPR-based knockouts, and pharmacological perturbations to probe the basis of neutrophil adaptation. We find that persistent, optogenetically driven phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production results in only transient activation of Rac, a hallmark feature of adaptive circuits. We further identify the guanine nucleotide exchange factor P-Rex1 as the primary PIP3-stimulated Rac activator, whereas actin polymerization and the GTPase-activating protein ArhGAP15 are essential for proper Rac turnoff. This circuit is masked by feedback and redundancy when chemoattractant is used as the input, highlighting the value of probing signaling networks at intermediate nodes to deconvolve complex signaling cascades.

Abstract: Lamellipodia are sheet-like cell protrusions driven by actin polymerization mainly through Rac1, a GTPase molecular switch. In Fcγ receptor-mediated phagocytosis of IgG-opsonized erythrocytes (IgG-Es), Rac1 activation is required for lamellipodial extension along the surface of IgG-Es. However, the significance of Rac1 deactivation in phagosome formation is poorly understood. Our live-cell imaging and electron microscopy revealed that RAW264 macrophages expressing a constitutively active Rac1 mutant showed defects in phagocytic cup formation, while lamellipodia were formed around IgG-Es. Because the activated Rac1 reduced the phosphorylation levels of myosin light chain, failure of the cup formation were probably due to inhibition of actin/myosin II contractility. Reversible photo-manipulation of the Rac1 switch in macrophages fed with IgG-Es could phenocopy two lamellipodial motilities: outward-extension and cup-constriction by Rac1 ON and OFF, respectively. In conjunction with FRET imaging of Rac1 activity, we provide a novel mechanistic model of phagosome formation spatiotemporally controlled by Rac1 switching within a phagocytic cup.

Abstract: Animal development is characterized by signaling events that occur at precise locations and times within the embryo, but determining when and where such precision is needed for proper embryogenesis has been a long-standing challenge. Here we address this question for extracellular signal regulated kinase (Erk) signaling, a key developmental patterning cue. We describe an optogenetic system for activating Erk with high spatiotemporal precision in vivo. Implementing this system in Drosophila, we find that embryogenesis is remarkably robust to ectopic Erk signaling, except from 1 to 4 hr post-fertilization, when perturbing the spatial extent of Erk pathway activation leads to dramatic disruptions of patterning and morphogenesis. Later in development, the effects of ectopic signaling are buffered, at least in part, by combinatorial mechanisms. Our approach can be used to systematically probe the differential contributions of the Ras/Erk pathway and concurrent signals, leading to a more quantitative understanding of developmental signaling.

Abstract: Calcium acts as a second messenger to regulate a myriad of cell functions, ranging from short-term muscle contraction and cell motility to long-term changes in gene expression and metabolism. To study the impact of Ca2+-modulated 'ON' and 'OFF' reactions in mammalian cells, pharmacological tools and 'caged' compounds are commonly used under various experimental conditions. The use of these reagents for precise control of Ca2+ signals, nonetheless, is impeded by lack of reversibility and specificity. The recently developed optogenetic tools, particularly those built upon engineered Ca2+ release-activated Ca2+ (CRAC) channels, provide exciting opportunities to remotely and non-invasively modulate Ca2+ signaling due to their superior spatiotemporal resolution and rapid reversibility. In this review, we briefly summarize the latest advances in the development of optogenetic tools (collectively termed as 'genetically encoded Ca2+ actuators', or GECAs) that are tailored for the interrogation of Ca2+ signaling, as well as their applications in remote neuromodulation and optogenetic immunomodulation. Our goal is to provide a general guide to choosing appropriate GECAs for optical control of Ca2+ signaling in cellulo, and in parallel, to stimulate further thoughts on evolving non-opsin-based optogenetics into a fully fledged technology for the study of Ca2+-dependent activities in vivo.

Abstract: Cell ablation is a strategy to study cell lineage and function during development. Optogenetic methods are an important cell-ablation approach, and we have previously developed a mini singlet oxygen generator (miniSOG) tool that works in the living Caenorhabditis elegans. Here, we use directed evolution to generate miniSOG2, an improved tool for cell ablation via photogenerated reactive oxygen species. We apply miniSOG2 to a far more complex model animal system, Drosophila melanogaster, and demonstrate that it can be used to kill a single neuron in a Drosophila larva. In addition, miniSOG2 is able to photoablate a small group of cells in one of the larval wing imaginal discs, resulting in an adult with one incomplete and one normal wing. We expect miniSOG2 to be a useful optogenetic tool for precision cell ablation at a desired developmental time point in live animals, thus opening a new window into cell origin, fate and function, tissue regeneration, and developmental biology.

Abstract: Phase transitions driven by intrinsically disordered protein regions (IDRs) have emerged as a ubiquitous mechanism for assembling liquid-like RNA/protein (RNP) bodies and other membrane-less organelles. However, a lack of tools to control intracellular phase transitions limits our ability to understand their role in cell physiology and disease. Here, we introduce an optogenetic platform that uses light to activate IDR-mediated phase transitions in living cells. We use this "optoDroplet" system to study condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1. Above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally definable liquid optoDroplets. FUS optoDroplet assembly is fully reversible even after multiple activation cycles. However, cells driven deep within the phase boundary form solid-like gels that undergo aging into irreversible aggregates. This system can thus elucidate not only physiological phase transitions but also their link to pathological aggregates.

Abstract: Cytokinesis, the final step of cell division, begins with the formation of a cleavage furrow. How the mitotic spindle specifies the furrow at the equator in animal cells remains unknown. Current models propose that the concentration of the RhoGEF ECT2 at the spindle midzone and the equatorial plasma membrane directs furrow formation. Using chemical genetic and optogenetic tools, we demonstrate that the association of ECT2 with the plasma membrane during anaphase is required and sufficient for cytokinesis. Local membrane targeting of ECT2 leads to unilateral furrowing, highlighting the importance of local ECT2 activity. ECT2 mutations that prevent centralspindlin binding compromise concentration of ECT2 at the midzone and equatorial membrane but sustain cytokinesis. While the association of ECT2 with the plasma membrane is essential for cytokinesis, our data suggest that ECT2 recruitment to the spindle midzone is insufficient to account for equatorial furrowing and may act redundantly with yet-uncharacterized signals.

Abstract: We describe a detailed procedure for the use of LOVTRAP, an approach to reversibly sequester and release proteins from cellular membranes using light. In the application described here, proteins that act at the plasma membrane are held at mitochondria in the dark, and reversibly released by irradiation. The technique relies on binding of an engineered Zdk domain to a LOV2 domain, with affinity <30 nM in the dark and >500 nM upon irradiation between 400 and 500 nm. LOVTRAP can be applied to diverse proteins, as it requires attaching only one member of the Zdk/LOV2 pair to the target protein, and the other to the membrane where the target protein is to be sequestered. Light-induced protein release occurs in less than a second, and the half-life of return can be adjusted using LOV point mutations (∼2 to 500 sec). © 2016 by John Wiley & Sons, Inc.

Abstract: Morphogenesis of multicellular organisms is driven by changes in cell behavior, which happen at precise locations and defined developmental stages. Therefore, the studying of morphogenetic events would greatly benefit from tools that allow the perturbation of cell activity with spatial and temporal precision. We recently developed an optogenetic approach to modulate cell contractility with cellular precision and on fast (seconds) timescales during Drosophila embryogenesis. We present here a protocol to handle genetically engineered photosensitive Drosophila embryos and achieve light-mediated inhibition of apical constriction during tissue invagination. The possibility to modulate the levels of optogenetic activation at different laser powers makes this method suited also for studying how mechanical stresses are sensed and interpreted in vivo. Given the conserved function of cell contractility during animal development, the application of this method to other morphogenetic processes will facilitate our understanding of tissue mechanics and cell-cell interaction during morphogenesis.

Abstract: In eukaryotes, protein kinase A (PKA) is a master regulator of cell proliferation and survival. The activity of PKA is subject to elaborate control and exhibits complex time dynamics. To probe the quantitative attributes of PKA dynamics in the yeast Saccharomyces cerevisiae, we developed an optogenetic strategy that uses a photoactivatable adenylate cyclase to achieve real-time regulation of cAMP and the PKA pathway. We capitalize on the precise and rapid control afforded by this optogenetic tool, together with quantitative computational modeling, to study the properties of feedback in the PKA signaling network and dissect the nonintuitive dynamic effects that ensue from perturbing its components. Our analyses reveal that negative feedback channeled through the Ras1/2 GTPase is delayed, pinpointing its time scale and its contribution to the dynamic features of the cAMP/PKA signaling network.

Abstract: Optogenetics is an emerging and powerful technique that allows the control of protein activity with light. The possibility of inhibiting or stimulating protein activity with the spatial and temporal precision of a pulse of laser light is opening new frontiers for the investigation of developmental pathways and cell biological bases underlying organismal development. With this powerful technique in hand, it will be possible to address old and novel questions about how cells, tissues, and organisms form. In this review, we focus on the applications of existing optogenetic tools for addressing issues in animal morphogenesis.