Showing 1 - 25 of 65 results
A guide to designing photocontrol in proteins: methods, strategies and applications.
Light is essential for various biochemical processes in all domains of life. In its presence certain proteins inside a cell are excited, which either stimulates or inhibits subsequent cellular processes. The artificial photocontrol of specifically proteins is of growing interest for the investigation of scientific questions on the organismal, cellular and molecular level as well as for the development of medicinal drugs or biocatalytic tools. For the targeted design of photocontrol in proteins, three major methods have been developed over the last decades, which employ either chemical engineering of small-molecule photosensitive effectors (photopharmacology), incorporation of photoactive non-canonical amino acids by genetic code expansion (photoxenoprotein engineering), or fusion with photoreactive biological modules (hybrid protein optogenetics). This review compares the different methods as well as their strategies and current applications for the light-regulation of proteins and provides background information useful for the implementation of each technique.
New developments in the biology of fibroblast growth factors.
The fibroblast growth factor (FGF) family is composed of 18 secreted signaling proteins consisting of canonical FGFs and endocrine FGFs that activate four receptor tyrosine kinases (FGFRs 1-4) and four intracellular proteins (intracellular FGFs or iFGFs) that primarily function to regulate the activity of voltage-gated sodium channels and other molecules. The canonical FGFs, endocrine FGFs, and iFGFs have been reviewed extensively by us and others. In this review, we briefly summarize past reviews and then focus on new developments in the FGF field since our last review in 2015. Some of the highlights in the past 6 years include the use of optogenetic tools, viral vectors, and inducible transgenes to experimentally modulate FGF signaling, the clinical use of small molecule FGFR inhibitors, an expanded understanding of endocrine FGF signaling, functions for FGF signaling in stem cell pluripotency and differentiation, roles for FGF signaling in tissue homeostasis and regeneration, a continuing elaboration of mechanisms of FGF signaling in development, and an expanding appreciation of roles for FGF signaling in neuropsychiatric diseases. This article is categorized under: Cardiovascular Diseases > Molecular and Cellular Physiology Neurological Diseases > Molecular and Cellular Physiology Congenital Diseases > Stem Cells and Development Cancer > Stem Cells and Development.
A nucleation barrier spring-loads the CBM signalosome for binary activation.
Immune cells activate in a binary, switch-like fashion that involves proteins polymerizing into large complexes known as signalosomes. The switch-like nature of signalosome formation has been proposed to result from large energy barriers to polymer nucleation. Whether such nucleation barriers indeed drive binary immune responses has not yet been shown. Here, we employed an in-cell biophysical approach to dissect the assembly mechanism of the CARD-BCL10-MALT1 (CBM) signalosome, a key determinant of transcription factor NF-κB activation in both innate and adaptive immunity. We found that the adaptor protein BCL10 encodes an intrinsic nucleation barrier, and that this barrier has been conserved from cnidaria to humans. Using optogenetic tools and a single-cell transcriptional reporter of NF-κB activity, we further revealed that endogenous human BCL10 is supersaturated even in unstimulated cells, indicating that the nucleation barrier operationally stores energy for subsequent activation. We found that upon stimulation, BCL10 nucleation by CARD9 multimers triggers self-templated polymerization that saturates NF-κB activation to produce a binary response. Pathogenic mutants of CARD9 that cause human immunodeficiencies eliminated nucleating activity. Conversely, a hyperactive cancer-causing mutation in BCL10 increased its spontaneous nucleation. Our results indicate that unassembled CBM signalosome components function analogously to a spring-loaded mousetrap, constitutively poised to activate NF-κB through irrevocable polymerization. This finding may inform our understanding of the root causes and progressive nature of pathogenic and age-associated inflammation.
Optophysiology: Illuminating cell physiology with optogenetics.
Optogenetics combines light and genetics to enable precise control of living cells, tissues, and organisms with tailored functions. Optogenetics has the advantages of noninvasiveness, rapid responsiveness, tunable reversibility, and superior spatiotemporal resolution. Following the initial discovery of microbial opsins as light-actuated ion channels, a plethora of naturally occurring or engineered photoreceptors or photosensitive domains that respond to light at varying wavelengths has ushered in the next chapter of optogenetics. Through protein engineering and synthetic biology approaches, genetically encoded photoswitches can be modularly engineered into protein scaffolds or host cells to control a myriad of biological processes, as well as to enable behavioral control and disease intervention in vivo. Here, we summarize these optogenetic tools on the basis of their fundamental photochemical properties to better inform the chemical basis and design principles. We also highlight exemplary applications of opsin-free optogenetics in dissecting cellular physiology (designated "optophysiology") and describe the current progress, as well as future trends, in wireless optogenetics, which enables remote interrogation of physiological processes with minimal invasiveness. This review is anticipated to spark novel thoughts on engineering next-generation optogenetic tools and devices that promise to accelerate both basic and translational studies.
Optogenetic activation of intracellular signaling based on light-inducible protein-protein homo-interactions.
Dynamic protein-protein interactions are essential for proper cell functioning. Homo-interaction events-physical interactions between the same type of proteins-represent a pivotal subset of protein-protein interactions that are widely exploited in activating intracellular signaling pathways. Capacities of modulating protein-protein interactions with spatial and temporal resolution are greatly desired to decipher the dynamic nature of signal transduction mechanisms. The emerging optogenetic technology, based on genetically encoded light-sensitive proteins, provides promising opportunities to dissect the highly complex signaling networks with unmatched specificity and spatiotemporal precision. Here we review recent achievements in the development of optogenetic tools enabling light-inducible protein-protein homo-interactions and their applications in optical activation of signaling pathways.
Light-Inducible Spatio-Temporal Control of TLR4 and NF-κB-Gluc Reporter in Human Pancreatic Cell Line.
Augmented Toll-like receptor 4 (TLR4) expression was found in nearly 70% of patients with pancreatic adenocarcinoma, which is correlated with increased tumorigenesis and progression. In this study, we engineered a new light-oxygen-voltage-sensing (LOV) domain-based optogenetic cell line (opto-TLR4 PANC-1) that enables time-resolved activation of the NF-κB and extracellular-signal regulated kinases (ERK)1/2 signalling pathway upon blue light-sensitive homodimerisation of the TLR4-LOV fusion protein. Continuous stimulation with light indicated strong p65 and ERK1/2 phosphorylation even after 24 h, whereas brief light exposure peaked at 8 h and reached the ground level 24 h post-illumination. The cell line further allows a voltage-dependent TLR4 activation, which can be continuously monitored, turned on by light or off in the dark. Using this cell line, we performed different phenotypic cell-based assays with 2D and 3D cultures, with the aim of controlling cellular activity with spatial and temporal precision. Light exposure enhanced cell attachment, the formation and extension of invadopodia, and cell migration in 3D spheroid cultures, but no significant changes in proliferation or viability could be detected. We conclude that the opto-TLR4 PANC-1 cell line is an ideal tool for investigating the underlying molecular mechanisms of TLR4, thereby providing strategies for new therapeutic options.
A Light-Oxygen-Voltage Receptor Integrates Light and Temperature.
Sensory photoreceptors enable organisms to adjust their physiology, behavior, and development in response to light, generally with spatiotemporal acuity and reversibility. These traits underlie the use of photoreceptors as genetically encoded actuators to alter by light the state and properties of heterologous organisms. Subsumed as optogenetics, pertinent approaches enable regulating diverse cellular processes, not least gene expression. Here, we controlled the widely used Tet repressor by coupling to light-oxygen-voltage (LOV) modules that either homodimerize or dissociate under blue light. Repression could thus be elevated or relieved, and consequently protein expression was modulated by light. Strikingly, the homodimeric RsLOV module from Rhodobacter sphaeroides not only dissociated under light but intrinsically reacted to temperature. The limited light responses of wild-type RsLOV at 37 °C were enhanced in two variants that exhibited closely similar photochemistry and structure. One variant improved the weak homodimerization affinity of 40 µM by two-fold and thus also bestowed light sensitivity on a receptor tyrosine kinase. Certain photoreceptors, exemplified by RsLOV, can evidently moonlight as temperature sensors which immediately bears on their application in optogenetics and biotechnology. Properly accounted for, the temperature sensitivity can be leveraged for the construction of signal-responsive cellular circuits.
Pathogenic ACVR1R206H activation by Activin A-induced receptor clustering and autophosphorylation.
Fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG) are debilitating diseases that share causal mutations in ACVR1, a TGF-β family type I receptor. ACVR1R206H is a frequent mutation in both diseases. Pathogenic signaling via the SMAD1/5 pathway is mediated by Activin A, but how the mutation triggers aberrant signaling is not known. We show that ACVR1 is essential for Activin A-mediated SMAD1/5 phosphorylation and is activated by two distinct mechanisms. Wild-type ACVR1 is activated by the Activin type I receptors, ACVR1B/C. In contrast, ACVR1R206H activation does not require upstream kinases, but is predominantly activated via Activin A-dependent receptor clustering, which induces its auto-activation. We use optogenetics and live-imaging approaches to demonstrate Activin A-induced receptor clustering and show it requires the type II receptors ACVR2A/B. Our data provide molecular mechanistic insight into the pathogenesis of FOP and DIPG by linking the causal activating genetic mutation to disrupted signaling.
Optogenetic Approaches for the Spatiotemporal Control of Signal Transduction Pathways.
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.
The Rise of Molecular Optogenetics.
Abstract not available.
Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease.
Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson's disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.
Lighting the way: Recent insights into the structure and regulation of phototropin blue light receptors.
The phototropins (phots) are light-activated kinases that are critical for plant physiology and the many diverse optogenetic tools that they have inspired. Phototropins combine two blue light sensing Light-Oxygen-Voltage (LOV) domains (LOV1 and LOV2) and a C-terminal serine/threonine kinase domain, using the LOV domains to control the catalytic activity of the kinase. While much is known about the structure and photochemistry of the light-perceiving LOV domains, particularly in how activation of the LOV2 domain triggers the unfolding of alpha helices that communicate the light signal to the kinase domain, many questions about phot structure and mechanism remain. Recent studies have made progress addressing these questions by utilizing small angle X-ray scattering (SAXS) and other biophysical approaches to study multidomain phots from Chlamydomonas and Arabidopsis, leading to models where the domains have an extended linear arrangement, with the activating LOV2 domain contacting the kinase domain N-lobe. We discuss this and other advances which have improved structural and mechanistic understanding of phot regulation in this review, along with the challenges that will have to be overcome to obtain high-resolution structural information on these exciting photoreceptors. Such information will be essential to advancing fundamental understanding of plant physiology while enabling engineering efforts at both the whole plant and molecular levels.
Light-regulated voltage-gated potassium channels for acute interrogation of channel function in neurons and behavior.
Voltage-gated potassium (Kv) channels regulate the membrane potential and conductance of excitable cells to control the firing rate and waveform of action potentials. Even though Kv channels have been intensely studied for over 70 year, surprisingly little is known about how specific channels expressed in various neurons and their functional properties impact neuronal network activity and behavior in vivo. Although many in vivo genetic manipulations of ion channels have been tried, interpretation of these results is complicated by powerful homeostatic plasticity mechanisms that act to maintain function following perturbations in excitability. To better understand how Kv channels shape network function and behavior, we have developed a novel optogenetic technology to acutely regulate Kv channel expression with light by fusing the light-sensitive LOV domain of Vaucheria frigida Aureochrome 1 to the N-terminus of the Kv1 subunit protein to make an Opto-Kv1 channel. Recording of Opto-Kv1 channels expressed in Xenopus oocytes, mammalian cells, and neurons show that blue light strongly induces the current expression of Opto-Kv1 channels in all systems tested. We also find that an Opto-Kv1 construct containing a dominant-negative pore mutation (Opto-Kv1(V400D)) can be used to down-regulate Kv1 currents in a blue light-dependent manner. Finally, to determine whether Opto-Kv1 channels can elicit light-dependent behavioral effect in vivo, we targeted Opto-Kv1 (V400D) expression to Kv1.3-expressing mitral cells of the olfactory bulb in mice. Exposure of the bulb to blue light for 2-3 hours produced a significant increase in sensitivity to novel odors after initial habituation to a similar odor, comparable to behavioral changes seen in Kv1.3 knockout animals. In summary, we have developed novel photoactivatable Kv channels that provide new ways to interrogate neural circuits in vivo and to examine the roles of normal and disease-causing mutant Kv channels in brain function and behavior.
Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives.
Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.
Optogenetic investigation of BMP target gene expression diversity.
Signaling molecules activate distinct patterns of gene expression to coordinate embryogenesis, but how spatiotemporal expression diversity is generated is an open question. In zebrafish, a BMP signaling gradient patterns the dorsal-ventral axis. We systematically identified target genes responding to BMP and found that they have diverse spatiotemporal expression patterns. Transcriptional responses to optogenetically delivered high- and low-amplitude BMP signaling pulses indicate that spatiotemporal expression is not fully defined by different BMP signaling activation thresholds. Additionally, we observed negligible correlations between spatiotemporal expression and transcription kinetics for the majority of analyzed genes in response to BMP signaling pulses. In contrast, spatial differences between BMP target genes largely collapsed when FGF and Nodal signaling were inhibited. Our results suggest that, similar to other patterning systems, combinatorial signaling is likely to be a major driver of spatial diversity in BMP-dependent gene expression in zebrafish.
The rise and shine of yeast optogenetics.
Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared to traditional chemically-inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, or protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast.
Optogenetic Control of the BMP Signaling Pathway.
Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGFβ) superfamily and have crucial roles during development; including mesodermal patterning and specification of renal, hepatic, and skeletal tissues. In vitro developmental models currently rely upon costly and unreliable recombinant BMP proteins that do not enable dynamic or precise activation of the BMP signaling pathway. Here, we report the development of an optogenetic BMP signaling system (optoBMP) that enables rapid induction of the canonical BMP signaling pathway driven by illumination with blue light. We demonstrate the utility of the optoBMP system in multiple human cell lines to initiate signal transduction through phosphorylation and nuclear translocation of SMAD1/5, leading to upregulation of BMP target genes including Inhibitors of DNA binding ID2 and ID4. Furthermore, we demonstrate how the optoBMP system can be used to fine-tune activation of the BMP signaling pathway through variable light stimulation. Optogenetic control of BMP signaling will enable dynamic and high-throughput intervention across a variety of applications in cellular and developmental systems.
Optogenetically Controlled TrkA Activity Improves the Regenerative Capacity of Hair-Follicle-Derived Stem Cells to Differentiate into Neurons and Glia.
Hair-follicle-derived stem cells (HSCs) originating from the bulge region of the mouse vibrissa hair follicle are able to differentiate into neuronal and glial lineage cells. The tropomyosin receptor kinase A (TrkA) receptor that is expressed on these cells plays key roles in mediating the survival and differentiation of neural progenitors as well as in the regulation of the growth and regeneration of different neural systems. In this study, the OptoTrkA system is introduced, which is able to stimulate TrkA activity via blue-light illumination in HSCs. This allows to determine whether TrkA signaling is capable of influencing the proliferation, migration, and neural differentiation of these somatic stem cells. It is found that OptoTrkA is able to activate downstream molecules such as ERK and AKT with blue-light illumination, and subsequently able to terminate this kinase activity in the dark. HSCs with OptoTrkA activity show an increased ability for proliferation and migration and also exhibited accelerated neuronal and glial cell differentiation. These findings suggest that the precise control of TrkA activity using optogenetic tools is a viable strategy for the regeneration of neurons from HSCs, and also provides a novel insight into the clinical application of optogenetic tools in cell-transplantation therapy.
Light control of RTK activity: from technology development to translational research.
Inhibition of receptor tyrosine kinases (RTKs) by small molecule inhibitors and monoclonal antibodies is used to treat cancer. Conversely, activation of RTKs with their ligands, including growth factors and insulin, is used to treat diabetes and neurodegeneration. However, conventional therapies that rely on injection of RTK inhibitors or activators do not provide spatiotemporal control over RTK signaling, which results in diminished efficiency and side effects. Recently, a number of optogenetic and optochemical approaches have been developed that allow RTK inhibition or activation in cells and in vivo with light. Light irradiation can control RTK signaling non-invasively, in a dosed manner, with high spatio-temporal precision, and without the side effects of conventional treatments. Here we provide an update on the current state of the art of optogenetic and optochemical RTK technologies and the prospects of their use in translational studies and therapy.
Syntaxin Clustering and Optogenetic Control for Synaptic Membrane Fusion.
Membrane fusion during synaptic transmission mediates the trafficking of chemical signals and neuronal communication. The fast kinetics of membrane fusion on the order of millisecond is precisely regulated by the assembly of SNAREs and accessory proteins. It is believed that the formation of the SNARE complex is a key step during membrane fusion. Little is known, however, about the molecular machinery that mediates the formation of a large pre-fusion complex, including multiple SNAREs and accessory proteins. Syntaxin, a transmembrane protein on the plasma membrane, has been observed to undergo oligomerization to form clusters. Whether this clustering plays a critical role in membrane fusion is poorly understood in live cells. Optogenetics is an emerging biotechnology armed with the capacity to precisely modulate protein-protein interaction in time and space. Here, we propose an experimental scheme that combines optogenetics with single-vesicle membrane fusion, aiming to gain a better understanding of the molecular mechanism by which the syntaxin cluster regulates membrane fusion. We envision that newly developed optogenetic tools could facilitate the mechanistic understanding of synaptic transmission in live cells and animals.
Novel culture system via wirelessly controllable optical stimulation of the FGF signaling pathway for human and pig pluripotency.
Stem cell fate is largely determined by cellular signaling networks and is heavily dependent on the supplementation of exogenous recombinant proteins into culture media; however, uneven distribution and inconsistent stability of recombinant proteins are closely associated with the spontaneous differentiation of pluripotent stem cells (PSCs) and result in significant costs in large-scale manufacturing. Here, we report a novel PSC culture system via wirelessly controllable optical activation of the fibroblast growth factor (FGF) signaling pathway without the need for supplementation of recombinant FGF2 protein, a key molecule for maintaining pluripotency of PSCs. Using a fusion protein between the cytoplasmic region of the FGF receptor-1 and a light-oxygen-voltage domain, we achieved tunable, blue light-dependent activation of FGF signaling in human and porcine PSCs. Our data demonstrate that a highly controllable optical stimulation of the FGF signaling pathway is sufficient for long-term maintenance of PSCs, without the loss of differentiation potential into three germ layers. This culture system will be a cost-effective platform for a large-scale stem cell culture.
Design and Application of Light-Regulated Receptor Tyrosine Kinases.
Understanding how the activity of membrane receptors and cellular signaling pathways shapes cell behavior is of fundamental interest in basic and applied research. Reengineering receptors to react to light instead of their cognate ligands allows for generating defined signaling inputs with high spatial and temporal precision and facilitates the dissection of complex signaling networks. Here, we describe fundamental considerations in the design of light-regulated receptor tyrosine kinases (Opto-RTKs) and appropriate control experiments. We also introduce methods for transient receptor expression in HEK293 cells, quantitative assessment of signaling activity in reporter gene assays, semiquantitative assessment of (in)activation time courses through Western blot (WB) analysis, and easy to implement light stimulation hardware.
Molecular Mechanism of Light-Induced Conformational Switching of the LOV Domain in Aureochrome-1.
Light oxygen voltage-sensing (LOV) domains are widely found in photoreceptor proteins of plants, algae, fungi, and bacteria. Structural studies of LOV domains suggest that Phe and Gln residues located in the proximity of the chromophore undergo conformational changes upon illumination; however, the molecular mechanism associated with activation of the effector domain remains to be elucidated. Photozipper (PZ) protein is an N-terminally truncated aureochrome-1 comprising a LOV domain and a basic leucine zipper domain. Blue light (BL) induces PZ dimerization and subsequently increases its affinity for target DNA. In this study, we prepared PZ mutants with substitutions of F298 and Q317 and performed quantitative analyses in dark and light states. Substitutions of Q317 significantly reduced the light-induced changes in PZ affinity for the target DNA, especially in the case of the high affinities observed in the dark state. Upon illumination, all PZ mutants showed increased affinity for the target sequence, which demonstrated a clear correlation with the dimer fraction of each PZ mutant. These results suggest the existence of a conformational equilibrium and that its shift by a synergistic interaction between the chromophore and protein moiety probably enables BL-regulated switching of aureochrome-1.
Optical Activation of TrkB Signaling.
Brain-derived neurotrophic factor (BDNF), via activation of tropomyosin receptor kinase B (TrkB), plays a critical role in neuronal proliferation, differentiation, survival, and death. Dysregulation of TrkB signaling is implicated in neurodegenerative disorders and cancers. Precise activation of TrkB signaling with spatial and temporal resolution is greatly desired to study the dynamic nature of TrkB signaling and its role in related diseases. Here we develop different optogenetic approaches that use light to activate TrkB signaling. Utilizing the photosensitive protein Arabidopsis thaliana cryptochrome 2 (CRY2), the light-inducible homo-interaction of the intracellular domain of TrkB (iTrkB) in the cytosol or on the plasma membrane is able to induce the activation of downstream MAPK/ERK and PI3K/Akt signaling as well as the neurite outgrowth of PC12 cells. Moreover, we prove that such strategies are generalizable to other optical homo-dimerizers by demonstrating the optical TrkB activation based on the light-oxygen-voltage domain of aureochrome 1 from Vaucheria frigida. The results open up new possibilities of many other optical platforms to activate TrkB signaling to fulfill customized needs. By comparing all the different strategies, we find that the CRY2-integrated approach to achieve light-induced cell membrane recruitment and homo-interaction of iTrkB is most efficient in activating TrkB signaling. The optogenetic strategies presented are promising tools to investigate BDNF/TrkB signaling with tight spatial and temporal control.
Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation.
Neuroligins (Nlgns) are adhesion proteins mediating trans-synaptic contacts in neurons. However, conflicting results around their role in synaptic differentiation arise from the various techniques used to manipulate Nlgn expression level. Orthogonally to these approaches, we triggered here the phosphorylation of endogenous Nlgn1 in CA1 mouse hippocampal neurons using a photoactivatable tyrosine kinase receptor (optoFGFR1). Light stimulation for 24 hr selectively increased dendritic spine density and AMPA-receptor-mediated EPSCs in wild-type neurons, but not in Nlgn1 knock-out neurons or when endogenous Nlgn1 was replaced by a non-phosphorylatable mutant (Y782F). Moreover, light stimulation of optoFGFR1 partially occluded LTP in a Nlgn1-dependent manner. Combined with computer simulations, our data support a model by which Nlgn1 tyrosine phosphorylation promotes the assembly of an excitatory post-synaptic scaffold that captures surface AMPA receptors. This optogenetic strategy highlights the impact of Nlgn1 intracellular signaling in synaptic differentiation and potentiation, while enabling an acute control of these mechanisms.