Showing 1 - 25 of 655 results
Dynamic organelle distribution initiates actin-based spindle migration in mouse oocytes.
Migration of meiosis-I (MI) spindle from the cell center to a sub-cortical location is a critical step for mouse oocytes to undergo asymmetric meiotic cell division. In this study, we investigate the mechanism by which formin-2 (FMN2) orchestrates the initial movement of MI spindle. By defining protein domains responsible for targeting FMN2, we show that spindle-periphery localized FMN2 is required for spindle migration. The spindle-peripheral FMN2 nucleates short actin bundles from vesicles derived likely from the endoplasmic reticulum (ER) and concentrated in a layer outside the spindle. This layer is in turn surrounded by mitochondria. A model based on polymerizing actin filaments pushing against mitochondria, thus generating a counter force on the spindle, demonstrated an inherent ability of this system to break symmetry and evolve directional spindle motion. The model is further supported through experiments involving spatially biasing actin nucleation via optogenetics and disruption of mitochondrial distribution and dynamics.
Optogenetic control of spine-head JNK reveals a role in dendritic spine regression.
In this study, we use an optogenetic inhibitor of JNK in dendritic spine sub-compartments of rat hippocampal neurons. JNK inhibition exerts rapid (within seconds) reorganisation of actin in the spine-head. Using real-time FRET to measure JNK activity, we find that either excitotoxic insult (NMDA) or endocrine stress (corticosterone), activate spine-head JNK causing internalization of AMPARs and spine retraction. Both events are prevented upon optogenetic inhibition of JNK, and rescued by JNK inhibition even 2 h after insult. Moreover, we identify that the fast-acting anti-depressant ketamine reduces JNK activity in hippocampal neurons suggesting that JNK inhibition may be a downstream mediator of its anti-depressant effect. In conclusion, we show that JNK activation plays a role in triggering spine elimination by NMDA or corticosterone stress, whereas inhibition of JNK facilitates regrowth of spines even in the continued presence of glucocorticoid. This identifies that JNK acts locally in the spine-head to promote AMPAR internalization and spine shrinkage following stress, and reveals a protective function for JNK inhibition in preventing spine regression.SIGNIFICANCE STATEMENT Identifying mechanisms that underlie dendritic spine elimination is important if we are to understand maladaptive changes that contribute to psychiatric disease. Compartment-specific, fast-acting tools can expedite this endeavor. Here we use a light-activated inhibitor of JNK to control kinase activity specifically in dendritic spines. Light-activation of the JNK inhibitor reduces AMPA receptor removal and spine regression in response to corticosterone and NMDA stress. Furthermore, we find that the anti-depressant drug ketamine lowers JNK activity in hippocampal neurons and prevents spine regression, though direct JNK inhibition is more effective. This study identifies a role for JNK in spine regression and may be relevant for endocrine control of synaptic strength and for conditions where chronic glucocorticoid stress leads to spine elimination.
Ras acts as a molecular switch between two forms of consolidated memory in Drosophila.
Long-lasting, consolidated memories require not only positive biological processes that facilitate long-term memories (LTM) but also the suppression of inhibitory processes that prevent them. The mushroom body neurons (MBn) in Drosophila melanogaster store protein synthesis-dependent LTM (PSD-LTM) as well as protein synthesis-independent, anesthesia-resistant memory (ARM). The formation of ARM inhibits PSD-LTM but the underlying molecular processes that mediate this interaction remain unknown. Here, we demonstrate that the Ras→Raf→rho kinase (ROCK) pathway in MBn suppresses ARM consolidation, allowing the formation of PSD-LTM. Our initial results revealed that the effects of Ras on memory are due to postacquisition processes. Ras knockdown enhanced memory expression but had no effect on acquisition. Additionally, increasing Ras activity optogenetically after, but not before, acquisition impaired memory performance. The elevated memory produced by Ras knockdown is a result of increased ARM. While Ras knockdown enhanced the consolidation of ARM, it eliminated PSD-LTM. We found that these effects are mediated by the downstream kinase Raf. Similar to Ras, knockdown of Raf enhanced ARM consolidation and impaired PSD-LTM. Surprisingly, knockdown of the canonical downstream extracellular signal-regulated kinase did not reproduce the phenotypes observed with Ras and Raf knockdown. Rather, Ras/Raf inhibition of ROCK was found to be responsible for suppressing ARM. Constitutively active ROCK enhanced ARM and impaired PSD-LTM, while decreasing ROCK activity rescued the enhanced ARM produced by Ras knockdown. We conclude that MBn Ras/Raf inhibition of ROCK suppresses the consolidation of ARM, which permits the formation of PSD-LTM.
Coupling delay controls synchronized oscillation in the segmentation clock.
Individual cellular activities fluctuate but are constantly coordinated at the population level via cell-cell coupling. A notable example is the somite segmentation clock, in which the expression of clock genes (such as Hes7) oscillates in synchrony between the cells that comprise the presomitic mesoderm (PSM)1,2. This synchronization depends on the Notch signalling pathway; inhibiting this pathway desynchronizes oscillations, leading to somite fusion3-7. However, how Notch signalling regulates the synchronicity of HES7 oscillations is unknown. Here we establish a live-imaging system using a new fluorescent reporter (Achilles), which we fuse with HES7 to monitor synchronous oscillations in HES7 expression in the mouse PSM at a single-cell resolution. Wild-type cells can rapidly correct for phase fluctuations in HES7 oscillations, whereas the absence of the Notch modulator gene lunatic fringe (Lfng) leads to a loss of synchrony between PSM cells. Furthermore, HES7 oscillations are severely dampened in individual cells of Lfng-null PSM. However, when Lfng-null PSM cells were completely dissociated, the amplitude and periodicity of HES7 oscillations were almost normal, which suggests that LFNG is involved mostly in cell-cell coupling. Mixed cultures of control and Lfng-null PSM cells, and an optogenetic Notch signalling reporter assay, revealed that LFNG delays the signal-sending process of intercellular Notch signalling transmission. These results-together with mathematical modelling-raised the possibility that Lfng-null PSM cells shorten the coupling delay, thereby approaching a condition known as the oscillation or amplitude death of coupled oscillators8. Indeed, a small compound that lengthens the coupling delay partially rescues the amplitude and synchrony of HES7 oscillations in Lfng-null PSM cells. Our study reveals a delay control mechanism of the oscillatory networks involved in somite segmentation, and indicates that intercellular coupling with the correct delay is essential for synchronized oscillation.
Light-mediated control of Gene expression in mammalian cells.
Taking advantage of the recent development of genetically-defined photo-activatable actuator molecules, cellular functions, including gene expression, can be controlled by exposure to light. Such optogenetic strategies enable precise temporal and spatial manipulation of targeted single cells or groups of cells at a level hitherto impossible. In this review, we introduce light-controllable gene expression systems exploiting blue or red/far-red wavelengths and discuss their inherent properties potentially affecting induced downstream gene expression patterns. We also discuss recent advances in optical devices that will extend the application of optical gene expression control technologies into many different areas of biology and medicine.
Stick-slip dynamics of cell adhesion triggers spontaneous symmetry breaking and directional migration of mesenchymal cells on one-dimensional lines.
Directional cell motility relies on the ability of single cells to establish a front-rear polarity and can occur in the absence of external cues. The initiation of migration has often been attributed to the spontaneous polarization of cytoskeleton components, while the spatiotemporal evolution of cell-substrate interaction forces has yet to be resolved. Here, we establish a one-dimensional microfabricated migration assay that mimics the complex in vivo fibrillar environment while being compatible with high-resolution force measurements, quantitative microscopy, and optogenetics. Quantification of morphometric and mechanical parameters of NIH-3T3 fibroblasts and RPE1 epithelial cells reveals a generic stick-slip behavior initiated by contractility-dependent stochastic detachment of adhesive contacts at one side of the cell, which is sufficient to trigger cell motility in 1D in the absence of pre-established polarity. A theoretical model validates the crucial role of adhesion dynamics, proposing that front-rear polarity can emerge independently of a complex self-polarizing system.
Optogenetic Control of Microtubule Dynamics.
Light can be controlled with high spatial and temporal accuracy. Therefore, optogenetics is an attractive experimental approach to modulate intracellular cytoskeleton dynamics at much faster timescales than by genetic modification. For example, in mammalian cells, microtubules (MTs) grow tens of micrometers per minute and many intracellular MT functions are mediated by a complex of +TIP proteins that dynamically associate with growing MT plus ends. EB1 is a central component of this +TIP protein network, and we recently developed a photo-inactivated π-EB1 by inserting a blue light-sensitive LOV2/Zdk1 module between the EB1 MT-binding domain and the +TIP adaptor domain. Blue light-induced π-EB1 photodissociation results in disassembly of the +TIP complex and strongly attenuates MT growth in mammalian cells.In this chapter, we discuss theoretical and practical aspects of how to perform high-resolution live-cell microscopy in combination with π-EB1 photodissociation. However, these techniques are broadly applicable to other LOV2-based and likely other blue light-sensitive optogenetics. In addition to being a tool to investigate +TIP functions acutely and with subcellular resolution, because of its dramatic and rapid change in intracellular localization, π-EB1 can serve as a powerful tool to test and characterize optogenetic illumination setups. We describe protocols on how to achieve micrometer-scale intracellular control of π-EB1 activity using patterned illumination, and we introduce a do-it-yourself LED cube design compatible with transmitted light microscopy in multiwell plates.
The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata.
Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. The crystal structures of two PACs, from Oscillatoria acuminata (OaPAC) and Beggiatoa sp. (bPAC), have been solved, and they show a high degree of similarity. However, the photoactivity of OaPAC is much lower than that of bPAC, and the regulatory mechanism of PAC photoactivity, which induces the difference in activity between OaPAC and bPAC, has not yet been clarified. Here, we investigated the role of the C-terminal region in OaPAC, the length of which is the only notable difference from bPAC. We found that the photoactivity of OaPAC was inversely proportional to the C-terminal length. However, the deletion of more than nine amino acids did not further increase the activity, indicating that the nine amino acids at the C-terminal critically affect the photoactivity. Besides, absorption spectral features of light-sensing domains (BLUF domains) of the C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. The study characterizes new PAC mutants with modified photoactivities, which could be useful as optogenetics tools.
A Computational Protocol for Regulating Protein Binding Reactions with a Light-Sensitive Protein Dimer.
Light-sensitive proteins can be used to perturb signaling networks in living cells and animals with high spatiotemporal resolution. We recently engineered a protein heterodimer that dissociates when irradiated with blue light and demonstrated that by fusing each half of the dimer to termini of a protein that it is possible to selectively block binding surfaces on the protein when in the dark. On activation with light, the dimer dissociates and exposes the binding surface, allowing the protein to bind its partner. Critical to the success of this system, called Z-lock, is that the linkers connecting the dimer components to the termini are engineered so that the dimer forms over the appropriate binding surface. Here, we develop and test a protocol in the Rosetta molecular modeling program for designing linkers for Z-lock. We show that the protocol can predict the most effective linker sets for three different light-sensitive switches, including a newly designed switch that binds the Rho-family GTPase Cdc42 on stimulation with blue light. This protocol represents a generalized computational approach to placing a wide variety of proteins under optogenetic control with Z-lock.
RhoA Mediates Epithelial Cell Shape Changes via Mechanosensitive Endocytosis.
Epithelial remodeling involves ratcheting behavior whereby periodic contractility produces transient changes in cell-cell contact lengths, which stabilize to produce lasting morphogenetic changes. Pulsatile RhoA activity is thought to underlie morphogenetic ratchets, but how RhoA governs transient changes in junction length, and how these changes are rectified to produce irreversible deformation, remains poorly understood. Here, we use optogenetics to characterize responses to pulsatile RhoA in model epithelium. Short RhoA pulses drive reversible junction contractions, while longer pulses produce irreversible junction length changes that saturate with prolonged pulse durations. Using an enhanced vertex model, we show this is explained by two effects: thresholded tension remodeling and continuous strain relaxation. Our model predicts that structuring RhoA into multiple pulses overcomes the saturation of contractility and confirms this experimentally. Junction remodeling also requires formin-mediated E-cadherin clustering and dynamin-dependent endocytosis. Thus, irreversible junction deformations are regulated by RhoA-mediated contractility, membrane trafficking, and adhesion receptor remodeling.
Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms.
Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. Here, we exploited proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, we revealed the landscape of interactions with RhoGEFs and RhoGAPs. We systematically defined effectors of Rho proteins to reveal candidates for classical and atypical Rho proteins. We used optogenetics to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. We identified that active RHOA binds the kinase SLK in Drosophila and mammalian cells to promote Ezrin-Radixin-Moesin phosphorylation. Our proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family.
Strategies for Engineering and Rewiring Kinase Regulation.
Eukaryotic protein kinases (EPKs) catalyze the transfer of a phosphate group onto another protein in response to appropriate regulatory cues. In doing so, they provide a primary means for cellular information transfer. Consequently, EPKs play crucial roles in cell differentiation and cell-cycle progression, and kinase dysregulation is associated with numerous disease phenotypes including cancer. Nonnative cues for synthetically regulating kinases are thus much sought after, both for dissecting cell signaling pathways and for pharmaceutical development. In recent years advances in protein engineering and sequence analysis have led to new approaches for manipulating kinase activity, localization, and in some instances specificity. These tools have revealed fundamental principles of intracellular signaling and suggest paths forward for the design of therapeutic allosteric kinase regulators.
Engineered BRET-Based Biologic Light Sources Enable Spatiotemporal Control over Diverse Optogenetic Systems.
Light-inducible optogenetic systems offer precise spatiotemporal control over a myriad of biologic processes. Unfortunately, current systems are inherently limited by their dependence on external light sources for their activation. Further, the utility of laser/LED-based illumination strategies are often constrained by the need for invasive surgical procedures to deliver such devices and local heat production, photobleaching and phototoxicity that compromises cell and tissue viability. To overcome these limitations, we developed a novel BRET-activated optogenetics (BEACON) system that employs biologic light to control optogenetic tools. BEACON is driven by self-illuminating bioluminescent-fluorescent proteins that generate "spectrally tuned" biologic light via bioluminescence resonance energy transfer (BRET). Notably, BEACON robustly activates a variety of commonly used optogenetic systems in a spatially restricted fashion, and at physiologically relevant time scales, to levels that are achieved by conventional laser/LED light sources.
SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors.
The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3'UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3'UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.
Primary Cilia Signaling Promotes Axonal Tract Development and Is Disrupted in Joubert Syndrome-Related Disorders Models.
Appropriate axonal growth and connectivity are essential for functional wiring of the brain. Joubert syndrome-related disorders (JSRD), a group of ciliopathies in which mutations disrupt primary cilia function, are characterized by axonal tract malformations. However, little is known about how cilia-driven signaling regulates axonal growth and connectivity. We demonstrate that the deletion of related JSRD genes, Arl13b and Inpp5e, in projection neurons leads to de-fasciculated and misoriented axonal tracts. Arl13b deletion disrupts the function of its downstream effector, Inpp5e, and deregulates ciliary-PI3K/AKT signaling. Chemogenetic activation of ciliary GPCR signaling and cilia-specific optogenetic modulation of downstream second messenger cascades (PI3K, AKT, and AC3) commonly regulated by ciliary signaling receptors induce rapid changes in axonal dynamics. Further, Arl13b deletion leads to changes in transcriptional landscape associated with dysregulated PI3K/AKT signaling. These data suggest that ciliary signaling acts to modulate axonal connectivity and that impaired primary cilia signaling underlies axonal tract defects in JSRD.
A time-dependent role for the transcription factor CREB in neuronal allocation to an engram underlying a fear memory revealed using a novel in vivo optogenetic tool to modulate CREB function.
The internal representation of an experience is thought to be encoded by long-lasting physical changes to the brain ("engrams") (Josselyn et al. Nat Rev Neurosci 16:521-534, 2015; Josselyn et al. J Neurosci 37:4647-4657, 2017; Schacter. 2001; Tonegawa et al. Neuron 87:918-931, 2015). Previously, we (Han et al. Science 316:457-460, 2007) and others (Zhou et al. Nat Neurosci 12:1438-1443, 2009) showed within the lateral amygdala (LA), a region critical for auditory conditioned fear, eligible neurons compete against one other for allocation to an engram. Neurons with relatively higher function of the transcription factor CREB were more likely to be allocated to the engram. In these studies, though, CREB function was artificially increased for several days before training. Precisely when increased CREB function is important for allocation remains an unanswered question. Here, we took advantage of a novel optogenetic tool (opto-DN-CREB) (Ali et al. Chem Biol 22:1531-1539, 2015) to gain spatial and temporal control of CREB function in freely behaving mice. We found increasing CREB function in a small, random population of LA principal neurons in the minutes-hours, but not 24 h, before training was sufficient to enhance memory, likely because these neurons were preferentially allocated to the underlying engram. However, similarly increasing CREB activity in a small population of random LA neurons immediately after training disrupted subsequent memory retrieval, likely by disrupting the precise spatial and temporal patterns of offline post-training neuronal activity and/or function required for consolidation. These findings reveal the importance of the timing of CREB activity in regulating allocation and subsequent memory retrieval, and further, highlight the potential of optogenetic approaches to control protein function with temporal specificity in behaving animals.
Blue light-triggered optogenetic system for treating uveal melanoma.
Uveal melanoma is the most common intraocular primary malignancy in adults and has been considered a fatal disease for decades. Optogenetics is an emerging technique that can control the activation of signaling components via irradiation with visible light. The clinical translation of optogenetics has been limited because of the need for surgical implantation of electrodes and relatively shallow tissue penetration. As visible light easily penetrates the eyes, we hypothesized that an optogenetics approach can be an effective treatment of uveal melanoma without surgery. In this study, we evaluated the feasibility of this strategy by using a genetically encoded optogenetic system based on reversible blue light-induced binding pairs between Fas-CIB1-EGFP and CRY2-mCherry-FADD. Subretinal injection of B16 cells was performed to create a uveal melanoma model. Plasmids pairs were co-transfected into B16 cells. We found that blue light irradiation dynamically controlled the translocation of FADD to Fas on the plasma membrane and induced the apoptosis of B16 cells transfected with the optogenetic nanosystem in vitro. Moreover, the blue light-controlled optogenetic nanosystem suppressed the growth of uveal melanoma in vivo by inducing apoptosis. These results suggest that light-controlled optogenetic therapy can be used as a potential novel therapeutic strategy for uveal melanoma.
Optimizing photoswitchable MEK.
Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both Drosophila and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.
A yeast optogenetic toolkit (yOTK) for gene expression control in Saccharomyces cerevisiae.
Optogenetic tools for controlling gene expression are ideal for tuning synthetic biological networks due to the exquisite spatiotemporal control available with light. Here we develop an optogenetic system for gene expression control integrated with an existing yeast toolkit allowing for rapid, modular assembly of light-controlled circuits in the important chassis organism Saccharomyces cerevisiae. We reconstitute activity of a split synthetic zinc-finger transcription factor (TF) using light-induced dimerization mediated by the proteins CRY2 and CIB1. We optimize function of this split TF and demonstrate the utility of the toolkit workflow by assembling cassettes expressing the TF activation domain and DNA-binding domain at different levels. Utilizing this TF and a synthetic promoter we demonstrate that light-intensity and duty-cycle can be used to modulate gene expression over the range currently available from natural yeast promoters. This work allows for rapid generation and prototyping of optogenetic circuits to control gene expression in Saccharomyces cerevisiae. This article is protected by copyright. All rights reserved.
The importance of cell-cell interaction dynamics in bottom-up tissue engineering: Concepts of colloidal self-assembly in the fabrication of multicellular architectures.
Building tissue from cells as the basic building block based on principles of self-assembly is a challenging and promising approach. Understanding how far principles of self-assembly and self-sorting known for colloidal particles apply to cells remains unanswered. In this study, we demonstrate that not just controlling the cell-cell interactions but also their dynamics is a crucial factor that determines the formed multicellular structure, using photoswitchable interactions between cells that are activated with blue light and reverse in the dark. Tuning dynamics of the cell-cell interactions by pulsed light activation, results in multicellular architectures with different sizes and shapes. When the interactions between cells are dynamic compact and round multicellular clusters under thermodynamic control form, while otherwise branched and lose aggregates under kinetic control assemble. These structures parallel what is known for colloidal assemblies under reaction and diffusion limited cluster aggregation, respectively. Similarly, dynamic interactions between cells are essential for cells to self-sort into distinct groups. Using four different cell types, which expressed two orthogonal cell-cell interaction pairs, the cells sorted into two separate assemblies. Bringing concepts of colloidal self-assembly to bottom-up tissue engineering provides a new theoretical framework and will help in the design of more predictable tissue-like structures.
Construction of light-activated neurotrophin receptors using the improved Light-Induced Dimerizer (iLID) .
Receptor tyrosine kinases (RTKs) play crucial roles in human health, and their misregulation is implicated in disorders ranging from neurodegenerative disorders to cancers. The highly conserved mechanism of activation of RTKs makes them especially appealing candidates for control via optogenetic dimerization methods. This work offers a strategy for using the improved Light-Induced Dimer (iLID) system with a constructed tandem-dimer of its binding partner nano (tdnano) to build light-activatable versions of RTKs. In the absence of light, the iLID-RTK is cytosolic, monomeric and inactive. Under blue light, the iLID + tdnano system recruits two copies of iLID-RTK to tdnano, dimerizing and activating the RTK. We demonstrate that iLID opto-iTrkA and opto-iTrkB are capable of reproducing downstream ERK and Akt signaling only in the presence of tdnano. We further show with our opto-iTrkA that the system is compatible with multi-day and population-level activation of TrkA in PC12 cells. By leveraging genetic targeting of tdnano, we achieve RTK activation at a specific subcellular location even with whole-cell illumination, allowing us to confidently probe the impact of context on signaling outcome.
Visualization of a blue light transmission area in living animals using light-induced nuclear translocation of fluorescent proteins.
Optical manipulations are widely used to analyze neuronal functions in vivo. Blue light is frequently used to activate channelrhodopsins or LOV domains, although the degrees of its absorption and scattering are higher than those of longer wavelength light. High spatial resolution of optical manipulation is easily achieved in vitro, while the light is unevenly scattered and absorbed in tissues due to many factors. It is difficult to spatially measure a blue light transmission area in vivo. Here, we propose a genetic method to visualize blue light transmission in the brain and other organs using light-induced nuclear translocation of fluorescent proteins with a LOV domain. A light-inducible nuclear localization signal (LINuS) consists of a LOV2 domain fused with a nuclear localization signal (NLS). We confirmed that blue light illumination induced reversible translocation of NES-tdTomato-LINuS from the cytosol to the nucleus within 30 min in HEK293 cells. By employing a PHP.eb capsid that can penetrate the blood-brain barrier, retro-orbital sinus injection of adeno-associated virus (AAV) vectors induced scattered expression of nuclear export signal (NES)-tdTomato-LINuS in the brain. We confirmed that 30-min transcranial blue light illumination induced nuclear translocation of NES-tdTomato-LINuS in the cortex, the hippocampus, and even the paraventricular nucleus of the thalamus. We also found that mice exposed to blue light in a shaved abdominal area exhibited a substantial increase in nuclear translocation in the ventral surface lobe of the liver. These results provide a simple way to obtain useful information on light transmission in tissues without any transgenic animals or skillful procedures.
Optogenetic control of cofilin and αTAT in living cells using Z-lock.
Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.
Optogenetic lac operon to control chemical and protein production in Escherichia coli with light.
Control of the lac operon with IPTG has been used for decades to regulate gene expression in E. coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities such as easy tunability, reversibility, dynamic induction strength, and spatial control that are difficult to obtain with chemical inducers. We developed an optogenetic lac operon in a series of circuits we call OptoLAC. With these circuits, we control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least 2L bioreactors. Furthermore, OptoLAC circuits enable light control of recombinant protein production, reaching yields comparable to IPTG induction, but with enhanced tunability of expression and spatial control. OptoLAC circuits are potentially useful to confer light controls over other cell functions originally engineered to be IPTG-inducible.
Elucidating cyclic AMP signaling in subcellular domains with optogenetic tools and fluorescent biosensors.
The second messenger 3',5'-cyclic nucleoside adenosine monophosphate (cAMP) plays a key role in signal transduction across prokaryotes and eukaryotes. Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision. To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited. Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively. In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell. This review provides an overview about optogenetic tools and biosensors to shed light on the subcellular organization of cAMP signaling.