Showing 1 - 25 of 25 results
A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch.
Optogenetics enables genome manipulations with high spatiotemporal resolution, opening exciting possibilities for fundamental and applied biological research. Here, we report the development of LiCre, a novel light-inducible Cre recombinase. LiCre is made of a single flavin-containing protein comprising the AsLOV2 photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizing mutations in its N-terminal and C-terminal domains. LiCre can be activated within minutes of illumination with blue light, without the need of additional chemicals. When compared to existing photoactivatable Cre recombinases based on two split units, LiCre displayed faster and stronger activation by light as well as a lower residual activity in the dark. LiCre was efficient both in yeast, where it allowed us to control the production of β-carotene with light, and in human cells. Given its simplicity and performances, LiCre is particularly suited for fundamental and biomedical research, as well as for controlling industrial bioprocesses.
Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces.
During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.
Optogenetic control of gut bacterial metabolism to promote longevity.
Gut microbial metabolism is associated with host longevity. However, because it requires direct manipulation of microbial metabolism in situ, establishing a causal link between these two processes remains challenging. We demonstrate an optogenetic method to control gene expression and metabolite production from bacteria residing in the host gut. We genetically engineer an Escherichia coli strain that secretes colanic acid (CA) under the quantitative control of light. Using this optogenetically-controlled strain to induce CA production directly in the Caenorhabditis elegans gut, we reveal the local effect of CA in protecting intestinal mitochondria from stress-induced hyper-fragmentation. We also demonstrate that the lifespan-extending effect of this strain is positively correlated with the intensity of green light, indicating a dose-dependent CA benefit on the host. Thus, optogenetics can be used to achieve quantitative and temporal control of gut bacterial metabolism in order to reveal its local and systemic effects on host health and aging.
Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos.
Ventral furrow formation, the first step in Drosophila gastrulation, is a well-studied example of tissue morphogenesis. Rho1 is highly active in a subset of ventral cells and is required for this morphogenetic event. However, it is unclear whether spatially patterned Rho1 activity alone is sufficient to recapitulate all aspects of this morphogenetic event, including anisotropic apical constriction and coordinated cell movements. Here, using an optogenetic probe that rapidly and robustly activates Rho1 in Drosophila tissues, we show that Rho1 activity induces ectopic deformations in the dorsal and ventral epithelia of Drosophila embryos. These perturbations reveal substantial differences in how ventral and dorsal cells, both within and outside the zone of Rho1 activation, respond to spatially and temporally identical patterns of Rho1 activation. Our results demonstrate that an asymmetric zone of Rho1 activity is not sufficient to recapitulate ventral furrow formation and reveal that additional, ventral-specific factors contribute to the cell- and tissue-level behaviors that emerge during ventral furrow formation.
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.
Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells.
Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single-excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28oC to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.
Optical control of ERK and AKT signaling promotes axon regeneration and functional recovery of PNS and CNS in Drosophila.
Neuroregeneration is a dynamic process synergizing the functional outcomes of multiple signaling circuits. Channelrhodopsin-based optogenetics shows the feasibility of stimulating neural repair but does not pin down specific signaling cascades. Here, we utilized optogenetic systems, optoRaf and optoAKT, to delineate the contribution of the ERK and AKT signaling pathways to neuroregeneration in live Drosophila larvae. We showed that optoRaf or optoAKT activation not only enhanced axon regeneration in both regeneration-competent and -incompetent sensory neurons in the peripheral nervous system but also allowed temporal tuning and proper guidance of axon regrowth. Furthermore, optoRaf and optoAKT differ in their signaling kinetics during regeneration, showing a gated versus graded response, respectively. Importantly in the central nervous system, their activation promotes axon regrowth and functional recovery of the thermonociceptive behavior. We conclude that non-neuronal optogenetics target damaged neurons and signaling subcircuits, providing a novel strategy in the intervention of neural damage with improved precision.
Light-Regulated allosteric switch enables temporal and subcellular control of enzyme activity.
Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.
Optogenetic activation of heterotrimeric G-proteins by LOV2GIVe, a rationally engineered modular protein.
Heterotrimeric G-proteins are signal transducers involved in mediating the action of many natural extracellular stimuli as well as of many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we developed LOV2GIVe, an engineered modular protein that allows the activation of heterotrimeric G-proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, i.e. metazoan opsins, which are light-activated GPCRs. Instead, LOV2GIVe consists of the fusion of a G-protein activating peptide derived from a non-GPCR regulator of G-proteins to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity.
Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium.
Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling has been challenging due to the lack of tools investigate ciliary signaling. Here, we describe a nanobody-based targeting approach for optogenetic tools in mammalian cells and in vivo in zebrafish to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after fusion to ciliary targeting sequences. We functionally localized modifiers of cAMP signaling, the photo-activated adenylate cyclase bPAC and the light-activated phosphodiesterase LAPD, and the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.
Application of optogenetic Amyloid-β distinguishes between metabolic and physical damage in neurodegeneration.
The brains of Alzheimer's Disease patients show a decrease in brain mass and a preponderance of extracellular Amyloid-β plaques. These plaques are formed by aggregation of polypeptides that are derived from the Amyloid Precursor Protein (APP). Amyloid-β plaques are thought to play either a direct or an indirect role in disease progression, however the exact role of aggregation and plaque formation in the aetiology of Alzheimer's Disease is subject to debate as the biological effects of soluble and aggregated Amyloid-β peptides are difficult to separate in vivo. To investigate the consequences of formation of Amyloid-β oligomers in living tissues, we developed a fluorescently tagged, optogenetic Amyloid-β peptide that oligomerizes rapidly in the presence of blue light. We applied this system to the crucial question of how intracellular Amyloid-β oligomers underlie the pathologies of Alzheimer's Disease. We use Drosophila, C. elegans and D. rerio to show that, although both expression and induced oligomerization of Amyloid-β were detrimental to lifespan and healthspan, we were able to separate the metabolic and physical damage caused by light-induced Amyloid-β oligomerization from Amyloid-β expression alone. The physical damage caused by Amyloid-β oligomers also recapitulated the catastrophic tissue loss that is a hallmark of late AD. We show that the lifespan deficit induced by Amyloid-β oligomers was reduced with Li+ treatment. Our results present the first model to separate different aspects of disease progression.
Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor.
The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.
Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling.
T cells are thought to discriminate self from foreign peptides by converting small differences in ligand binding half-life into large changes in cell signaling. Such a kinetic proofreading model has been difficult to test directly, as existing methods of altering ligand binding half-life also change other potentially important biophysical parameters, most notably the mechanical stability of the receptor-ligand interaction. Here we develop an optogenetic approach to specifically tune the binding half-life of a chimeric antigen receptor without changing other binding parameters and provide direct evidence of kinetic proofreading in T cell signaling. This half-life discrimination is executed in the proximal signaling pathway, downstream of ZAP70 recruitment and upstream of diacylglycerol accumulation. Our methods represent a general tool for temporal and spatial control of T cell signaling and extend the reach of optogenetics to probe pathways where the individual molecular kinetics, rather than the ensemble average, gates downstream signaling.
Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions.
Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested 'AND' gate design of SPARK2-in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest-yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.
Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology.
Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors. Here we introduce a light-inducible SG system, termed OptoGranules, based on optogenetic multimerization of G3BP1, which is an essential scaffold protein for SG assembly. In this system, which permits experimental control of SGs in living cells in the absence of exogenous stressors, we demonstrate that persistent or repetitive assembly of SGs is cytotoxic and is accompanied by the evolution of SGs to cytoplasmic inclusions that recapitulate the pathology of ALS-FTD.
RalB directly triggers invasion downstream Ras by mobilizing the Wave complex.
The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anti-cancer strategies.
Functionally asymmetric motor neurons contribute to coordinating locomotion of Caenorhabditis elegans.
Locomotion circuits developed in simple animals, and circuit motifs further evolved in higher animals. To understand locomotion circuit motifs, they must be characterized in many models. The nematode Caenorhabditis elegans possesses one of the best-studied circuits for undulatory movement. Yet, for 1/6th of the cholinergic motor neurons (MNs), the AS MNs, functional information is unavailable. Ventral nerve cord (VNC) MNs coordinate undulations, in small circuits of complementary neurons innervating opposing muscles. AS MNs differ, as they innervate muscles and other MNs asymmetrically, without complementary partners. We characterized AS MNs by optogenetic, behavioral and imaging analyses. They generate asymmetric muscle activation, enabling navigation, and contribute to coordination of dorso-ventral undulation as well as anterio-posterior bending wave propagation. AS MN activity correlated with forward and backward locomotion, and they functionally connect to premotor interneurons (PINs) for both locomotion regimes. Electrical feedback from AS MNs via gap junctions may affect only backward PINs.
Optogenetic dissection of mitotic spindle positioning in vivo.
The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα-GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα-GDP, Gα-GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2 Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.
Shining light on spindle positioning.
Optogenetic approaches are leading to a better understanding of the forces that determine the plane of cell division.
Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble.
To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA's N-terminal long arm, dynein-based astral microtubule gliding, and NuMA's direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.
Time-gated detection of protein-protein interactions with transcriptional readout.
Transcriptional assays, such as yeast two-hybrid and TANGO, that convert transient protein-protein interactions (PPIs) into stable expression of transgenes are powerful tools for PPI discovery, screens, and analysis of cell populations. However, such assays often have high background and lose information about PPI dynamics. We have developed SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics), in which proteolytic release of a membrane-tethered transcription factor (TF) requires both a PPI to deliver a protease proximal to its cleavage peptide and blue light to uncage the cleavage site. SPARK was used to detect 12 different PPIs in mammalian cells, with 5 min temporal resolution and signal ratios up to 37. By shifting the light window, we could reconstruct PPI time-courses. Combined with FACS, SPARK enabled 51 fold enrichment of PPI-positive over PPI-negative cells. Due to its high specificity and sensitivity, SPARK has the potential to advance PPI analysis and discovery.
Decoding temporal interpretation of the morphogen Bicoid in the early Drosophila embryo.
Morphogen gradients provide essential spatial information during development. Not only the local concentration but also duration of morphogen exposure is critical for correct cell fate decisions. Yet, how and when cells temporally integrate signals from a morphogen remains unclear. Here, we use optogenetic manipulation to switch off Bicoid-dependent transcription in the early Drosophila embryo with high temporal resolution, allowing time-specific and reversible manipulation of morphogen signalling. We find that Bicoid transcriptional activity is dispensable for embryonic viability in the first hour after fertilization, but persistently required throughout the rest of the blastoderm stage. Short interruptions of Bicoid activity alter the most anterior cell fate decisions, while prolonged inactivation expands patterning defects from anterior to posterior. Such anterior susceptibility correlates with high reliance of anterior gap gene expression on Bicoid. Therefore, cell fates exposed to higher Bicoid concentration require input for longer duration, demonstrating a previously unknown aspect of Bicoid decoding.
Cell cycle entry triggers a switch between two modes of Cdc42 activation during yeast polarization.
Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42•GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold, and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment.
Near-infrared photoactivatable control of Ca(2+) signaling and optogenetic immunomodulation.
The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed 'Opto-CRAC') that selectively and remotely controls Ca(2+) oscillations and Ca(2+)-responsive gene expression to regulate the function of non-excitable cells, including T lymphocytes, macrophages and dendritic cells. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless photoactivation of Ca(2+)-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded 'photoactivatable adjuvant' to improve antigen-specific immune responses to specifically destruct tumor cells. Our study represents a solid step forward towards the goal of achieving remote and wireless control of Ca(2+)-modulated activities with tailored function.
Controlling fertilization and cAMP signaling in sperm by optogenetics.
Optogenetics is a powerful technique to control cellular activity by light. The light-gated Channelrhodopsin has been widely used to study and manipulate neuronal activity in vivo, whereas optogenetic control of second messengers in vivo has not been examined in depth. In this study, we present a transgenic mouse model expressing a photoactivated adenylyl cyclase (bPAC) in sperm. In transgenic sperm, bPAC mimics the action of the endogenous soluble adenylyl cyclase (SACY) that is required for motility and fertilization: light-stimulation rapidly elevates cAMP, accelerates the flagellar beat, and, thereby, changes swimming behavior of sperm. Furthermore, bPAC replaces endogenous adenylyl cyclase activity. In mutant sperm lacking the bicarbonate-stimulated SACY activity, bPAC restored motility after light-stimulation and, thereby, enabled sperm to fertilize oocytes in vitro. We show that optogenetic control of cAMP in vivo allows to non-invasively study cAMP signaling, to control behaviors of single cells, and to restore a fundamental biological process such as fertilization.