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Showing 1 - 25 of 2049 results
1.
Synthetic Toll-Like Receptors for Control of Innate Immunity With Far-Red Light.
Abstract:
Toll-like receptors (TLRs) are single-pass transmembrane proteins that initiate innate immune responses through recognition of pathogen-associated molecular patterns, including lipopolysaccharide, flagellin, and microbial nucleic acids. In mammals, TLRs are expressed in both immune and non-immune cells, where they activate cytokine expression through the myeloid differentiation primary response 88 (MyD88) signaling pathway and subsequently engage the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) or interferon regulatory factor 3 / interferon regulatory factor 7 (IRF3/IRF7) pathways. To enable optical control of TLR function, the extracellular domains of several homodimeric TLRs, including TLR3, TLR4, and TLR5, are replaced with the photosensory core module of the bacterial phytochrome DrBphP. The resulting chimeric receptors activate the NF-κB and IRF3/IRF7 pathways in mammalian cells in a far-red-light-dependent manner. The MyD88 pathway is further reprogrammed to induce caspase activation instead of cytokine production, thereby creating a synthetic system that links TLR stimulation to caspase signaling. This strategy establishes a versatile optogenetic platform for far-red-light control of innate immune and cell death pathways.
2.
Synthetic yeast-bacterium consortium enables co-inducible relayed synthesis of chemicals.
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Xu, M
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Yu, J
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Chen, X
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Li, X
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Guo, Y
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Qian, Z
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Liu, Q
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Ren, Y
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Lu, J
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Chen, X
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Yang, Y
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Cai, M
Abstract:
Microbial coculture can integrate advantages and overcome the metabolic imbalance of individual species. Programming strain interactions represents a common routine for synthetic microbial communities with distinct species, which causes difficulties and redundant workloads in interaction construction before being available as chassis hosts. This study explores yeast-bacterium consortium without engineered interactions for the co-inducible relayed synthesis of natural products. The Komagataella phaffii-Escherichia coli consortium is explored for co-growth under selected conditions. Low-level glucose- and blue light-responsive transcriptional systems are rebuilt separately for each host, allowing single-signal co-induced activation of compound synthesis in coculture. Pathway redirection, genome mining, and rewiring of key targets for acyl donor degradation result in efficient production of the reporter molecule simvastatin (26.2 mg l-1) through living consortium cultured on simple carbon source. Inducible biosynthesis of another reporter compound (2S)-naringenin (165.6 mg l-1) further validates the extendibility of this community. The described platform represents a breakthrough in engineering microbial consortium for biosynthesis.
3.
GCL pruning of PIP3 establishes the soma-germline boundary.
Abstract:
Primordial germ cells (PGCs) are the first cells specified in the Drosophila embryo and are precursors to the germline. Their formation requires suppression of somatic fates, achieved by degrading the receptor tyrosine kinase Torso at the posterior pole through the ubiquitin ligase adaptor germ cell-less (GCL). Although Torso is known to antagonize PGC formation, the underlying mechanisms remained unclear. Here, we combine optogenetic Ras activation and Ras effector loop mutants to show that Ras suppresses PGC formation independently of the canonical Raf/MEK/ERK pathway. We identify an unexpected early role for Torso in activating phosphoinositide 3-kinase (PI3K), generating membrane domains enriched in phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Elevated PI3K activity disrupts PGC formation, while reduced PI3K activity creates ectopic PGCs. We demonstrate that GCL remodels the posterior pole membrane by suppressing Torso-dependent PI3K activation. Clearing PIP3 enables myosin II enrichment, allowing for PGC formation. Together, our findings reveal how antagonistic Torso and GCL activities establish the soma-germline boundary by organizing cortical lipids.
4.
Delineation of signaling microdomains from live-cell movies.
Abstract:
Many molecular signals are known to be organized in subcellular regions termed microdomains. One example entails Rho GTPases, which control a range of cell behaviors through their functional patterning in spatiotemporal units. While fluorescent biosensors have allowed precise measurement of Rho GTPase activity in living cells, computational tools to delineate and track signaling microdomains in time-lapse image sequences of such biosensors do not exist. Here, we introduce a method that centers on the notion of activation time series coordination to identify signaling microdomains in space and time. After validating the algorithm with simulated microdomains, we show that our method identifies domains that support current hypotheses on the signaling architecture governing Rho GTPase organization and also respond to perturbation with optogenetics. A record of this paper's transparent peer review process is included in the supplemental information.
5.
A single-component optogenetic toolkit for reversible visualization and programmable control of microtubule dynamics.
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Lan, TH
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Ma, G
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Liu, X
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Duong, T
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Price, A
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Chiu, M
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Du, D
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Reiner, DJ
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Huang, Y
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Zhou, Y
Abstract:
Microtubules form dynamic cytoskeletal scaffolds essential for intracellular transport, organelle positioning, and spatial organization of signaling. Their architecture and function are continuously remodeled through the concerted actions of microtubule-associated proteins (MAPs), post-translational modifications (PTMs), and molecular motors. To precisely interrogate these processes in living systems, we developed a genetically encoded, single-component optogenetic platform for spatiotemporal control of microtubule organization and dynamics. By harnessing light-induced oligomerization to regulate microtubule association, this system supports reversible microtubule labeling and plus-end tracking, localized control of tubulin PTMs, optically regulated kinesin-driven cargo transport, and inducible microtubule severing within a unified design strategy. Using these tools, we reveal how local microtubule integrity governs lysosomal trafficking and endoplasmic reticulum (ER)-associated signaling dynamics. Collectively, this modular optogenetic toolkit bridges molecular design with cytoskeletal function, offering a versatile platform to dissect how dynamic cytoskeletal architectures coordinate intracellular organization, transport, and signaling.
6.
Light-Regulated Cancer Immunotherapy Using Individually Encapsulated Synthetic Circuit-Engineered Cells.
Abstract:
Cell therapy holds great promise for cancer immunotherapy, but its clinical efficacy is severely hindered by poor post-transplant cell survival, low homing efficiency, and host immune clearance. To address these challenges, this study develops a novel light-controlled immunotherapy strategy that integrates a red/far-red light genetic switch with single-cell encapsulation engineering. The red/far-red light (660/730 nm) reversible regulatory system enables precise spatiotemporal control over the expression of therapeutic proteins in engineered cells (e.g., CAR-T or engineered HEK 293T cells), allowing on-demand activation of anti-tumor immune responses. On this basis, a mild enzyme-mediated single-cell encapsulation technique is further employed to rapidly form a protective hydrogel coating in situ on the cell surface, thereby enhancing the survival of transplanted cells under hostile in vivo microenvironments. This strategy combines precise gene expression regulation with physical protection, improving therapeutic outcomes without the need for genomic modification of the cells. It provides a new paradigm for developing safe, controllable, and efficient cancer immunotherapy. Key features • Using a 660/730 nm red/far-red light reversible switch, deep tissue penetration enables spatiotemporal precise control of tumor-targeted therapeutic proteins. • Achieving rapid and gentle in situ gelation encapsulation of single-cell surfaces through HRP-pHLIP membrane anchoring and HA-dopamine enzymatic crosslinking. • Targeted strategies to overcome post-transplant hypoxia, inflammatory stress, and pulmonary first-pass entrapment, physically enhancing early cell survival prior to reaching the target tissue. • This experimental protocol requires at least three days.
7.
Multi-level precise regulation of gene transcription in the yeast Saccharomyces cerevisiae based on light-sensitive CRISPR/Cas systems.
Abstract:
Regulation of gene transcription based on clustered regularly interspaced short palindromic repeats (CRISPR) is a powerful tool for constructing synthetic gene circuits in Saccharomyces cerevisiae. The current CRISPR-based regulatory approaches primarily focus on inhibiting the binding of dCas9 protein to single guide RNA (sgRNA) or blocking target site recognition. However, these regulation strategies are often at a single level, and their sensitivity still needs to be improved. In this study, the gene regulatory approaches at the translational and post-translational levels were integrated with optogenetic control patterns to attain very sensitive multi-level precision regulation of the dCas9 protein, thereby facilitating flexible regulation of transcription levels of target genes. This strategy was used to regulate the transcription levels of fluorescent proteins, resulting in up to 2.58-fold increase in the fluorescence intensity of mCherry compared to that without regulation. This CRISPR-based multi-level optogenetic system should be extremely helpful in understanding gene regulatory networks and in designing robust genetic circuits for synthetic biology.
8.
An optogenetic smart microscopy platform reveals signaling dynamics-dependent control over collective cell migration.
Abstract:
In cell biology, optical techniques can measure cells' internal states (biosensors) and stimulate cellular responses (optogenetics). Yet the design of all-optical experiments is often manual: a predetermined stimulus pattern is applied to cells, biosensors are measured over time, and data are processed offline. Here, we develop PyCLM, a Python-based suite enabling closed-loop measurement, image segmentation, and optogenetic control of thousands of cells per experiment. We showcase PyCLM on diverse applications, including performing feedback control on single cells and delivering developmental signaling patterns to Drosophila embryos. We compare single-cell versus tissue-scale optogenetic control of epithelial migration, revealing that fast and slow waves of receptor tyrosine kinase activity determine the direction of tissue movement, matching prior in vivo observations in zebrafish and mouse. PyCLM enables simple setup of dynamic experiments to probe cell and tissue properties and provides a first step toward real-time control of single-cell states at the tissue scale.
9.
Temporal and spatial control of phosphatidylinositides using optogenetics ameliorates behavioral deficits in an Alzheimer's disease mouse model.
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Costa, AP
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Acquarone, E
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Lazarian, A
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Herman, M
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Wartchow, KM
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Bartelo, N
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Dartora, WJ
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Krumsiek, J
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Li, XL
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Baskin, JM
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Arancio, O
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Hussaini, SA
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McIntire, LB
Abstract:
Deficits in levels of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] mediate Alzheimer's disease (AD) pathogenesis and etiology. The depletion of PI(4,5)P2 has been reported in AD in both human brain and animal models. Repletion of the synaptic pool of PI(4,5)P2, through haploinsufficiency of the degrading enzyme, Synaptojanin 1, ameliorated behavioral deficits in a mouse model of AD, in spite of accumulating amyloid. In order to refine the contribution of PI(4,5)P2 to AD, we used optogenetic translocation of the PI(4,5)P2-synthesizing enzyme, phosphoinositide phosphate 4 kinase2A (PIP4K2A) to the plasma membrane using light inducible dimerizable cryptochrome 2 (CRY-2) and the transcription factor CRY2-binding domain (CIBN) fused to the plasma membrane-targeting motif (CAAX). Spatiotemporally controlled production of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] at the plasma membrane induced amelioration of behavioral deficits in a mouse model of AD. Imaging mass spectrometry confirmed alteration of specific PI(4,5)P2 acyl species, di-oleate, indicating that precise PI(4,5)P2 species may ultimately be leveraged for therapeutic intervention.
10.
Mapping the dynamic plant interactome: from in vitro assays to in vivo quantitative approaches.
Abstract:
Protein-protein interactions underpin virtually all biological processes in plants, from signal transduction and immune responses to development and stress adaptation. Despite their fundamental importance, the plant interactome remains far from complete, and existing maps are systematically biased by the technical limitations inherent to conventional detection platforms.
11.
An intrinsic cytoskeletal oscillator establishes neuronal polarity.
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Lin, TC
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Coles, CH
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Alfadil, E
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Fäßler, F
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Husch, A
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Dupraz, S
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Pietralla, T
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Narita, A
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Schelski, M
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Flynn, KC
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Stern, S
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Möhl, C
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Hilton, BJ
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Vauti, F
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Arnold, HH
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Schur, FKM
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Bradke, F
Abstract:
Neurons acquire polarity by specifying one neurite as the axon, whereas the others become dendrites. But how this fundamental asymmetry is established remains unclear1. Neuronal polarization has been thought to rely primarily on growth cones that sense external cues2. Here we show that growth cones alone do not direct this process and that the soma acts as a central organizer of neuronal polarization. Using live imaging and genetic loss-of-function approaches in vivo, combined with optogenetic control and local cytoskeletal perturbations in cultured neurons, we uncover a soma-initiated oscillatory program that primes axon selection. Periodic actin branching that depends on the actin-related protein 2/3 (ARP2/3) complex at the soma remodels a global actomyosin network, thereby generating an actin wave that retracts neurites before propagating into a single neurite tip. Exposure to this wave relaxes local actomyosin contractility, which drives a transient microtubule-based protrusion and biases this neurite towards axon fate. As the cell exits this oscillatory stage, this neurite can overcome global inhibition and extend independently of ARP2/3, whereas actomyosin activity suppresses axon formation in the remaining neurites so that they subsequently become dendrites. This soma-driven mechanism ensures the emergence of a single axon independent of environmental cues and underpins the unidirectional information flow in neuronal circuits.
12.
Astrocytic ankyrin-2 enables memory persistence in the mouse hippocampus.
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Kim, H
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Lim, J
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Kim, J
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Ozkan, E
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Kim, GH
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Park, H
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Park, MG
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Joo, B
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Lee, S
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Lee, KJ
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Kaang, BK
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Lee, CJ
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Koh, W
Abstract:
Memory persistence, the ability to retain information over time, is a fundamental feature of long-term memory. Although astrocytes contribute to synaptic plasticity, the molecular mechanisms by which they support memory persistence remain unclear. Here we show that astrocytic ankyrin-2 (Ank2) is required for memory persistence in adult mice. Astrocyte-specific deletion of Ank2 impaired remote memory without affecting recent memory and disrupted the maintenance of long-term potentiation. Loss of Ank2 reduced astrocyte contacts with engram neurons and impaired astrocyte morphogenesis driven by brain-derived neurotrophic factor (BDNF) signaling through the truncated tropomyosin receptor kinase B receptor (TrkB.T1) and inositol 1,4,5-trisphosphate receptor type 2 (IP3R2). Consistent with this mechanism, astrocytic Ank2 was required for the enhancement of memory persistence by hippocampal BDNF infusion. Furthermore, selective optogenetic activation of astrocytic TrkB.T1 signaling enhanced remote memory, demonstrating that astrocytic BDNF signaling is sufficient to promote memory persistence. These findings identify astrocytic Ank2 as a key regulator of long-term memory persistence.
13.
OpenEvo: An Open-Source Platform for Automated Evolution and Analysis.
Abstract:
Here we introduce OpenEvo, a fully open-source, low-cost turbidostat platform for automated continuous culture and directed evolution experiments. Existing tools are expensive, complex, or lack open-source hardware; OpenEvo addresses this gap with a complete, fully automated evolution platform with detailed, illustrated construction instructions for beginners, open-source software and firmware, priced around $300. An optional PC-based interface offers enhanced functionality, including remote access, programmable evolution cycles, programmable LED stimulation, and a data visualization tool. OpenEvo can cycle through three types of media for positive, negative, and neutral selection conditions, supporting a wide range of experimental designs. We validate the use of OpenEvo by evolving Haloferax volcanii to grow from 15% to 12% salt over ~150 cycles, ~1,000 hours. Evolved cells grew 55% faster than wild-type at 12% salt. Whole-genome sequencing of adapted cells found SNPs and large deletions. We also demonstrate positive and negative selection using the OpenEvo LEDs to drive optogenetics via a Phytochrome B-based optogenetic tool, with light as the selection stimulus during over 4000 hours of growth. OpenEvo lowers the technical and cost barriers for continuous evolution experiments, serves as a teaching tool, and is designed to grow an open community of users who share modifications.
14.
CD44 restricts EGFR mobility to polarize cytoskeletal signalling modules driving bleb-based migration.
Abstract:
Cells under high confinement migrate efficiently in low-adhesion environments by forming stable, polarized, hydrostatic pressure-driven leader blebs. Here we investigated the basis of polarized bleb morphology in metastatic melanoma cells migrating under low-adhesive and highly confined microenvironments. Using high-resolution live imaging, molecular perturbations and biosensors, we show that EGF signalling through PI3K stabilizes and maintains polarized leader blebs. EGFR and PI3K activities form a gradient within leader blebs that decreases from rear to front, promoting phosphatidylinositol 3,4,5-trisphosphate and Rac1-GTP accumulation at the bleb rear, whereas phosphatidylinositol 4,5-bisphosphate and RhoA-GTP concentrate at the bleb tip, the inverse of the organization observed in integrin-dependent mesenchymal migration. Optogenetic disruption of this gradient triggers bleb retraction, underscoring its functional importance. Mathematical modelling and experiments identified a mechanism whereby during bleb initiation, CD44 and ERM proteins restrict EGFR mobility within a membrane-apposed cortical actin meshwork at the bleb rear, establishing the EGFR-PI3K-Rac gradient. Together, these findings define the biophysical and molecular mechanisms that underlie polarity in bleb-based migration and highlight how alternative spatial organization of signalling modules supports distinct migration modes in different microenvironments.
15.
Red-Shifted Epac-Based FRET cAMP Sensors for All-Optical cAMP Control and Multiparameter Imaging.
Abstract:
Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger downstream of G protein-coupled receptors (GPCRs) and a central regulator of cellular signaling. Genetically encoded exchange proteins directly activated by cAMP (Epac)-based Förster resonance energy transfer (FRET) biosensors enable real-time monitoring of cAMP dynamics in living cells, but commonly used cyan/yellow FRET pairs require short-wavelength excitation, limiting compatibility with multiplex imaging and blue-light optogenetic tools such as bacterial photoactivated adenylyl cyclases (bPACs). Here, we engineered and systematically characterized four red-shifted Epac-based single-chain FRET cAMP sensors combining yellow or orange FRET donors with red fluorescent FRET acceptors. Using ratiometric live-cell imaging, we quantified stimulus-evoked FRET responses and identified Epacred4 as the best-performing variant, showing an approximately 55% decrease in normalized FRET after forskolin stimulation. Epacred4 also reliably detected Gi/o-mediated decreases in cAMP following μ-opioid receptor activation. Brief 405 nm light pulses induced graded and reversible cAMP elevations using the low dark-activity variant bPAC-F198Y. Furthermore, Epacred4 enabled analysis of cAMP recovery kinetics during phosphodiesterase inhibition and multiplex imaging of cAMP and intracellular Ca2+ using Fura-2 with minimal spectral and pH-related interference under physiological imaging conditions. Together, Epacred4 represents a robust red-shifted cAMP sensor for optogenetic and multiplex signaling studies.
16.
PIP2 stabilizes NaV1.5 gating and links receptor signaling to cardiac late sodium current.
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Gada, KD
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Kamuene, JM
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Cruz, AS
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Meng, Z
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Connolly, JG
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Ng, F
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Ma, X
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Chandrashekar, A
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Xu, Y
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Cui, M
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Plant, LD
Abstract:
The cardiac sodium channel NaV1.5 initiates each heartbeat by generating the rapid depolarizing upstroke of the action potential. Dysregulation of NaV1.5 gating can produce cardiac arrhythmias by slowing inactivation, increasing late sodium current (INa,L), and impairing electrical stability. Here, we show that phosphatidylinositol-4,5-bisphosphate (PIP2) is a critical membrane cofactor that stabilizes NaV1.5 gating. Acute PIP2 depletion in human iPSC-derived cardiomyocytes, produced by activation of endogenous AT1 receptors, activation of an engineered M3q-DREADD, or optogenetic recruitment of CRY2-pseudojanin, shifted voltage dependence, slowed fast inactivation, and increased INa,L. These effects were prevented by augmenting intracellular PIP2, required PLC activity when driven by Gq-coupled receptors, and were independent of downstream Ca2 or PKC signaling. Unlike the skeletal-muscle isoform NaV1.4, NaV1.5 displayed PIP2-dependent shifts in both activation and steady-state inactivation, indicating isoform-specific lipid coupling. Induced-fit docking and molecular dynamics simulations identified a PIP2-interaction interface between the domain IV voltage sensor and pore that contains disease-linked residues. The disease-reported variant R1644C weakened and redistributed the predicted PIP2-contact network, produced elevated basal INa,L, showed enhanced sensitivity to PIP2 depletion, and caused an approximately 30-fold reduction in apparent functional PIP2 sensitivity in excised patches. These findings define a lipid-dependent mechanism that stabilizes NaV1.5 gating and reveal how physiological Gq signaling and inherited channel variants can converge on the channel-PIP2 axis to promote proarrhythmic late sodium current.
17.
Light-activated insulin receptor modulates neuronal plasticity and cerebellar-driven behavior.
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Preissing, B
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Sackel, F
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Trajkovic-Arsic, M
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Rohr, L
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Linke, AL
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Bewerunge-Hudler, M
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Liffers, ST
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Siveke, JT
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Herlitze, S
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Siveke, I
Abstract:
We have developed an optogenetic tool called InLOV-an insulin receptor fused to a LOV domain-that enables activation of the insulin receptor signaling pathway via light-induced phosphorylation of its intracellular domains. Light activation of InLOV promotes insulin-induced neuronal plasticity in the mouse cerebellum and enhances cerebellar-driven self-motion behavior. Thus, InLOV enables optogenetic modulation of insulin receptor phosphorylation, opening new possibilities for disease modeling and therapeutic strategies for pathological insulin signaling in humans.
18.
Dimer asymmetry in signaling of blue light sensor histidine kinases.
Abstract:
Photoreceptor sensory histidine kinases (SHKs) couple light absorption to conformational changes regulating two-component signaling. Despite their importance and widespread use in optogenetics, the underlying structural signaling mechanisms remain poorly understood. Here, we engineered dimeric SHKs based on Pseudomonas putida short light-oxygen-voltage (LOV) proteins, determined their crystal structures, and investigated their signaling mechanisms. Regardless of illumination, the structures adopted a light-state like LOV-LOV dimer with symmetric/straight kinase modules. In contrast, small-angle x-ray scattering together with functional assays revealed pronounced light-dependent rearrangements in solution and allowed the assignment of the kinase-ON dark state to an asymmetric/kinked conformation, whereas the light state adopts a symmetric/straight structure. Comparative analyses of natural and engineered SHKs identified conserved motifs linking light-induced LOV domain rotation to kinase activity. The findings highlight the central role of dimer asymmetry and flexibility in SHK signaling, thereby not least informing the engineering of new light-responsive signaling systems.
19.
Light-controlled CRISPR-dCas9 epigenome editing: advanced drug-delivery strategies and oncology applications.
Abstract:
Cancer is increasingly recognized as a disease of the dysregulated epigenome; however, current epi-drugs are blunt, systemically toxic instruments. Catalytically dead CRISPR nucleases (dCas9) linked to chromatin effectors have now made it possible not only to write and erase epigenetic marks at specified loci without double-strand breaks but also to add an element of optogenetics, or reversible and light-encoded control over the timing and localization of the editors. In this review, the technological underpinnings of light-controlled CRISPR-dCas9 epigenome editing, which include architectures of dCas9 scaffold and guide, blue-to-near-infrared photoswitches, and high-gain epigenetic effector designs, are synthesized, and viral, non-viral, and stimuli-responsive delivery platforms, which have to be co-optimized with clinical light interfaces, are discussed. We then outline four functional routes by which opto-epigenome editors may be used therapeutically in cancer: tumor suppressor reactivation; oncogene and super-enhancer repression with metabolic rewiring; control of cancer stem cell differentiation; and immunomodulation of the tumor microenvironment. Lastly, a translational roadmap is defined in terms of preclinical model tiers, biomarker strategies, regulatory and manufacturing factors, and future directions, including NIR and bioluminescent actuation, implantable μLED devices, and AI-guided closed-loop illumination. Together, these aspects constitute design principles for advancing light-addressable epigenome editors toward first-in-human studies and for integrating them into combination regimens as a new class of precision cancer therapeutics.
20.
Enhancing Optogenetics-Based Cancer Therapy Via Nanotechnology.
Abstract:
Optogenetics represents a promising frontier in precision cancer therapy by enabling spatiotemporal control over cellular behavior. However, its clinical application is limited by inefficient delivery of optogenetic components and poor tissue penetration of visible light. Recent advances in nanotechnology offer solutions to these challenges. Nanoscale drug delivery systems enhance the targeted delivery of optogenetic tools, while light-conversion nanomaterials enable deep-tissue activation. Besides, the integration of nanotechnology with optogenetics further facilitates the development of engineered living therapeutics, including immune cells and bacteria, allowing programmable and localized antitumor responses. Despite promising preclinical progress, key challenges remain in long-term biosafety, immunogenicity, and precise light dosing. Future progress will depend on interdisciplinary efforts combining biocompatible nanomaterials, protein engineering, and artificial intelligence to advance clinically viable optogenetic therapies and pave the way toward personalized cancer treatment. Collectively, the synergistic integration of optogenetics and nanotechnology holds potential for overcoming longstanding barriers in cancer treatment, paving the way for precision cancer therapeutics.
21.
Linear ubiquitin chain assembly complex contributes to NLRP3-mediated pyroptotic cell death.
Abstract:
Activation of the NLRP3 inflammasome by infectious or sterile insults culminates in pyroptosis, a lytic and highly inflammatory form of programmed cell death. A safeguarded two-step process tightly regulates pyroptosis: priming, which drives NF-κB signaling, followed by execution, ultimately leading to plasma membrane rupture. Linear (Met1-linked) ubiquitination, catalyzed by the E3 ligase complex LUBAC, was previously shown to participate in pyroptosis, but the underlying mechanisms are not fully understood. In this study, we show that Met1-linked ubiquitin chains can assemble during both priming and execution phases, independently of the inflammasome sensor NLRP3. Genetic deletion of the LUBAC enzymes or pharmacological inhibition impairs pyroptosis. Conversely, cell death is enhanced without the deubiquitinase OTULIN, which selectively removes linear ubiquitination. Finally, using an optogenetic model to bypass priming, we demonstrate that Met-1-linked ubiquitination is required for the execution phase of pyroptosis. These findings offer insights into the regulation of pyroptotic cell death by linear ubiquitination.
22.
Gene expression in synthetic biology: Going with the light.
Abstract:
Inducible expression of endogenous and foreign genes has been a pivotal driving force behind a lot many seminal breakthroughs in biotechnology. Synthetic biology, a very promising field, largely relies on transgene expression platforms which facilitate convenient and conditional regulation. Optogenetic approaches that exploit light to steer biological events, e.g., gene expression, with excellent spatiotemporal control, are often more precise compared to chemical induction. Light being an omnipresent environmental stimulus, serves as the ideal cue, and enables high spatiotemporal accuracy with respect to gene expression. In this review, we focus on different elements relevant to light-inducible gene expression - light-responsive promoters, light-regulated transcription factors, and photocaged inducers. Using light as a binary input function, we explore the essence of logic gates towards the development of gene expression circuits - thereby understanding the entanglement between optogenetics and synthetic biology. We primarily focus on prokaryotes, but also draw comparisons with analogous eukaryotic gene expression systems.
23.
Long-range mutual activation establishes Rho and Rac polarity during cell migration.
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De Belly, H
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Gallén, AF
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Strickland, E
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Estrada, DC
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Godinez, DS
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Neiva, E
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Zager, PJ
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Nagy, TL
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Burkhardt, JK
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Turlier, H
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Weiner, OD
Abstract:
In migrating cells, the GTPase Rac organizes a protrusive front, whereas Rho organizes a contractile back. How these GTPases are positioned at opposite poles remains unclear. We leverage optogenetics, mechanical perturbations, and mathematical modelling to reveal a surprising mechanochemical long-range mutual activation between front and back polarity programmes that complements their well-known local mutual inhibition. Rac-based protrusions elevate membrane tension, stimulating an mTORC2-dependent activation of Rho at the opposite side of the cell. Conversely, Rho-mediated contractility induces cortical-flow-based regulation of phosphoinositide signalling that triggers Rac activation distally. We develop a minimal mechanochemical model to explain how long-range facilitation, together with local inhibition, enables robust Rho and Rac partitioning. Our findings demonstrate how the actin cortex and plasma membrane interact as an integrated mechanochemical system for long-range Rac-Rho patterning. This circuit is required for efficient polarity and migration in primary human T cells and is conserved in epithelial cells, highlighting the generality of this mechanism.
24.
Design and Implementation of a Blue-Light-Controlled Gene-Switch System.
Abstract:
Synthetic biology seeks to build predictable, programmable biological systems. We developed a blue-light-inducible T7RNAP system with dual-input regulation to enable precise spatiotemporal gene control, which is vital for biomanufacturing, therapy, and microbial engineering. We optimized it by replacing RBS sequences, testing tandem T7 promoters, and evaluating split-T7RNAP variants. Expression and bactericidal efficacy were assessed via fluorescent output and real-time growth curves under blue light. RBS variants caused up to 50-fold differences in expression. Three tandem T7 promoters provided the best balance between yield and fidelity. Integration of a benzoate-responsive module enabled 4.5-fold repression at 3 mM benzoate, demonstrating effective chemical off-switching without compromising light induction. This system combines blue light precision with environmental responsiveness, offering non-invasive, on-demand activation for antimicrobial therapy or spatial bioproduction. The benzoate-triggered off-switch is especially valuable for ecological applications such as biocontainment or bioremediation, where gene expression must shut down upon detection of pollutants, for example, aromatic hydrocarbons. Its orthogonal, modular design supports context-dependent control, making it ideal for environmental biosensors, programmable probiotics, and smart antimicrobial delivery in complex ecosystems.
25.
Optogenetic control of plasma membrane O-GlcNAcylation regulates WNK1 condensates and cellular signaling.
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Zhu, Q
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Liu, Q
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Fan, Z
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Shi, Y
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Liu, X
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Guo, Y
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Luo, J
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Zhao, J
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Qin, W
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Wang, Y
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Wang, P
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Ye, H
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Yi, W
Abstract:
Glycosylation plays a pivotal role in regulating diverse biological processes. However, the lack of tools capable of controlling the spatiotemporal dynamics of glycosylation has largely hindered its functional elucidation. Here, we introduce an optogenetic approach that employs red/far-red light to dynamically and reversibly control the plasma membrane localization of O-linked N-acetylglucosamine transferase (OGT) in living systems. Red-light-induced translocation of OGT suppresses insulin signaling in both cells and mice. Glycoproteomic and phosphoproteomic analyses reveal a global impact of OGT-mediated glycosylation on signal transduction. Moreover, using protein semisynthesis, cell-based assays, and molecular dynamics simulations, we demonstrate that red-light-induced O-GlcNAcylation of WNK1 at S1949 inhibits downstream cell volume response signaling pathways by suppressing WNK1 biomolecular condensate formation. Together, our findings provide a valuable tool to modulate subcellular O-GlcNAcylation and control cellular signaling in living systems, with broad applicability to the study of glycosylation in cells.
