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Showing 76 - 100 of 2068 results
76.
Light-inducible FLPase reconstitution enables temporal control of gene expression in Drosophila melanogaster.
Abstract:
Precise temporal control of gene expression is a prerequisite for disentangling timing-specific effects of gene function within the life cycle of Drosophila melanogaster. Here, we implement light-inducible FLPase reconstitution (LIFR) as a conditional gene expression system in flies, which combines blue light-responsive Magnet photoswitches and split-FLPase to remove an FRT-flanked stop cassette and irreversibly switch on transgene expression in response to light. This system is highly efficient, has virtually no transgene leakage, and a single light pulse is sufficient to induce long-term transgene expression. We demonstrate that LIFR in adulthood overcomes the developmental lethality elicited by constitutive pan-neuronal overexpression of neurodegeneration-causing mutants TDP43G298S and HTTQ97. We also illustrate that LIFR can help trace specific cell-type fates across developmental stages. Thus, we demonstrate proof of principle that LIFR is a versatile platform to conditionally activate long-lasting gene expression without the side effects of existing systems, thereby extending the Drosophila melanogaster genetic toolbox.
77.
Dynamic heterogeneity in an E. coli stress response regulon mediates gene activation and antimicrobial peptide tolerance.
Abstract:
The bacterial stress response is an intricately regulated system that plays a critical role in cellular resistance to drug treatment. Stress response genes are organized into networks with transcriptional regulators controlling downstream genes, but how variability in these regulators affects the downstream response remains unclear. We investigate how heterogeneity in the virulence regulator PhoP propagates to its diverse regulon. Using optogenetic control of PhoP expression and information theory, we find that downstream genes differ in mean expression, sensitivity to PhoP levels, and signal transmission reliability. These response functions also vary between individual cells, increasing population heterogeneity. We tie these variations to cell survival upon exposure to a clinically relevant antimicrobial peptide, showing that while high expression of the PhoP-regulon gene pmrD provides protection, cell survival is best determined by the integrated PhoP and pmrD expression levels. Our findings demonstrate that heterogeneous expression of stress response regulators has clear consequences for bacterial stress survival.
78.
Epigenome regulators imbue a single eukaryotic promoter with diverse gene expression dynamics.
Abstract:
Biological information can be encoded in signaling dynamics, which have been implicated in many physiological processes; yet the diversity of dynamic expression profiles driven by a single gene remains unclear. To explore this, we screen 80 chromatin-associated proteins (CAPs) for their potential to drive diverse dynamic gene expression profiles from the same genome-integrated reporter in yeast. Using locus-specific optogenetic recruitment and live-cell microscopy, we measure dynamic expression profiles within single cells. CAP recruitment elicits a range of responses varying in activation delay, strength, production rate, and noise. We find that promoter activity is characterized by graded, rather than switch-like, transitions. A kinetic model with three promoter states and a positive feedback loop successfully captures the key features of expression driven by each CAP. These results reveal the rich dynamic landscape possible from a single gene, offering insights into native cellular processes and enhancing gene expression control in synthetic biology.
79.
Phage-assisted evolution of allosteric protein switches.
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Southern, NT
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von Bachmann, A
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Hovsepyan, A
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Griebl, M
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Wolf, B
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Lemmen, N
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Kroell, AS
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Westermann, S
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Mathony, J
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Niopek, D
Abstract:
Allostery, the transmission of locally induced conformational changes to distant functional sites, is a key mechanism for protein regulation. Artificial allosteric effectors enable remote manipulation of cell function; their engineering, however, is hampered by our limited understanding of allosteric residue networks. Here, we introduce a phage-assisted evolution platform for in vivo optimization of allosteric proteins. It applies opposing selection pressures to enhance activity and switchability of phage-encoded effectors and leverages retron-based recombineering to broadly explore fitness landscapes, introducing point mutations, insertions, and deletions. Applying this framework to the transcription factor AraC yielded near-binary optogenetic switches, with light-controlled activity spanning ~1000-fold dynamic range. Long-read sequencing across selection cycles enabled high-resolution tracking of evolving variant pools, revealing adaptive trajectories and context-dependent residue interactions. Mechanistically, we find that linker mutations promoting α-helix extension at the sensor-effector junction enhance conformational coupling between LOV2 and AraC. These variants emerge consistently across independently evolved pools, underscoring their functional relevance. Together, we develop a framework for the directed evolution of programmable allosteric switches in vivo. By coupling dynamic selection with deep mutational scanning and temporal sequencing, it enables both functional optimization and mechanistic insight into allosteric networks.
80.
A Single-Chain Light-Activatable Transcriptional Reporter for Fluorescently Tagging Mammalian Cells In Vitro.
Abstract:
Optogenetic tools have revolutionized the control of gene expression with high spatial and temporal resolution. Here we present a Single-chain Light-Activatable Transcriptional Reporter (SLATR), a system capable of fluorescently tagging target cells with minutes of white light stimulation. In its inactive, or dark state, a transcriptional factor is cytosolically bound, preventing nuclear translocation. White light irradiation triggers its release through the protease cleavage of a site that is sterically caged by the circularly permuted Avena sativa LOV2 (cpAsLOV2) domain. We discovered that cpAsLOV2 cages the cleavage site more efficiently than AsLOV2, achieving low background in the SLATR design. We demonstrate that SLATR exhibits a signal-to-background ratio between 3.4 and 36 and achieves reporter activation within 60 min of light stimulation. Furthermore, SLATR outperforms the only other single-chain light-activatable transcriptional reporter, LAUNCHER, with faster kinetics, greater light sensitivity, and markedly lower background under identical stimulation conditions. Our single-chain light-activatable transcriptional system expands the optogenetic toolkit though providing a simpler system for regulating gene expression with precise spatiotemporal control.
81.
Engineering of genetically encoded programmable calcium channel inhibitory binders.
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Liu, X
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Ali, S
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Lan, TH
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Wang, D
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McKee, B
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Nonomura, T
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Liu, S
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Zhao, F
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Zhu, MX
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Huang, Y
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Deng, Q
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Ma, G
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Zhou, Y
Abstract:
Store-operated Ca2+ release-activated Ca2+ (CRAC) channels, composed of STIM and ORAI, are essential for immune and developmental processes, and their dysregulation underlies channelopathies such as Stormorken syndrome. Here, we report the engineering of genetically encoded CRAC channel inhibitory binders (CRABs) derived from the ORAI C-terminal tail. Guided by deep mutational scanning, we optimize a membrane-anchored CRAB variant that potently inhibits Ca2+ influx and NFAT signaling, and rescues thrombocytopenia-like phenotypes in a zebrafish model of Stormorken syndrome. To enable tunable inhibition, we further design oligomeric, optogenetic (Opto-CRAB), and chemogenetic (Chemo-CRAB) variants, providing graded and real-time control of CRAC activity. Chemo-CRAB further suppresses Ca2+ signaling downstream of RTKs, GPCRs, and CAR-T cell activation, establishing broad applicability across physiological and synthetic contexts. Together, these programmable peptide-based inhibitors provide a versatile platform to dissect SOCE dynamics and hold promise as a therapeutic strategy against autoimmune, inflammatory, and neoplastic disorders driven by CRAC channel hyperactivity.
82.
GTPase-activating protein DLC1 spatio-temporally regulates Rho signaling.
Abstract:
Emerging evidence suggests that Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) bind to the cytoskeleton or focal adhesions (FAs), controlling spatio-temporal Rho GTPase activity through feedback mechanisms. We explore such feedback in the Rho-specific GAP Deleted in Liver Cancer 1 (DLC1), which binds to FAs through mechanosensitive interactions. Using a FRET biosensor, we show that DLC1 loss of function leads to globally increased Rho activity and contractility in fibroblasts. Although Rho activity appears macroscopically steady, individual molecules undergo 'signaling flux'-a dynamic cycle of activation and deactivation. To measure this flux, we built a genetic circuit that enables both optogenetic activation of Rho and simultaneous readout of Rho activity. In cells at mechanical steady state, this reveals that DLC1 globally controls the rate of Rho deactivation, both at FAs and at the plasma membrane. Transient induction of local contractility, however, shows DLC1 associating with and dissociating from FAs during their reinforcement and relaxation, which might provide local positive feedback on Rho activity for robust FA disassembly. Together, our results indicate that DLC1 regulates Rho activity both globally at steady state and locally at FAs under tension, highlighting the complexity of spatio-temporal Rho GTPase signaling.
83.
Rationally designed light-inducible RNA-releasing protein for translational regulation and optogenetic control of gene therapies.
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Li, S
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Gao, T
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Wang, Y
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He, Q
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Ma, H
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Ye, H
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Shen, Y
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Wang, Y
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Wang, Y
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Li, X
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Fang, S
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Chen, Q
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Liu, Q
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Sun, G
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Xin, H
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Xue, J
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Liu, S
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Zhu, M
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Xu, W
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Wang, Z
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Shao, J
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Xie, M
Abstract:
In this study, we describe a rationally designed light-inducible RNA-releasing protein (LIRP) capable of inhibiting mRNA translation in the dark while permitting gene expression upon exposure to blue or ambient light. This LIRP-dependent gene switch is compatible with various delivery routes of gene- and cell-based therapy, such as subcutaneous implantation of microencapsulated light-sensitive cells or expression in various light-accessible body sites using single adeno-associated virus (AAV) vectors. To exemplify a gene therapy approach that directly harnesses ambient light as a natural illumination source to induce therapeutic action, we show how intradermal delivery of AAV2 vectors carrying a LIRP-regulated gene switch controlling murine thymic stromal lymphopoietin expression was effective in enabling light-dependent prevention and treatment of diet-induced obesity. To describe another therapeutic scenario, we engineered AAV2 vectors for LIRP-dependent expression of Vascular endothelial growth factor (VEGF) inhibitors for the treatment of retinal neovascular diseases. Upon intravitreal delivery into mice suffering from wet macular degeneration, VEGF inhibitors were constantly produced when animals were exposed to daylight, but therapeutic actions could be flexibly interrupted either by exposure to dark environments or by administration of a selective blue light filter at any point in time. When compared to conventional treatment strategies based on constitutive VEGF inhibition over the course of 3 months, we show that a regulated gene therapy approach through LIRP-dependent optogenetics was advantageous in maintaining a normal retina thickness. This work not only provides a valuable addition to the optogenetic toolbox but also offers a perspective to translate light-dependent gene switches toward therapeutic usage.
84.
WDR44 drives de novo α-synuclein aggregation at the lysosomal membrane and promotes neuronal dysfunction in Parkinson's Disease.
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Teixeira, M
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Sheta, R
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Bérard, M
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Insinna, C
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Mahul-Mellier, AL
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Delmas, CVL
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Lépinay, E
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Idi, W
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Ricard, A
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Del Cid-Pellitero, E
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Trabolsi, C
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Gobeil, S
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Canron, MH
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Bézard, E
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Rajput, A
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Cicchetti, F
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Parent, M
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Fon, EA
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Ramalingam, N
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Dettmer, U
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Calon, F
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Westlake, C
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Lashuel, HA
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Oueslati, A
Abstract:
The aggregation of α-synuclein (α-SYN) into Lewy bodies (LBs) is a central event in the pathogenesis of Parkinson's disease (PD) and related synucleinopathies1,2. Despite significant advances in understanding α-SYN self-assembly, the precise sequence of early aggregation steps has not been directly visualized in living neurons. Here, we use an optogenetic-induced protein aggregation system with a high temporal resolution to monitor the onset of α-SYN assembly in neurons. We found that the initiation and accumulation of α-SYN aggregates occur predominantly at the lysosomal membrane, an event driven by the α-SYN N-terminus and modulated by the membrane-associated adaptor protein WD repeat-containing protein 44 (WDR44). Remarkably, we demonstrate that WDR44 knockdown markedly reduced de novo α-SYN aggregation in both neuronal cultures and in vivo, whereas WDR44 overexpression enhances α-SYN aggregation in PD patient-derived iPSC neurons. Consistent with its potential pathogenic involvement, WDR44 aberrantly accumulates in vivo and in the brains of PD patients, where it colocalizes with LB inclusions. Finally, we show that lysosome-associated α-SYN aggregates compromised lysosomal structure and function, leading to neuronal impairment, a phenotype worsened by WDR44 overexpression, linking early aggregation events to downstream toxicity. Together, these findings reveal the earliest dynamic stages of α-SYN oligomerization in living neurons and identify the WDR44-α-SYN interaction as a promising therapeutic target for reducing α-SYN pathology and enabling early intervention in PD.
85.
Long-term quantification and brief-pulse optogenetic perturbation of nucleocytoplasmic GtaC dynamics during Dictyostelium development.
Abstract:
Transcription factor nucleocytoplasmic dynamics play a key role in developmental gene regulation. In Dictyostelium , the transcription factor GtaC exhibits nucleocytoplasmic shuttling, but its shuttling trajectory across multicellular aggregation has not been systematically quantified. Using a knock-in strain, we tracked GtaC dynamics from starvation through aggregation and quantified developmental changes in shuttling period, amplitude, and synchrony. Notably, brief-pulse optogenetic activation of cAMP at higher input frequencies reproduced the reported attenuation of GtaC shuttling amplitude with high temporal precision and minimal phototoxicity. Together, long-term quantification and brief-pulse optogenetic cAMP perturbation show that GtaC nucleocytoplasmic shuttling is developmentally tuned in a frequency-dependent manner.
86.
Photoactivated probiotic micro-reactor synchronizes STING/TLRs agonists to spatiotemporally synergize antitumor immunotherapy.
Abstract:
Reprogramming tumor-associated macrophages (TAMs) from the pro-tumoral M2-like state to the immunostimulatory M1-like phenotype has emerged as a promising strategy for tumor therapy. However, most M2-like TAMs are preferentially located in hypoxic regions of the tumor, which are poorly accessible to many advanced drug delivery systems, posing a significant challenge to effective TAM reprogramming. Here, leveraging the tropism of facultative anaerobic bacteria to localize and propagate in the hypoxic tumor, an optogenetically engineered Escherichia coli Nissle 1917 strain conjugated with murine STING agonist (EcNflaB@UPD) was developed for cancer-specific immunotherapy. Upon near-infrared light illumination, the blue and UV emissions from upconversion nanoparticles (UCNPs) simultaneously activate the expression of Toll-like receptor (TLR) agonist, flaB, from EcNflaB, and the release of photocaged murine STING agonist, DMXAA, respectively. This spatiotemporally synchronized dual release ensures co-localized STING and TLR5 agonists inside the hypoxic niche, repolarizing TAMs from the M2 to the M1 phenotype via synergistic TLR5-MAPK1-NF-κB and STING-NF-κB signaling. The polarization of TAMs enhances their antigen-presenting capacity and, more importantly, activates the cytotoxic, stem-like and memory CD8+ T cells responses. This subsequently inhibits tumor growth, relapse, and metastasis in the murine 4T1 tumor model. Collectively, our work introduces the bacteria-based system that uses near-infrared light to dual-release immunotherapeutics for systemic anti-tumor immunity, opening new avenues for precise and effective cancer immunotherapy.
87.
Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling.
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Prahl, LS
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Canlla, R
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Huang, AZ
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Alber, DS
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Shefter, SL
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Davis, SN
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Grindel, SH
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Huang, ZD
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Mumford, TR
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Benman, W
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Bugaj, LJ
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McCracken, KW
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Hughes, AJ
Abstract:
The mammalian kidney relies on a branched network of collecting ducts for fluid transport and homeostasis. Replicating this network in vitro would parallelize function in synthetic replacement kidneys, yet current organoids have limited branching capacity. Here, we establish a developmentally-informed strategy to control organoid budding through optogenetic control of a receptor tyrosine kinase, RET. We first show pharmacological manipulation of RET signaling controls the extent of branching in mouse embryonic kidneys and human stem cell-derived kidney organoids. Next, we develop an optogenetic RET receptor (optoRET) that signals in a ligand-independent manner via blue light-mediated clustering. Epithelial cells expressing optoRET reproduce stereotyped RET signaling, scattering, and symmetry breaking in response to blue light. Human kidney organoids undergo budding with controllable orientation in response to spatially patterned optoRET stimulation. Our results establish ligand-free optogenetic control of branching and inspire new synthetic biology strategies for epithelial organoid design.
88.
Optogenetic Control of the Integrated Stress Response Limits Glioblastoma Invasion.
Abstract:
The integrated stress response (ISR) is a highly conserved signaling network, allowing cells to adapt and respond to various stressors. With its aggressive spread and high recurrence rates, glioblastoma multiforme (GBM) is one of the toughest cancers to date, yet the role of the ISR is still to be well understood, whether activation may suppress or promote this disease, and drug-treatment of GBM has thus far shown inconclusive results. In this work, we use an optogenetic tool, opto-PKR, to specifically trigger ISR activation via light-induced oligomerizing PKR-kinases, offering high spatiotemporal and reversible control, while avoiding potential upstream damage or side effects from drugs. Using immunofluorescence and RNA-sequencing, we show that targeted ISR activation reaching levels where both adaptive (ATF4) and terminal responses (CHOP) are activated results in subsequent downregulation of genes associated with the extracellular environment and glial cell migration, further supported by ECM-stain and scratch assays. Next, we show inhibition of aggressive spread for ISR-activated GBM spheroids in collagen 3D culture. Photopatterning of ISR activation in spheroids demonstrates a cell-intrinsic effect at the tissue scale, and recovery studies indicate a tunable, non-ablative intervention space. These findings suggest a route to containment and motivate ISR-activating small molecule screening in GBM models.
89.
Dilating the aging clock with light.
Abstract:
Precise and rapid modeling of aging remains a significant challenge. This review explores the potential of emerging light-based technologies-including direct light exposure, photodynamic therapy (PDT), and optogenetics-as accelerated, mechanistically targeted models for interrogating aging processes. Unlike conventional methods that rely on chronologically aged organisms or non-specific stressors, these light-driven approaches can induce specific hallmark aging phenotypes within days. This offers unprecedented temporal and spatial control, allowing researchers to precisely trigger and define molecular pathways, such as localized oxidative bursts or programmed protein interactions. Such capabilities provide a powerful platform for testing causal hypotheses about aging, especially organ-specific aging. The review also outlines a framework connecting each light modality to aging, including cellular senescence, telomere attrition, proteostasis loss, and epigenetic drift. It discusses a mechanistic matrix linking each light modality to specific molecular targets and age-associated outcomes. Light-based platforms, when combined with genetic and pharmacological tools, are poised to accelerate discovery in aging by enabling high-throughput and organelle-specific perturbation of aging biology. These approaches could ultimately redefine how we experimentally dissect and potentially modulate the aging process either by rapidly recapitulating aging features for study or by revealing leverage points to slow aging.
90.
Optogenetic mediated contractility enables reversible control of microglial morphology and migration in vivo.
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Biermeier, CM
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Albert, M
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Pal, AA
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Gallo, E
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Wittmann, J
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Hartmann, J
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Bill, RT
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Julmi, J
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Gilmour, D
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Peri, F
Abstract:
Directed migration and rapid process extension-retraction allow microglia to continuously survey the brain and efficiently identify and phagocytose apoptotic neurons. Defining how cytoskeletal regulators coordinate these behaviors could guide strategies for targeted modulation of microglial activity. To this aim, using in vivo imaging in zebrafish, we identified a mechanistic framework in which myosin II-dependent contractility governs transitions between surveillance, migratory, and phagocytic states. Building on this, we engineered an optogenetic RhoA actuator (opto-ArhGEF25) that enables reversible, spatiotemporally precise manipulation of microglial behavior in vivo. We show that patterned RhoA activation can modulate process dynamics and induce front-rear polarity that drives rapid repulsive migration away from the applied light source, overriding injury-evoked cues and preventing migration toward lesions. Together, these results establish optogenetic control of the cytoskeleton as a powerful approach to probe and ultimately modulate microglial function in the living brain.
91.
Optogenetic Tools for Spatiotemporal Interrogation of Cytoskeletal Dynamics.
Abstract:
The cytoskeleton is a dynamic intracellular network that governs cell shape, migration, division, and mechanotransduction. Precise spatiotemporal control of cytoskeletal regulation is essential for understanding how these processes are coordinated in physiology and disease, yet conventional pharmacological and genetic approaches often lack sufficient resolution or reversibility. Optogenetic technologies provide a powerful alternative by enabling light-controlled, noninvasive manipulation of cytoskeletal regulators with high temporal precision and subcellular specificity. This review summarizes recent advances in genetically encoded optogenetic tools for interrogating cytoskeletal dynamics. We discuss core design strategies, including allosteric regulation, light-induced oligomerization, heterodimerization, and dissociation, and highlight representative applications targeting actin filaments, microtubules, and upstream signaling pathways such as Rho family GTPases. We conclude by outlining current limitations and emerging directions, including improved tissue penetration, reduced phototoxicity, and multiplexed optical control, which are expected to further expand the utility of optogenetics in cytoskeleton research.
92.
Local RhoA activation induces anillin-independent septin recruitment in interphase cells.
Abstract:
The regulation of the actin cytoskeleton is key to controlling cell shape and structure. While the Rho GTPase RhoA is well known to regulate the actomyosin cytoskeleton, its function in controlling the septin cytoskeleton remains unclear. As RhoA interactions can vary in both time and space, they can be challenging to discern from traditional bulk biochemical assays. Here, we use multiple optogenetic tools to spatially and temporally increase myosin localization, stimulate contractile force, and activate RhoA to investigate how RhoA and its downstream effector myosin impact the septin cytoskeleton. We find that neither local accumulation of myosin nor increased activity of myosin is sufficient to alter septin architecture. Local activation of RhoA, however, results in a local increase in septin accumulation. Importantly, this septin increase is independent of the scaffolding protein anillin, which can directly bind both septin and RhoA. Together, these data expand the potential role of septins in mediating RhoA signaling by stimulating the remodeling of the septin cytoskeleton.
93.
Structural insights into photo-state-specific binding of affibody Aff6 to the photosensory core module of DrBphP.
Abstract:
Light-inducible heterodimerization systems offer precise, reversible control of protein interactions in living cells. Leveraging the high tissue-penetration of red/far-red light, the MagRed system, composed of a bacteriophytochrome Deinococcus radiodurans BphP (DrBphP) and its engineered affibody binder Aff6, achieves robust photoswitchable dimerization. This makes MagRed well-suited for in vivo and deep-tissue optogenetic application. However, the structural mechanism underlying Aff6's photo-state-specific recognition of DrBphP remains elusive. Here, we combine solution NMR spectroscopy, surface plasmon resonance (SPR), molecular docking and mutational analysis to elucidate the light-dependent interaction between a monomeric photosensory core module of DrBphP (DrBphP-PCMmono) and Aff6. We show that DrBphP-PCMmono alone is sufficient for light-inducible heterodimerization with Aff6, exhibiting a ∼ 23-fold affinity difference between the Pfr and Pr states. NMR titration reveals that Aff6 binds primarily to the PHY domain and the C-terminal region of the helical spine. Furthermore, docking and mutagenesis identify a key aromatic interaction (involving F327/H334 of DrBphP and F18 of Aff6) as the molecular basis for this conformational selectivity. Additionally, Aff6 binding stabilizes the Pfr state and retards the Pfr-to-Pr reversion of DrBphP-PCMmono. These findings not only provide critical structural insight into MagRed function but also establish a foundation for rationally engineering next-generation phytochrome-based optogenetic tools.
94.
OptoTAT reveals microtubule acetylation as a rapid trigger for GEF-H1-mediated cell migration.
Abstract:
Microtubule acetylation is implicated in regulating cell motility, yet its physiological role in directional migration and the underlying molecular mechanisms have remained unclear. This knowledge gap has persisted primarily due to a lack of tools capable of rapidly manipulating microtubule acetylation in actively migrating cells. To overcome this limitation and elucidate the causal relationship between microtubule acetylation and cell migration, we developed a novel optogenetic actuator, optoTAT, which enables precise induction of microtubule acetylation within minutes in live cells. Implementing optoTAT in migration assays, we observed striking and rapid responses at both molecular and cellular levels. First, microtubule acetylation triggers release of the RhoA activator GEF-H1 from sequestration on microtubules. This release subsequently enhances actomyosin contractility and drives focal adhesion maturation. These subcellular processes collectively promote sustained directional migration. Our findings position GEF-H1 as a critical molecular responder to microtubule acetylation, enabling a dynamic crosstalk between the actin and microtubule cytoskeletal networks in the coordination of cellular motility.
95.
STIM1 and endoplasmic reticulum-plasma membrane contact sites oscillate independently of calcium-induced calcium release.
Abstract:
Calcium (Ca²+) release from intracellular stores, Ca²+ entry across the plasma membrane and their coordination via store-operated Ca²+ entry (SOCE) are critical for receptor-activated Ca²+ oscillations. However, the precise mechanism of Ca²+ oscillations and whether their control loop resides at the plasma membrane or intracellularly remains unresolved. By examining the dynamics of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER)-localized Ca²+ sensor that activates the Orai1 channel on the plasma membrane for SOCE, in mast cells, we found that a significant proportion of cells exhibited STIM1 oscillations with the same periodicity as Ca²+ oscillations. These cortical oscillations, shared with ER-plasma membrane (ER-PM) contact site proteins, were only detectable using total internal reflection fluorescence microscopy. Notably, STIM1 oscillations could occur independently of Ca²+ oscillations. Simultaneous imaging of cytoplasmic Ca²+ and ER Ca²+ with CEPIA1er revealed that receptor activation does not deplete ER Ca²+, whereas receptor activation without extracellular Ca²+ influx induces cyclic ER Ca²+ depletion. However, under such non-physiological conditions, cyclic ER Ca²+ oscillations lead to sustained STIM1 recruitment, indicating that oscillatory Ca²+ release is neither necessary nor sufficient for STIM1 oscillations. Using optogenetic tools to manipulate ER-PM contact site dynamics, we found that persistent ER-PM contact sites reduced the amplitude of Ca²+ oscillations without alteration of oscillation frequency. Together, these findings suggest an active cortical mechanism governs the rapid dissociation of ER-PM contact sites, thereby controlling amplitude of oscillatory Ca²+ dynamics during receptor-induced Ca²+ oscillations.
96.
Enhancing the performance of Magnets photosensors.
Abstract:
Photosensory protein domains, derived from nature, are foundational for optogenetic protein engineering. Tailoring their properties enables their full exploitation for optogenetic regulation in basic research and applied bioengineering applications. Here, we present a simple, yet powerful strategy based on random mutagenesis coupled to high-throughput screening that allowed altering the most fundamental properties of the widely used nMag/pMag photodimerization system: its light sensitivity and activation. Variants were characterized in vivo in bacteria by flow cytometry and during the entire growth curve by spectrofluorometry. We identify mutations that either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the light activation and dark-to-light fold change. Notably, light sensitivity and activation levels could be changed independently. In addition, we demonstrated that the shapes of the dose-response curves can be finely tuned. This broadens the applicability of the Magnets photosensors for optogenetic regulation strategies.
97.
Red-light-activated living bacterial electron generator for on-demand drug release in colonic inflammation.
Abstract:
Inflammatory bowel disease demands spatiotemporally precise drug delivery, yet the variable gut redox environment limits stimuli-responsive nanocarriers. Here we report a living biohybrid platform in which optogenetically engineered Shewanella oneidensis MR-1 is electrostatically conjugated with azo-bond covalent organic frameworks (TA-COFs) loaded with anti-inflammatory drugs magnolol or 4-iodobenzoic acid. Under intestinal conditions and non-invasive red-light irradiation (660 nm), light-induced restoration of the metal-reducing pathway promotes extracellular electron transfer, thereby cleaving azo bonds in the COF. This triggers rapid structural disassembly and a 2.8-fold increase in drug release. Although wild-type Shewanella is thermally inactivated at 37 °C and cannot utilize abundant colonic acetate, expression of heat-shock genes (groES/thiF) and an acetate-to-TCA pathway (ato1/ato2/gltA) confers 37 °C tolerance and robust metabolism in the gut. In DSS-induced colitis mice, oral administration of the biohybrid significantly alleviates inflammation, restores epithelial barrier integrity, rebalances gut microbiota (enrichment of Akkermansia, Muribaculaceae, and Lachnospiraceae). This work presents a generalizable strategy for constructing electroactive living composites by integrating microbial electron generation with stimuli-responsive nanomaterials, offering a new paradigm for light-programmed smart therapeutics and programmable living materials in biomedical applications.
98.
Optimizing information transmission in optogenetic Wnt signaling.
Abstract:
Populations of cells regulate gene expression in response to external signals, but their ability to make reliable collective decisions is limited by both intrinsic noise in molecular signaling and variability between individual cells. In this work, we use optogenetic control of the canonical Wnt pathway as an example to study how reliably information about an external signal is transmitted to a population of cells, and determine an optimal encoding strategy to maximize information transmission from Wnt signals to gene expression. We find that it is possible to reach an information capacity beyond 1 bit only through an appropriate, discrete encoding of signals: using no Wnt, a short Wnt pulse, or a sustained Wnt signal. By averaging over an increasing number of outputs, we systematically vary the effective noise in the pathway. As the effective noise decreases, the optimal encoding comprises more discrete input signals. These signals do not need to be fine-tuned to achieve near-optimal information transmission. The optimal code transitions into a continuous code in the small-noise limit, which can be shown to be consistent with the Jeffreys prior. We visualize the performance of different signal encodings using decoding maps. Our results suggest that optogenetic Wnt signaling allows for regulatory control beyond a simple binary switch and provide a framework to apply ideas from information processing to single-cell in vitro experiments.
99.
Myosin II-driven contractions of supporting cap cells promote sensory adaptation of Drosophila proprioceptors.
Abstract:
Mechanoreceptors can be motile and actively amplify their mechanical input.1,2,3,4 We here found that the responses of mechanoreceptor cells can also be shaped actively by contractile supporting cells. Drosophila larvae monitor body movements with pentascolopidial chordotonal (lch5) organs that are stretched out between cuticular attachment sites.5,6,7,8 These proprioceptive organs contain five stretch-receptor neurons each that receive mechanical stimuli from supporting cap cells. The elastic cap cells are surrounded by extracellular matrix and contain actin cables and non-muscle myosin II motors, suggesting that the cells might be motile.9,10 We show that the supporting cap cells are pre-strained at rest to about twice their relaxed length, and that the force they transmit is modulated by myosin II in the cap cells. Cap cells contracted upon optogenetic activation of myosin II. Cap cell-specific knockdown of the regulatory light chain of myosin II relieved tension and converted the spiking responses of the stretch receptors from phasic to more tonic, impairing adaptation to sustained stimuli. Our findings thus illustrate that mechanoreceptor responses can be actively tailored by contractile neighboring cells.
100.
Actin-membrane interface stress regulates Arp2/3-branched actin density during lamellipodial protrusion.
Abstract:
Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells. Here, we utilize mouse embryonic fibroblast CRISPR knock-in lines with labeled ARP2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that ARP2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of ARP2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility through cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity.
