Qr: journal:"Nat Commun"
Showing 1 - 25 of 131 results
1.
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.
2.
Dual-channel optogenetics in yeast for multiplexed light-based control of cellular processes and pathways.
Abstract:
Optogenetics which involves the use of light to control cell functions on a genetic level has found utility in studying cell physiology, biomaterials and metabolic engineering. S. cerevisiae is an industrially relevant model organism that is used in many applications, but due to the large number of genes required and issues relating to cross-activation between different colours, optogenetics for different wavelengths of light have not been multiplexed in S. cerevisiae. In this paper, we develop a compact red light responsive optogenetic system for S. cerevisiae that requires only a single gene and no exogenous cofactors. Through engineering modular protein domains, we reduce the cross-activation of our system by blue light. We integrate our red light optogenetic system with EL222 blue light optogenetics to establish dual channel optogenetics in S. cerevisiae and demonstrate its utility for engineering biology through the light-based control of flavonoid luteolin synthesis and flocculation for ease of product extraction. We also demonstrate our system's potential for the development of living materials by producing dual-coloured optogenetic patterns using S. cerevisiae. This work expands optogenetic applications in S. cerevisiae from single-light to multi-light systems, introducing the potential to multiplex different colours of light for dynamic, orthogonal control of separate cell processes.
3.
Light- and chemical-induced ciliary signaling governs dorsal/ventral regionalization of human telencephalic organoids.
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Shimada, IS
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Goto, A
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Hashimoto, Y
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Inoue, H
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Sugawara, T
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Doura, T
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Fujita, T
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Iwata, T
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Shimmoto, R
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Takase, H
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Itoh, M
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Kiyonaka, S
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Kato, Y
Abstract:
Neural stem/progenitor cells (NPCs) have primary cilia, which are critical organelles for Sonic hedgehog signaling. However, little is known about the components of primary cilia in NPCs and whether manipulating signaling in the cilia is sufficient to alter dorsal/ventral regional identity. Using a human telencephalic organoid model, we perform comprehensive proteomic profiling of NPC cilia and find enrichment in GTPase signaling. Deletion of the ciliary GTPase ARL13B reduces ciliary localization of GPR161, an orphan G protein-coupled receptor 161 that negatively regulates Sonic hedgehog, resulting in ventralization of NPCs. GPR161 deletion also induces ventralization. To investigate whether manipulation of ciliary signaling is sufficient to restore dorsal identity in this context, we optogenetically elevate ciliary cAMP, rescuing dorsal fate in GPR161 KO organoids. Furthermore, chemogenetic induction of GPR161 removal from cilia is sufficient to increase ventral NPCs. These data indicate that ciliary signaling functions as a critical switch regulating dorsal/ventral fate decisions.
4.
Photoactivatable CRISPR/Cas13d via upconversion nanoparticles for deep tissue RNA engineering and orthopedic therapy.
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Zhao, J
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Zhang, J
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Gao, M
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Miao, Z
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Zhang, Y
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Guo, Y
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Fan, Z
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Tian, J
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Yang, L
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Jiang, N
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Ma, J
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Jiao, J
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Pan, J
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Ma, X
Abstract:
Spatiotemporal control of RNA therapeutics remains a fundamental challenge limiting clinical translation. Here, we develop a photoactivatable CRISPR/Cas13d (paCas13d) system that enables non-invasive, light-controlled RNA manipulation in deep tissues. Through structure-guided engineering, we identify optimal split sites within RfxCas13d and create light-switchable fragments using CRY2PHR/CIBN optogenetic dimerization. To overcome the limited tissue penetration of blue light, we engineer polyethylenimine-functionalized upconversion nanoparticles (UCNPs-PEI) that serve dual roles as gene carriers and photon transducers, converting tissue-penetrating near-infrared (NIR) to blue light. The UCNPs-PEI@paCas13d system achieves precise spatiotemporal control of RNA targeting within bone tissue in vivo. In a murine steroid-associated osteonecrosis model, NIR-activated paCas13d achieves robust TET3 knockdown, disrupting the TET3-5hmC-PTEN axis that drives glucocorticoid-induced osteocyte apoptosis. This targeted intervention prevents bone deterioration, with treated mice showing preserved trabecular architecture, enhanced bone volume, and favorable shifts in bone turnover markers, while maintaining systemic glucocorticoid efficacy. Our platform combines the programmability of CRISPR/Cas13d with non-invasive optical control, offering a versatile approach for treating diseases requiring localized RNA modulation while minimizing systemic effects.
5.
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.
6.
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.
7.
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.
8.
Closed-loop optogenetic control of cell biology enables outcome-driven microscopy.
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Passmore, JB
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Rates, A
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Schröder, J
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van Laarhoven, MTP
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Hellebrekers, VJW
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van Hoef, HG
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Geurts, AJM
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van Straaten, W
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Nijenhuis, W
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Berger, F
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Smith, CS
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Smal, I
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Kapitein, LC
Abstract:
Smart microscopy is transforming biological imaging by integrating real-time analysis with adaptive acquisition to enhance imaging efficiency. Whereas many emerging implementations are event-driven and focus on on-demand data acquisition to reduce phototoxicity, we here present 'outcome-driven' microscopy, a framework combining smart microscopy with optogenetics to control cell biological processes and achieve predefined outcomes. We validate this approach using light-based control of cell migration and nucleocytoplasmic transport, demonstrating robust spatiotemporal control of cellular behaviour in single cells and in cell populations.
9.
Photoswitchable intein for light control of covalent protein binding and cleavage.
Abstract:
Precise control of covalent protein binding and cleavage in mammalian cells is crucial for manipulating cellular processes but remains challenging due to dark background, poor stability, low efficiency, or requirement of unnatural amino acids in current optogenetic tools. We introduce a photoswitchable intein (PS Intein) engineered by allosterically modulating a small autocatalytic gp41-1 intein with tandem Vivid photoreceptor. PS Intein exhibits superior functionality and low background in cells compared to existing tools. PS Intein-based systems enable light-induced covalent binding, cleavage, and release of proteins for regulating gene expression and cell fate. The high responsiveness and ability to integrate multiple inputs allow for intersectional cell targeting using cancer- and tumor microenvironment-specific promoters. PS Intein tolerates various fusions and insertions, facilitating its application in diverse cellular contexts. This versatile technology offers efficient light-controlled protein manipulation, providing a powerful tool for adding functionalities to proteins and precisely controlling protein networks in living cells.
10.
Optogenetic actin network assembly on lipid bilayer uncovers the network density-dependent functions of actin-binding proteins.
Abstract:
The actin cytoskeleton forms a meshwork that drives cellular deformation. Network properties, determined by density and actin-binding proteins, are crucial, yet how density governs protein penetration and dynamics remains unclear. Here, we report an in vitro optogenetic system, named OptoVCA, enabling Arp2/3 complex-mediated actin assembly on lipid membranes. By tuning illumination power, duration, and pattern, OptoVCA flexibly manipulates the density, thickness, and shape of the actin network. Taking these advantages, we examine how network density affects two actin-binding proteins: myosin and ADF/cofilin. We find that even modest increases in density strictly inhibit myosin filament penetration by steric hindrance. Penetrated myosin filaments generate directional actin flow in networks with density gradients. In contrast, ADF/cofilin accesses networks regardless of density, yet network disassembly is markedly reduced by increased density. Thus, OptoVCA reveals that network density differentially regulates actin-binding protein penetration and activity. These findings advance understanding of cell mechanics through precise, light-based manipulation of cytoskeletal structure.
11.
Endogenous OptoRhoGEFs reveal biophysical principles of epithelial tissue furrowing.
Abstract:
During development, epithelia function as malleable sheets that undergo extensive remodeling to shape developing embryos. Optogenetic control of Rho signaling provides an avenue to investigate mechanisms of epithelial morphogenesis, but transgenic optogenetic tools can be limited by variability in expression levels and deleterious effects of transgenic overexpression on development. Here, we use CRISPR/Cas9 to tag Drosophila RhoGEF2 and Cysts/Dp114RhoGEF with components of the iLID/SspB optogenetic heterodimer, permitting light-dependent control over endogenous protein activities. Using quantitative optogenetic perturbations, we uncover a dose-dependence of tissue furrow depth and bending behavior on RhoGEF recruitment, revealing mechanisms by which developing embryos can shape tissues into particular morphologies. We show that at the onset of gastrulation, furrows formed by cell lateral contraction are oriented and size-constrained by basal actomyosin. Our findings demonstrate the use of quantitative, 3D-patterned perturbations of cell contractility to precisely shape tissue structures and interrogate developmental mechanics.
12.
De novo designed protein guiding targeted protein degradation.
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Li, Z
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Qiao, G
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Wang, X
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Wang, M
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Cheng, J
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Hu, G
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Li, X
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Wu, J
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Liu, J
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Gao, C
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Liu, L
Abstract:
Targeted protein degradation is a powerful tool for biological research, cell therapy, and synthetic biology. However, conventional methods often depend on pre-fused degrons or chemical degraders, limiting their wider applications. Here we develop a guided protein labeling and degradation system (GPlad) in Escherichia coli, using de novo designed guide proteins and arginine kinase (McsB) for precise degradation of various proteins, including fluorescent proteins, metabolic enzymes, and human proteins. We expand GPlad into versatile tools such as antiGPlad, OptoGPlad, and GPTAC, enabling reversible inhibition, optogenetic regulation, and biological chimerization. The combination of GPlad and antiGPlad allows for programmable circuit construction, including ON/OFF switches, signal amplifiers, and oscillators. OptoGPlad-mediated degradation of MutH accelerates E. coli evolution under protocatechuic acid stress, reducing the required generations from 220 to 100. GPTAC-mediated degradation of AroE enhanced the titer of 3-dehydroshikimic acid to 92.6 g/L, a 23.8% improvement over the conventional CRISPR interference method. We provide a tunable, plug-and-play strategy for straightforward protein degradation without the need for pre-fusion, with substantial implications for synthetic biology and metabolic engineering.
13.
Deep-tissue high-sensitivity multimodal imaging and optogenetic manipulation enabled by biliverdin reductase knockout.
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Kasatkina, LA
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Ma, C
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Sheng, H
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Lowerison, M
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Menozzi, L
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Baloban, M
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Tang, Y
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Xu, Y
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Humayun, L
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Vu, T
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Song, P
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Yao, J
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Verkhusha, VV
Abstract:
Performance of near-infrared probes and optogenetic tools derived from bacterial phytochromes is limited by availability of their biliverdin chromophore. To address this, we use a biliverdin reductase-A knock-out mouse model (Blvra-/-), which elevates endogenous biliverdin levels. We show that Blvra⁻/⁻ significantly enhances function of bacterial phytochrome-based systems. Light-controlled transcription using iLight optogenetic tool improves ~25-fold in Blvra-/- cells, compared to wild-type controls, and achieves ~100-fold activation in neurons. Light-induced insulin production in Blvra-/- mice reduces blood glucose by ~60% in diabetes model. To overcome depth limitations in imaging, we employ 3D photoacoustic, ultrasound, and two-photon fluorescence microscopy. This enables simultaneous photoacoustic imaging of DrBphP in neurons and super-resolution ultrasound localization microscopy of brain vasculature at depths of ~7 mm through intact scalp and skull. Two-photon microscopy achieves cellular resolution of miRFP720-expressing neurons at ~2.2 mm depth. Overall, Blvra-/- model represents powerful platform for improving efficacy of biliverdin-dependent tools for deep-tissue imaging and optogenetic manipulation.
14.
Optogenetic storage and release of protein and mRNA in live cells and animals.
Abstract:
Cells compartmentalize biomolecules in membraneless structures called biomolecular condensates. While their roles in regulating cellular processes are increasingly understood, tools for their synthetic manipulation remain limited. Here, we introduce RELISR (Reversible Light-Induced Store and Release), an optogenetic condensate system that enables reversible storage and release of proteins or mRNAs. RELISR integrates multivalent scaffolds, optogenetic switches, and cargo-binding domains to trap cargo in the dark and release it upon blue-light exposure. We demonstrate its utility in primary neurons and show that light-triggered release of signaling proteins can modulate fibroblast morphology. Furthermore, light-induced release of cargo mRNA results in protein translation both in vitro and in live mice. RELISR thus provides a versatile platform for spatiotemporal control of protein activity and mRNA translation in complex biological systems, with broad potential for research and therapeutic applications.
15.
Stimulation of corticospinal neurons by optogenetic cAMP inductions promotes motor recovery after spinal cord injury in female rats via raphespinal tract modulation.
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Martínez-Rojas, B
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Martín-Pérez, S
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Giraldo, E
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Lopez-Mocholi, E
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Alastrue, A
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Andrade-Talavera, Y
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Prius-Mengual, J
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Paniagua, G
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Pedraza, M
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Hingorani, S
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Rost, BR
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Schmitz, D
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Llansola, M
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Felipo, V
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Rodríguez-Moreno, A
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Moreno-Manzano, V
Abstract:
After spinal cord injury (SCI), cyclic adenosine monophosphate (cAMP) levels drop in the spinal cord, cortex and brainstem, unlike in regenerating peripheral neurons. To address SCI recovery, we expressed photoactivatable adenylate cyclase (bPAC) in corticospinal neurons of female rats with dorsal hemisection for on-demand cAMP inductions. bPAC stimulation restored passive and firing properties of corticospinal neurons, promoted early and sustained locomotor recovery and increased corticospinal tract plasticity. Additionally, bPAC enhanced sparing of lumbar-projecting brainstem neurons after SCI, accompanied by activation of cAMP signaling in the raphe-reticular formation and increased excitatory/inhibitory neurotransmitter balance. Accordingly, augmented density of serotonergic tracts was found caudal to the injury in bPAC rats, correlating with enhanced functional performance. Serotonergic implication in motor recovery was further evidenced by selective depletion, resulting in the abrogation of bPAC-mediated recovery. Overall, our findings underscore that cAMP induction in corticospinal neurons enhances locomotion after SCI, through a cortical rerouting pathway via the serotonergic descending tract.
16.
Engineered depalmitoylases enable selective manipulation of protein localization and function.
Abstract:
S-Palmitoylation is a reversible post-translational modification that tunes the localization, stability, and function of an impressive array of proteins including ion channels, G-proteins, and synaptic proteins. Indeed, altered protein palmitoylation is linked to various human diseases including cancers, neurodevelopmental and neurodegenerative diseases. As such, strategies to selectively manipulate protein palmitoylation with enhanced temporal and subcellular precision are sought after to both delineate physiological functions and as potential therapeutics. Here, we develop chemogenetically and optogenetically inducible engineered depalmitoylases to manipulate the palmitoylation status of target proteins. We demonstrate that this strategy is programmable allowing selective depalmitoylation in specific organelles, triggered by cell-signaling events, and of individual protein complexes. Application of this methodology revealed bidirectional tuning of neuronal excitability by distinct depalmitoylases. Overall, this strategy represents a versatile and powerful method for manipulating protein palmitoylation in live cells, providing insights into their regulation in distinct physiological contexts.
17.
Ferroptosis spreads to neighboring cells via plasma membrane contacts.
Abstract:
Ferroptosis is a lytic, iron-dependent form of regulated cell death characterized by excessive lipid peroxidation and associated with necrosis spread in diseased tissues through unknown mechanisms. Using a novel optogenetic system for light-driven ferroptosis induction via degradation of the anti-ferroptotic protein GPX4, we show that lipid peroxidation and ferroptotic death can spread to neighboring cells through their closely adjacent plasma membranes. Ferroptosis propagation is dependent on cell distance and completely abolished by disruption of α-catenin-dependent intercellular contacts or by chelation of extracellular iron. Remarkably, bridging cells with a lipid bilayer or increasing contacts between neighboring cells enhances ferroptosis spread. Reconstitution of iron-dependent spread of lipid peroxidation between pure lipid, contacting liposomes provides evidence for the physicochemical mechanism involved. Our findings support a model in which iron-dependent lipid peroxidation propagates across proximal plasma membranes of neighboring cells, thereby promoting the transmission of ferroptotic cell death with consequences for pathological tissue necrosis spread.
18.
Light-dependent modulation of protein localization and function in living bacteria cells.
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McQuillen, R
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Perez, AJ
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Yang, X
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Bohrer, CH
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Smith, EL
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Chareyre, S
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Tsui, HT
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Bruce, KE
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Hla, YM
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McCausland, JW
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Winkler, ME
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Goley, ED
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Ramamurthi, KS
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Xiao, J
Abstract:
Most bacteria lack membrane-enclosed organelles and rely on macromolecular scaffolds at different subcellular locations to recruit proteins for specific functions. Here, we demonstrate that the optogenetic CRY2-CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations with varying efficiencies in live Escherichia coli cells, including the nucleoid, the cell pole, the membrane, and the midcell division plane. Such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. We further show that CRY2-CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system. Finally, we test this optogenetic system in three additional bacterial species, Bacillus subtilis, Caulobacter crescentus, and Streptococcus pneumoniae, providing important considerations for this system's applicability in bacterial cell biology.
19.
Genetically-stable engineered optogenetic gene switches modulate spatial cell morphogenesis in two- and three-dimensional tissue cultures.
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Beyer, HM
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Kumar, S
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Nieke, M
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Diehl, CMC
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Tang, K
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Shumka, S
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Koh, CS
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Fleck, C
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Davies, JA
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Khammash, M
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Zurbriggen, MD
Abstract:
Recent advances in tissue engineering have been remarkable, yet the precise control of cellular behavior in 2D and 3D cultures remains challenging. One approach to address this limitation is to genomically engineer optogenetic control of cellular processes into tissues using gene switches that can operate with only a few genomic copies. Here, we implement blue and red light-responsive gene switches to engineer genomically stable two- and three-dimensional mammalian tissue models. Notably, we achieve precise control of cell death and morphogen-directed patterning in 2D and 3D tissues by optogenetically regulating cell necroptosis and synthetic WNT3A signaling at high spatiotemporal resolution. This is accomplished using custom-built patterned LED systems, including digital mirrors and photomasks, as well as laser techniques. These advancements demonstrate the capability of precise spatiotemporal modulation in tissue engineering and open up new avenues for developing programmable 3D tissue and organ models, with significant implications for biomedical research and therapeutic applications.
20.
Image-guided optogenetic spatiotemporal tissue patterning using μPatternScope.
Abstract:
In the field of tissue engineering, achieving precise spatiotemporal control over engineered cells is critical for sculpting functional 2D cell cultures into intricate morphological shapes. In this study, we engineer light-responsive mammalian cells and target them with dynamic light patterns to realize 2D cell culture patterning control. To achieve this, we developed μPatternScope (μPS), a modular framework for software-controlled projection of high-resolution light patterns onto microscope samples. μPS comprises hardware and software suite governing pattern projection and microscope maneuvers. Together with a 2D culture of the engineered cells, we utilize μPS for controlled spatiotemporal induction of apoptosis to generate desired 2D shapes. Furthermore, we introduce interactive closed-loop patterning, enabling a dynamic feedback mechanism between the measured cell culture patterns and the light illumination profiles to achieve the desired target patterning trends. Our work offers innovative tools for advanced tissue engineering applications through seamless fusion of optogenetics, optical engineering, and cybernetics.
21.
A sensitive red/far-red photoswitch for controllable gene therapy in mouse models of metabolic diseases.
Abstract:
Red light optogenetic systems are in high demand for the precise control of gene expression for gene- and cell-based therapies. Here, we report a red/far-red light-inducible photoswitch (REDLIP) system based on the chimeric photosensory protein FnBphP (Fn-REDLIP) or PnBphP (Pn-REDLIP) and their interaction partner LDB3, which enables efficient dynamic regulation of gene expression with a timescale of seconds without exogenous administration of a chromophore in mammals. We use the REDLIP system to establish the REDLIP-mediated CRISPR-dCas9 (REDLIPcas) system, enabling optogenetic activation of endogenous target genes in mammalian cells and mice. The REDLIP system is small enough to support packaging into adeno-associated viruses (AAVs), facilitating its therapeutic application. Demonstrating its capacity to treat metabolic diseases, we show that an AAV-delivered Fn-REDLIP system achieved optogenetic control of insulin expression to effectively lower blood glucose levels in type 1 diabetes model mice and control an anti-obesity therapeutic protein (thymic stromal lymphopoietin, TSLP) to reduce body weight in obesity model mice. REDLIP is a compact and sensitive optogenetic tool for reversible and non-invasive control that can facilitate basic biological and biomedical research.
22.
Oncogenic EML4-ALK assemblies suppress growth factor perception and modulate drug tolerance.
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Gonzalez-Martinez, D
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Roth, L
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Mumford, TR
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Guan, J
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Le, A
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Doebele, RC
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Huang, B
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Tulpule, A
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Niewiadomska-Bugaj, M
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Bivona, TG
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Bugaj, LJ
Abstract:
Drug resistance remains a challenge for targeted therapy of cancers driven by EML4-ALK and related fusion oncogenes. EML4-ALK forms cytoplasmic protein condensates, which result from networks of interactions between oncogene and adapter protein multimers. While these assemblies are associated with oncogenic signaling, their role in drug response is unclear. Here, we use optogenetics and live-cell imaging to find that EML4-ALK assemblies suppress transmembrane receptor tyrosine kinase (RTK) signaling by sequestering RTK adapter proteins including GRB2 and SOS1. Furthermore, ALK inhibition, while suppressing oncogenic signaling, simultaneously releases the sequestered adapters and thereby resensitizes RTK signaling. Resensitized RTKs promote rapid and pulsatile ERK reactivation that originates from paracrine ligands shed by dying cells. Reactivated ERK signaling promotes cell survival, which can be counteracted by combination therapies that block paracrine signaling. Our results identify a regulatory role for RTK fusion assemblies and uncover a mechanism of tolerance to targeted therapies.
23.
Optogenetic patterning generates multi-strain biofilms with spatially distributed antibiotic resistance.
Abstract:
Spatial organization of microbes in biofilms enables crucial community function such as division of labor. However, quantitative understanding of such emergent community properties remains limited due to a scarcity of tools for patterning heterogeneous biofilms. Here we develop a synthetic optogenetic toolkit 'Multipattern Biofilm Lithography' for rational engineering and orthogonal patterning of multi-strain biofilms, inspired by successive adhesion and phenotypic differentiation in natural biofilms. We apply this toolkit to profile the growth dynamics of heterogeneous biofilm communities, and observe the emergence of spatially modulated commensal relationships due to shared antibiotic protection against the beta-lactam ampicillin. Supported by biophysical modeling, these results yield in-vivo measurements of key parameters, e.g., molecular beta-lactamase production per cell and length scale of antibiotic zone of protection. Our toolbox and associated findings provide quantitative insights into the spatial organization and distributed antibiotic protection within biofilms, with direct implications for future biofilm research and engineering.
24.
Optogenetic dissection of transcriptional repression in a multicellular organism.
Abstract:
Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can repress or activate specific genes, how these functions are implemented at the molecular level has remained elusive, particularly in the endogenous context of developing animals. Here, we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible (~1 min) and memoryless. Furthermore, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.
25.
From resonance to chaos by modulating spatiotemporal patterns through a synthetic optogenetic oscillator.
Abstract:
Oscillations are a recurrent phenomenon in biological systems across scales, but deciphering their fundamental principles is very challenging. Here, we tackle this challenge by redesigning the wellcharacterised synthetic oscillator known as “repressilator” in Escherichia coli and controlling it using optogenetics, creating the “optoscillator”. Bacterial colonies manifest oscillations as spatial ring patterns. When we apply periodic light pulses, the optoscillator behaves as a forced oscillator and we systematically investigate the properties of the rings under various light conditions. Combining experiments with mathematical modeling, we demonstrate that this simple oscillatory circuit can generate complex dynamics that are transformed into distinct spatial patterns. We report the observation of synchronisation, resonance, subharmonic resonance and period doubling. Furthermore, we present evidence of a chaotic regime. This work highlights the intricate spatiotemporal patterns accessible by synthetic oscillators and underscores the potential of our approach in revealing fundamental principles of biological oscillations.
