Qr: host:"HEK293"
Showing 1 - 25 of 403 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.
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.
3.
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.
4.
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.
5.
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.
6.
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.
7.
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.
8.
Optogenetic Regulation of Localization and Function of Serotonin Transporter by Modulating Its Interaction with Soluble Guanylate Cyclase.
Abstract:
Serotonin (5-HT) signaling is strictly controlled by the serotonin transporter (SERT). The present study aims to establish optogenetic approaches for the control of SERT localization and function by modulating the interaction between SERT and its regulatory protein, soluble guanylate cyclase (sGC). We generated several cell lines that stably express blue light-inducible optogenetic elements fused to sGC or the fourth internal loop (IL4) motif of SERT. Our results indicated that blue light-induced SERT-sGC interaction by heterodimerizing SsrA embedded in the membrane-associated improved light-induced dimer (iLID) and SspB-sGCβ1 decreased SERT localization in the plasma membrane, thus reducing the maximum transport velocity of SERT without affecting its Km for substrate. The light-induced subcellular redistribution of SERT was shown to be attributable to an interference of the SERT-sGC interaction with SERT trafficking but not PKC-mediated internalization. In addition, the light-induced SERT-sGC interaction was blocked by the IL4 peptide or a mutation in the IL4 motif. Furthermore, light-induced exposure of the IL4 motif in iLID decreased the SERT-sGC interaction by displacing SERT from the SERT-sGC complex, thus increasing SERT localization in the membrane and elevating its ability for substrate uptake. This study achieved light-inducible modulation of the protein-protein interaction that allows for the study of biochemical and cellular processes in live cells.
9.
Target degradation of CASPASE-1 for alleviation of inflammation in sepsis via optogenetically engineered extracellular vesicles.
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Du, Y
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Xiao, D
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Li, H
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Fan, L
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Shen, K
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Zhang, B
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Zhang, L
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Guo, L
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Li, Q
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Zheng, J
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Wang, J
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Yao, L
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Yang, G
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Yang, X
Abstract:
Sepsis is a comprehensive ailment of systemic inflammatory response syndrome arising from infection. Activation of CASPASE-1 plays a central role in initiating the inflammatory cascade during sepsis. Herein, we construct optogenetically engineered extracellular vesicles (EVs) that achieve the specific degradation of CASPASE-1 and inhibit sepsis-associated inflammation. Specifically, blue light (460 nm)-induced CRY2/CIBN heterodimerization was applied during the EVs production stage to selectively load GCE-CTM fusion proteins into EVs by EXPLORs technology, yielding EVsGCE-CTM loading efficiency compared to conventional methods. Upon systemic delivery, EVsGCE-C TM preferentially accumulated in macrophages, where the GCE domain selectively bound activated CASPASE-1. The CTM motif then facilitated its lysosomal degradation by chaperone-mediated autophagy, resulting in potent inhibition of CASPASE-1 activity. In a murine model of sepsis, treatment with EVsGCE-CTM effectively attenuated systemic inflammation, reduced multi-organ damage, and significantly improved survival outcomes. This approach enables highly efficient, ubiquitin-independent degradation of intracellular target proteins through macrophage-directed EVs delivery, offering a potential therapeutic approach to address sepsis and other inflammation-related diseases.
10.
RhoG, Rac1 and Cdc42 cooperation in cell protrusion revealed by multiplexed optogenetics and biosensor imaging.
Abstract:
The small GTPase Rac1 controls cell protrusion for a wide variety of critical cell functions. Its regulation by upstream guanine exchange factors (GEFs) has been the focus of multiple studies, but regulation by the GTPase RhoG remains poorly understood. RhoG is known to activate the ELMO/DOCK180 GEF complex, which in turn interacts with Rac1. It is unclear which aspects of protrusion are controlled by RhoG, and which of RhoG’s effects on protrusion are mediated by Rac1. To address these questions, we developed biosensors and optogenetic tools to activate one GTPase while observing another, and to simultaneously visualize the activity of two GTPases. New tools included a photoactivable RhoG, a RhoG biosensor, and red shifted biosensors of RhoG and Rac1. RhoG and Rac1 activation events in protrusions were spatio-temporally correlated with one another and with protrusion velocity. Causal inference indicated that RhoG indeed unidirectionally activated Rac1. Photoactivation of RhoG and Rac1 indicated that specific aspects of protrusion behavior were controlled by RhoG, and only some via Rac1. Further dissection of RhoG to Rac1 signaling through simultaneous GTPase activation and biosensor visualization showed that PA-RhoG activates Rac1 predominantly through DOCK180 and that PA-RhoG can activate Cdc42 independently of Rac1.
11.
Short RNA chaperones promote aggregation-resistant TDP-43 conformers to mitigate neurodegeneration.
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Copley, KE
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Mauna, JC
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Danielson, HL
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Chen, Q
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Ozguney, B
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Ngo, M
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Xie, L
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Smirnov, A
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Davis, M
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Mayne, L
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Linsenmeier, M
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Rubien, JD
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Bergmann, CA
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Portz, B
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Lee, BL
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Odeh, HM
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Lai, L
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Chang, YW
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Hallegger, M
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Ule, J
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Pasinelli, P
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Poon, Y
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Mittal, J
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Fawzi, NL
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Black, BE
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Donnelly, CJ
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Jensen, BK
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Shorter, J
Abstract:
Aberrant aggregation of the prion-like RNA binding protein TDP-43 drives several fatal neurodegenerative proteinopathies, including amyotrophic lateral sclerosis (ALS). In this work, we define how short, specific RNAs solubilize TDP-43. These short RNAs engage and stabilize the TDP-43 RNA recognition motifs, which allosterically destabilizes a conserved helical region in the prion-like domain, thereby promoting aggregation-resistant conformers. Sequence-space mining identified short RNA chaperones with enhanced activity against TDP-43 and disease-linked variants. Enhanced short RNA chaperones mitigated aberrant TDP-43 phenotypes in optogenetic models and in ALS patient-derived and control motor neurons. In mice with cytoplasmic TDP-43 aggregation and motor neuron loss, an enhanced short RNA chaperone reduced pathological aggregation, restored TDP-43 function, and conferred neuroprotection. These results define a mechanistic and therapeutic framework for RNA-based strategies to counter TDP-43 proteinopathies.
12.
An extracellular, optogenetic antibody platform for stimulus-gated antigen recognition and modulation of cell behavior.
Abstract:
Here, we present extrabody, an activatable, modular antibody platform that enables optogenetic or chemical reassembly of split antibody fragments for inducible extracellular antigen recognition. We demonstrate compatibility across diverse targets, including GFP, mCherry, and the tumor-associated antigens EGFR and HER2, and show that both nanobody- and scFv-derived fragments support light-dependent reconstitution. Extrabody enables input-gated cell-cell interactions and antigen transfer, providing external control over intercellular communication. Integration with synNotch receptors and chimeric antigen receptors (CARs) further allows dual-input regulation of downstream responses, including gene expression, cytokine release, and cytotoxicity. Together, these results establish extrabody as a versatile and generalizable interface for externally controlled cellular communication and synthetic signaling.
13.
Optimized optogenetic anti-CRISPR for endogenous gene regulation in Drosophila.
Abstract:
Optogenetic tools-light-responsive proteins that enable to regulate specific cellular activities, study biological processes, and develop new therapies-are attractive approaches for achieving endogenous gene regulation under minimally invasive conditions. Our first step in constructing an optogenetic system to regulate endogenous Drosophila gene expression was to identify inhibitory anti-CRISPR (Acr) proteins that block CRISPRa-mediated activation. Next, we inserted optogenetic protein LOV2 into these Acrs, tested for their ability to optogenetically modulate endogenous gene upregulation through the CRISPRa-based flySAM system in Drosophila, and found that the photoswitchability of these prototypes was weak. We therefore engineered an optimized Acr-LOV2 fusion module by refining length of intrinsically disordered and ordered regions (IDR and IOR) of Acrs. This optimization yielded a variant with significantly greater sensitivity to blue-light-induced endogenous gene upregulation than the prototypes, leading to new in vivo discoveries. In addition, this work provides insights for in vivo functional characterization of the IDR and the IOR of these small-sized proteins. Together, these findings establish a robust optogenetic toolbox for precise, light-controlled endogenous gene regulation in Drosophila.
14.
BMAL1 regulates circadian rhythms via phase separation-mediated transcriptional hub formation.
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Gao, W
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Zhu, L
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Wei, Y
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Luo, G
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Wang, J
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Wang, L
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Peng, Z
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Li, X
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Wu, Z
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Li, J
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Wu, Y
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Wang, X
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Jing, J
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Zou, S
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Zhao, Q
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Fan, Y
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Yuan, Q
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Zhou, C
Abstract:
The mechanisms by which core clock components are spatially organized to ensure robust oscillations in mammals remain unclear. Here, we identify the positive limb factor BMAL1 as a phase-separating protein that forms dynamic biomolecular condensates essential for circadian transcription and behavior. Endogenous BMAL1 forms nuclear puncta that oscillate in sync with the circadian cycle. Deletion analysis and optogenetic clustering identify an N-terminal 90-amino acid intrinsically disordered region whose phosphorylation state tunes BMAL1 phase separation. Besides, BMAL1 condensates behave as multi-molecular assemblies that selectively recruit CLOCK, p300, MED1, and are specifically promoted by E-box DNA. Functionally, an IDR-deleted BMAL1 mutant fails to rescue rhythmic transcription in Bmal1-KO cells and cannot restore locomotor rhythms when reintroduced into SCN-specific Bmal1‑KO mice. These findings establish BMAL1 condensates as dynamic transcriptional hubs that couple phase separation to circadian rhythm in cells and in vivo.
15.
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.
16.
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.
17.
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.
18.
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.
19.
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.
20.
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.
21.
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.
22.
Optogenetic manipulation of estrogen receptor signaling to improve estrogen deficiency.
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Liu, J
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Xie, L
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Wang, J
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Chen, Q
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Zhu, M
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Zhang, L
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Xie, S
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Lu, B
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Chen, X
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Xu, Y
Abstract:
Estrogen receptor (ER)-mediated genomic actions are crucial for maintaining various physiological functions, and their dysfunction is associated with numerous human diseases. Traditional estrogen replacement therapy (ERT) is commonly used to manage estrogen deficiency-related conditions, such as vulvovaginal atrophy during menopause, but its systemic effects pose notable risks. This study introduces OptoER, an optogenetic tool engineered to precisely modulate ER-mediated genomic pathways through light-induced transcription regulation, offering spatial-temporal control over ER-dependent gene expression. Our in vitro studies demonstrate that OptoER significantly enhances ER-specific gene transcription and protein synthesis, leading to improved cell proliferation and migration. In a proof-of-principle study using ovariectomized (OVX) mice, OptoER demonstrated considerable therapeutic potential for vaginal atrophy, with observed improvement in epithelial thickness and keratinization. These findings suggest that OptoER provides a targeted therapeutic strategy for estrogen deficiency conditions, with significant implications for treating vaginal atrophy and promoting regenerative healing in estrogen-deprived tissues.
23.
An orthogonal CRISPR/Cpf1 platform for precise spatiotemporal gene regulation and osteoporotic fracture repair.
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Zhao, J
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Wang, Z
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Lu, L
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Bu, G
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Miao, Z
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Zhang, Y
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Guo, Y
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Yang, Z
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Ma, J
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Jiao, J
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Ma, X
Abstract:
CRISPR-Cas systems enable powerful gene editing and regulation, yet single-modality control often fails to achieve orthogonal, spatiotemporally precise regulation of multiple endogenous genes. We engineered OREC, an orthogonal platform integrating chemogenetic and optogenetic modalities for precise, reversible, multiplex gene control. OREC comprises two components: ORECC regulated by doxycycline (Dox) and ORECo controlled by light. By assembling catalytically dead Cpf1 (dCpf1), gene regulatory elements, and crRNA arrays on single transcripts, OREC enables robust simultaneous manipulation of multiple genes. We demonstrated OREC's therapeutic potential in vitro for osteoblast function modulation and in vivo for osteoporotic fracture repair. OREC effectively activated Bmp2 while inhibiting Dkk1, significantly enhancing bone formation and fracture healing in mouse models. These results establish OREC as a versatile platform for precise multiplex gene regulation, offering significant advancement for CRISPR-based gene therapy applications in complex tissues where coordinated control of multiple therapeutic targets is essential.
24.
Rapid optogenetic manipulation of autophagy reveals that the nuclear pore complex is a robust autophagy substrate.
Abstract:
Autophagy, a conserved recycling process, manages intracellular quality control to mitigate stress. To determine the rapid effects of autophagy perturbation, we developed the first optogenetic tool to rapidly inhibit autophagy, termed ASAP. Our approach selectively inhibits autophagy within 5 minutes, providing a precise and dynamic approach to study autophagy regulation. Proteomic profiling with ASAP revealed the most tightly regulated autophagy substrates along with novel, previously unidentified substrates, including nuclear pore complex (NPC) proteins. Interestingly, autophagy regulates quality control of incomplete NPCs still in the cytoplasm via specific LC3-interacting regions (LIRs), sparing NPCs embedded in the nuclear envelope. Upon rapid autophagy inhibition, incomplete NPCs accumulate and instead of undergoing autophagic degradation, cytoplasmic NPCs aggregate in processing bodies. Using ASAP, we demonstrate rapid and specific inhibition of autophagy, revealing that the nuclear pore complex is a tightly regulated autophagy substrate.
25.
p62/SQSTM1 Condensation Modulates Mitochondrial Clustering to Participate in Mitochondrial Quality Control.
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Sun, S
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Xin, J
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Zhang, Y
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Yang, B
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Su, D
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Ni, R
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Ma, Q
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Li, N
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Ma, G
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Peng, Q
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Chen, S
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Prehn, JHM
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Tam, KY
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Wang, H
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Ying, Z
Abstract:
Mitochondrial quality control is tightly associated with aging-related neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Previous studies reported that ALS/FTD-associated protein p62 drives "mitochondrial clustering" (perinuclear clustering of fragmented and swollen mitochondria) during PINK1/Parkin-mediated mitophagy, but the underlying molecular mechanism, especially the precise role of p62 in mitochondrial clustering during mitophagy and the potential relationship between the mitochondrial quality control mediated by p62 and disease pathogenesis of ALS/FTD, remains unclear. Here, using cell biology in combination with an optogenetic tool, we show that the phase separation (condensation) of p62 mediates the clustering of damaged mitochondria to form "grape-like" clusters during PINK1/Parkin-mediated mitophagy, which is tightly associated with aging-related neurodegenerative diseases. In addition, our data suggest this mitochondrial clustering process is an arrest mechanism driven by p62 condensation (beyond the function of other autophagy receptors in mitophagy), which acts as a "brake" to reduce the surface area of dysfunctional mitochondria within cytoplasm for minimizing mitochondrial turnover in cells. Moreover, ALS/FTD-related pathological mutations perturb p62 condensation, thereby inhibiting mitochondrial clustering and destroying the "brake" machinery of mitochondrial quality control. Together, our data highlight how p62 condensation modulates organelle quality control in cell biology, and the important role of p62 condensation in both physiology and pathology.
