Qr: switch:"AsLOV2"
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1.
Chromatin context shapes SPT5 regulation of promoter-proximal Pol II, fine-tuning gene expression changes during Drosophila embryogenesis.
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Dulja, A
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Mayer, M
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Engel, N
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Golov, AK
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Bender, K
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Forneris, M
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Kherdjemil, Y
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Feng, S
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Viales, RR
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Furlong, EEM
Abstract:
Transcription involves initiation, pausing, elongation, and termination. Suppressor of Ty5 (SPT5) regulates promoter-proximal pausing and elongation, but how it orchestrates both steps during dynamic developmental changes in gene expression remains unclear. Here, using rapid optogenetic depletion in Drosophila embryos, we uncover different consequences of SPT5 removal at different developmental stages. In early embryos, SPT5 depletion causes a shift of RNA polymerase II (Pol II) from the canonical pausing site to the +1 nucleosome, which is strongly positioned. In late embryos, SPT5 depletion similarly reduces pausing at the canonical site, but the transcriptional machinery can overcome the +1 nucleosome-which appears more labile at this time point-moving into the gene body. This results in lethality and both up- and downregulation of expression, depending on the balance between Pol II entering the gene body and defective elongation. This is intensified for genes naturally increasing or decreasing their expression, indicating that SPT5 contributes to fine-tuning dynamic expression changes.
2.
Tunable Chemical and Optical Modulation of ER-Plasma Membrane Contact-Site Geometry, Dynamics, and Protein Recruitment with High-Fidelity Visualization.
Abstract:
Endoplasmic reticulum-plasma membrane (ER-PM) contact sites are dynamic membrane interfaces that regulate essential cellular processes, including calcium signaling and lipid homeostasis. Emerging evidence suggests that contact-site geometry and organization critically influence these functions, yet existing tools cannot systematically manipulate individual geometric parameters while providing high-fidelity visualization in living cells. Here, we develop complementary chemical and optical platforms for inducible ER-PM contact-site reconstitution. A nontoxic, reversible abscisic acid-inducible system based on the plant-derived ABIcs/PYLcs pair and a rapidly reversible optogenetic iLID/SspB system enable robust visualization and dose-dependent control of contact-site assembly and disassembly. Increasing inducer dose selectively increases contact-site density and total contact area per cell without substantially changing the average size of individual contact sites. In contrast, systematic variation of tether length or tether abundance selectively increases average contact-site size and total contact area without altering contact-site density, providing orthogonal strategies to modulate contact-site density and size. Importantly, engineered contact sites recruit ER-PM contact-site-associated proteins, including MAPPER, Kv2.1, and Stromal Interaction Molecule 1 (STIM1), demonstrating key organizational features of native ER-PM contacts. MAPPER recruitment is maintained across tether lengths tested, whereas Kv2.1/STIM1 recruitment depends strongly on tether length, with longer tethers promoting recruitment by increasing intermembrane spacing. In contrast, varying tether abundance has little effect on protein recruitment, revealing distinct roles for intermembrane spacing and tether density in organizing ER-PM contact sites. Together, this work establishes a quantitative platform for engineering ER-PM contact sites with defined geometric properties and provides a framework for investigating how membrane contact-site architecture regulates cellular function.
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.
An intrinsic cytoskeletal oscillator establishes neuronal polarity.
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Lin, TC
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Coles, CH
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Alfadil, E
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Fäßler, F
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Husch, A
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Dupraz, S
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Pietralla, T
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Narita, A
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Schelski, M
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Flynn, KC
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Stern, S
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Möhl, C
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Hilton, BJ
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Vauti, F
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Arnold, HH
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Schur, FKM
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Bradke, F
Abstract:
Neurons acquire polarity by specifying one neurite as the axon, whereas the others become dendrites. But how this fundamental asymmetry is established remains unclear1. Neuronal polarization has been thought to rely primarily on growth cones that sense external cues2. Here we show that growth cones alone do not direct this process and that the soma acts as a central organizer of neuronal polarization. Using live imaging and genetic loss-of-function approaches in vivo, combined with optogenetic control and local cytoskeletal perturbations in cultured neurons, we uncover a soma-initiated oscillatory program that primes axon selection. Periodic actin branching that depends on the actin-related protein 2/3 (ARP2/3) complex at the soma remodels a global actomyosin network, thereby generating an actin wave that retracts neurites before propagating into a single neurite tip. Exposure to this wave relaxes local actomyosin contractility, which drives a transient microtubule-based protrusion and biases this neurite towards axon fate. As the cell exits this oscillatory stage, this neurite can overcome global inhibition and extend independently of ARP2/3, whereas actomyosin activity suppresses axon formation in the remaining neurites so that they subsequently become dendrites. This soma-driven mechanism ensures the emergence of a single axon independent of environmental cues and underpins the unidirectional information flow in neuronal circuits.
5.
Adhesion-driven rigidity transition decoupled from density-driven jamming triggers epithelial organization in embryonic tissues.
Abstract:
The active regulation of tissue material properties via phase transitions is central in morphogenesis. Transitions occur abruptly at critical points in different control parameters, such as cell density, shape or adhesion. Whether these parameters are interdependent, and perform redundant or distinct functions, is unknown. Here we show that depending on the co-regulation of multiple control parameters, a tissue not only tunes its deformability but also its morphogenetic trajectory. We theoretically define a phase diagram capturing the material states of zebrafish pluripotent tissues undergoing epiboly-a tissue movement occurring during gastrulation-and show that they simultaneously cross critical points in cell density, connectivity and adhesion strength. We then combine optogenetics, biophysical measurements and quantitative morphometrics to independently modulate each parameter in vivo, and identify adhesion as the main determinant of tissue rheology. Further decoupling adhesion from density and inducing adhesion-driven rigidification in unjammed pluripotent tissues is sufficient to switch their morphogenetic program and trigger epithelial organization. This switch in tissue reorganization is achieved via tricellular junction formation, followed by lumenogenesis and the initiation of apical polarity. Our work reveals that the nonlinear dynamics of emergent tissue mechanics are mechanisms of tissue organization and morphogenesis.
6.
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.
7.
Kinetic properties of optogenetic site-specific DNA recombination by LiCre-loxP.
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Dufour, A
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Duplus-Bottin, H
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Boukéké-Lesplulier, T
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Casassa, E
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Triqueneaux, G
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Tarbouriech, L
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Darthenay-Kiennemann, C
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Dumont, A
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Moali, C
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Vittoz, F
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Jost, D
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Yvert, G
Abstract:
Advances in optogenetics now allow specific modifications to the DNA of live cells with light. However, successfully using these technologies requires knowing their properties in terms of sensitivity, efficiency, kinetics and mechanism. We previously developed an optogenetic tool made of a single chimeric protein called LiCre that enables the induction of specific changes in the genome with blue light via DNA recombination between loxP sites ( Duplus-Bottin et al., 2021). Here, we used in vitro and in vivo experiments combined with kinetic modeling to provide a deeper characterization of the photoactivated LiCre-loxP recombination reaction. We find that LiCre binds DNA with high affinity in the absence of a light stimulus and that this binding is cooperative, although not as much as for the Cre recombinase from which LiCre was derived. In yeast, the addition of riboflavin to the culture medium had no effect on LiCre's efficiency, even when cells over-expressed riboflavin kinase, suggesting that the abundance of the flavin mononucleotide cofactor is not limiting for the reaction. However, LiCre's efficiency in yeast gradually increased when raising the temperature from 20°C to 37°C. The recombination kinetics observed in live cells are best explained by a model where the photoactivation of two or more DNA-bound LiCre units (happening in seconds) can produce (in several minutes) a functional recombination synapse. This model was able to capture the effect of a point mutation altering LiCre's light cycle. This deeper understanding of the LiCre-loxP system provides additional knowledge for designing experiments where specific genetic changes are induced in live cells with light.
8.
Tissue rigidity phase transition shapes morphogen gradients.
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Autorino, C
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Khoromskaia, D
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Harari, L
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Floris, E
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Booth, H
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Pallares-Cartes, C
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Petrasiunaite, V
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Dorrity, M
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Corominas-Murtra, B
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Hadjivasiliou, Z
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Petridou, NI
Abstract:
During development, local mechanochemical cues within the cell microenvironment are translated into signalling pathways that drive cell fate decisions. Yet, as cells differentiate collectively, how global tissue-level properties shape these instructive cues remains unclear. Here we show that a tissue-scale rigidity transition guides patterning by tuning the length scales and timescales of morphogen signalling. By combining rigidity percolation theory, reaction-diffusion modelling, quantitative imaging and optogenetics in zebrafish, we uncover dynamical global tissue rigidity patterns that actively shape the Nodal morphogen gradient by locally changing its concentration and accelerating its signalling activity. In this self-generated mechanism, Nodal, besides instructing meso-endoderm fate specification, increases cell-cell adhesion strength via regulating planar cell polarity genes. Once the adhesion strength reaches a critical point, it triggers a rigidity transition which, in turn, induces the collapse of tissue porosity. The abrupt tissue reorganization negatively feeds back on Nodal signalling, impacting both its length scales, by restricting Nodal diffusivity, and its timescales, by speeding up the expression of its antagonist Lefty, thereby ensuring timely signal termination and robust patterning. Overall, we uncover a multiscale regulatory mechanism by which positional information and tissue material properties dynamically tune one another.
9.
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.
10.
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.
11.
The local mechanostructural properties of protein cargoes regulate nucleocytoplasmic transport.
Abstract:
The nuclear pore complex regulates nucleocytoplasmic transport. It was recently shown that the global mechanical stability of proteins regulates their nuclear import rate. On the basis of these findings, we hypothesize that the main principles governing protein translocation through narrow biological pores-in which locally unstructured and unfolded regions determine cargo orientation and translocation kinetics-can help rationalize protein trafficking across the nuclear pore complex. Inspired by single-molecule studies showing that proteins exhibit different mechanical stability when pulled from different termini, here we show that the rate of both nuclear import and export is enhanced when the translocating protein is threaded through the nuclear pore from the specific region exhibiting lower local nanomechanical stability and increased structural disorder. We demonstrate this for a range of model proteins with different folds and stabilities by combining single-molecule magnetic tweezers with single-cell optogenetic experiments, complemented by steered molecular dynamics simulations and biochemical binding assays. Our bioinformatics survey then shows that in human transcription factors, the termini containing the nuclear localization signal sequence exhibit a higher degree of structural disorder. We propose that protein orientation might offer an additional layer of structural and mechanical control of the kinetics of nuclear transport.
12.
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.
13.
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.
14.
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.
15.
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.
16.
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.
17.
Modification of the BphP1-QPAS1 optogenetic system for gene expression regulation in Nicotiana benthamiana tobacco leaves using near-infrared light.
Abstract:
In plants, the regulation of transgene transcription is typically achieved using chemical agents. A safe alternative to chemically induced systems may be optogenetic systems. The BphP1-QPAS1 system has distinct advantages over other optogenetic systems, as it is activated by near-infrared (NIR, 780 nm) light, which is beyond the spectrum of plant photoreceptors. This system is based on the use of a split transcription factor (TF), consisting of the DNA-binding and dimerization domains of the yeast TF Gal4, fused to the QPAS1 component, along with the transactivation domain VP16 fused to BphP1. Under NIR light, BphP1 interacts with QPAS1, leading to the formation of the functional TF Gal4-VP16. A primary obstacle to using optogenetic systems in plants is their undesired activation under white light, which is vital for normal plant growth. A potential solution to this issue is temporarily removing one component of the split TF from the nucleus under white light. We modified the BphP1-QPAS1 system to activate reporter gene expression in Nicotiana benthamiana leaves using NIR light. We combined BphP1-QPAS1 with several variants of LOV domain-containing proteins activated by blue light (460-480 nm). The best results were achieved by combining the BphP1-QPAS1 system with the AsLOV2 domain, which carries the degron sequence RRRG at the C-terminal Jα helix and initiates the degradation of the chimeric protein NES-Gal4-QPAS1-AsLOV2-RRRG under white light. This modification induced the BphP1-QPAS1 system in tobacco leaves only under NIR light, but not in the dark or under white light. We believe that, in the future, the BphP1-QPAS1 system could be applied to enhance plant resistance to adverse environmental conditions, pests, and viral diseases.
18.
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.
19.
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.
20.
Dynamic Control of Cell Signaling with Optogenetic Modulation of Protein Abundance in Living Mammalian Cells.
Abstract:
Precise modulation of protein levels is essential for investigating the dynamics of signaling pathways. Degradation-based approaches allow rapid reduction of protein abundance, and their combination with optogenetic regulation enables controlled and reversible manipulation. In this chapter, we describe an optogenetic strategy that stabilizes a protein of interest by inhibiting its degradation. As a proof of concept, we demonstrate light-dependent stabilization of MKP3 and constitutively active MEK and examine the resulting changes in ERK signaling. We also provide a step-by-step protocol for measuring protein stabilization kinetics. This method integrates a potent degradation tag with optogenetic control to achieve tunable and reversible regulation of protein levels in living cells.
21.
Optogenetic Proximity Labeling Maps Spatially Resolved Mitochondrial Surface Proteomes and a Locally Regulated Ribosome Pool.
Abstract:
Outer mitochondrial membranes (OMM) function as dynamic hubs for inter-organelle communication, integrating bidirectional signals, and coordinating organelle behavior in a context-dependent manner. However, tools for mapping mitochondrial surface proteomes with high spatial and temporal resolution remain limited. Here, we introduce an optogenetic proximity labeling strategy using LOV-Turbo, a light-activated biotin ligase, to profile mitochondrial surface proteomes with improved precision, temporal control, and reduced background. By fusing LOV-Turbo to a panel of variants of an OMM-anchored protein, Miro1, we generate spatially distinct baits that resolve modular architectures and regulatory states of the OMM proteomes across diverse conditions, a database we name MitoSurf. Building on this proteomic map, we present RiboLOOM, a platform that defines LOV-Turbo labeled ribosomes and their bound mRNAs at the mitochondrial surface. MitoSurf and RiboLOOM uncover a spatially distinct ribosome pool at the OMM that is maintained by Miro1, enabling local mRNA engagement and translation of mitochondria-related proteins. These findings establish Miro1 as a key organizer of mitochondrial protein biogenesis through spatial confinement of surface-associated ribosomes. Our platform reveals an uncharted layer of mitochondrial surface biology and provides a generalizable strategy to dissect dynamic RNA-protein-organelle interfaces in living cells.
22.
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.
23.
Engineered AcrIIA5 for optogenetic control of CRISPR-Cas9-based genome editing.
Abstract:
The CRISPR-Cas9 system has been proven to be a powerful tool for gene editing in living cells and shows great potential in genetic disease treatment. Anti-CRISPR (Acr)-based optogenetic tools could spatiotemporally regulate the activity of CRISPR-Cas9, thereby improving the precision and safety of gene editing. However, these tools could only regulate a certain Cas9 protein because of the high specificity of Acr used, limiting their further application. In this study, we developed a new optogenetic tool named CASANOVA-A5 (CRISPR-Cas9 activity switching via a novel optogenetic variant of AcrIIA5) by inserting the blue light sensor AsLOV2 into AcrIIA5 with a broad inhibition spectrum. We proved that the CASANOVA-A5 could regulate the gene editing activity of SpCas9, SaCas9, NmeCas9, and St1Cas9 in a blue light-dependent manner. Additionally, we engineered AcrIIA5-LOV9 by integrating the blue light-dependent degron module LOV9, showing obvious optical regulation for SpCas9. Together, our work demonstrates two feasible methods to engineer the Acrs to potent optogenetic tools and suggests systematic strategies for further optimization.
24.
Optogenetic Control the Activity of Pyruvate Decarboxylase in Saccharomyces cerevisiae for Tunable Ethanol Production.
Abstract:
Saccharomyces cerevisiae is a widely used chassis in metabolic engineering. Due to the Crabtree effect, it preferentially produces ethanol under high-glucose conditions, limiting the synthesis of other valuable metabolites. Conventional metabolic engineering approaches typically rely on irreversible genetic modifications, making it insufficient for dynamic metabolic control. In contrast, optogenetics offers a reversible and tunable method for regulating cellular metabolism with high temporal precision. In this study, we engineered the pyruvate decarboxylase isozyme 1 (Pdc1) by inserting the photosensory modules (AsLOV2 and cpLOV2 domains) into rationally selected positions within the enzyme. Through a growth phenotype-based screening system, we identified two blue light-responsive variants, OptoPdc1D1 and OptoPdc1D2, which enable light-dependent control of enzymatic activity. Leveraging these OptoPdc1 variants, we developed opto-S. cerevisiae strains, MLy-9 and MLy-10, which demonstrated high efficiency in modulating both cell growth and ethanol production. These strains allow reliable regulation of ethanol biosynthesis in response to blue light, achieving a dynamic control range of approximately 20- to 120-fold. The opto-S. cerevisiae strains exhibited dose-dependent production in response to blue light intensity and pulse patterns, confirming their potential for precise metabolic control. This work establishes a novel protein-level strategy for regulating metabolic pathways in S. cerevisiae and introduces an effective method for controlling ethanol metabolism via optogenetic regulation.
25.
A single-component optogenetic toolkit for programmable control of microtubule.
Abstract:
Microtubules (MTs) 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 optogenetic toolkit for spatiotemporal control of MT organization and dynamics. By replacing native multimerization motifs with a blue light-responsive oligoermization domain, we have engineered single-component probes, OptoMT and OptoTIP, that reversibly label MT polymers or track plus-ends with tunable kinetics from seconds to minutes. When coupled to enzymatic effectors, these modules enable localized tubulin acetylation or detyrosination, directly linking PTMs to MT stability. We further engineered OptoMotor, a light-activatable kinesin platform that reconstitutes tail-dependent cargo transport along MTs, and OptoSAW, a light-triggered severing actuator for controlled MT disassembly. Using these tools, we reveal how local MT integrity governs lysosomal trafficking and ER-associated signaling dynamics. Collectively, this versatile single-component toolkit bridges molecular design with cytoskeletal function, offering new avenues to illuminate how dynamic cytoskeletal architectures coordinate intracellular organization, transport, and signaling.
