Qr: journal:"Cell Rep Methods"
Showing 1 - 6 of 6 results
1.
Rapid and reversible regulation of cell cycle progression in budding yeast using optogenetics.
Abstract:
The complexity of the eukaryotic cell cycle complicates experiment design and data interpretation, limiting our understanding of how cells coordinate cell cycle processes. Traditional perturbation methods, including knockouts, deletions, and arrest-inducing chemicals, are limited by compensatory feedback interactions or pleiotropic side effects. Inducible synthetic systems offer greater specificity but often rely on external inducers, making rapid reversibility difficult. Here, we developed OPTO-Cln2, an optogenetic tool for light-controlled and reversible regulation of G1 progression in budding yeast. Using time-lapse microscopy, we show that OPTO-Cln2-strains rapidly switch between normal and altered G1 progression. Combining OPTO-Cln2 with a readout of TORC1 and PKA activity, we find that oscillatory signaling dynamics is coordinated with G1 progression. Finally, we show that OPTO-Cln2 enables at least two cycles of synchronous arrest and release in liquid cultures. This system provides a powerful approach for studying cell cycle dynamics and the coordination of cell growth with division.
2.
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.
3.
Light-inducible FLPase reconstitution enables temporal control of gene expression in Drosophila melanogaster.
Abstract:
Precise temporal control of gene expression is a prerequisite for disentangling timing-specific effects of gene function within the life cycle of Drosophila melanogaster. Here, we implement light-inducible FLPase reconstitution (LIFR) as a conditional gene expression system in flies, which combines blue light-responsive Magnet photoswitches and split-FLPase to remove an FRT-flanked stop cassette and irreversibly switch on transgene expression in response to light. This system is highly efficient, has virtually no transgene leakage, and a single light pulse is sufficient to induce long-term transgene expression. We demonstrate that LIFR in adulthood overcomes the developmental lethality elicited by constitutive pan-neuronal overexpression of neurodegeneration-causing mutants TDP43G298S and HTTQ97. We also illustrate that LIFR can help trace specific cell-type fates across developmental stages. Thus, we demonstrate proof of principle that LIFR is a versatile platform to conditionally activate long-lasting gene expression without the side effects of existing systems, thereby extending the Drosophila melanogaster genetic toolbox.
4.
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.
5.
An improved FLARE system for recording and manipulating neuronal activity.
Abstract:
To address the need for methods for tagging and manipulating neuronal ensembles underlying specific behaviors, we present an improved version of FLARE, termed cytoFLARE (cytosol-expressed FLARE). cytoFLARE incorporates cytosolic tethering of a transcription factor and expression of a more sensitive pair of calcium-sensing domains. We show that cytoFLARE captures more calcium- and light-dependent signals in HEK293T cells and higher signal-to-background ratios in neuronal cultures. We further establish cytoFLARE transgenic Drosophila models and apply cytoFLARE to label activated neurons upon sensory or optogenetic stimulation within a defined time window. Notably, through the cytoFLARE-driven expression of optogenetic actuators, we successfully reactivated and inhibited neurons involved in the larval nociceptive system. Our findings demonstrate the characterization and application of time-gated calcium integrators for both recording and manipulating neuronal activity in Drosophila larvae.
6.
Computational framework for single-cell spatiotemporal dynamics of optogenetic membrane recruitment.
Abstract:
We describe a modular computational framework for analyzing cell-wide spatiotemporal signaling dynamics in single-cell microscopy experiments that accounts for the experiment-specific geometric and diffractive complexities that arise from heterogeneous cell morphologies and optical instrumentation. Inputs are unique cell geometries and protein concentrations derived from confocal stacks and spatiotemporally varying environmental stimuli. After simulating the system with a model of choice, the output is convolved with the microscope point-spread function for direct comparison with the observable image. We experimentally validate this approach in single cells with BcLOV4, an optogenetic membrane recruitment system for versatile control over cell signaling, using a three-dimensional non-linear finite element model with all parameters experimentally derived. The simulations recapitulate observed subcellular and cell-to-cell variability in BcLOV4 signaling, allowing for inter-experimental differences of cellular and instrumentation origins to be elucidated and resolved for improved interpretive robustness. This single-cell approach will enhance optogenetics and spatiotemporally resolved signaling studies.
