Showing 1 - 10 of 10 results
Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice.
Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.
Engineering a far-red light–activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors.
It is widely understood that CRISPR-Cas9 technology is revolutionary, with well-recognized issues including the potential for off-target edits and the attendant need for spatiotemporal control of editing. Here, we describe a far-red light (FRL)–activated split-Cas9 (FAST) system that can robustly induce gene editing in both mammalian cells and mice. Through light-emitting diode–based FRL illumination, the FAST system can efficiently edit genes, including nonhomologous end joining and homology-directed repair, for multiple loci in human cells. Further, we show that FAST readily achieves FRL-induced editing of internal organs in tdTomato reporter mice. Finally, FAST was demonstrated to achieve FRL-triggered editing of the PLK1 oncogene in a mouse xenograft tumor model. Beyond extending the spectrum of light energies in optogenetic toolbox for CRISPR-Cas9 technologies, this study demonstrates how FAST system can be deployed for programmable deep tissue gene editing in both biological and biomedical contexts toward high precision and spatial specificity.
Optogenetic stimulation of phosphoinositides reveals a critical role of primary cilia in eye pressure regulation.
Glaucoma is a group of progressive optic neuropathies that cause irreversible vision loss. Although elevated intraocular pressure (IOP) is associated with the development and progression of glaucoma, the mechanisms for its regulation are not well understood. Here, we have designed CIBN/CRY2-based optogenetic constructs to study phosphoinositide regulation within distinct subcellular compartments. We show that stimulation of CRY2-OCRL, an inositol 5-phosphatase, increases aqueous humor outflow and lowers IOP in vivo, which is caused by a calcium-dependent actin rearrangement of the trabecular meshwork cells. Phosphoinositide stimulation also rescues defective aqueous outflow and IOP in a Lowe syndrome mouse model but not in IFT88fl/fl mice that lack functional cilia. Thus, our study is the first to use optogenetics to regulate eye pressure and demonstrate that tight regulation of phosphoinositides is critical for aqueous humor homeostasis in both normal and diseased eyes.
Dynamic Fas signaling network regulates neural stem cell proliferation and memory enhancement.
Activation of Fas (CD95) is observed in various neurological disorders and can lead to both apoptosis and prosurvival outputs, yet how Fas signaling operates dynamically in the hippocampus is poorly understood. The optogenetic dissection of a signaling network can yield molecular-level explanations for cellular responses or fates, including the signaling dysfunctions seen in numerous diseases. Here, we developed an optogenetically activatable Fas that works in a physiologically plausible manner. Fas activation in immature neurons of the dentate gyrus triggered mammalian target of rapamycin (mTOR) activation and subsequent brain-derived neurotrophic factor secretion. Phosphorylation of extracellular signal-regulated kinase (Erk) in neural stem cells was induced under prolonged Fas activation. Repetitive activation of this signaling network yielded proliferation of neural stem cells and a transient increase in spatial working memory in mice. Our results demonstrate a novel Fas signaling network in the dentate gyrus and illuminate its consequences for adult neurogenesis and memory enhancement.
Nuclear actin regulates inducible transcription by enhancing RNA polymerase II clustering.
Gene expression in response to external stimuli underlies a variety of fundamental cellular processes. However, how the transcription machinery is regulated under these scenarios is largely unknown. Here, we discover a novel role of nuclear actin in inducible transcriptional regulation using next-generation transcriptome sequencing and super-resolution microscopy. The RNA-seq data reveal that nuclear actin is required for the establishment of the serum-induced transcriptional program. Using super-resolution imaging, we found a remarkable enhancement of RNA polymerase II (Pol II) clustering upon serum stimulation and this enhancement requires the presence of nuclear actin. To study the molecular mechanisms, we firstly observed that Pol II clusters co-localized with the serum-response genes and nuclear actin polymerized in adjacent to Pol II clusters upon serum stimulation. Furthermore, N-WASP and Arp2/3 are reported to interact with Pol II, and we demonstrated N-WASP is required for serum-enhanced Pol II clustering. Importantly, using an optogenetic tool, we revealed that N-WASP phase-separated with the carboxy-terminal domain of Pol II and nuclear actin. In addition to serum stimulation, we found nuclear actin also essential in enhancing Pol II clustering upon interferon-γ treatment. Taken together, our work unveils nuclear actin promotes the formation of transcription factory on inducible genes, acting as a general mechanism underlying the rapid response to environmental cues.
Exosome-based delivery of super-repressor IκBα relieves sepsis-associated organ damage and mortality.
As extracellular vesicles that play an active role in intercellular communication by transferring cellular materials to recipient cells, exosomes offer great potential as a natural therapeutic drug delivery vehicle. The inflammatory responses in various disease models can be attenuated through introduction of super-repressor IκB (srIκB), which is the dominant active form of IκBα and can inhibit translocation of nuclear factor κB into the nucleus. An optogenetically engineered exosome system (EXPLOR) that we previously developed was implemented for loading a large amount of srIκB into exosomes. We showed that intraperitoneal injection of purified srIκB-loaded exosomes (Exo-srIκBs) attenuates mortality and systemic inflammation in septic mouse models. In a biodistribution study, Exo-srIκBs were observed mainly in the neutrophils, and in monocytes to a lesser extent, in the spleens and livers of mice. Moreover, we found that Exo-srIκB alleviates inflammatory responses in monocytic THP-1 cells and human umbilical vein endothelial cells.
Engineering light-controllable CAR T cells for cancer immunotherapy.
T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation. We first demonstrated light-controllable gene expression and functional modulation in human embryonic kidney 293T and Jurkat T cell lines. We then improved the LINTAD system to achieve optimal efficiency in primary human T cells. The results showed that pulsed light stimulations can activate LINTAD CAR T cells with strong cytotoxicity against target cancer cells, both in vitro and in vivo. Therefore, our LINTAD system can serve as an efficient tool to noninvasively control gene activation and activate inducible CAR T cells for precision cancer immunotherapy.
Shape-morphing living composites.
This work establishes a means to exploit genetic networks to create living synthetic composites that change shape in response to specific biochemical or physical stimuli. Baker's yeast embedded in a hydrogel forms a responsive material where cellular proliferation leads to a controllable increase in the composite volume of up to 400%. Genetic manipulation of the yeast enables composites where volume change on exposure to l-histidine is 14× higher than volume change when exposed to d-histidine or other amino acids. By encoding an optogenetic switch into the yeast, spatiotemporally controlled shape change is induced with pulses of dim blue light (2.7 mW/cm2). These living, shape-changing materials may enable sensors or medical devices that respond to highly specific cues found within a biological milieu.
Stick-slip dynamics of cell adhesion triggers spontaneous symmetry breaking and directional migration of mesenchymal cells on one-dimensional lines.
Directional cell motility relies on the ability of single cells to establish a front-rear polarity and can occur in the absence of external cues. The initiation of migration has often been attributed to the spontaneous polarization of cytoskeleton components, while the spatiotemporal evolution of cell-substrate interaction forces has yet to be resolved. Here, we establish a one-dimensional microfabricated migration assay that mimics the complex in vivo fibrillar environment while being compatible with high-resolution force measurements, quantitative microscopy, and optogenetics. Quantification of morphometric and mechanical parameters of NIH-3T3 fibroblasts and RPE1 epithelial cells reveals a generic stick-slip behavior initiated by contractility-dependent stochastic detachment of adhesive contacts at one side of the cell, which is sufficient to trigger cell motility in 1D in the absence of pre-established polarity. A theoretical model validates the crucial role of adhesion dynamics, proposing that front-rear polarity can emerge independently of a complex self-polarizing system.
Engineering a light-responsive, quorum quenching biofilm to mitigate biofouling on water purification membranes.
Quorum quenching (QQ) has been reported to be a promising approach for membrane biofouling control. Entrapment of QQ bacteria in porous matrices is required to retain them in continuously operated membrane processes and to prevent uncontrollable biofilm formation by the QQ bacteria on membrane surfaces. It would be more desirable if the formation and dispersal of biofilms by QQ bacteria could be controlled so that the QQ bacterial cells are self-immobilized, but the QQ biofilm itself still does not compromise membrane performance. In this study, we engineered a QQ bacterial biofilm whose growth and dispersal can be modulated by light through a dichromatic, optogenetic c-di-GMP gene circuit in which the bacterial cells sense near-infrared (NIR) light and blue light to adjust its biofilm formation by regulating the c-di-GMP level. We also demonstrated the potential application of the engineered light-responsive QQ biofilm in mitigating biofouling of water purification forward osmosis membranes. The c-di-GMP-targeted optogenetic approach for controllable biofilm development we have demonstrated here should prove widely applicable for designing other controllable biofilm-enabled applications such as biofilm-based biocatalysis.