Showing 1 - 19 of 19 results
Optogenetic control of integrin-matrix interaction.
Optogenetic approaches have gathered momentum in precisely modulating and interrogating cellular signalling and gene expression. The use of optogenetics on the outer cell surface to interrogate how cells receive stimuli from their environment, however, has so far not reached its full potential. Here we demonstrate the development of an optogenetically regulated membrane receptor-ligand pair exemplified by the optically responsive interaction of an integrin receptor with the extracellular matrix. The system is based on an integrin engineered with a phytochrome-interacting factor domain (OptoIntegrin) and a red light-switchable phytochrome B-functionalized matrix (OptoMatrix). This optogenetic receptor-ligand pair enables light-inducible and -reversible cell-matrix interaction, as well as the controlled activation of downstream mechanosensory signalling pathways. Pioneering the application of optogenetic switches in the extracellular environment of cells, this OptoMatrix–OptoIntegrin system may serve as a blueprint for rendering matrix–receptor interactions amendable to precise control with light.
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.
High-resolution Patterned Biofilm Deposition Using pDawn-Ag43.
Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms into arbitrary spatial patterns at high spatial resolution. The technique uses a genetically encoded optogenetic construct-pDawn-Ag43-that couples biofilm formation in E. coli to optical stimulation by blue light. We detail the process for transforming E. coli with pDawn-Ag43, preparing the required optical set-up, and the protocol for culturing patterned biofilms using pDawn-Ag43 bacteria. Using this protocol, biofilms with a spatial resolution below 25 μm can be patterned on various surfaces and environments, including enclosed chambers, without requiring microfabrication, clean-room facilities, or surface pretreatment. The technique is convenient and appropriate for use in applications that investigate the effect of biofilm structure, providing tunable control over biofilm patterning. More broadly, it also has potential applications in biomaterials, education, and bio-art.
Cyclic Stiffness Modulation of Cell‐Laden Protein–Polymer Hydrogels in Response to User‐Specified Stimuli Including Light.
Although mechanical signals presented by the extracellular matrix are known to regulate many essential cell functions, the specific effects of these interactions, particularly in response to dynamic and heterogeneous cues, remain largely unknown. Here, a modular semisynthetic approach is introduced to create protein–polymer hydrogel biomaterials that undergo reversible stiffening in response to user‐specified inputs. Employing a novel dual‐chemoenzymatic modification strategy, fusion protein‐based gel crosslinkers are created that exhibit stimuli‐dependent intramolecular association. Linkers based on calmodulin yield calcium‐sensitive materials, while those containing the photosensitive light, oxygen, and voltage sensing domain 2 (LOV2) protein give phototunable constructs whose moduli can be cycled on demand with spatiotemporal control about living cells. These unique materials are exploited to demonstrate the significant role that cyclic mechanical loading plays on fibroblast‐to‐myofibroblast transdifferentiation in 3D space. The moduli‐switchable materials should prove useful for studies in mechanobiology, providing new avenues to probe and direct matrix‐driven changes in 4D cell physiology.
Reversible hydrogels with tunable mechanical properties for optically controlling cell migration.
Synthetic hydrogels are widely used as biomimetic in vitro model systems to understand how cells respond to complex microenvironments. The mechanical properties of hydrogels are deterministic for many cellular behaviors, including cell migration, spreading, and differentiation. However, it remains a major challenge to engineer hydrogels that recapture the dynamic mechanical properties of native extracellular matrices. Here, we provide a new hydrogel platform with spatiotemporally tunable mechanical properties to assay and define cellular behaviors under light. The change in the mechanical properties of the hydrogel is effected by a photo-induced switch of the cross-linker fluorescent protein, Dronpa145N, between the tetrameric and monomeric states, which causes minimal changes to the chemical properties of the hydrogel. The mechanical properties can be rapidly and reversibly tuned for multiple cycles using visible light, as confirmed by rheological measurements and atomic force microscopybased nano-indentation. We further demonstrated real-time and reversible modulation of cell migration behaviors on the hydrogels through photo-induced stiffness switching, with minimal invasion to the cultured cells. Hydrogels with a programmable mechanical history and a spatially defined mechanical hierarchy might serve as an ideal model system to better understand complex cellular functions.
Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast.
Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus Neurospora crassa When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene FLO1, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor TUP1 provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV's ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.IMPORTANCE Optogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Here, we report a novel optogenetic switch (FUN-LOV) based on the LOV domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes, heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.
Independent Control over Multiple Cell Types in Space and Time Using Orthogonal Blue and Red Light Switchable Cell Interactions.
Independent control over multiple cell–material interactions with high spatiotemporal resolution is a key for many biomedical applications and understanding cell biology, as different cell types can perform different tasks in a multicellular context. In this study, the binding of two different cell types to materials is orthogonally controlled with blue and red light providing independent regulation in space and time. Cells expressing the photoswitchable protein cryptochrome 2 (CRY2) on cell surface bind to N‐truncated CRY‐interacting basic helix–loop–helix protein 1 (CIBN)‐immobilized substrates under blue light and cells expressing the photoswitchable protein phytochrome B (PhyB ) on cell surface bind to phytochrome interaction factor 6 (PIF6)‐immobilized substrates under red light, respectively. These light‐switchable cell interactions provide orthogonal and noninvasive control using two wavelengths of visible light. Moreover, both cell–material interactions are dynamically switched on under light and reversible in the dark. The specificity of the CRY2/CIBN and PhyB/PIF6 interactions and their response to different wavelengths of light allow selectively activating the binding of one cell type with blue and the other cell type with red light in the presence of the other cell type.
Bioprinting Living Biofilms through Optogenetic Manipulation.
In this paper, we present a new strategy for microprinting dense bacterial communities with a prescribed organization on a substrate. Unlike conventional bioprinting techniques that require bioinks, through optogenetic manipulation, we directly manipulated the behaviors of Pseudomonas aeruginosa to allow these living bacteria to autonomically form patterned biofilms following prescribed illumination. The results showed that through optogenetic manipulation, patterned bacterial communities with high spatial resolution (approximately 10 μm) could be constructed in 6 h. Thus, optogenetic manipulation greatly increases the range of available bioprinting techniques.
A Rac1-FMNL2 signaling module affects cell-cell contact formation independent of Cdc42 and membrane protrusions.
De novo formation of epithelial cell-cell contacts relies on actin-based protrusions as well as tightly controlled turnover of junctional actin once cells encounter each other and adhesion complexes assemble. The specific contributions of individual actin regulators on either protrusion formation or junctional actin turnover remain largely unexplored. Based on our previous findings of Formin-like 2 (FMNL2)-mediated control of junctional actin dynamics, we investigated its potential role in membrane protrusions and impact on newly forming epithelial contacts. CRISPR/Cas9-mediated loss of FMNL2 in human MCF10A cells combined with optogenetic control of Rac1 activity confirmed its critical function in the establishment of intercellular contacts. While lamellipodial protrusion rates remained unaffected, FMNL2 knockout cells were characterized by impaired filopodia formation similar to depletion of the Rho GTPase Cdc42. Silencing of Cdc42, however, failed to affect FMNL2-mediated contact formation. Hence, we propose a cell-cell contact-specific and Rac1-mediated function of FMNL2 entirely independent of Cdc42. Consistent with this, direct visualizations of native epithelial junction formation revealed a striking and specifically Rac1- and not Cdc42-dependent recruitment of FMNL2 to newly forming junctions as well as established cell-cell contacts within epithelial sheets.
Biofilm Lithography enables high-resolution cell patterning via optogenetic adhesin expression.
Bacterial biofilms represent a promising opportunity for engineering of microbial communities. However, our ability to control spatial structure in biofilms remains limited. Here we engineerEscherichia coliwith a light-activated transcriptional promoter (pDawn) to optically regulate expression of an adhesin gene (Ag43). When illuminated with patterned blue light, long-term viable biofilms with spatial resolution down to 25 μm can be formed on a variety of substrates and inside enclosed culture chambers without the need for surface pretreatment. A biophysical model suggests that the patterning mechanism involves stimulation of transiently surface-adsorbed cells, lending evidence to a previously proposed role of adhesin expression during natural biofilm maturation. Overall, this tool-termed "Biofilm Lithography"-has distinct advantages over existing cell-depositing/patterning methods and provides the ability to grow structured biofilms, with applications toward an improved understanding of natural biofilm communities, as well as the engineering of living biomaterials and bottom-up approaches to microbial consortia design.
Optogenetics reprogramming of planktonic cells for biofilm formation.
Single-cell behaviors play essential roles during early-stage biofilms formation. In this study, we evaluated whether biofilm formation could be guided by precisely manipulating single cells behaviors. Thus, we established an illumination method to precisely manipulate the type IV pili (TFP) mediated motility and microcolony formation of Pseudomonas aeruginosa by using a combination of a high-throughput bacterial tracking algorithm, optogenetic manipulation and adaptive microscopy. We termed this method as Adaptive Tracking Illumination (ATI). We reported that ATI enables the precise manipulation of TFP mediated motility and microcolony formation during biofilm formation by manipulating bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) levels in single cells. Moreover, we showed that the spatial organization of single cells in mature biofilms can be controlled using ATI. Thus, the established method (i.e., ATI) can markedly promote ongoing studies of biofilms.
Optogenetics Manipulation Enables Prevention of Biofilm Formation of Engineered Pseudomonas aeruginosa on Surfaces.
Synthetic biologists have attempted to solve real-world problems, such as those of bacterial biofilms, that are involved in the pathogenesis of many clinical infections and difficult to eliminate. To address this, we employed a blue light responding system and integrated it into the chromosomes of Pseudomonas aeruginosa. With making rational adaptions and improvements of the light-activated system, we provided a robust and convenient means to spatiotemporally control gene expression and manipulate biological processes with minimal perturbation in P. aeruginosa. It increased the light-induced gene expression up to 20-fold. Moreover, we deliberately introduced a functional protein gene PA2133 containing an EAL domain to degrade c-di-GMP into the modified system, and showed that the optimally engineered optogenetic tool inhibited the formation of P. aeruginosa biofilms through the induction of blue light, resulting in much sparser and thinner biofilms. Our approach establishes a methodology for leveraging the tools of synthetic biology to guide biofilm formation and engineer biofilm patterns with unprecedented spatiotemporal resolution. Furthermore, the findings suggest that the synthetic optogenetic system may provide a promising strategy that could be applied to control and fight biofilms.
Blue Light Switchable Bacterial Adhesion as a Key Step toward the Design of Biofilms.
The control of where and when bacteria adhere to a substrate is a key step toward controlling the formation and organization in biofilms. This study shows how we engineer bacteria to adhere specifically to substrates with high spatial and temporal control under blue light, but not in the dark, by using photoswitchable interaction between nMag and pMag proteins. For this, we express pMag proteins on the surface of E. coli so that the bacteria can adhere to substrates with immobilized nMag protein under blue light. These adhesions are reversible in the dark and can be repeatedly turned on and off. Further, the number of bacteria that can adhere to the substrate as well as the attachment and detachment dynamics are adjustable by using different point mutants of pMag and altering light intensity. Overall, the blue light switchable bacteria adhesions offer reversible, tunable and bioorthogonal control with exceptional spatial and temporal resolution. This enables us to pattern bacteria on substrates with great flexibility.
B12-dependent photoresponsive protein hydrogels for controlled stem cell/protein release.
Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive "smart" hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) proteins using genetically encoded SpyTag-SpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarHC hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.
Cell-matrix adhesion and cell-cell adhesion differentially control basal myosin oscillation and Drosophila egg chamber elongation.
Pulsatile actomyosin contractility, important in tissue morphogenesis, has been studied mainly in apical but less in basal domains. Basal myosin oscillation underlying egg chamber elongation is regulated by both cell-matrix and cell-cell adhesions. However, the mechanism by which these two adhesions govern basal myosin oscillation and tissue elongation is unknown. Here we demonstrate that cell-matrix adhesion positively regulates basal junctional Rho1 activity and medio-basal ROCK and myosin activities, thus strongly controlling tissue elongation. Differently, cell-cell adhesion governs basal myosin oscillation through controlling medio-basal distributions of both ROCK and myosin signals, which are related to the spatial limitations of cell-matrix adhesion and stress fibres. Contrary to cell-matrix adhesion, cell-cell adhesion weakly affects tissue elongation. In vivo optogenetic protein inhibition spatiotemporally confirms the different effects of these two adhesions on basal myosin oscillation. This study highlights the activity and distribution controls of basal myosin contractility mediated by cell-matrix and cell-cell adhesions, respectively, during tissue morphogenesis.
Manipulating leukocyte interactions in vivo through optogenetic chemokine release.
Light-mediated release of signaling ligands, such as chemoattractants, growth factors, and cytokines is an attractive strategy for investigation and therapeutic targeting of leukocyte communication and immune responses. We introduce a versatile optogenetic method to control ligand secretion, combining UV-conditioned endoplasmic reticulum-to-Golgi trafficking and a furin-processing step. As proof of principle, we achieved light-triggered chemokine secretion and demonstrated that a brief pulse of chemokine release can mediate a rapid flux of leukocyte contacts with target cells in vitro and in vivo. This approach opens new possibilities for dynamic investigation of leukocyte communication in vivo and may confer the potential to control the local release of soluble mediators in the context of immune cell therapies.
Development of a light-regulated cell-recovery system for non-photosynthetic bacteria.
Recent advances in the understanding of photosensing in biological systems have enabled the use of photoreceptors as novel genetic tools. Exploiting various photoreceptors that cyanobacteria possess, a green light-inducible gene expression system was previously developed for the regulation of gene expression in cyanobacteria. However, the applications of cyanobacterial photoreceptors are not limited to these bacteria but are also available for non-photosynthetic microorganisms by the coexpression of a cyanobacterial chromophore with a cyanobacteria-derived photosensing system. An Escherichia coli-derived self-aggregation system based on Antigen 43 (Ag43) has been shown to induce cell self-aggregation of various bacteria by exogenous introduction of the Ag43 gene.
Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1.
Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell-cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell-cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell-cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell-cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1.
Multi-chromatic control of mammalian gene expression and signaling.
The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical applications.