Showing 276 - 300 of 1687 results
276.
Optogenetic control of GGGGCC repeat-containing RNA phase transition.
Abstract:
The GGGGCC (G4C2) hexanucleotide repeat expansion in the C9ORF72 gene is a major cause of both hereditary amyotrophic lateral sclerosis and familial frontotemporal dementia. Recent studies have shown that G4C2 hexanucleotide repeat-containing RNA transcripts ((G4C2)n RNA) could go through liquid-liquid phase separation to form RNA foci, which may elicit neurodegeneration. However, the direct causality between these abnormal RNA foci and neuronal toxicity remains to be demonstrated. Here we introduce an optogenetic control system that can induce the assembly and phase separation of (G4C2)n RNA foci with blue light illumination in human cells, by fusing a specific (G4C2)n RNA binding protein as the linker domain to Cry2, a protein that oligomerizes in response to blue light. Our results demonstrate that a higher number of G4C2 repeats have the potential to be induced into more RNA foci in the cells. Both spontaneous and induced RNA foci display liquid-like properties according to FRAP measurements. Computational simulation shows strong consistency with the experimental results and supports the effect of our system to promote the propensity of (G4C2)n RNA towards phase separation. This system can thus be used to investigate whether (G4C2)n RNA foci would disrupt normal cellular processes and lead to pathological phenotypes relevant to repeat expansion disorders.
277.
Spatiotemporal control of ERK pulse frequency coordinates fate decisions during mammary acinar morphogenesis.
Abstract:
The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the average frequency of non-periodic ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility and proliferation. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated multicellular ERK waves emerge, correlating with high and low ERK pulse frequencies in outer surviving and inner dying cells, respectively. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Acini harboring the PIK3CA H1047R mutation display increased ERK pulse frequency and inner cell survival. Thus, fate decisions during acinar morphogenesis are coordinated by different spatiotemporal modalities of ERK pulse frequency.
278.
Nano-optogenetic immunotherapy.
Abstract:
Chimeric antigen receptor (CAR) T cell-based immunotherapy has been increasingly used in the clinic for cancer intervention over the past 5 years. CAR T-cell therapy takes advantage of genetically-modified T cells to express synthetic CAR molecules on the cell surface. To date, up to six CAR T cell therapy products have been approved by the Food and Drug Administration for the treatment of leukaemia, lymphoma, and multiple myeloma. In addition, hundreds of CAR-T products are currently under clinical trials to treat solid tumours. In both the fundamental research and clinical applications, CAR T cell immunotherapy has achieved exciting progress with remarkable remission or suppression of cancers. However, CAR T cell-based immunotherapy still faces significant safety issues, as exemplified by "on-target off-tumour" cytotoxicity due to lack of strict antigen specificity. In addition, uncontrolled massive activation of infused CAR T cells may create severe systemic inflammation with cytokine release syndrome and neurotoxicity. These challenges call for a need to combine nanotechnology and optogenetics with immunoengineering to develop spatiotemporally-controllable CAR T cells, which enable wireless photo-tunable activation of therapeutic immune cells to deliver personalised therapy in the tumour microenvironment.
279.
Optogenetic Control of Bacterial Expression by Red Light.
-
Multamäki, E
-
García de Fuentes, A
-
Sieryi, O
-
Bykov, A
-
Gerken, U
-
Ranzani, AT
-
Köhler, J
-
Meglinski, I
-
Möglich, A
-
Takala, H
Abstract:
In optogenetics, as in nature, sensory photoreceptors serve to control cellular processes by light. Bacteriophytochrome (BphP) photoreceptors sense red and far-red light via a biliverdin chromophore and, in response, cycle between the spectroscopically, structurally, and functionally distinct Pr and Pfr states. BphPs commonly belong to two-component systems that control the phosphorylation of cognate response regulators and downstream gene expression through histidine kinase modules. We recently demonstrated that the paradigm BphP from Deinococcus radiodurans exclusively acts as a phosphatase but that its photosensory module can control the histidine kinase activity of homologous receptors. Here, we apply this insight to reprogram two widely used setups for bacterial gene expression from blue-light to red-light control. The resultant pREDusk and pREDawn systems allow gene expression to be regulated down and up, respectively, uniformly under red light by 100-fold or more. Both setups are realized as portable, single plasmids that encode all necessary components including the biliverdin-producing machinery. The triggering by red light affords high spatial resolution down to the single-cell level. As pREDusk and pREDawn respond sensitively to red light, they support multiplexing with optogenetic systems sensitive to other light colors. Owing to the superior tissue penetration of red light, the pREDawn system can be triggered at therapeutically safe light intensities through material layers, replicating the optical properties of the skin and skull. Given these advantages, pREDusk and pREDawn enable red-light-regulated expression for diverse use cases in bacteria.
280.
Shedding light on current trends in molecular optogenetics.
Abstract:
Molecular optogenetics is a highly dynamic research field. In the past two years, the field was characterized by the development of new allosteric switches as well as the forward integration of optogenetics research towards application. Further, two areas of research have significantly gathered momentum, the use of optogenetics to control liquid-liquid phase separation as well as the application of optogenetic tools in the extracellular space. Here, we review these areas and discuss future directions.
281.
CRY-BARs: Versatile light-gated molecular tools for the remodeling of membrane architectures.
Abstract:
BAR (Bin, Amphiphysin and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR (inverse-BAR) domain containing tools derived from the fusion of the A. thaliana Cryptochrome 2 photoreceptor and I-BAR protein domains ('CRY-BARs') with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate here that the CRY-BAR optogenetic tool evokes membrane dynamics changes associated with cellular activity. Moreover, we provide evidence that ezrin, an actin and PIP2 binding protein, acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, we propose that CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.
282.
Dynamic cybergenetic control of bacterial co-culture composition via optogenetic feedback.
Abstract:
Communities of microbes play important roles in natural environments and hold great potential for deploying division-of-labor strategies in synthetic biology and bioproduction. However, the difficulty of controlling the composition of microbial consortia over time hinders their optimal use in many applications. Here, we present a fully automated, high-throughput platform that combines real-time measurements and computer-controlled optogenetic modulation of bacterial growth to implement precise and robust compositional control of a two-strain E. coli community. In addition, we develop a general framework for dynamic modeling of synthetic genetic circuits in the physiological context of E. coli and use a host-aware model to determine the optimal control parameters of our closed-loop compositional control system. Our platform succeeds in stabilizing the strain ratio of multiple parallel co-cultures at arbitrary levels and in changing these targets over time, opening the door for the implementation of dynamic compositional programs in synthetic bacterial communities.
283.
Illuminating bacterial behaviors with optogenetics.
Abstract:
Optogenetic approaches enable light-mediated control of cellular activities using genetically encoded photoreceptors. While optogenetic technology is already well established in neuroscience and fundamental research, the implementation of optogenetic tools in bacteriology is still emerging. Engineered bacteria with the specific optogenetic system that function at the transcriptional or post-translational level can sense and respond to light, allowing optogenetic control of bacterial behaviors. In this review, we give a brief overview of available optogenetic systems, including their mode of action, classification, and engineering strategies, and focus on optogenetic control of bacterial behaviors with the highlight of strategies for use of optogenetic systems.
284.
Optogenetic control of RelA reveals effect of transcription factor dynamics on downstream gene expression.
Abstract:
Many transcription factors (TFs) translocate to the nucleus with varied dynamic patterns in response to different inputs. A notable example of such behavior is RelA, a subunit of NF-κB, which translocates to the nucleus with either pulsed or sustained dynamics, depending on the stimulus. Our understanding of how these dynamics are interpreted by downstream genes has remained incomplete, partly because ubiquitously used environmental inputs activate other transcriptional regulators in addition to RelA. Here, we use an optogenetic tool, CLASP (controllable light-activated shuttling and plasma membrane sequestration), to control RelA spatiotemporal dynamics in mouse fibroblasts and quantify their effect on downstream genes using RNA-seq. Using RelA-CLASP, we show for the first time that nuclear translocation of RelA, without post-translational modifications or activation of other transcriptional regulators, is sufficient to activate downstream genes. Furthermore, we find that TNFα, a common endogenous input, regulates many genes independently of RelA, and that this gene regulation is different from that induced by RelA-CLASP. Genes responsive to RelA-CLASP show a wide range of dynamics in response to a constant RelA input. We use a simple promoter model to recapitulate these diverse dynamic responses, as well as data collected in response to a pulsed RelA-CLASP input, and extract features of many RelA-responsive promoters. We also pinpoint many genes for which more complex models, involving feedback or multi-step promoters, may be needed to explain their response to constant and pulsed TF inputs. This study introduces a new robust tool for studying mammalian transcriptional regulation and demonstrates the power of optogenetic tools in dissecting the quantitative features of important cellular pathways.
285.
Implementation of a Novel Optogenetic Tool in Mammalian Cells Based on a Split T7 RNA Polymerase.
Abstract:
Optogenetic tools are widely used to control gene expression dynamics both in prokaryotic and eukaryotic cells. These tools are used in a variety of biological applications from stem cell differentiation to metabolic engineering. Despite some tools already available in bacteria, no light-inducible system currently exists to control gene expression independently from mammalian transcriptional and/or translational machineries thus working orthogonally to endogenous regulatory mechanisms. Such a tool would be particularly important in synthetic biology, where orthogonality is advantageous to achieve robust activation of synthetic networks. Here we implement, characterize, and optimize a new optogenetic tool in mammalian cells based on a previously published system in bacteria called Opto-T7RNAPs. The tool is orthogonal to the cellular machinery for transcription and consists of a split T7 RNA polymerase coupled with the blue light-inducible magnets system (mammalian OptoT7-mOptoT7). In our study we exploited the T7 polymerase's viral origins to tune our system's expression level, reaching up to an almost 20-fold change activation over the dark control. mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition, and Pepper aptamer for RNA visualization. Moreover, we show that mOptoT7 can mitigate the gene expression burden when compared to another optogenetic construct. These properties make mOptoT7 a powerful new tool to use when orthogonality and viral RNA species (that lack endogenous RNA modifications) are desired.
286.
A genetically encoded photo-proximity labeling approach for mapping protein territories.
Abstract:
Studying dynamic biological processes requires approaches compatible with the lifetimes of the biochemical transactions under investigation, which can be very short. We describe a genetically encoded system that allows protein interactomes to be captured using visible light. Our approach involves fusing an engineered flavoprotein to a protein of interest. Brief excitation of the fusion protein leads to local generation of reactive radical species within cell-permeable probes. When combined with quantitative proteomics, the system generates ‘snapshots’ of protein interactions with high temporal resolution. The intrinsic fluorescence of the fusion domain permits correlated imaging and proteomics analyses, a capability that is exploited in several contexts, including defining the protein clients of the major vault protein (MVP). The technology should be broadly useful in the biomedical area.
287.
LITOS: a versatile LED illumination tool for optogenetic stimulation.
Abstract:
Optogenetics has become a key tool to manipulate biological processes with high spatio-temporal resolution. Recently, a number of commercial and open-source multi-well illumination devices have been developed to provide throughput in optogenetics experiments. However, available commercial devices remain expensive and lack flexibility, while open-source solutions require programming knowledge and/or include complex assembly processes. We present a LED Illumination Tool for Optogenetic Stimulation (LITOS) based on an assembled printed circuit board controlling a commercially available 32 × 64 LED matrix as illumination source. LITOS can be quickly assembled without any soldering, and includes an easy-to-use interface, accessible via a website hosted on the device itself. Complex light stimulation patterns can easily be programmed without coding expertise. LITOS can be used with different formats of multi-well plates, petri dishes, and flasks. We validated LITOS by measuring the activity of the MAPK/ERK signaling pathway in response to different dynamic light stimulation regimes using FGFR1 and Raf optogenetic actuators. LITOS can uniformly stimulate all the cells in a well and allows for flexible temporal stimulation schemes. LITOS's affordability and ease of use aims at democratizing optogenetics in any laboratory.
288.
Defunctionalizing intracellular organelles such as mitochondria and peroxisomes with engineered phospholipase A/acyltransferases.
Abstract:
Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we design a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid defunctionalization of organelles via remodeling of the membrane phospholipids. In particular, we identify catalytically active PLAAT truncates with minimal unfavorable characteristics. Chemically-induced translocation of the optimized PLAAT to the mitochondria surface results in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapt the molecular tool in peroxisomes, and observe leakage of matrix-resident functional proteins. The technique is compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should prove useful in studying organelle biology of diverse contexts.
289.
Optogenetic control of YAP cellular localisation and function.
Abstract:
YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attach a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression and cell proliferation. Similarly, optofYap can be used in zebrafish embryos to modulate target genes. We demonstrate that optoYAP can override a cell's response to substrate stiffness to generate anchorage-independent growth. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.
290.
Light-activated mitochondrial fission through optogenetic control of mitochondria-lysosome contacts.
-
Qiu, K
-
Zou, W
-
Fang, H
-
Hao, M
-
Mehta, K
-
Tian, Z
-
Guan, JL
-
Zhang, K
-
Huang, T
-
Diao, J
Abstract:
Mitochondria are highly dynamic organelles whose fragmentation by fission is critical to their functional integrity and cellular homeostasis. Here, we develop a method via optogenetic control of mitochondria-lysosome contacts (MLCs) to induce mitochondrial fission with spatiotemporal accuracy. MLCs can be achieved by blue-light-induced association of mitochondria and lysosomes through various photoactivatable dimerizers. Real-time optogenetic induction of mitochondrial fission is tracked in living cells to measure the fission rate. The optogenetic method partially restores the mitochondrial functions of SLC25A46-/- cells, which display defects in mitochondrial fission and hyperfused mitochondria. The optogenetic MLCs system thus provides a platform for studying mitochondrial fission and treating mitochondrial diseases.
291.
Optogenetics for light control of biological systems
-
Emiliani, V
-
Entcheva, E
-
Hedrich, R
-
Hegemann, P
-
Konrad, K R
-
Lüscher, C
-
Mahn, M
-
Pan, Z
-
Sims, R R
-
Vierock, J
-
Yizhar, O
Abstract:
The H2 + H2 system has long been considered a benchmark system for ro-vibrational energy transfer in bimolecular collisions. However, most studies thus far have focused on collisions involving H2 molecules in the ground vibrational level or in the first excited vibrational state. While H2 + H2/HD collisions have received wide attention due to the important role they play in astrophysics, D2 + D2 collisions have received much less attention. Recently, Zhou et al. [ Nat. Chem. 2022, 14, 658-663, DOI: 10.1038/s41557-022-00926-z] examined stereodynamic aspects of rotational energy transfer in collisions of two aligned D2 molecules prepared in the v = 2 vibrational level and j = 2 rotational level. Here, we report quantum calculations of rotational and vibrational energy transfer in collisions of two D2 molecules prepared in vibrational levels up to v = 2 and identify key resonance features that contribute to the angular distribution in the experimental results of Zhou et al. The quantum scattering calculations were performed in full dimensionality and using the rigid-rotor approximation using a recently developed highly accurate six-dimensional potential energy surface for the H4 system that allows descriptions of collisions involving highly vibrationally excited H2 and its isotopologues.
292.
Emerging molecular technologies for light-mediated modulation of pancreatic beta-cell function.
Abstract:
Optogenetic modalities as well as optochemical and photopharmacological strategies, collectively termed optical methods, have revolutionized the control of cellular functions via light with great spatiotemporal precision. In comparison to the major advances in the photomodulation of signaling activities noted in neuroscience, similar applications to endocrine cells of the pancreas, particularly insulin-producing β-cells, have been limited. The availability of tools allowing light-mediated changes in the trafficking of ions such as K+ and Ca2+ and signaling intermediates such as cyclic adenosine monophosphate (cAMP), renders β-cells and their glucose-stimulated insulin secretion (GSIS) amenable to optoengineering for drug-free control of blood sugar.
293.
Programming the lifestyles of engineered bacteria for cancer therapy.
Abstract:
Bacteria can be genetically engineered to act as therapeutic delivery vehicles in the treatment of tumors, killing cancer cells or activating the immune system. This is known as Bacteria-Mediated Cancer Therapy (BMCT). Tumor invasion, colonization and tumor regression are major biological events, which are directly associated with antitumor effects and are uncontrollable due to the influence of tumor microenvironments during the BMCT process. Here, we developed a genetic circuit for dynamically programming bacterial lifestyles (planktonic, biofilm or lysis), to precisely manipulate the process of bacterial adhesion, colonization and drug release in BMCT process, via hierarchical modulation of the lighting power density (LPD) of near-infrared (NIR) light. The deep tissue penetration of NIR offers us a modality for spatiotemporal and noninvasive control of bacterial genetic circuits in vivo. By combining computational modeling with high throughput characterization device, we optimized the genetic circuits in engineered bacteria to program the process of bacterial lifestyle transitions by altering the illumination scheme of NIR. Our results showed that programming intratumoral bacterial lifestyle transitions allows precise control of multiple key steps throughout the BMCT process, and therapeutic efficacy can be greatly improved by controlling the localization and dosage of therapeutic agents via optimizing the illumination scheme.
294.
Recent advances in cellular optogenetics for photomedicine.
Abstract:
Since the successful introduction of exogenous photosensitive proteins, channelrhodopsin, to neurons, optogenetics has enabled substantial understanding of profound brain function by selectively manipulating neural circuits. In an optogenetic system, optical stimulation can be precisely delivered to brain tissue to achieve regulation of cellular electrical activity with unprecedented spatio-temporal resolution in living organisms. In recent years, the development of various optical actuators and novel light-delivery techniques has greatly expanded the scope of optogenetics, enabling the control of other signal pathways in non-neuronal cells for different biomedical applications, such as phototherapy and immunotherapy. This review focuses on the recent advances in optogenetic regulation of cellular activities for photomedicine. We discuss emerging optogenetic tools and light-delivery platforms, along with a survey of optogenetic execution in mammalian and microbial cells.
295.
Visual detection of submicroscopic protein clusters with a phase-separation-based fluorescent reporter.
Abstract:
Protein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often small and fall below the detection limits of conventional fluorescence microscopy. Existing techniques to visualize such aggregates require specialized microscopy and may require overexpression of the protein of interest, which can introduce clustering artifacts that are not representative of the endogenous protein. Here we describe a fluorescent reporter strategy that detects endogenous protein clustering with high sensitivity, called CluMPS (Clusters Magnified by Phase Separation). A CluMPS reporter detects and visually amplifies even small clusters of a binding partner, generating large, easily quantifiable phase-separated condensates as a readout. We use optogenetic clustering to show that the CluMPS approach can reliably report on target clusters as small as tetramers. Experiments and simulations showed that CluMPS activation depends on the affinity for the target, the target cluster size, and the cluster size distribution. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. Uniquely, CluMPS permitted visualization of clusters of endogenous proteins, allowing the measurement of drug response kinetics of oncogenic condensates in patient-derived cancer cells. Finally, CluMPS could be multiplexed to report on distinct clustered species in the same cell. CluMPS thus provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context.
296.
GPCR-dependent spatiotemporal cAMP generation confers functional specificity in cardiomyocytes and cardiac responses.
Abstract:
Cells interpret a variety of signals through G protein-coupled receptors (GPCRs), and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different responses, despite generating similar levels of cAMP. We previously showed that GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here, we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to cue different outputs. We show that generating cAMP from the Golgi by an optogenetic approach or activated GPCR leads to regulation of a specific PKA target that increases rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We validated the physiological consequences of these observations in intact zebrafish and mice. Thus, the same GPCR regulates distinct molecular and physiological pathways depending on its subcellular location despite generating cAMP in each case.
297.
Automatic detection of spatio-temporal signalling patterns in cell collectives.
Abstract:
An increasing experimental evidence points to physiological importance of space-time correlations in signalling of cell collectives. From wound healing to epithelial homeostasis to morphogenesis, coordinated activation of bio-molecules between cells allows the collectives to perform more complex tasks and better tackle environmental challenges. To understand this information exchange and to advance new theories of emergent phenomena, we created ARCOS, a computational method to detect and quantify collective signalling. We demonstrate ARCOS on cell and organism collectives with space-time correlations on different scales in 2D and 3D. We make a new observation that oncogenic mutations in the MAPK/ERK and PIK3CA/Akt pathways of MCF10A epithelial cells induce ERK activity waves with different size, duration, and frequency. The open-source implementations of ARCOS are available as R and Python packages, and as a plugin for napari image viewer to interactively quantify collective phenomena without prior programming experience.
298.
Dimerization of iLID Optogenetic Proteins Observed Using 3D Single-Molecule Tracking in Live Bacterial Cells.
Abstract:
3D single molecule tracking microscopy has enabled measurements of protein diffusion in living cells, offering information about protein dynamics and the cellular environment. For example, different diffusive states can be resolved and assigned to protein complexes of different size and composition. However, substantial statistical power and biological validation, often through genetic deletion of binding partners, are required to support diffusive state assignments. When investigating some cellular processes, transient perturbation to protein spatial distributions is preferable to permanent genetic deletion of an essential protein. Optogenetic dimerization systems can be used to manipulate protein spatial distributions which could offer a means to deplete specific diffusive states observed in single molecule tracking experiments. Here, we evaluate the performance of the iLID optogenetic system in living E. coli cells using diffraction-limited microscopy and 3D single molecule tracking. We observed a robust optogenetic response in protein spatial distribution after 488 nm laser activation. Surprisingly, 3D single molecule tracking results indicate activation of the optogenetic response at high intensity wavelengths for which there is evidence of minimal photon absorbance by the LOV2 domain. However, the preactivation response was minimized through the use of iLID system mutants, and titration of protein expression levels.
299.
Engineering of optogenetic devices for biomedical applications in mammalian synthetic biology.
Abstract:
Gene- and cell-based therapies are the next frontiers in the field of medicine. Both are transformative and innovative therapies; however, a lack of safety data limits the translation of such promising technologies to the clinic. Improving the safety and promoting the clinical translation of these therapies can be achieved by tightly regulating the release and delivery of therapeutic outputs. In recent years, the rapid development of optogenetic technology has provided opportunities to develop precision-controlled gene- and cell-based therapies, in which light is introduced to precisely and spatiotemporally manipulate the behaviour of genes and cells. This review focuses on the development of optogenetic tools and their applications in biomedicine, including photoactivated genome engineering and phototherapy for diabetes and tumours. The prospects and challenges of optogenetic tools for future clinical applications are also discussed.
300.
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.