Showing 1 - 11 of 11 results
T cells selectively filter oscillatory signals on the minutes timescale.
T cells experience complex temporal patterns of stimulus via receptor-ligand-binding interactions with surrounding cells. From these temporal patterns, T cells are able to pick out antigenic signals while establishing self-tolerance. Although features such as duration of antigen binding have been examined, our understanding of how T cells interpret signals with different frequencies or temporal stimulation patterns is relatively unexplored. We engineered T cells to respond to light as a stimulus by building an optogenetically controlled chimeric antigen receptor (optoCAR). We discovered that T cells respond to minute-scale oscillations of activation signal by stimulating optoCAR T cells with tunable pulse trains of light. Systematically scanning signal oscillation period from 1 to 150 min revealed that expression of CD69, a T cell activation marker, reached a local minimum at a period of ∼25 min (corresponding to 5 to 15 min pulse widths). A combination of inhibitors and genetic knockouts suggest that this frequency filtering mechanism lies downstream of the Erk signaling branch of the T cell response network and may involve a negative feedback loop that diminishes Erk activity. The timescale of CD69 filtering corresponds with the duration of T cell encounters with self-peptide-presenting APCs observed via intravital imaging in mice, indicating a potential functional role for temporal filtering in vivo. This study illustrates that the T cell signaling machinery is tuned to temporally filter and interpret time-variant input signals in discriminatory ways.
Single-component optogenetic tools for inducible RhoA GTPase signaling.
We created optogenetic tools to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activating GEF effectors, were fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these effectors induced potent contractile signaling sufficient to separate adherens junctions in response to as little as one pulse of blue light. Cytoskeletal morphology changes were dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA-mediated cytoskeletal activation induced YAP nuclear localization within minutes and subsequent mechanotransduction, verified by YAP-TEAD transcriptional activity. These single-component tools, which do not require protein binding partners, offer spatiotemporally precise control over RhoA signaling that will advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.
β-Catenin signaling dynamics regulate cell fate in differentiating neural stem cells.
Stem cells undergo differentiation in complex and dynamic environments wherein instructive signals fluctuate on various timescales. Thus, cells must be equipped to properly respond to the timing of signals, for example, to distinguish sustained signaling from transient noise. However, how stem cells respond to dynamic variations in differentiation cues is not well characterized. Here, we use optogenetic activation of β-catenin signaling to probe the dynamic responses of differentiating adult neural stem cells (NSCs). We discover that, while elevated, sustained β-catenin activation sequentially promotes proliferation and differentiation, transient β-catenin induces apoptosis. Genetic perturbations revealed that the neurogenic/apoptotic fate switch was mediated through cell-cycle regulation by Growth Arrest and DNA Damage 45 gamma (Gadd45γ). Our results thus reveal a role for β-catenin dynamics in NSC fate decisions and may suggest a role for signal timing to minimize cell-fate errors, analogous to kinetic proofreading of stem-cell differentiation.
Optogenetic Control Reveals Differential Promoter Interpretation of Transcription Factor Nuclear Translocation Dynamics.
Gene expression is thought to be affected not only by the concentration of transcription factors (TFs) but also the dynamics of their nuclear translocation. Testing this hypothesis requires direct control of TF dynamics. Here, we engineer CLASP, an optogenetic tool for rapid and tunable translocation of a TF of interest. Using CLASP fused to Crz1, we observe that, for the same integrated concentration of nuclear TF over time, changing input dynamics changes target gene expression: pulsatile inputs yield higher expression than continuous inputs, or vice versa, depending on the target gene. Computational modeling reveals that a dose-response saturating at low TF input can yield higher gene expression for pulsatile versus continuous input, and that multi-state promoter activation can yield the opposite behavior. Our integrated tool development and modeling approach characterize promoter responses to Crz1 nuclear translocation dynamics, extracting quantitative features that may help explain the differential expression of target genes.
Optogenetic Rac1 engineered from membrane lipid-binding RGS-LOV for inducible lamellipodia formation.
We report the construction of a single-component optogenetic Rac1 (opto-Rac1) to control actin polymerization by dynamic membrane recruitment. Opto-Rac1 is a fusion of wildtype human Rac1 small GTPase to the C-terminal region of BcLOV4, a LOV (light-oxygen-voltage) photoreceptor that rapidly binds the plasma membrane upon blue-light activation via a direct electrostatic interaction with anionic membrane phospholipids. Translocation of the fused wildtype Rac1 effector permits its activation by GEFs (guanine nucleotide exchange factors) and consequent actin polymerization and lamellipodia formation, unlike in existing single-chain systems that operate by allosteric photo-switching of constitutively active Rac1 or the heterodimerization-based (i.e. two-component) membrane recruitment of a Rac1-activating GEF. Opto-Rac1 induction of lamellipodia formation was spatially restricted to the patterned illumination field and was efficient, requiring sparse stimulation duty ratios of ∼1-2% (at the sensitivity threshold for flavin photocycling) to cause significant changes in cell morphology. This work exemplifies how the discovery of LOV proteins of distinct signal transmission modes can beget new classes of optogenetic tools for controlling cellular function.
High-throughput multicolor optogenetics in microwell plates.
Optogenetic probes can be powerful tools for dissecting complexity in cell biology, but there is a lack of instrumentation to exploit their potential for automated, high-information-content experiments. This protocol describes the construction and use of the optoPlate-96, a platform for high-throughput three-color optogenetics experiments that allows simultaneous manipulation of common red- and blue-light-sensitive optogenetic probes. The optoPlate-96 enables illumination of individual wells in 96-well microwell plates or in groups of wells in 384-well plates. Its design ensures that there will be no cross-illumination between microwells in 96-well plates, and an active cooling system minimizes sample heating during light-intensive experiments. This protocol details the steps to assemble, test, and use the optoPlate-96. The device can be fully assembled without specialized equipment beyond a 3D printer and a laser cutter, starting from open-source design files and commercially available components. We then describe how to perform a typical optogenetics experiment using the optoPlate-96 to stimulate adherent mammalian cells. Although optoPlate-96 experiments are compatible with any plate-based readout, we describe analysis using quantitative single-cell immunofluorescence. This workflow thus allows complex optogenetics experiments (independent control of stimulation colors, intensity, dynamics, and time points) with high-dimensional outputs at single-cell resolution. Starting from 3D-printed and laser-cut components, assembly and testing of the optoPlate-96 can be accomplished in 3-4 h, at a cost of ~$600. A full optoPlate-96 experiment with immunofluorescence analysis can be performed within ~24 h, but this estimate is variable depending on the cell type and experimental parameters.
Cancer mutations and targeted drugs can disrupt dynamic signal encoding by the Ras-Erk pathway.
The Ras-Erk (extracellular signal-regulated kinase) pathway encodes information in its dynamics; the duration and frequency of Erk activity can specify distinct cell fates. To enable dynamic encoding, temporal information must be accurately transmitted from the plasma membrane to the nucleus. We used optogenetic profiling to show that both oncogenic B-Raf mutations and B-Raf inhibitors can cause corruption of this transmission, so that short pulses of input Ras activity are distorted into abnormally long Erk outputs. These changes can reshape downstream transcription and cell fates, resulting in improper decisions to proliferate. These findings illustrate how altered dynamic signal transmission properties, and not just constitutively increased signaling, can contribute to cell proliferation and perhaps cancer, and how optogenetic profiling can dissect mechanisms of signaling dysfunction in disease.
Modular engineering of cellular signaling proteins and networks.
Living cells respond to their environment using networks of signaling molecules that act as sensors, information processors, and actuators. These signaling systems are highly modular at both the molecular and network scales, and much evidence suggests that evolution has harnessed this modularity to rewire and generate new physiological behaviors. Conversely, we are now finding that, following nature's example, signaling modules can be recombined to form synthetic tools for monitoring, interrogating, and controlling the behavior of cells. Here we highlight recent progress in the modular design of synthetic receptors, optogenetic switches, and phospho-regulated proteins and circuits, and discuss the expanding role of combinatorial design in the engineering of cellular signaling proteins and networks.
Regulation of endogenous transmembrane receptors through optogenetic Cry2 clustering.
Transmembrane receptors are the predominant conduit through which cells sense and transduce extracellular information into intracellular biochemical signals. Current methods to control and study receptor function, however, suffer from poor resolution in space and time and often employ receptor overexpression, which can introduce experimental artefacts. We report a genetically encoded approach, termed Clustering Indirectly using Cryptochrome 2 (CLICR), for spatiotemporal control over endogenous transmembrane receptor activation, enabled through the optical regulation of target receptor clustering and downstream signalling using noncovalent interactions with engineered Arabidopsis Cryptochrome 2 (Cry2). CLICR offers a modular platform to enable photocontrol of the clustering of diverse transmembrane receptors including fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR) and integrins in multiple cell types including neural stem cells. Furthermore, light-inducible manipulation of endogenous receptor tyrosine kinase (RTK) activity can modulate cell polarity and establish phototaxis in fibroblasts. The resulting spatiotemporal control over cellular signalling represents a powerful new optogenetic framework for investigating and controlling cell function and fate.
Light-inducible activation of target mRNA translation in mammalian cells.
A genetically encoded optogenetic system was constructed that activates mRNA translation in mammalian cells in response to light. Blue light induces the reconstitution of an RNA binding domain and a translation initiation domain, thereby activating target mRNA translation downstream of the binding sites.
Optogenetic protein clustering and signaling activation in mammalian cells.
We report an optogenetic method based on Arabidopsis thaliana cryptochrome 2 for rapid and reversible protein oligomerization in response to blue light. We demonstrated its utility by photoactivating the β-catenin pathway, achieving a transcriptional response higher than that obtained with the natural ligand Wnt3a. We also demonstrated the modularity of this approach by photoactivating RhoA with high spatiotemporal resolution, thereby suggesting a previously unknown mode of activation for this Rho GTPase.