Vertebrate cells differentially interpret ciliary and extraciliary cAMP.

Hedgehog pathway components and select G protein-coupled receptors (GPCRs) localize to the primary cilium, an organelle specialized for signal transduction. We investigated whether cells distinguish between ciliary and extraciliary GPCR signaling. To test whether ciliary and extraciliary cyclic AMP (cAMP) convey different information, we engineered optogenetic and chemogenetic tools to control the subcellular site of cAMP generation. Generating equal amounts of ciliary and cytoplasmic cAMP in zebrafish and mammalian cells revealed that ciliary cAMP, but not cytoplasmic cAMP, inhibited Hedgehog signaling. Modeling suggested that the distinct geometries of the cilium and cell body differentially activate local effectors. The search for effectors identified a ciliary pool of protein kinase A (PKA). Blocking the function of ciliary PKA, but not extraciliary PKA, activated Hedgehog signal transduction and reversed the effects of ciliary cAMP. Therefore, cells distinguish ciliary and extraciliary cAMP using functionally and spatially distinct pools of PKA, and different subcellular pools of cAMP convey different information.

Optogenetic clustering and membrane translocation of the BcLOV4 photoreceptor.

Optogenetic tools respond to light through one of a small number of behaviors including allosteric changes, dimerization, clustering, or membrane translocation. Here, we describe a new class of optogenetic actuator that simultaneously clusters and translocates to the plasma membrane in response to blue light. We demonstrate that dual translocation and clustering of the BcLOV4 photoreceptor can be harnessed for novel single-component optogenetic tools, including for control of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are mechanistically linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~threefold-to-fourfold, and decreased the expression levels needed for strong signal activation. Thus light-induced clustering of BcLOV4 provides a strategy to generate a new class of optogenetic tools and to enhance existing ones.

Temperature-responsive optogenetic probes of cell signaling.

We describe single-component optogenetic probes whose activation dynamics depend on both light and temperature. We used the BcLOV4 photoreceptor to stimulate Ras and phosphatidyl inositol-3-kinase signaling in mammalian cells, allowing activation over a large dynamic range with low basal levels. Surprisingly, we found that BcLOV4 membrane translocation dynamics could be tuned by both light and temperature such that membrane localization spontaneously decayed at elevated temperatures despite constant illumination. Quantitative modeling predicted BcLOV4 activation dynamics across a range of light and temperature inputs and thus provides an experimental roadmap for BcLOV4-based probes. BcLOV4 drove strong and stable signal activation in both zebrafish and fly cells, and thermal inactivation provided a means to multiplex distinct blue-light sensitive tools in individual mammalian cells. BcLOV4 is thus a versatile photosensor with unique light and temperature sensitivity that enables straightforward generation of broadly applicable optogenetic tools.

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.

Enhancing Single-Cell Western Blotting Sensitivity Using Diffusive Analyte Blotting and Antibody Conjugate Amplification.

While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein content often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity Western blots on single cells (scWesterns) are attractive because they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by the affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address the sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in the limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates, which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 1000 molecules, a 120-fold improvement. This enables us to detect 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies compared to 84.5% of cells using in-gel detection. These results suggest the compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagents─not previously accessible for in-gel use─for further signal amplification and detection of low-abundance targets.

ß-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 ß-catenin signaling to probe the dynamic responses of differentiating adult neural stem cells (NSCs). We discover that, while elevated, sustained ß-catenin activation sequentially promotes proliferation and differentiation, transient ß-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 ß-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.

Cytosolic Delivery of Small Protein Scaffolds Enables Efficient Inhibition of Ras and Myc.

The ability to deliver small protein scaffolds intracellularly could enable the targeting and inhibition of many therapeutic targets that are not currently amenable to inhibition with small-molecule drugs. Here, we report the engineering of small protein scaffolds with anionic polypeptides (ApPs) to promote electrostatic interactions with positively charged nonviral lipid-based delivery systems. Proteins fused with ApPs are either complexed with off-the-shelf cationic lipids or encapsulated within ionizable lipid nanoparticles for highly efficient cytosolic delivery (up to 90%). The delivery of protein inhibitors is used to inhibit two common proto-oncogenes, Ras and Myc, in two cancer cell lines. This report demonstrates the feasibility of combining minimally engineered small protein scaffolds with tractable nanocarriers to inhibit intracellular proteins that are generally considered "undruggable" with current small molecule drugs and biologics.

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.

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 ß-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.

Age-Associated Increase in BMP Signaling Inhibits Hippocampal Neurogenesis.

Hippocampal neurogenesis, the product of resident neural stem cell proliferation and differentiation, persists into adulthood but decreases with organismal aging, which may contribute to the age-related decline in cognitive function. The mechanisms that underlie this decrease in neurogenesis are not well understood, although evidence in general indicates that extrinsic changes in an aged stem cell niche can contribute to functional decline in old stem cells. Bone morphogenetic protein (BMP) family members are intercellular signaling proteins that regulate stem and progenitor cell quiescence, proliferation, and differentiation in various tissues and are likewise critical regulators of neurogenesis in young adults. Here, we establish that BMP signaling increases significantly in old murine hippocampi and inhibits neural progenitor cell proliferation. Furthermore, direct in vivo attenuation of BMP signaling via genetic and transgenic perturbations in aged mice led to elevated neural stem cell proliferation, and subsequent neurogenesis, in old hippocampi. Such advances in our understanding of mechanisms underlying decreased hippocampal neurogenesis with age may offer targets for the treatment of age-related cognitive decline.

Rapid Optogenetic Clustering in the Cytoplasm with BcLOVclust.

Protein clustering is a powerful form of optogenetic control, yet remarkably few proteins are known to oligomerize with light. Recently, the photoreceptor BcLOV4 was found to form protein clusters in mammalian cells in response to blue light, although clustering coincided with its translocation to the plasma membrane, potentially constraining its application as an optogenetic clustering module. Herein we identify key amino acids that couple BcLOV4 clustering to membrane binding, allowing us to engineer a variant that clusters in the cytoplasm and does not associate with the membrane in response to blue light. This variant-called BcLOVclust-clustered over many cycles with substantially faster clustering and de-clustering kinetics compared to the widely used optogenetic clustering protein Cry2. The magnitude of clustering could be strengthened by appending an intrinsically disordered region from the fused in sarcoma (FUS) protein, or by selecting the appropriate fluorescent protein to which it was fused. Like wt BcLOV4, BcLOVclust activity was sensitive to temperature: light-induced clusters spontaneously dissolved at a rate that increased with temperature despite constant illumination. At low temperatures, BcLOVclust and Cry2 could be multiplexed in the same cells, allowing light control of independent protein condensates. BcLOVclust could also be applied to control signaling proteins and stress granules in mammalian cells. While its usage is currently best suited in cells and organisms that can be cultured below ∼30 °C, a deeper understanding of BcLOVclust thermal response will further enable its use at physiological mammalian temperatures.

Multiplexed dynamic control of temperature to probe and observe mammalian cells.

Temperature is a critical parameter for biological function, yet there is a lack of approaches to modulate the temperature of biological specimens in a dynamic and high-throughput manner. We present the thermoPlate, a device for programmable control of temperature in each well of a 96-well plate, in a manner compatible with mammalian cell culture and live cell imaging. The thermoPlate maintains precise feedback control of temperature patterns independently in each well, with minutes-scale heating and cooling through ΔT ~15-20°C. A computational model that predicts thermal diffusion guides optimal design of heating protocols. The thermoPlate allowed systematic characterization of both synthetic and natural thermo-responsive systems. We first used the thermoPlate in conjunction with live-cell microscopy to characterize the rapid temperature-dependent phase separation of a synthetic elastin-like polypeptide (ELP53). We then measured stress granule (SG) formation in response to heat stress, observing differences in SG dynamics with each increasing degree of stress. We observed adaptive formation of SGs, whereby SGs formed but then dissolved in response to persistent heat stress (≥ 42°C). SG adaptation revealed a biochemical memory of stress that depended on both the time and temperature of heat shock. Stress memories continued to form even after the removal of heat and persisted for 6-9 hours before dissipating. The capabilities and open-source nature of the thermoPlate will empower the study and engineering of a wide range of thermoresponsive phenomena.

A temperature-inducible protein module for control of mammalian cell fate.

Inducible protein switches are used throughout the biosciences to allow on-demand control of proteins in response to chemical or optical inputs. However, these inducers either cannot be controlled with precision in space and time or cannot be applied in optically dense settings, limiting their application in tissues and organisms. Here we introduce a protein module whose active state can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization through temperature), exists as a monomer in the cytoplasm at elevated temperatures but both oligomerizes and translocates to the plasma membrane when temperature is lowered. Using custom devices for rapid and high-throughput temperature control during live-cell microscopy, we find that the original Melt variant fully switches states between 28-32°C, and state changes can be observed within minutes of temperature changes. Melt was highly modular, permitting thermal control over diverse intracellular processes including signaling, proteolysis, and nuclear shuttling through straightforward end-to-end fusions with no further engineering. Melt was also highly tunable, giving rise to a library of Melt variants with switch point temperatures ranging from 30-40°C. The variants with higher switch points allowed control of molecular circuits between 37°C-41°C, a well-tolerated range for mammalian cells. Finally, Melt could thermally regulate important cell decisions over this range, including cytoskeletal rearrangement and apoptosis. Thus Melt represents a versatile thermogenetic module that provides straightforward, temperature-based, real-time control of mammalian cells with broad potential for biotechnology and biomedicine.

Lipid-mediated intracellular delivery of recombinant bioPROTACs for the rapid degradation of undruggable proteins.

Recently, targeted degradation has emerged as a powerful therapeutic modality. Relying on "event-driven" pharmacology, proteolysis targeting chimeras (PROTACs) can degrade targets and are superior to conventional inhibitors against undruggable proteins. Unfortunately, PROTAC discovery is limited by warhead scarcity and laborious optimization campaigns. To address these shortcomings, analogous protein-based heterobifunctional degraders, known as bioPROTACs, have been developed. Compared to small-molecule PROTACs, bioPROTACs have higher success rates and are subject to fewer design constraints. However, the membrane impermeability of proteins severely restricts bioPROTAC deployment as a generalized therapeutic modality. Here, we present an engineered bioPROTAC template able to complex with cationic and ionizable lipids via electrostatic interactions for cytosolic delivery. When delivered by biocompatible lipid nanoparticles, these modified bioPROTACs can rapidly degrade intracellular proteins, exhibiting near-complete elimination (up to 95% clearance) of targets within hours of treatment. Our bioPROTAC format can degrade proteins localized to various subcellular compartments including the mitochondria, nucleus, cytosol, and membrane. Moreover, substrate specificity can be easily reprogrammed, allowing modular design and targeting of clinically-relevant proteins such as Ras, Jnk, and Erk. In summary, this work introduces an inexpensive, flexible, and scalable platform for efficient intracellular degradation of proteins that may elude chemical inhibition.

Simple visualization of submicroscopic protein clusters with a phase-separation-based fluorescent reporter.

Protein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often too small to appear as puncta in conventional fluorescence microscopy. Here, we describe a fluorescent reporter strategy that detects protein clusters 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, quantifiable fluorescence condensates. We use computational modeling and optogenetic clustering to demonstrate that CluMPS can detect small oligomers and behaves rationally according to key system parameters. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. CluMPS also detected and tracked clusters of unmodified and tagged endogenous proteins, and orthogonal CluMPS probes could be multiplexed in cells. CluMPS provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context. A record of this paper's transparent peer review process is included in the supplemental information.

Rapid optogenetic clustering of a cytoplasmic BcLOV4 variant.

Protein clustering is a powerful form of optogenetic control, yet there is currently only one protein -Cry2-whose light-induced clustering has been harnessed for these purposes. Recently, the photoreceptor BcLOV4 was found to form protein clusters in mammalian cells in response to blue light, although clustering coincided with its translocation to the plasma membrane, potentially constraining its application as an optogenetic clustering module. Herein we identify key amino acids that couple clustering to membrane binding, allowing us to engineer a variant of BcLOV4 that clusters in the cytoplasm and does not associate with the membrane in response to blue light. This variant, BcLOVclust, clustered over many cycles with dramatically faster clustering and de-clustering kinetics compared to Cry2. The magnitude of BcLOVclust clustering could be strengthened by appending an intrinsically disordered region from the fused in sarcoma (FUS) protein, or by optimizing the fluorescent protein to which it was fused. BcLOVclust retained the temperature sensitivity of BcLOV4 such that light induced clustering was transient, and the rate of spontaneous declustering increased with temperature. At low temperatures, BcLOVclust and Cry2 could be multiplexed in the same cells, allowing light control of independent protein condensates. BcLOVclust could also be applied to control signaling proteins and stress granules in mammalian cells. Thus BcLOVclust provides an alternative to Cry2 for optogenetic clustering and a method for multiplexed clustering. While its usage is currently suited for organisms that can be cultured below ~30 °C, a deeper understanding of BcLOVclust thermal response will further enable its use at physiological mammalian temperatures.

Optogenetic control of YAP reveals a dynamic communication code for stem cell fate and proliferation.

YAP is a transcriptional regulator that controls pluripotency, cell fate, and proliferation. How cells ensure the selective activation of YAP effector genes is unknown. This knowledge is essential to rationally control cellular decision-making. Here we leverage optogenetics, live-imaging of transcription, and cell fate analysis to understand and control gene activation and cell behavior. We reveal that cells decode the steady-state concentrations and timing of YAP activation to control proliferation, cell fate, and expression of the pluripotency regulators Oct4 and Nanog. While oscillatory YAP inputs induce Oct4 expression and proliferation optimally at frequencies that mimic native dynamics, cellular differentiation requires persistently low YAP levels. We identify the molecular logic of the Oct4 dynamic decoder, which acts through an adaptive change sensor. Our work reveals how YAP levels and dynamics enable multiplexing of information transmission for the regulation of developmental decision-making and establishes a platform for the rational control of these behaviors.

Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification.

While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein contents often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity western blots on single cells (scWesterns) are attractive since they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 103 molecules, a 520-fold improvement. This enables us to detect 85% and 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies respectively, compared to 47% of cells using in-gel detection. These results suggest compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagents - not previously accessible for in-gel use - for further signal amplification and detection of low abundance targets.

High-throughput feedback-enabled optogenetic stimulation and spectroscopy in microwell plates.

The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.

Upregulation of arginase-II contributes to decreased age-related myocardial contractile reserve.

Arginase-II (Arg-II) reciprocally regulates nitric oxide synthase (NOS) and offsets basal myocardial contractility. Furthermore, decreased or absent myocardial NOS activity is associated with a depression in myocardial contractile reserve. We therefore hypothesized that upregulation of Arg-II might in part be responsible for depressed myocardial contractility associated with age. We studied arginase activity/expression, NOS expression, NO production in the presence and absence of the arginase inhibitor S-(2-boronoethyl)-L: -cysteine (BEC) in old (22 months) and young (3 months) rat hearts and myocytes. The spatial confinement of Arg-II and NOS was determined with immuno-electron-miocrographic (IEM) and immuno-histochemical studies. We tested the effect of BEC on the force frequency response (FFR) in myocytes, as well as NOS abundance and activity. Arginase activity and Arg-II expression was increased in old hearts (2.27 ± 0.542 vs. 0.439 ± 0.058 nmol urea/mg protein, p = 0.02). This was associated with a decrease in NO production, which was restored with BEC (4.54 ± 0.582 vs. 12.88 ± 0.432 µmol/mg, p < 0.01). IEM illustrates increased mitochondrial density in old myocytes (51.7 ± 1.8 vs. 69 ± 2.2 × 10(6)/cm(2), p < 0.01), potentially contributing to increased Arg-II abundance and activity. Immunohistochemistry revealed an organized pattern of mitochondria and Arg-II that appears disrupted in old myocytes. The FFR was significantly depressed in old myocytes (61.42 ± 16.04 vs. -5.15 ± 5.65%), while inhibition of Arg-II restored the FFR (-5.15 ± 5.65 vs. 70.98 ± 6.10%). NOS-2 is upregulated sixfold in old hearts contributing to increased production of reactive oxygen species which is attenuated with NOS-2 inhibition by 1400 W (4,735 ± 427 vs. 4,014 ± 314 RFU/min/mg protein, p = 0.005). Arg-II upregulation in aging rat hearts contributes to age-related decreased contractile function.