Biology subjects

Toettcher, J. E.

Publications and source records attributed to Toettcher, J. E..

29 records · Page 2Linked to original sources

bioRxiv2020

Clustering-based positive feedback between a kinase and its substrate enables effective T-cell receptor signaling

Protein clusters and condensates are pervasive in mammalian signaling. Yet how the signaling capacity of higher-order assemblies differs from simpler forms of molecular organization is still poorly understood. Here, we present an optogenetic approach to switch between light-induced clusters and simple protein heterodimers with a single point mutation. We apply this system to study how clustering affects signaling from the kinase Zap70 and its substrate LAT, proteins that normally form membrane-localized clusters during T cell activation. We find that light-induced clusters of LAT and Zap70 trigger potent activation of downstream signaling pathways even in non-T cells, whereas one-to-one dimers do not. We provide evidence that clusters harbor a local positive feedback loop between three components: Zap70, LAT, and Src-family kinases that bind to phosphorylated LAT and further activate Zap70. Overall, our study provides evidence for a specific role of protein condensates in cell signaling, and identifies a simple biochemical circuit that can robustly sense protein oligomerization state. Highlights- A general system for studying the role of protein clusters versus dimers. - Membrane clusters of the kinase Zap70 and its substrate LAT trigger potent downstream signaling. - Clustering Zap70 with LAT is required for full activation of Zap70 kinase activity. - A positive feedback loop connects phosphorylated LAT to Zap70 activation via Src-family kinases.

cell biology↗

bioRxiv2020

Light-responsive monobodies for dynamic control of customizable protein binding

Customizable, high affinity protein-protein interactions, such as those mediated by antibodies and antibody-like molecules, are invaluable to basic and applied research and have become pillars for modern therapeutics. The ability to reversibly control the binding activity of these proteins to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, a light-controlled monobody (OptoMB) that works in vitro and in vivo, whose affinity for its SH2-domain target exhibits a 300-fold shift in binding affinity upon illumination. We demonstrate that our SH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. This OptoMB belongs to a new class of light-sensitive protein binders we call OptoBinders (OptoBNDRs) which, by virtue of their ability to be designed to bind any protein of interest, have the potential to find new powerful applications as light-switchable binders of untagged proteins with high affinity and selectivity, and with the temporal and spatial precision afforded by light.

biochemistry↗

bioRxiv2020

Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix

Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (J-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to J helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 s, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between J helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

biophysics↗

bioRxiv2019

Engineering combinatorial and dynamic decoders using synthetic immediate-early genes

For tissues to grow and function properly, cells must coordinate actions such as proliferation, differentiation and apoptosis. This coordination is achieved in part by the activation of intracellular signaling pathways that trigger the expression of context-specific target genes. While the function of these natural circuits has been actively studied, synthetic biology provides additional powerful tools for deconstructing, repurposing, and designing novel signal-decoding circuits. Here we report the construction of synthetic immediate-early genes (synIEGs), target genes of the Erk signaling pathway that implement complex, user-defined regulation and can be monitored through the use of live-cell biosensors to track transcription and translation. We demonstrate the power and flexibility of this approach by confirming Erk duration-sensing by the FOS immediate-early gene, elucidating how the BTG2 gene is regulated by transcriptional activation and translational repression after growth-factor stimulation, and by designing a synthetic immediate-early gene that responds with AND-gate logic to the combined presence of growth factor and DNA damage stimuli. Our work paves the way to defining the molecular circuits that link signaling pathways to specific target genes, highlighting an important role for post-transcriptional regulation in signal decoding that may be masked by analyses of RNA abundance alone.

synthetic biology↗

bioRxiv2019

Optogenetic rescue of a developmental patterning mutant

Animal embryos are partitioned into spatial domains by complex patterns of signaling and gene expression, yet it is still largely unknown what features of a developmental signal are essential. Part of the challenge arises because it has been impossible to \"paint\" arbitrary signaling patterns on an embryo to test their sufficiency. Here we demonstrate exactly this capability by combining optogenetic control of Ras/Erk signaling with the genetic loss of terminal signaling in early Drosophila embryos. Simple all-or-none light inputs at the embryonic termini were able to completely rescue normal development, generating viable larvae and fertile adults from this otherwise-lethal genetic mutant. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered by different cumulative doses of Erk. These results open the door to the targeted design of complex morphogenetic outcomes as well as the ability to correct patterning errors that underlie developmental defects.\n\nSummarySpatial gradients of protein activity pattern all developing embryos. But which features of these patterns are essential and which are dispensable for development? Answering this question has been deceptively difficult, due to the difficulty of shaping spatial gradients using chemical or genetic perturbations. Johnson and colleagues use optogenetics to fully restore normal embryo development with a pattern of light, opening the door to complete control over spatial organization in developing and engineered tissues.

developmental biology↗

bioRxiv2019

Optogenetic control of protein binding using light-switchable nanobodies

A growing number of optogenetic tools have been developed to control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in vivo or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications: modulating intracellular protein localization and signaling pathway activity and controlling target protein binding to surfaces and in protein separation columns. This work represents a first step towards programmable photoswitchable regulation of untagged, endogenous target proteins.\n\nHighlightsO_LIOpto-Nanobodies (OptoNBs) enable light-regulated binding to a wide range of protein targets.\nC_LIO_LIWe identify an optimized LOV domain and two loop insertion sites for light-regulated binding.\nC_LIO_LIOptoNBs function in vivo: when expressed in cells and fused to signaling domains, OptoNBs enable light-activated and light-inhibited Ras/Erk signaling.\nC_LIO_LIOptoNBs function in vitro: Target proteins can be reversibly bound to OptoNB-coated beads and separated through size-exclusion chromatography.\nC_LI

biochemistry↗

bioRxiv2019

A live-cell screen for altered Erk dynamics reveals principles of proliferative control

Complex, time-varying responses have been observed widely in cell signaling, but how specific dynamics are generated or regulated is largely unknown. One major obstacle has been that high-throughput screens for identifying pathway components are typically incompatible with the live-cell assays used to monitor dynamics. Here, we address this challenge by performing a drug screen for altered Erk signaling dynamics in primary mouse keratinocytes. We screened a library of 429 kinase inhibitors, monitoring Erk activity over 5 h in more than 80,000 single live cells. The screen revealed both known and uncharacterized modulators of Erk dynamics, including inhibitors of non-EGFR receptor tyrosine kinases (RTKs) that increased Erk pulse frequency and overall activity. Using drug treatment and direct optogenetic control, we demonstrate that drug-induced changes to Erk dynamics alter the conditions under which cells proliferate. Our work opens the door to high-throughput screens using live-cell biosensors and reveals that cell proliferation integrates information from Erk dynamics as well as additional permissive cues.

cell biology↗

bioRxiv2018

Signaling dynamics control cell fate in the early Drosophila embryo

The Erk mitogen-activated protein kinase plays diverse roles in animal development, where its activity is associated with phenomena including cell migration, proliferation and differentiation. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? Here, we combine precise optogenetic control with genetic perturbations to dissect Erk-dependent cellular responses in the early Drosophila embryo. We find that light-stimulated Erk activity is sufficient to posterior-ize the majority of the embryo, leading to massive apical constriction through expression of the autocrine receptor-ligand pair mist and fog. Ectopic contraction at non-terminal positions requires at least 1 h of high-amplitude Erk signaling, whereas a 30 min pulse of Erk activity patterns non-contractile neurogenic ectoderm during the same time window. In contrast to the canonical transient versus sustained model, the cell fate switch is triggered by the cumulative load of Erk signaling, not the duration of a single persistent pulse. Our results reveal that the early fly embryo harbors a classic example of dynamic cell fate control, where the total dose of Erk activity selects between two distinct physiological outcomes.

developmental biology↗

bioRxiv2018

Mapping local and global liquid-liquid phase behavior in living cells using light-activated multivalent seeds

Recent studies show that liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here we introduce a biomimetic optogenetic system, \"Corelets\", and utilize its rapid and quantitative tunability to map the first full intracellular phase diagrams, which dictate whether phase separation occurs, and if so by nucleation and growth or spinodal decomposition. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes.

biophysics↗

bioRxiv2018

Protein phase separation provides long-term memory of transient spatial stimuli

Protein/RNA clusters arise frequently in spatially-regulated biological processes, from the asymmetric distribution of P granules and PAR proteins in developing embryos to localized receptor oligomers in migratory cells. This co-occurrence suggests that protein clusters might possess intrinsic properties that make them a useful substrate for spatial regulation. Here, we demonstrate that protein droplets show a robust form of spatial memory, maintaining the spatial pattern of an inhibitor of droplet formation long after it has been removed. Despite this persistence, droplets can be highly dynamic, continuously exchanging monomers with the diffuse phase. We investigate the principles of biophysical spatial memory in three contexts: a computational model of phase separation; a novel optogenetic system where light can drive rapid, localized dissociation of liquid-like protein droplets; and membrane-localized signal transduction from clusters of receptor tyrosine kinases. Our results suggest that the persistent polarization underlying many cellular and developmental processes could arise through a simple biophysical process, without any additional requirement for biochemical positive and negative feedback loops.\n\nHighlightsO_LIWe introduce PixELLs, an optogenetic system for protein droplet disassembly.\nC_LIO_LIModeling and experiments demonstrate long-term memory of local droplet dissociation.\nC_LIO_LIDroplets remember spatial stimuli in nuclei, the cytosol and on cell membranes.\nC_LIO_LIFGFR-optoDroplets convert transient local inputs to persistent cytoskeletal responses.\nC_LI

systems biology↗

bioRxiv2017

A size-invariant bud-length timer enables robustness in yeast cell size control

Cell populations across nearly all forms of life generally maintain a characteristic cell type-dependent size, but how size control is achieved has been a long-standing question. Prior work has uncovered diverse size control strategies operating at distinct cell cycle stages, but it is unclear how these numerous pathways are integrated to provide robust, systems-level cell size control for any organism. Here, we probe cell growth and size control in budding yeast that can be reversibly blocked from bud initiation. While blocked, cells continue to grow isotropically, increasing their volume by more than an order of magnitude over unperturbed cells. Upon release, these giant yeast resume budding and the population returns to its initial volume distribution within a few cell division cycles. Size control under these conditions does not require an explicit molecular size sensor. Instead, our observations are consistent with a size-invariant bud growth timer specifying the duration of S/G2/M to limit daughter cell size.

cell biology↗