Publications by authors named "Jared E Toettcher"

46 Publications

Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch.

Cell Rep 2021 Jun;35(12):109280

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA; IRCC International Research Collaboration Center, National Institutes of Natural Sciences, 4-3-13 Toranomon, Minato-ku, Tokyo 105-0001, Japan. Electronic address:

Protein clustering is pervasive in cell signaling, yet how signaling from higher-order assemblies differs from simpler forms of molecular organization is still poorly understood. We present an optogenetic approach to switch between oligomers and heterodimers with a single point mutation. We apply this system to study signaling from the kinase Zap70 and its substrate linker for activation of T cells (LAT), proteins that normally form membrane-localized condensates during T cell activation. We find that fibroblasts expressing synthetic Zap70:LAT clusters activate downstream signaling, whereas one-to-one heterodimers do not. We provide evidence that clusters harbor a positive feedback loop among Zap70, LAT, and Src-family kinases that binds phosphorylated LAT and further activates Zap70. Finally, we extend our optogenetic approach to the native T cell signaling context, where light-induced LAT clustering is sufficient to drive a calcium response. Our study reveals a specific signaling function for protein clusters and identifies a biochemical circuit that robustly senses protein oligomerization state.
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http://dx.doi.org/10.1016/j.celrep.2021.109280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292983PMC
June 2021

Temporal integration of inductive cues on the way to gastrulation.

Proc Natl Acad Sci U S A 2021 Jun;118(23)

Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544;

Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells' ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in , a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo-which normally only receives a mesoderm-inducing cue-reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene () as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning.
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http://dx.doi.org/10.1073/pnas.2102691118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201965PMC
June 2021

Optogenetic Amplification Circuits for Light-Induced Metabolic Control.

ACS Synth Biol 2021 05 9;10(5):1143-1154. Epub 2021 Apr 9.

Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey 08544, United States.

Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 23-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light duty cycles as low as ∼1%, and strong homogeneous light-induction in bioreactors of at least 5 L, with limited illumination at cell densities above 40 OD. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.
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http://dx.doi.org/10.1021/acssynbio.0c00642DOI Listing
May 2021

Signaling, Deconstructed: Using Optogenetics to Dissect and Direct Information Flow in Biological Systems.

Annu Rev Biomed Eng 2021 Jul 15;23:61-87. Epub 2021 Mar 15.

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.

Cells receive enormous amounts of information from their environment. How they act on this information-by migrating, expressing genes, or relaying signals to other cells-comprises much of the regulatory and self-organizational complexity found across biology. The "parts list" involved in cell signaling is generally well established, but how do these parts work together to decode signals and produce appropriate responses? This fundamental question is increasingly being addressed with optogenetic tools: light-sensitive proteins that enable biologists to manipulate the interaction, localization, and activity state of proteins with high spatial and temporal precision. In this review, we summarize how optogenetics is being used in the pursuit of an answer to this question, outlining the current suite of optogenetic tools available to the researcher and calling attention to studies that increase our understanding of and improve our ability to engineer biology.
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http://dx.doi.org/10.1146/annurev-bioeng-083120-111648DOI Listing
July 2021

Dynamical Modeling of Optogenetic Circuits in Yeast for Metabolic Engineering Applications.

ACS Synth Biol 2021 02 25;10(2):219-227. Epub 2021 Jan 25.

Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey 08544, United States.

Dynamic control of engineered microbes using light via optogenetics has been demonstrated as an effective strategy for improving the yield of biofuels, chemicals, and other products. An advantage of using light to manipulate microbial metabolism is the relative simplicity of interfacing biological and computer systems, thereby enabling control of the microbe. Using this strategy for control and optimization of product yield requires an understanding of how the microbe responds in real-time to the light inputs. Toward this end, we present mechanistic models of a set of yeast optogenetic circuits. We show how these models can predict short- and long-time response to varying light inputs and how they are amenable to use with model predictive control (the industry standard among advanced control algorithms). These models reveal dynamics characterized by time-scale separation of different circuit components that affect the steady and transient levels of the protein under control of the circuit. Ultimately, this work will help enable real-time control and optimization tools for improving yield and consistency in the production of biofuels and chemicals using microbial fermentations.
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http://dx.doi.org/10.1021/acssynbio.0c00372DOI Listing
February 2021

Roadmap on biology in time varying environments.

Phys Biol 2021 May 17;18(4). Epub 2021 May 17.

Department of Physics, University of Illinois at Urbana-Champaign, Champaign, Illinois 61801, United States of America.

Biological organisms experience constantly changing environments, from sudden changes in physiology brought about by feeding, to the regular rising and setting of the Sun, to ecological changes over evolutionary timescales. Living organisms have evolved to thrive in this changing world but the general principles by which organisms shape and are shaped by time varying environments remain elusive. Our understanding is particularly poor in the intermediate regime with no separation of timescales, where the environment changes on the same timescale as the physiological or evolutionary response. Experiments to systematically characterize the response to dynamic environments are challenging since such environments are inherently high dimensional. This roadmap deals with the unique role played by time varying environments in biological phenomena across scales, from physiology to evolution, seeking to emphasize the commonalities and the challenges faced in this emerging area of research.
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http://dx.doi.org/10.1088/1478-3975/abde8dDOI Listing
May 2021

Design and Characterization of Rapid Optogenetic Circuits for Dynamic Control in Yeast Metabolic Engineering.

ACS Synth Biol 2020 12 24;9(12):3254-3266. Epub 2020 Nov 24.

Department of Chemical and Biological Engineering, Hoyt Laboratory 101, Princeton University, William Street, Princeton, New Jersey 08544, United States.

The use of optogenetics in metabolic engineering for light-controlled microbial chemical production raises the prospect of utilizing control and optimization techniques routinely deployed in traditional chemical manufacturing. However, such mechanisms require well-characterized, customizable tools that respond fast enough to be used as real-time inputs during fermentations. Here, we present OptoINVRT7, a new rapid optogenetic inverter circuit to control gene expression in . The circuit induces gene expression in only 0.6 h after switching cells from light to darkness, which is at least 6 times faster than previous OptoINVRT optogenetic circuits used for chemical production. In addition, we introduce an engineered inducible promoter (P), which is stronger than any constitutive or inducible promoter commonly used in yeast. Combining OptoINVRT7 with P achieves strong and light-tunable levels of gene expression with as much as 132.9 ± 22.6-fold induction in darkness. The high performance of this new optogenetic circuit in controlling metabolic enzymes boosts production of lactic acid and isobutanol by more than 50% and 15%, respectively. The strength and controllability of OptoINVRT7 and P open the door to applying process control tools to engineered metabolisms to improve robustness and yields in microbial fermentations for chemical production.
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http://dx.doi.org/10.1021/acssynbio.0c00305DOI Listing
December 2020

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

ACS Chem Biol 2020 10 18;15(10):2752-2765. Epub 2020 Sep 18.

Department of Chemistry, Stony Brook University, New York, 11794, United States.

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 (Jα-helix) which is coupled to the output domain. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, 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 a 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 Jα 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.
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http://dx.doi.org/10.1021/acschembio.0c00543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572778PMC
October 2020

Engineering combinatorial and dynamic decoders using synthetic immediate-early genes.

Commun Biol 2020 08 13;3(1):436. Epub 2020 Aug 13.

Department of Molecular Biology, Princeton University, Princeton, NJ, 08544, USA.

Many cell- and tissue-level functions are coordinated by intracellular signaling pathways that trigger the expression of context-specific target genes. Yet the input-output relationships that link pathways to the genes they activate are incompletely understood. Mapping the pathway-decoding logic of natural target genes could also provide a basis for engineering novel signal-decoding circuits. Here we report the construction of synthetic immediate-early genes (SynIEGs), target genes of Erk signaling that implement complex, user-defined regulation and can be monitored by using live-cell biosensors to track their transcription and translation. We demonstrate the power of this approach by confirming Erk duration-sensing by FOS, elucidating how the BTG2 gene is differentially regulated by external stimuli, and designing a synthetic immediate-early gene that selectively responds to the combination of growth factor and DNA damage stimuli. SynIEGs pave the way toward engineering molecular circuits that decode signaling dynamics and combinations across a broad range of cellular contexts.
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http://dx.doi.org/10.1038/s42003-020-01171-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426417PMC
August 2020

Optogenetic control of protein binding using light-switchable nanobodies.

Nat Commun 2020 08 13;11(1):4044. Epub 2020 Aug 13.

Department of Molecular Biology, Princeton University, Princeton, NJ, 08544, USA.

A growing number of optogenetic tools have been developed to reversibly 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 cells 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 including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.
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http://dx.doi.org/10.1038/s41467-020-17836-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426870PMC
August 2020

Development of light-responsive protein binding in the monobody non-immunoglobulin scaffold.

Nat Commun 2020 08 13;11(1):4045. Epub 2020 Aug 13.

Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, 08544, USA.

Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-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. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light.
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http://dx.doi.org/10.1038/s41467-020-17837-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427095PMC
August 2020

Optogenetic Rescue of a Patterning Mutant.

Curr Biol 2020 09 23;30(17):3414-3424.e3. Epub 2020 Jul 23.

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. Electronic address:

Animal embryos are patterned by a handful of highly conserved inductive signals. Yet, in most cases, it is unknown which pattern features (i.e., spatial gradients or temporal dynamics) are required to support normal development. An ideal experiment to address this question would be to "paint" arbitrary synthetic signaling patterns on "blank canvas" embryos to dissect their requirements. Here, we demonstrate exactly this capability by combining optogenetic control of Ras/extracellular signal-related kinase (ERK) signaling with the genetic loss of the receptor tyrosine-kinase-driven terminal signaling patterning in early Drosophila embryos. Blue-light illumination at the embryonic termini for 90 min was sufficient to rescue normal development, generating viable larvae and fertile adults from an otherwise lethal terminal signaling mutant. Optogenetic rescue was possible even using a simple, all-or-none light input that reduced the gradient of Erk activity and eliminated spatiotemporal differences in terminal gap gene expression. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered at different signaling thresholds and that the morphogenetic movements of gastrulation are robust to a 3-fold variation in the posterior pattern width. These results open the door to controlling tissue organization with simple optical stimuli, providing new tools to probe natural developmental processes, create synthetic tissues with defined organization, or directly correct the patterning errors that underlie developmental defects.
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http://dx.doi.org/10.1016/j.cub.2020.06.059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730203PMC
September 2020

A Live-Cell Screen for Altered Erk Dynamics Reveals Principles of Proliferative Control.

Cell Syst 2020 03 18;10(3):240-253.e6. Epub 2020 Mar 18.

Department of Molecular Biology, Princeton University, Princeton, NJ 08544. Electronic address:

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 are typically incompatible with the live-cell assays used to monitor dynamics. Here, we address this challenge by screening a library of 429 kinase inhibitors and monitoring extracellular-regulated kinase (Erk) activity over 5 h in more than 80,000 single primary mouse keratinocytes. Our screen reveals both known and uncharacterized modulators of Erk dynamics, including inhibitors of non-epidermal growth factor receptor (EGFR) receptor tyrosine kinases (RTKs) that increase 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.
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http://dx.doi.org/10.1016/j.cels.2020.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540725PMC
March 2020

Deconstructing and repurposing the light-regulated interplay between phytochromes and interacting factors.

Commun Biol 2019 2;2:448. Epub 2019 Dec 2.

1Lehrstuhl für Biochemie, Universität Bayreuth, 95447 Bayreuth, Germany.

Phytochrome photoreceptors mediate adaptive responses of plants to red and far-red light. These responses generally entail light-regulated association between phytochromes and other proteins, among them the phytochrome-interacting factors (PIF). The interaction with phytochrome B (PhyB) localizes to the bipartite APB motif of the PIFs (PIF). To address a dearth of quantitative interaction data, we construct and analyze numerous PIF3/6 variants. Red-light-activated binding is predominantly mediated by the APB N-terminus, whereas the C-terminus modulates binding and underlies the differential affinity of PIF3 and PIF6. We identify PIF variants of reduced size, monomeric or homodimeric state, and with PhyB affinities between 10 and 700 nM. Optogenetically deployed in mammalian cells, the PIF variants drive light-regulated gene expression and membrane recruitment, in certain cases reducing basal activity and enhancing regulatory response. Moreover, our results provide hitherto unavailable quantitative insight into the PhyB:PIF interaction underpinning vital light-dependent responses in plants.
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http://dx.doi.org/10.1038/s42003-019-0687-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888877PMC
July 2020

Optimizing photoswitchable MEK.

Proc Natl Acad Sci U S A 2019 12 3;116(51):25756-25763. Epub 2019 Dec 3.

Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544;

Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.
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http://dx.doi.org/10.1073/pnas.1912320116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926043PMC
December 2019

Light-based control of metabolic flux through assembly of synthetic organelles.

Nat Chem Biol 2019 06 13;15(6):589-597. Epub 2019 May 13.

Department of Molecular Biology, Princeton University, Princeton, NJ, USA.

To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.
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http://dx.doi.org/10.1038/s41589-019-0284-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755918PMC
June 2019

Signaling Dynamics Control Cell Fate in the Early Drosophila Embryo.

Dev Cell 2019 02;48(3):361-370.e3

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. Electronic address:

The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? We combine optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. We find that Erk activity is sufficient to "posteriorize" 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. We find that the endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes.
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http://dx.doi.org/10.1016/j.devcel.2019.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6394837PMC
February 2019

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

PLoS One 2018 21;13(12):e0209301. Epub 2018 Dec 21.

Marine Biological Laboratory, Woods Hole, MA, United States of America.

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. The G1/S boundary of the cell cycle serves as a major point of size control, and mechanisms operating here restrict passage of cells to Start if they are too small. In contrast, it is less clear how size is regulated post-Start, during S/G2/M. To gain further insight into post-Start size control, we prepared 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 from their block, giant mothers reenter the cell cycle and their progeny rapidly return to the original unperturbed size. We found this behavior to be consistent with a size-invariant 'timer' specifying the duration of S/G2/M. These results indicate that yeast use at least two distinct mechanisms at different cell cycle phases to ensure size homeostasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209301PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303054PMC
May 2019

A bright future: optogenetics to dissect the spatiotemporal control of cell behavior.

Curr Opin Chem Biol 2019 02 5;48:106-113. Epub 2018 Dec 5.

Department of Molecular Biology, Princeton University, Princeton NJ 08544, United States. Electronic address:

Cells sense, process, and respond to extracellular information using signaling networks: collections of proteins that act as precise biochemical sensors. These protein networks are characterized by both complex temporal organization, such as pulses of signaling activity, and by complex spatial organization, where proteins assemble structures at particular locations and times within the cell. Yet despite their ubiquity, studying these spatial and temporal properties has remained challenging because they emerge from the entire protein network rather than a single node, and cannot be easily tuned by drugs or mutations. These challenges are being met by a new generation of optogenetic tools capable of directly controlling the activity of individual signaling nodes over time and the assembly of protein complexes in space. Here, we outline how these recent innovations are being used in conjunction with engineering-influenced experimental design to address longstanding questions in signaling biology.
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http://dx.doi.org/10.1016/j.cbpa.2018.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382565PMC
February 2019

Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.

Cell 2018 11;175(6):1467-1480.e13

Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA; Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA; Howard Hughes Medical Institute, Princeton University, Princeton, NJ 08544, USA. Electronic address:

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 an oligomerizing biomimetic system, "Corelets," and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs and the transition mechanism, in a protein sequence dependent manner. 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. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2018.10.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724719PMC
November 2018

P-Rex1 is dispensable for Erk activation and mitogenesis in breast cancer.

Oncotarget 2018 Jun 19;9(47):28612-28624. Epub 2018 Jun 19.

Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6160, USA.

Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 1 (P-Rex1) is a key mediator of growth factor-induced activation of Rac1, a small GTP-binding protein widely implicated in actin cytoskeleton reorganization. This Guanine nucleotide Exchange Factor (GEF) is overexpressed in human luminal breast cancer, and its expression associates with disease progression, metastatic dissemination and poor outcome. Despite the established contribution of P-Rex1 to Rac activation and cell locomotion, whether this Rac-GEF has any relevant role in mitogenesis has been a subject of controversy. To tackle the discrepancies among various reports, we carried out an exhaustive analysis of the potential involvement of P-Rex1 on the activation of the mitogenic Erk pathway. Using a range of luminal breast cancer cellular models, we unequivocally showed that silencing P-Rex1 (transiently, stably, using multiple siRNA sequences) had no effect on the phospho-Erk response upon stimulation with growth factors (EGF, heregulin, IGF-I) or a GPCR ligand (SDF-1). The lack of involvement of P-Rex1 in Erk activation was confirmed at the single cell level using a fluorescent biosensor of Erk kinase activity. Depletion of P-Rex1 from breast cancer cells failed to affect cell cycle progression, cyclin D1 induction, Akt activation and apoptotic responses. In addition, mammary-specific P-Rex1 transgenic mice (MMTV-P-Rex1) did not show any obvious hyperproliferative phenotype. Therefore, despite its crucial role in Rac1 activation and cell motility, P-Rex1 is dispensable for mitogenic or survival responses in breast cancer cells.
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http://dx.doi.org/10.18632/oncotarget.25584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033363PMC
June 2018

Four Key Steps Control Glycolytic Flux in Mammalian Cells.

Cell Syst 2018 07 27;7(1):49-62.e8. Epub 2018 Jun 27.

Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA; Department of Chemistry, Princeton University, Princeton, NJ 08544, USA. Electronic address:

Altered glycolysis is a hallmark of diseases including diabetes and cancer. Despite intensive study of the contributions of individual glycolytic enzymes, systems-level analyses of flux control through glycolysis remain limited. Here, we overexpress in two mammalian cell lines the individual enzymes catalyzing each of the 12 steps linking extracellular glucose to excreted lactate, and find substantial flux control at four steps: glucose import, hexokinase, phosphofructokinase, and lactate export (and not at any steps of lower glycolysis). The four flux-controlling steps are specifically upregulated by the Ras oncogene: optogenetic Ras activation rapidly induces the transcription of isozymes catalyzing these four steps and enhances glycolysis. At least one isozyme catalyzing each of these four steps is consistently elevated in human tumors. Thus, in the studied contexts, flux control in glycolysis is concentrated in four key enzymatic steps. Upregulation of these steps in tumors likely underlies the Warburg effect.
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http://dx.doi.org/10.1016/j.cels.2018.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062487PMC
July 2018

Preclinical assessment of antiviral combination therapy in a genetically humanized mouse model for hepatitis delta virus infection.

Sci Transl Med 2018 06;10(447)

Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Washington Road, Princeton, NJ 08544, USA.

Chronic delta hepatitis, caused by hepatitis delta virus (HDV), is the most severe form of viral hepatitis, affecting at least 20 million hepatitis B virus (HBV)-infected patients worldwide. HDV/HBV co- or superinfections are major drivers for hepatocarcinogenesis. Antiviral treatments exist only for HBV and can only suppress but not cure infection. Development of more effective therapies has been impeded by the scarcity of suitable small-animal models. We created a transgenic (tg) mouse model for HDV expressing the functional receptor for HBV and HDV, the human sodium taurocholate cotransporting peptide NTCP. Both HBV and HDV entered hepatocytes in these mice in a glycoprotein-dependent manner, but one or more postentry blocks prevented HBV replication. In contrast, HDV persistently infected hNTCP tg mice coexpressing the HBV envelope, consistent with HDV dependency on the HBV surface antigen (HBsAg) for packaging and spread. In immunocompromised mice lacking functional B, T, and natural killer cells, viremia lasted at least 80 days but resolved within 14 days in immunocompetent animals, demonstrating that lymphocytes are critical for controlling HDV infection. Although acute HDV infection did not cause overt liver damage in this model, cell-intrinsic and cellular innate immune responses were induced. We further demonstrated that single and dual treatment with myrcludex B and lonafarnib efficiently suppressed viremia but failed to cure HDV infection at the doses tested. This small-animal model with inheritable susceptibility to HDV opens opportunities for studying viral pathogenesis and immune responses and for testing novel HDV therapeutics.
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http://dx.doi.org/10.1126/scitranslmed.aap9328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337727PMC
June 2018

Protein Phase Separation Provides Long-Term Memory of Transient Spatial Stimuli.

Cell Syst 2018 06 30;6(6):655-663.e5. Epub 2018 May 30.

Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ 08544, USA. Electronic address:

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 biochemical feedback loops.
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http://dx.doi.org/10.1016/j.cels.2018.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023754PMC
June 2018

Optogenetic regulation of engineered cellular metabolism for microbial chemical production.

Nature 2018 03 21;555(7698):683-687. Epub 2018 Mar 21.

Department of Chemical and Biological Engineering, Hoyt Laboratory, Princeton University, 25 William Street, Princeton, New Jersey 08544, USA.

The optimization of engineered metabolic pathways requires careful control over the levels and timing of metabolic enzyme expression. Optogenetic tools are ideal for achieving such precise control, as light can be applied and removed instantly without complex media changes. Here we show that light-controlled transcription can be used to enhance the biosynthesis of valuable products in engineered Saccharomyces cerevisiae. We introduce new optogenetic circuits to shift cells from a light-induced growth phase to a darkness-induced production phase, which allows us to control fermentation with only light. Furthermore, optogenetic control of engineered pathways enables a new mode of bioreactor operation using periodic light pulses to tune enzyme expression during the production phase of fermentation to increase yields. Using these advances, we control the mitochondrial isobutanol pathway to produce up to 8.49 ± 0.31 g l of isobutanol and 2.38 ± 0.06 g l of 2-methyl-1-butanol micro-aerobically from glucose. These results make a compelling case for the application of optogenetics to metabolic engineering for the production of valuable products.
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http://dx.doi.org/10.1038/nature26141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876151PMC
March 2018

Illuminating developmental biology with cellular optogenetics.

Curr Opin Biotechnol 2018 08 2;52:42-48. Epub 2018 Mar 2.

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, United States. Electronic address:

In developmental biology, localization is everything. The same stimulus-cell signaling event or expression of a gene-can have dramatically different effects depending on the time, spatial position, and cell types in which it is applied. Yet the field has long lacked the ability to deliver localized perturbations with high specificity in vivo. The advent of optogenetic tools, capable of delivering highly localized stimuli, is thus poised to profoundly expand our understanding of development. We describe the current state-of-the-art in cellular optogenetic tools, review the first wave of major studies showcasing their application in vivo, and discuss major obstacles that must be overcome if the promise of developmental optogenetics is to be fully realized.
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http://dx.doi.org/10.1016/j.copbio.2018.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082700PMC
August 2018

Optogenetic Reconstitution for Determining the Form and Function of Membraneless Organelles.

Biochemistry 2018 05 26;57(17):2432-2436. Epub 2018 Jan 26.

Department of Molecular Biology , Princeton University , Princeton , New Jersey 08544 , United States.

It has recently become clear that large-scale macromolecular self-assembly is a rule, rather than an exception, of intracellular organization. A growing number of proteins and RNAs have been shown to self-assemble into micrometer-scale clusters that exhibit either liquid-like or gel-like properties. Given their proposed roles in intracellular regulation, embryo development, and human disease, it is becoming increasingly important to understand how these membraneless organelles form and to map their functional consequences for the cell. Recently developed optogenetic systems make it possible to acutely control cluster assembly and disassembly in live cells, driving the separation of proteins of interest into liquid droplets, hydrogels, or solid aggregates. Here we propose that these approaches, as well as their evolution into the next generation of optogenetic biophysical tools, will allow biologists to determine how the self-assembly of membraneless organelles modulates diverse biochemical processes.
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http://dx.doi.org/10.1021/acs.biochem.7b01173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972035PMC
May 2018
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