Publications

2024

Brumbaugh-Reed, EH, K Aoki, and JE Toettcher. “Rapid and Reversible Dissolution of Biomolecular Condensates Using Light-Controlled Recruitment of a Solubility Tag..” bioRxiv (2024): n. pag.
Kim-Yip*, RP et al. “Efficient Prime Editing in Two-Cell Mouse Embryos Using PEmbryo.” Nature Biotechnology (2024): n. pag.

2023

Mesev, EV et al. “Synthetic Heterodimers of Type III Interferon Receptors Require TYK2 for STAT Activation..” Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 43.9 (2023): 414–426.
Underhill, EJ, and JE Toettcher. “Control of Gastruloid Patterning and Morphogenesis by the Erk and Akt Signaling Pathways..” Development (Cambridge, England) 150.16 (2023): n. pag.
Ho, EK et al. “Dynamics of an Incoherent Feedforward Loop Drive ERK-Dependent Pattern Formation in the Early Drosophila Embryo..” Development (Cambridge, England) 150.17 (2023): n. pag.
McNamara, HM, B Ramm, and JE Toettcher. “Synthetic Developmental Biology: New Tools to Deconstruct and Rebuild Developmental Systems..” Seminars in cell & developmental biology 141 (2023): 33–42.
Zhu, L, HM McNamara, and Jared E Toettcher. “Light-Switchable Transcription Factors Obtained by Direct Screening in Mammalian Cells..” Nature communications 14.1 (2023): 3185.
McNamara, HM et al. “Recording Morphogen Signals Reveals Origins of Gastruloid Symmetry Breaking..” bioRxiv (2023): n. pag.

2022

Ravindran, PT et al. “A Synthetic Gene Circuit for Imaging-Free Detection of Signaling Pulses..” Cell systems 13.2 (2022): 131–142.e13.
McFann, SE, SY Shvartsman, and JE Toettcher. “Putting in the Erk: Growth Factor Signaling and Mesoderm Morphogenesis..” Current topics in developmental biology 149 (2022): 263–310.

2021

Farahani, PE et al. “Substratum Stiffness Regulates Erk Signaling Dynamics through Receptor-Level Control..” Cell reports 37.13 (2021): 110181.
Palmer, MA et al. “Stress Ball Morphogenesis: How the Lizard Builds Its Lung..” Science advances 7.52 (2021): eabk0161.

2020

Ravindran, PT et al. “Engineering Combinatorial and Dynamic Decoders Using Synthetic Immediate-Early Genes.” Commun Biol (2020): n. pag.
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.
Gil, AA et al. “Optogenetic Control of Protein Binding Using Light-Switchable Nanobodies.” Nat Commun (2020): n. pag.
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.
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.
Johnson, HE et al. “Optogenetic Rescue of a Patterning Mutant.” Curr Biol (2020): n. pag.
Goglia, Alexander et al. “A Live-Cell Screen for Altered Erk Dynamics Reveals Principles of Proliferative Control.” Cell Systems 10.3 (2020): 240–253.

2019

Zhao, Evan et al. “Light-Based Control of Metabolic Flux through Assembly of Synthetic Organelles.” Nature chemical biology 15 (2019): 589–597.

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.

Johnson, Heath, and Jared Toettcher. “Signaling Dynamics Control Cell Fate in the Early Drosophila Embryo.” Developmental Cell 48.3 (2019): 361–370. Print.
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.
Goglia, Alexander, and Jared Toettcher. “A Bright Future: Optogenetics to Dissect the Spatiotemporal Control of Cell Behavior.” Current opinion in chemical biology 48 (2019): 106–113. Print.

2018

Bracha, Dan et al. “Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.” Cell 175.6 (2018): 1467–1480. Print.
Allard, Corey AH et al. “A Size-Invariant Bud-Duration Timer Enables Robustness in Yeast Cell Size Control.” PloS one 13.12 (2018): e0209301. Print.
Signaling pathways, such as the Ras-Erk pathway, encode information through both their amplitude and dynamics. Differences in signal duration and frequency can lead to distinct cellular output decisions. Thus, temporal signals must be faithfully transmitted from the plasma membrane (Ras) to the nucleus (Erk) to properly control the cell’s response. Because the Ras-Erk pathway regulates important cell decisions such as proliferation, changes to dynamic signal transduction properties could result in improper cell decisions and dysfunction. However, it has been difficult to examine whether corruption of signal transmission dynamics is associated with diseases such as cancer.
Barrio-Real et al. “P-Rex1 Is Dispensable for Erk Activation and Mitogenesis in Breast Cancer.” Oncotarget 9.47 (2018): 28612.
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.

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.

Tanner, LB et al. “Flux Control in Mammalian Glycolysis Resides in a Few Key Pathway Steps.” Cell Systems 7 (2018): 1–14.

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.

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−1 of isobutanol and 2.38 ± 0.06 g l−1 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.

Dine, EA et al. “Protein Phase Separation Provides Long-Term Memory of Transient Spatial Stimuli.” Cell Systems 6 (2018): 663.
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.
Johnson, HE, and Toettcher JE. “Illuminating Developmental Biology With Cellular Optogenetics.” Current opinion in biotechnology 52 (2018): 42–48.
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.
Dine, EA, and Toettcher JE. “Optogenetic Reconstitution for Determining the Form and Function of Membraneless Organelles.” Biochemistry 57.17 (2018): 2432–2436.

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.

2017

Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene’s transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.

Goglia, Alexander G. et al. “Optogenetic Control of Ras Erk Signaling Using the Phy-PIF System.” Kinase Signaling Networks. New York, NY: Humana Press, 2017. 3–20. Print.

The Ras/Erk signaling pathway plays a central role in diverse cellular processes ranging from development to immune cell activation to neural plasticity to cancer. In recent years, this pathway has been widely studied using live-cell fluorescent biosensors, revealing complex Erk dynamics that arise in many cellular contexts. Yet despite these high-resolution tools for measurement, the field has lacked analogous tools for control over Ras/Erk signaling in live cells. Here, we provide detailed methods for one such tool based on the optical control of Ras activity, which we call "Opto-SOS." Expression of the Opto-SOS constructs can be coupled with a live-cell reporter of Erk activity to reveal highly quantitative input-to-output maps of the pathway. Detailed herein are protocols for expressing the Opto-SOS system in cultured cells, purifying the small molecule cofactor necessary for optical stimulation, imaging Erk responses using live-cell microscopy, and processing the imaging data to quantify Ras/Erk signaling dynamics.

Johnson, Heath E. et al. “The Spatiotemporal Limits of Developmental Erk Signaling.” Developmental Cell 40.2 (2017): 185–192.

Animal development is characterized by signaling events that occur at precise locations and times within the embryo, but determining when and where such precision is needed for proper embryogenesis has been a long-standing challenge. Here we address this question for extracellular signal regulated kinase (Erk) signaling, a key developmental patterning cue. We describe an optogenetic system for activating Erk with high spatiotemporal precision in vivo. Implementing this system in Drosophila, we find that embryogenesis is remarkably robust to ectopic Erk signaling, except from 1 to 4 hr post-fertilization, when perturbing the spatial extent of Erk pathway activation leads to dramatic disruptions of patterning and morphogenesis. Later in development, the effects of ectopic signaling are buffered, at least in part, by combinatorial mechanisms. Our approach can be used to systematically probe the differential contributions of the Ras/Erk pathway and concurrent signals, leading to a more quantitative understanding of developmental signaling.

Phase transitions driven by intrinsically disordered protein regions (IDRs) have emerged as a ubiquitous mechanism for assembling liquid-like RNA/protein (RNP) bodies and other membrane-less organelles. However, a lack of tools to control intracellular phase transitions limits our ability to understand their role in cell physiology and disease. Here, we introduce an optogenetic platform that uses light to activate IDR-mediated phase transitions in living cells. We use this “optoDroplet” system to study condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1. Above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally definable liquid optoDroplets. FUS optoDroplet assembly is fully reversible even after multiple activation cycles. However, cells driven deep within the phase boundary form solid-like gels that undergo aging into irreversible aggregates. This system can thus elucidate not only physiological phase transitions but also their link to pathological aggregates.

2016

The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses.

Gordley, Russell et al. “Engineering Dynamical Control of Cell Fate Switching Using Synthetic Phospho-Regulons..” Proc Natl Acad Sci U S A 113.47 (2016): 13528–13533.
Many cells can sense and respond to time-varying stimuli, selectively triggering changes in cell fate only in response to inputs of a particular duration or frequency. A common motif in dynamically controlled cells is a dual-timescale regulatory network: although long-term fate decisions are ultimately controlled by a slow-timescale switch (e.g., gene expression), input signals are first processed by a fast-timescale signaling layer, which is hypothesized to filter what dynamic information is efficiently relayed downstream. Directly testing the design principles of how dual-timescale circuits control dynamic sensing, however, has been challenging, because most synthetic biology methods have focused solely on rewiring transcriptional circuits, which operate at a single slow timescale. Here, we report the development of a modular approach for flexibly engineering phosphorylation circuits using designed phospho-regulon motifs. By then linking rapid phospho-feedback with slower downstream transcription-based bistable switches, we can construct synthetic dual-timescale circuits in yeast in which the triggering dynamics and the end-state properties of the ON state can be selectively tuned. These phospho-regulon tools thus open up the possibility to engineer cells with customized dynamical control.
Johnson, Heath, and Jared Toettcher. “The Duty of an Intracellular Signal: Illuminating Calcium’s Role in Transcriptional Control..” Cell Syst 2.4 (2016): 223–4.
An optogenetic approach reveals how cells encode external information in complex patterns of protein activity.
Hoeller, Oliver et al. “Gβ Regulates Coupling Between Actin Oscillators for Cell Polarity and Directional Migration..” PLoS Biol 14.2 (2016): e1002381.
For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP3) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gβ and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gβ. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gβ regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gβ and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well.

2013

Toettcher, Jared E. “Cell Cycle Arrest After DNA Damage.” Encyclopedia of Systems Biology. New York, NY: Springer, 2013. 249–254.
Cell cycle arrest after DNA damage describes the interconnection between two complex signaling processes – DNA damage sensing and the cell cycle – by
a variety of biochemical interactions. Damage may arise from various sources, including radiation, chemical agents, or errors during DNA synthesis or cell division. The resulting damage is sensed by a signaling network that halts the cell cycle by modulating cyclin/Cdk activity. Cell cycle arrest can be transient to allow repair of DNA damage, or can persist indefinitely as a senescence-like state. This essay describes mechanisms of DNA damage-induced cell cycle arrest, their dynamics, and their effect on eventual cell fate. It also discusses mathematical modeling approaches used to gain insight into these processes.
Toettcher, Jared, Orion Weiner, and Wendell Lim. “Using Optogenetics to Interrogate the Dynamic Control of Signal Transmission by the Ras Erk Module..” Cell 155.6 (2013): 1422–34.
The complex, interconnected architecture of cell-signaling networks makes it challenging to disentangle how cells process extracellular information to make decisions. We have developed an optogenetic approach to selectively activate isolated intracellular signaling nodes with light and use this method to follow the flow of information from the signaling protein Ras. By measuring dose and frequency responses in single cells, we characterize the precision, timing, and efficiency with which signals are transmitted from Ras to Erk. Moreover, we elucidate how a single pathway can specify distinct physiological outcomes: by combining distinct temporal patterns of stimulation with proteomic profiling, we identify signaling programs that differentially respond to Ras dynamics, including a paracrine circuit that activates STAT3 only after persistent (>1 hr) Ras activation. Optogenetic stimulation provides a powerful tool for analyzing the intrinsic transmission properties of pathway modules and identifying how they dynamically encode distinct outcomes.

2011

Toettcher, Jared E. et al. “Recycling Circuit Simulation Techniques for Mass-Action Biochemical Kinetics.” Advanced Simulation and Verification of Electronic and Biological Systems. New York, NY: Springer Science & Business Media, 2011. 115–136.

Many numerical techniques developed for analyzing circuits can be “recycled”—that is, they can be used to analyze mass-action kinetics (MAK) models of biological processes. But the recycling must be judicious, as the differences in behavior between typical circuits and typical MAK models can impact a numerical technique’s accuracy and efficiency. In this chapter, we compare circuits and MAK models from this numerical perspective, using illustrative examples, theoretical comparisons of properties such as conservation and invariance of the non-negative orthant, as well as computational results from biological system models.

Toettcher, Jared E. et al. “Biochemical Oscillator Sensitivity Analysis in the Presence of Conservation Constraints.” Proceedings of the 48th Design Automation Conference 2011: 806–811.

Computing parametric sensitivities for oscillators has a now well-understood subtlety associated with the indeterminacy of phase. A less universal, but still vexing, subtlety arises when an oscillator is described by a system of differential equations with "hidden" conservation constraints (HCC's); defined as weighted sums of state variables that are time-invariant. If there are HCC's, as is commonly the case for models of biochemical oscillators but rarely the case for practical circuit oscillators, the now-standard approach to computing parametric sensitivities can yield incorrect results. In addition, the monodromy matrix (the matrix of state sensitivities over one oscillation period), is often defective in a way that interferes with the usual approach to computing oscillator phase noise. In this paper we analyze the HCC case, and show that by augmenting the standard sensitivity approach with explicit HCC's, one can recover the correct parametric sensitivities. In addition, we prove that there is a typically satisfied condition that guarantees that a system with HCCs will have a defective monodromy matrix. A deliberately "flawed" ring oscillator circuit and a cyanobacterial circadian clock biochemical oscillator are used to demonstrate the parametric sensitivity problem and its resolution, and to show the issue of the defective monodromy matrix.

Toettcher, Jared et al. “Light-Based Feedback for Controlling Intracellular Signaling Dynamics..” Nat Methods 8.10 (2011): 837–9.
The ability to apply precise inputs to signaling species in live cells would be transformative for interrogating and understanding complex cell-signaling systems. Here we report an 'optogenetic' method for applying custom signaling inputs using feedback control of a light-gated protein-protein interaction. We applied this strategy to perturb protein localization and phosphoinositide 3-kinase activity, generating time-varying signals and clamping signals to buffer against cell-to-cell variability or changes in pathway activity.