Biology subjects

Toettcher, J. E.

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

At least 19 recordsLinked to original sources

bioRxiv2026

The regulatory logic of a dose-dependent developmental fate decision

In canonical developmental patterning, the embryo is exposed to gradients of signaling activators that elicit different cellular responses depending on the activators concentration. Recent optogenetic studies of terminal ERK signaling downstream of Torso receptor tyrosine kinase in the early Drosophila embryo reveal that even a brief, 5-minute ERK stimulus is sufficient to rescue the development of larval "tail" structures. Here, we reveal components of the molecular network that defines this sensitive developmental fate response. We find that low ERK doses produce sustained Abdominal-B (Abd-B) expression comparable to that of wild-type embryos. Abd-B expression is adjacent to, but non-overlapping with, two other transcriptional repressors: the ERK effector Tailless (Tll) and the gap gene Giant (Gt). Analysis of gene expression patterns in response to optogenetic perturbations suggests that the Tll-dependent repression of gt constitutes the sensitive ERK-responsive step: even low tll expression leads to potent repression of gt in nearby regions, with Abd-B expression arising in a stripe between the tll and gt domains. Our work suggests that the spectrum of phenotypes produced through optogenetic manipulation can be used to define how robust patterning can arise from low doses of inductive signals. HighlightsO_LIA very low dose of receptor tyrosine kinase signaling in the early embryo induces tail formation many hours later. C_LIO_LITransient ERK activity results in stable Abd-B expression, beginning as a stripe in nuclear cycle 14. C_LIO_LIOptogenetic ERK inputs induce a spectrum of ectopic phenotypes that reveal mutually exclusive expression of Tailless, Giant, and Abd-B in the posterior. C_LIO_LITailless-dependent repression of giant is the sensitive ERK-responsive step. C_LI

developmental biology↗

bioRxiv2025

PyCLM: programming-free, closed-loop microscopy for real-time measurement, segmentation, and optogenetic stimulation

In cell biology, optical techniques are increasingly used to measure cells internal states (biosensors) and to stimulate cellular responses (optogenetics). Yet the design of all-optical experiments is often manual: a pre-determined stimulus pattern is applied to cells, biosensors are measured over time, and the resulting data is processed off-line. With the advent of machine learning for segmentation and tracking, it becomes possible to envision closed-loop experiments where real-time information about cells positions and states are used to dynamically determine optogenetic stimuli to alter or control their behavior. Here, we develop PyCLM, a Python-based suite of tools to enable real-time measurement, image segmentation, and optogenetic control of thousands of cells per experiment. PyCLM is designed to be as simple for the end user as possible, and multipoint experiments can be set up that combine a wide variety of imaging, image processing, and stimulation modalities without any programming. We showcase PyCLM on diverse applications: studying the effect of epidermal growth factor receptor activity waves on epithelial tissue movement, simultaneously stimulating [~]1,000 single cells to guide tissue flows, and performing real-time feedback control of cell-to-cell fluorescence heterogeneity. This tool will enable the next generation of dynamic experiments to probe cell and tissue properties, and provides a first step toward precise control of cell states at the tissue scale.

systems biology↗

bioRxiv2025

Multimodal control of Cas13 activity through domain insertion at an allosteric hotspot

CRISPR-Cas13 RNA nucleases have emerged as powerful tools for programmable RNA targeting. A light-controlled RNA nuclease could be transformative by enabling researchers to selectively knock down transcripts at desired positions in a cell or tissue or at timepoints of interest. Here, we develop a set of multimodal RfxCas13d tools that can be controlled by either light or small molecule addition. Screening an RfxCas13d library containing insertions of the AsLOV2 photoswitchable domain revealed an OptoCas13d-off variant that induced target RNA cleavage in the dark and switched to an inactive state under blue light. Insertion at this same allosteric hotspot could be further exploited to generate an OptoCas13d-on with the opposite light dependence and a ChemoCas13d that is activated upon the addition of rapamycin analogs. Through biochemical assays, we showed that AsLOV2 domain switching did not substantially affect Cas13d-RNA complex formation, indicating allosteric control over Cas13d catalytic activity. We applied the OptoCas13d-on system to target several endogenous transcripts and showed that it exhibited efficient mRNA knockdown only upon blue light illumination. Overall, our results demonstrate that engineered OptoCas13d can achieve cellular RNA modulation with high spatial and temporal precision.

bioengineering↗

bioRxiv2025

Design and optimization of a kinase-controlled allosteric switch

Post-translational control enables rapid and precise regulation of cell behavior. Despite these advantages, general strategies to build phosphorylation-based synthetic circuits are limited. Here, we reasoned that engineered allostery, a technique that has been applied to design light- and chemically-gated protein switches, could also be used to engineer phosphorylation-controlled protein switches (phospho-switches). Using an allosterically controllable Gal4 transcription factor as a scaffold, we show that a classic kinase FRET biosensor architecture can be used as a starting point for phospho-switch design. We optimize all features of the phospho-switch to develop an ERK-controlled transcription factor with a 20-fold phosphorylation-dependent change in transcriptional output. Our switch outperforms the c-fos promoter, a classic ERK-responsive transcriptional biosensor, in selectivity and sensitivity. We further show that our switch architecture can be generalized to other input kinases and allosterically controlled targets. This work provides a general platform for a new generation of kinase-responsive tools for biosensing and synthetic biology applications.

synthetic biology↗

bioRxiv2025

In vivo measurements of receptor tyrosine kinase activity reveal feedback regulation of a developmental gradient

A lack of tools for detecting receptor activity in vivo has limited our ability to fully explore receptor-level control of developmental patterning. Here, we extend a new class of biosensors for receptor tyrosine kinase (RTK) activity, the pYtag system, to visualize endogenous RTK activity in Drosophila. We build biosensors for three Drosophila RTKs that function across developmental stages and tissues. By characterizing Torso::pYtag during terminal patterning in the early embryo, we find that Torso activity differs from downstream ERK activity in two surprising ways: Torso activity is narrowly restricted to the poles but produces a broader gradient of ERK, and Torso activity decreases over developmental time while ERK activity is sustained. This decrease in Torso activity is driven by ERK pathway-dependent negative feedback. Our results suggest an updated model of terminal patterning where a narrow domain of Torso activity, tuned in amplitude by negative feedback, locally activates signaling effectors which diffuse through the syncytial embryo to form the ERK gradient. Altogether, this work highlights the usefulness of pYtags for investigating receptor-level regulation of developmental patterning.

developmental biology↗

bioRxiv2025

CD44 and Ezrin restrict EGF receptor mobility to generate a novel spatial arrangement of cytoskeletal signaling modules driving bleb-based migration

Cells under high confinement form highly polarized hydrostatic pressure-driven, stable leader blebs that enable efficient migration in low adhesion, environments. Here we investigated the basis of the polarized bleb morphology of metastatic melanoma cells migrating in non-adhesive confinement. Using high-resolution time-lapse imaging and specific molecular perturbations, we found that EGF signaling via PI3K stabilizes and maintains a polarized leader bleb. Protein activity biosensors revealed a unique EGFR/PI3K activity gradient decreasing from rear-to-front, promoting PIP3 and Rac1-GTP accumulation at the bleb rear, with its antagonists PIP2 and RhoA-GTP concentrated at the bleb tip, opposite to the front-to-rear organization of these signaling modules in integrin-mediated mesenchymal migration. Optogenetic experiments showed that disrupting this gradient caused bleb retraction, underscoring the role of this signaling gradient in bleb stability. Mathematical modeling and experiments identified a mechanism where, as the bleb initiates, CD44 and ERM proteins restrict EGFR mobility in a membrane-apposed cortical actin meshwork in the bleb rear, establishing a rear-to-front EGFR-PI3K-Rac activity gradient. Thus, our study reveals the biophysical and molecular underpinnings of cell polarity in bleb-based migration of metastatic cells in non-adhesive confinement, and underscores how alternative spatial arrangements of migration signaling modules can mediate different migration modes according to the local microenvironment.

cell biology↗

bioRxiv2024

Efficient Genome Editing with Chimeric Oligonucleotide-Directed Editing

Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report new nCas9-DNA polymerase fusion proteins to create chimeric oligonucleotide-directed editing (CODE) systems for search-and-replace genome editing. Through successive rounds of engineering, we developed CODEMax and CODEMax(exo+) editors that achieve efficient genome modifications in human cells with low unintended edits. CODEMax and CODEMax(exo+) contain an engineered Bst DNA polymerase derivative known for its robust strand displacement ability. Additionally, CODEMax(exo+) features a 5 to 3 exonuclease activity that promotes effective strand invasion and repair outcomes favoring the incorporation of the desired edit. We demonstrate CODEs can perform small insertions, deletions, and substitutions with improved efficiency compared to PEMax at many loci. Overall, CODEs complement existing prime editors to expand the toolbox for genome manipulations without double-stranded breaks.

bioengineering↗

bioRxiv2024

Large-scale control over collective cell migration using light-controlled epidermal growth factor receptors

Receptor tyrosine kinases (RTKs) are thought to play key roles in coordinating cell movement at single-cell and tissue scales. The recent development of optogenetic tools for controlling RTKs and their downstream signaling pathways suggested these responses may be amenable to engineering-based control for sculpting tissue shape and function. Here, we report that a light-controlled EGF receptor (OptoEGFR) can be deployed in epithelial cell lines for precise, programmable control of long-range tissue movements. We show that in OptoEGFR-expressing tissues, light can drive millimeter-scale cell rearrangements to densify interior regions or produce rapid outgrowth at tissue edges. Light-controlled tissue movements are driven primarily by PI 3-kinase signaling, rather than diffusible signals, tissue contractility, or ERK kinase signaling as seen in other RTK-driven migration contexts. Our study suggests that synthetic, light-controlled RTKs could serve as a powerful platform for controlling cell positions and densities for diverse applications including wound healing and tissue morphogenesis.

bioengineering↗

bioRxiv2024

Rapid and reversible dissolution of biomolecular condensates using light-controlled recruitment of a solubility tag

Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function--dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles, a major question in cell biology and drug development. Here we report an optogenetic approach to selectively dissolve a condensate of interest in a reversible and spatially controlled manner. We show that light-gated recruitment of maltose-binding protein (MBP), a commonly used solubilizing domain in protein purification, results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP, showing that disrupting condensation of the oncogenic fusion protein FUS-CHOP results in reversion of FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.

bioengineering↗

bioRxiv2023

Recording morphogen signals reveals origins of gastruloid symmetry breaking

When cultured in three dimensional spheroids, mammalian stem cells can reproducibly self-organize a single anterior-posterior axis and sequentially differentiate into structures resembling the primitive streak and tailbud. Whereas the embryos body axes are instructed by spatially patterned extra-embryonic cues, it is unknown how these stem cell gastruloids break symmetry to reproducibly define a single anterior-posterior (A-P) axis. Here, we use synthetic gene circuits to trace how early intracellular signals predict cells future anterior-posterior position in the gastruloid. We show that Wnt signaling evolves from a homogeneous state to a polarized state, and identify a critical 6-hour time period when single-cell Wnt activity predicts future cellular position, prior to the appearance of polarized signaling patterns or morphology. Single-cell RNA sequencing and live-imaging reveal that early Wnt-high and Wnt-low cells contribute to distinct cell types and suggest that axial symmetry breaking is driven by sorting rearrangements involving differential cell adhesion. We further extend our approach to other canonical embryonic signaling pathways, revealing that even earlier heterogeneity in TGF{beta} signaling predicts A-P position and modulates Wnt signaling during the critical time period. Our study reveals a sequence of dynamic cellular processes that transform a uniform cell aggregate into a polarized structure and demonstrates that a morphological axis can emerge out of signaling heterogeneity and cell movements even in the absence of exogenous patterning cues. HighlightsO_LISymmetry-breaking gastruloid protocol where Wnt signaling evolves from a uniform high state to a single posterior domain. C_LIO_LISynthetic gene circuits record Wnt, Nodal and BMP signaling with high time resolution. C_LIO_LIHeterogeneity in Wnt signaling at 96 h predicts cells future positions and types. C_LIO_LIWnt activity differences are influenced by earlier heterogeneity in Nodal/BMP activity. C_LI

developmental biology↗

bioRxiv2023

Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo

Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. Here we use optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe of brachyenteron (byn) expression in the early Drosophila embryo. This byn stripe depends on interpretation of an upstream signal - a gradient of ERK kinase activity - and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce either transient or sustained byn expression, respectively. These ERK stimuli also regulate tll and hkb expression with distinct dynamics: tll transcription is rapidly induced under both low and high stimuli, whereas hkb transcription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustained byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that an all-or-none stimulus can be blurred through intracellular diffusion to non-locally produce a stripe of byn gene expression. Overall, our study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.

developmental biology↗

bioRxiv2023

Control of gastruloid patterning and morphogenesis by the Erk and Akt signaling pathways

Fibroblast growth factor (FGF) dependent elongation along an anterior-posterior (A-P) axis is a conserved feature of vertebrate embryogenesis. A-P axis elongation can also be reproduced in 3D cell culture models termed gastruloids, enabling dissection of this process in a controlled, minimal context. Here, we set out to determine how gastruloid posterior elongation depends on the Erk and Akt pathways, canonical downstream effectors of FGF signaling. We find that gastruloids exhibit reproducible posterior-to-anterior gradients in Erk and Akt phosphorylation that are generated independently and correlate with distinct zones of tissue movement, cell proliferation, and expression of cell motility and adhesion regulators. Pharmacological inhibition of FGFR, Erk, or Akt signaling impairs gastruloid elongation, and quantification of signaling gradients reveals how these patterns interact and scale with A-P axis length. Using global inhibitors and activators of each pathway, we find that a gradient of Ras/Erk signaling is required for the establishment of appropriately localized domains of E-cadherin, Snail, and Brachyury expression, whereas perturbing PI3K/Akt signaling alters proliferation but not patterning. Taken together, our data demonstrate that graded PI3K/Akt and Ras/Erk signaling provide spatial information to control proliferation and cell-cell adhesion during gastruloid elongation.

developmental biology↗

bioRxiv2022

pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells

Receptor tyrosine kinases (RTKs) are major signaling hubs in metazoans, playing crucial roles in cell proliferation, migration, and differentiation. However, few tools are available to measure the activity of a specific RTK in individual living cells. Here, we present pYtags, a modular approach for monitoring the activity of a user-defined RTK by live-cell microscopy. pYtags consist of an RTK modified with a tyrosine activation motif that, when phosphorylated, recruits a fluorescently labeled tandem SH2 domain with high specificity. We show that pYtags enable the monitoring of a specific RTK on seconds-to-minutes time scales and across subcellular and multicellular length scales. Using a pYtag biosensor for epidermal growth factor receptor (EGFR), we quantitively characterize how signaling dynamics vary with the identity and dose of activating ligand. We show that orthogonal pYtags can be used to monitor the dynamics of EGFR and ErbB2 activity in the same cell, revealing distinct phases of activation for each RTK. The specificity and modularity of pYtags opens the door to robust biosensors of multiple tyrosine kinases and may enable engineering of synthetic receptors with orthogonal response programs. HighlightsO_LIpYtags report on signaling of user-defined RTKs in living cells C_LIO_LIEGFR signaling dynamics depend on ligand identity and dimer strength C_LIO_LIrthogonal pYtags enable reporter multiplexing C_LIO_LIpYtags can report on signaling of endogenously expressed RTKs C_LI

cell biology↗

bioRxiv2021

Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation

Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. Here we show that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal quantitative regulatory dynamics from a complex genetic network in vivo. We engineer light-controlled variants of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. Our results recapitulate known relationships, including rapid Bicoid-dependent expression of giant and hunchback and delayed repression of Kruppel. In contrast, we find that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a previously unreported role for Bicoid in suppressing knirps expression. Acute modulation of transcription factor concentration while simultaneously recording output gene activity represents a powerful approach for studying how gene circuit elements are coupled to cell identification and complex body pattern formation in vivo.

developmental biology↗

bioRxiv2021

Dynamics and heterogeneity of Erk-induced immediate-early gene expression

Many canonical signaling pathways exhibit complex time-varying responses, yet how minutes-timescale pulses of signaling interact with the dynamics of transcription and gene expression remains poorly understood. Erk-induced immediate early gene (IEG) expression is a model of this interface, exemplifying both dynamic pathway activity and a rapid, potent transcriptional response. Here, we quantitatively characterize IEG expression downstream of dynamic Erk stimuli in individual cells. We find that IEG expression responds rapidly to acute changes in Erk activity, but only in a sub-population of stimulus-responsive cells. We find that while Erk activity partially predicts IEG expression, a majority of response heterogeneity is independent of Erk and can be rapidly tuned by different mitogenic stimuli and parallel signaling pathways. We extend our findings to an in vivo context, the mouse epidermis, where we observe heterogenous immediate-early gene accumulation in both fixed tissue and single-cell RNA-sequencing data. Our results demonstrate that signaling dynamics can be faithfully transmitted to gene expression and suggest that the signaling-responsive population is an important parameter for interpreting gene expression responses.

cell biology↗

bioRxiv2021

A synthetic gene circuit for imaging-free detection of dynamic cell signaling

Cells employ intracellular signaling pathways to sense and respond to changes in their external environment. In recent years, live-cell biosensors have revealed complex pulsatile dynamics in many pathways, but studies of these signaling dynamics are limited by the necessity of live-cell imaging at high spatiotemporal resolution1. Here, we describe an approach to infer pulsatile signaling dynamics from just a single measurement in fixed cells using a pulse-detecting gene circuit. We computationally screened for circuit with pulse detecting capability, revealing an incoherent feedforward topology that robustly performs this computation. We then implemented the motif experimentally for the Erk signaling pathway using a single engineered transcription factor and fluorescent protein reporter. Our recorder of Erk activity dynamics (READer) responds sensitively to both spontaneous and stimulus-driven Erk pulses. READer circuits thus open the door to permanently labeling transient, dynamic cell populations to elucidate the mechanistic underpinnings and biological consequences of signaling dynamics.

systems biology↗

bioRxiv2020

Optogenetic Amplification Circuits for Light-Induced Metabolic Control

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 21.8-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 doses as low as ~1%, and strong homogeneous light-induction in bioreactors of at least 5L, with limited illumination at cell densities above 40 OD600. 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.

synthetic biology↗

bioRxiv2020

A self-exciting point process to study multi-cellular spatial signaling patterns

Multi-cellular organisms rely on spatial signaling among cells to drive their organization, development, and response to stimuli. Several models have been proposed to capture the behavior of spatial signaling in multi-cellular systems, but existing approaches fail to capture both the autonomous behavior of single cells and the interactions of a cell with its neighbors simultaneously. We propose a spatiotemporal model of dynamic cell signaling based on Hawkes processes--self-exciting point processes--that model the signaling processes within a cell and spatial couplings between cells. With this cellular point process (CPP) model, we capture both the single-cell protein bursting rate and the magnitude and duration of signaling between cells relative to spatial locations. Furthermore, our model captures tissues composed of heterogeneous cell types with different bursting rates and signaling behaviors across multiple signaling proteins. We apply our model to epithelial cell systems that exhibit a range of autonomous and spatial signaling behaviors basally and under pharmacological exposure. Our model identifies known drug-induced signaling deficits, characterizes differences in signaling across a wound front, and generalizes to multi-channel observations.

cell biology↗