#Icircuit free free#

Curves represent single cells (gray lines, n = 11 cells) and the mean of all cells (black line) from a single experiment. Blue bars indicate periods of optogenetic illumination. ( b) Single-cell traces showing nuclear ErkKTR-iRFP during cycles of optogenetic stimulation. Phosphorylation of the ErkKTR-irFP biosensor by Erk leads to its export from the nucleus. Upon recruitment to the membrane, SOScat activates Ras/Erk signaling. Blue light drives heterodimerization between tagRFP-SSPB-SOScat and membrane-localized iLID-CAAX. ( a) Diagram of the OptoSOS system and ErkKTR biosensor. Inset shows simulated results from the model from h, with (green) or without (gray) negative feedback. ( i) Quantification of flow cytometry data in g, showing data from 2 experimental replicates. An input u (gold) stimulates intermediate node x 1 (blue), which produces a negative regulator x 3 (purple) that inhibits the production of x 1. ( h) An extended mathematical model of the READer circuit incorporating negative feedback on Erk target gene induction. ( g) GFP histograms obtained by flow cytometry for the experimental workflow in f. Serum inputs of varying duration were applied to cells, which were fixed 3 h after the end of the pulse. ( f) Mapping how pulse duration affects READer circuit output. Data is quantified from 2 wells each for 3 independent replicates. ( e) Quantification of data from d in all three conditions.

#Icircuit free free#

( d) Flow cytometry histograms of GFP from parental NIH3T3 (gray) or READer cells (green) incubated in growth factor free media (constant OFF), 10% serum (constant ON) or a one-hour pulse of 10% serum. ( c) Images of NIH3T3 READer cells exposed to constant serum or a 1 h serum pulse. In response to an input pulse, KGV is first expressed and then imported into the nucleus, leading to high GFP expression. A constant-on stimulus drives KGV expression and nuclear export, preventing GFP production. Under constant-off stimuli, KGV and GFP levels remain low. ( b) Illustration of READer circuit logic. Destabilization (PEST) sequences are added to both components to ensure rapid degradation and ‘resetting’ in the absence of stimulus. An Erk-responsive promoter drives expression of a KTR-Gal4-VP64 (KGV) fusion protein, which in turn triggers expression of a GFP reporter. ( a) Schematic of the Recorder of Erk Activity Dynamics (READer) circuit. ( e) Representative time course of Circuit 7 simulated with either a constant-on (left) or pulsed input (right), along with the model equations representing Circuit 7. Each point represents a single parameterization of an FFL circuit colored as in b (left), or with only Circuit 7 colored (right). ( d) Responses of all circuits from the computational screen. Circuits exhibiting pulse detection would lie in quadrant 3, where responses to pulsed inputs exceed those of either ON or OFF stimuli. ( c) Computational screen workflow: constant-off, constant-on and pulsed inputs were applied to 10,000 random parameterizations of each network shown in b. ( b) Coherent and incoherent feed-forward loop (FFL) topologies screened for pulse detection. In an alternative workflow, dynamics could be inferred from a single fluorescence measurement, enabling rapid and scalable isolation of dynamic cells for downstream analyses. Currently, signaling dynamics are typically obtained from live-imaging of individual cells, limiting throughput. ( a) Workflows for studying signaling dynamics. READer circuits open the door to permanently labeling transient, dynamic cell populations to elucidate the mechanistic underpinnings and biological consequences of signaling dynamics.Ĭell signaling network motifs signaling dynamics synthetic biology.Ĭopyright © 2021. Our 'recorder of Erk activity dynamics' (READer) responds sensitively to spontaneous and stimulus-driven Erk pulses. We implemented the motif experimentally for the Erk signaling pathway using a single engineered transcription factor and fluorescent protein reporter. We computationally screened for circuits with the capability to selectively detect signaling pulses, revealing an incoherent feedforward topology that robustly performs this computation. Here, we describe an approach to infer pulsatile signaling dynamics from a single measurement in fixed cells using a pulse-detecting gene circuit. 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 resolution. Cells employ intracellular signaling pathways to sense and respond to changes in their external environment.