We 1st exclude TrackMate-tracked nuclei on either of two conditions: (1) they do not express both H2B and KTR, or (2) if H2B intensity changes dramatically on the imaging timecourse, indicating a tracking error (Number S1ACB). for 4 h under continuous tradition in GF-free press supplemented with different doses of EGF (indicated above). Timer shows hh:mm; scale pub shows 30 m. NIHMS1572046-product-3.avi (3.7M) GUID:?9EAD23AB-0F11-428B-A21C-1C540DF12BCD 4: FGD4 Movie S3, related to Number 3. Time-lapse imaging of main mouse keratinocytes expressing the H2B-RFP nuclear marker (not demonstrated) and KTR-BFP Erk activity biosensor (demonstrated). Cells were imaged using a 10X air flow objective every 3 min for 5 h under continuous tradition in GF-free press supplemented with the kinase inhibitors indicated at a concentration of 2.5 M. Timer shows hh:mm; scale pub shows 30 m. NIHMS1572046-product-4.avi (5.4M) GUID:?C95D8BBE-8885-4200-A294-E33A54E849DB 5: Movie S4, related to Number 4. Time-lapse imaging of main mouse keratinocytes expressing the H2B-RFP nuclear marker (not demonstrated) and KTR-BFP Erk activity biosensor (demonstrated). Cells were imaged using a 20X air flow objective every 3 min for 16 h under continuous tradition in GF-free press supplemented the kinase inhibitors indicated at a concentration of 2.5 M or neutralizing antibodies against Met or VEGFR2. Timer shows hh:mm; scale pub shows 30 m. NIHMS1572046-product-5.avi (15M) GUID:?E9F69BCB-E49C-4317-9ABE-A41578986F21 6: Movie S5, related to Number 6. Time-lapse imaging of main mouse keratinocytes expressing the OptoSOS system (not demonstrated), H2B-RFP nuclear marker (not demonstrated) and KTR-iRFP Erk activity biosensor (demonstrated). Cells were imaged using a 20X air flow objective every 90 sec for 15 h under continuous tradition in GF-free press. At t = 2 h (as indicated from the +EGFRi label) cells were treated with 2.5 M lapatinib. At t = 3 h, cells were stimulated with 15 min pulses of 450 nm blue light. Blue package indicates instances of light delivery. Timer shows hh:mm; scale pub shows 30 m. NIHMS1572046-product-6.avi (5.3M) GUID:?9E6CD927-98D0-4189-90D2-6B24127FDB5C 7. NIHMS1572046-product-7.pdf (2.3M) GUID:?C4843C25-E436-4998-8148-857B25EDC4DA Data Availability StatementAll Jython and MATLAB code is definitely available on Github (github.com/toettchlab/Goglia2019). All time-lapse microscopy data from your small-molecule display will be available at the Image Data Source (idr.openmicroscopy.org/; accession quantity forthcoming). Abstract Complex, time-varying reactions have been observed widely in cell signaling, isoquercitrin but how specific dynamics are generated or regulated is largely unknown. One major obstacle has been that high-throughput screens are typically incompatible with isoquercitrin the live-cell assays used to monitor dynamics. Here, we address this challenge by screening a library of 429 kinase inhibitors and isoquercitrin monitoring Erk activity over 5 hours in more than 80,000 single main mouse keratinocytes. Our screen revealed both known and uncharacterized modulators of Erk dynamics, including inhibitors of non-EGFR receptor tyrosine kinases (RTKs) that increased Erk pulse frequency and overall activity. Using drug treatment and direct optogenetic control, we demonstrate that drug-induced changes to Erk dynamics alter the conditions under which cells proliferate. Our work opens the door to high-throughput screens using live-cell biosensors and reveals that cell proliferation integrates information from Erk dynamics as well as additional permissive cues. eTOC blurb Goglia et al. recognized modulators of ERK dynamics by screening a library of 429 kinase inhibitors and monitoring Erk activity over 5 hours in more than 80,000 single main mouse keratinocytes. They recognized both known and uncharacterized modulators, including inhibitors of non-EGFR receptor tyrosine kinases (RTKs) that increased Erk pulse frequency and overall activity. Their 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. Graphical Abstract Introduction Animal cells must respond to a large number of external cues to function isoquercitrin appropriately during development and adult tissue homeostasis. To that end, a typical mammalian cell is usually endowed with hundreds of unique receptors, yet only a few signaling pathways downstream of these receptors are tasked with responding to these many inputs. For instance, the 58 human receptor tyrosine kinases (RTKs) activate around the order of ten intracellular pathways (e.g., Ras/Erk, PI3K/Akt, Src, PLC, calcium), yet can trigger diverse downstream cellular responses in developing and adult tissues (Downward, 2001; Lemmon and Schlessinger, 2010). Cells are thus faced with the challenge of accurately transmitting information from many upstream inputs using only a few wires or transmission transduction pathways. One resolution to this paradox comes in the form of dynamic regulation. Two receptors may trigger different time-varying responses from a single pathway, which can then be interpreted into unique fates (Marshall, 1995). Indeed, many core mammalian signaling pathways have now been observed to generate complex, time-varying signaling behaviors in response to certain input stimuli (Purvis and Lahav, 2013). A growing body of evidence suggests that these dynamics are relevant to normal cell function: Erk and p53 pulses have been observed with.