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Cell Biology

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

Department of Chemical & Biological Engineering, Princeton University, United States
Department of Bioengineering, Rice University, United States
Program in Systems, Synthetic, and Physical Biology, Rice University, United States
Department of Molecular Biology, Princeton University, United States
IRCC International Research Collaboration Center, National Institutes of Natural Sciences, Japan
Department of Biosciences, Rice University, United States

May 22, 2023

https://doi.org/10.7554/eLife.82863

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Figures
Tables
Additional files

6 figures, 3 tables and 1 additional file

Figures

Figure 1 with 5 supplements

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pYtags: a biosensing strategy to monitor receptor tyrosine kinase (RTK) activity in living cells.

(A) The T-cell receptor complex contains six immunoreceptor tyrosine-based activation motifs (ITAMs) from CD3 chains that, when phosphorylated, bind to the tSH2 domain of ZAP70 (ZtSH2). (B) Three repeats of CD3 ITAMs were appended to the C-terminus of epidermal growth factor receptor (EGFR) and clearance of ZtSH2 from the cytosol was assessed. (C) Timelapse images of NIH3T3 cells expressing EGFR pYtag (CD3ε variant), treated first with EGF (100 ng/mL) and then with gefitinib (10 µM). Scale bar, 20 µm. (D) Mean clearance of cytosolic ZtSH2 in cells co-expressing iRFP-ZtSH2 and EGFR C-terminally labeled with one of six CD3 ITAMs. EGF (100 ng/mL) and gefitinib (10 µM) were sequentially added at times denoted by arrows. n = 2 independent experiments. (E) Clearance of cytosolic ZtSH2 10 min post-EGF treatment and 40 min post-gefitinib treatment from (D). Lines denote mean values, boxes denote 25–75th percentiles, and whiskers denote minima and maxima. n ≥ 14 cells from two independent experiments. n.s., not significant, ***p<0.001 by Kolmogorov–Smirnov test with cells expressing no additional EGFR 10 min post-EGF. (F) Immunoblots of NIH3T3 cells expressing either WT EGFR or EGFR pYtag treated with EGF (100 ng/mL). (G) Mean ± SEM levels of EGFR, Akt, and Erk phosphorylation from (F). n = 3 independent experiments. (H) The EGFR pYtag was tested in SYF cells to determine whether SFKs are required for ITAM phosphorylation. (I) Representative images of NIH3T3 and SYF cells expressing EGFR pYtag, treated with EGF (100 ng/mL). Scale bars, 40 µm. (J) Mean clearance of cytosolic ZtSH2 in SYF and NIH3T3 cells 10 min after treatment with EGF. For each condition, n > 20 cells from three independent experiments. See also Figure 1—video 1.

Figure 1—source data 1 Uncropped gels for Figure 1F.: https://cdn.elifesciences.org/articles/82863/elife-82863-fig1-data1-v1.zip
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Figure 1—figure supplement 1

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Grb2 fails to discriminate between immunoreceptor tyrosine-based activation motif (ITAM)-labeled and unlabeled RTKs.

(A) ZtSH2- and Grb2-based reporters of receptor tyrosine kinase (RTK) signaling were co-expressed in cells expressing either ITAM-labeled or WT epidermal growth factor receptor (EGFR). (B) NIH3T3 cells before and 5 min after treatment with EGF (100 ng/mL). Scale bars, 20 µm. (C) Mean ± SD clearance of ZtSH2 and Grb2 reporters from the cytosol following treatment with EGF (100 ng/mL). n = 3 independent experiments. (D) Clearance of reporters from the cytosol 5 min after treatment with EGF in (C). Lines denote mean values, boxes denote 25–75th percentiles, and whiskers denote minima and maxima. For each condition, n > 16 cells from three independent experiments. (E) ZtSH2 reports the signaling of ITAM-labeled RTKs, while Grb2 fails to discriminate between RTKs displaying or lacking ITAMs.

Figure 1—figure supplement 2

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Effects of the expression levels of pYtag components in NIH3T3 cells.

Images of EGFR-pYtag expressing NIH3T3 cells expressing various levels of (A) EGFR-CD3ε-FusionRed or (B) iRFP-ZtSH2, treated with EGF (20 ng/mL). Scale bars, 20 µm. (C) Maximum % clearance of ZtSH2 from the cytosol in EGFR-pYtag expressing NIH3T3 cells treated with EGF (20 ng/mL). (D) Maximum % clearance of ZtSH2 from the cytosol as a function of the ratio of EGFR-CD3ε-FusionRed to iRFP-ZtSH2 (EGFR:ZtSH2 ratio). (**E, F**) Heatmaps of single-cell trajectories for all cells quantified in (C) and sorted in order of increasing EGFR:ZtSH2 ratio. (E) shows the % clearance of ZtSH2 from the cytosol; (F) shows min–max normalized responses to compare activation kinetics. Panels (**C–E**): points denote individual cells. n = 86 cells from three independent experiments.

Figure 1—figure supplement 3

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Effects of the expression levels of pYtag components in HEK293T cells.

Images of EGFR-pYtag expressing HEK293T cells expressing variable levels of (A) EGFR-CD3ε-mNeonGreen or (B) mScarlet-ZtSH2, treated with EGF (100 ng/mL). Scale bars, 20 µm. (C) Maximum % clearance of ZtSH2 from the cytosol in EGFR-pYtag expressing HEK293T cells treated with EGF (100 ng/mL). (D) Maximum % clearance of ZtSH2 from the cytosol as a function of the ratio of EGFR-CD3ε-mNeonGreen to mScarlet-ZtSH2 (EGFR:ZtSH2 ratio). (**E, F**) Heatmaps of single-cell trajectories for all cells quantified in (C) and sorted in order of increasing EGFR:ZtSH2 ratio. (E) shows the % clearance of ZtSH2 from the cytosol; (F) shows min–max normalized responses to compare activation kinetics. Panels (**C, D**): points denote individual cells. n = 84 cells from three independent experiments.

Figure 1—figure supplement 4

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Mathematical modeling of the effects of pYtag component expression levels on biosensor readout.

(A) Heatmap of maximum % clearance of ZtSH2 in response to 20 ng/mL EGF. For each cell, rows and columns denote levels of EGFR-CD3ε and ZtSH2, respectively. (B) Each cell from (A) was plotted as a function of the ratio of EGFR to ZtSH2 (EGFR:ZtSH2 ratio). Right plot presents zoomed-in results for EGFR:ZtSH2 ratios between 0.5 and 2.

Figure 1—video 1

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posterframe for video — Timelapse of iRFP-ZtSH2 in NIH3T3 cells co-expressing iRFP-ZtSH2 and EGFR-CD3ε-FusionRed.

Cells were first treated with EGF (100 ng/mL) then treated with gefitinib (10 µM) at the times denoted in the video. Related to Figure 1C.

Figure 2 with 2 supplements

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Monitoring epidermal growth factor receptor (EGFR) signaling at subcellular and multicellular length scales.

(A) Images of EGFR pYtag-expressing NIH3T3 cells treated with EGF (20 ng/mL). Scale bar, 20 µm. (B) Mean ± SD clearance of cytosolic ZtSH2 from (A). n = 3 independent experiments. (C) EGFR pYtag-expressing NIH3T3 cells treated with EGF (20 ng/mL) were monitored for internalized ZtSH2-positive vesicles, and then treated with gefitinib (10 µM). Scale bar, 20 µm. (D) Timelapse images from the region denoted by the blue dashed border from (C). Scale bar, 10 µm. (E) MCF10A human mammary epithelial cells cultured on soft substrata form round, multilayered clusters. EGFR pYtag and ErkKTR were used to spatiotemporally monitor both EGFR and Erk responses after stimulation with EGF. (F) Images of MCF10A cells cultured on soft substrata and treated with EGF (100 ng/mL). Scale bar, 25 µm. (G) Apical and lateral enrichment of ZtSH2 was quantified by line scans denoted by magenta and orange vectors, respectively. Scale bar, 10 µm. (H) Heatmaps of EGFR pYtag and ErkKTR responses from (F). Rows denote individual cells. For each cell, signaling responses of each biosensor were normalized to their respective minima and maxima. (I) Time to half maximal response for EGFR pYtag and ErkKTR for cells from (H). n = 19 (periphery) and n = 11 (interior) cells from three biological replicates. (J) Time to half maximal response for EGFR pYtag and ErkKTR. Responses of individual cells are denoted by points and connected by lines. n > 30 cells from three biological replicates. ***p<0.001 by Kolmogorov–Smirnov test. See also Figure 2—video 1.

Figure 2—figure supplement 1

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Analysis of epidermal growth factor receptor (EGFR) and ZtSH2 internalization.

(A) Images of control (EGFR-FusionRed) and EGFR pYtag (EGFR-CD3ε-FusionRed; iRFP-ZtSH2) NIH3T3 cells immunostained for EEA1 after treatment with EGF (100 ng/mL) for varying durations. Scale bars, 20 µm. (B) Intensity of EGFR-CD3ε-FusionRed at the cell membrane from (A). For each condition, n > 70 cells from three independent experiments. (C) Volume of pixels doubly positive for EGFR-CD3ε-FusionRed and EEA1 in individual cells. For each condition, n > 36 cells from three independent experiments. (D) Image of EGFR pYtag-expressing NIH3T3 cell treated with EGF (100 ng/mL) for 30 min and immunostained for ZtSH2 and EEA1. Scale bars, 20 µm (top) and 5 µm (bottom). (E) Fluorescence measurements along the red vector in (D). For each protein, intensities were normalized to the minimum and maximum values measured. (F) Image of EGFR pYtag-expressing HEK293T cell treated with EGF (100 ng/mL) for 5 min and immunostained for EEA1. Scale bars, 10 µm (top) and 3 µm (bottom). (G) Fluorescence measurements along the red vector in (F). For each protein, intensities were normalized to the minimum and maximum values measured.

Figure 2—video 1

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Figure 3 with 2 supplements

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Epidermal growth factor receptor (EGFR) pYtag reveals dose- and ligand-dependent signaling dynamics.

(A) Mean ± SD responses of EGFR pYtag-expressing NIH3T3 cells to varying doses of EGF, epiregulin (EREG), and epigen (EPGN). The same 0 ng/mL control was used for each ligand. Data were collected from 475 cells across four independent experiments with each dose tested at least twice. (B) Dose–response profiles from (A) were analyzed using a mathematical model of EGFR pYtag. (C) Simulations of EGFR pYtag responses to ligand of varying doses for different values of β and γ. (D) GBM-associated mutants of EGFR that form strong EREG-bound dimers were predicted to exhibit stronger pYtag responses to 20 ng/mL EREG compared to WT EGFR. (E) Mean ± SD pYtag response of WT and GBM-associated mutant EGFRs in NIH3T3 cells after EREG treatment (20 ng/mL). n = 3 independent experiments.

Figure 3—figure supplement 1

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ZtSH2 and epidermal growth factor receptor (EGFR) localization in response to different ligands.

Representative images of EGFR pYtag-expressing NIH3T3 cells treated with different concentrations of EGF, EREG, or EPGN. Scale bars, 20 µm.

Figure 3—figure supplement 2

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Ligand-free dimers in mathematical model recapitulate biphasic signaling response of epidermal growth factor receptor (EGFR).

(A) EGFR pYtag response to EGF (20 ng/mL) shown in Figure 2B. (B) The effect of ligand-free dimers on EGFR pYtag responses was simulated by tuning the dissociation rate of ligand-free dimers k₆. (C) Percentage of receptors existing as ligand-free dimers before ligand stimulation as a function of k₆. Color of data points corresponds to color of curves in (D). (D) Simulated EGFR pYtag responses to EGF for varying values of k₆.

Figure 4 with 1 supplement

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Monitoring distinct receptor tyrosine kinases (RTKs) in heterodimeric complexes.

(A) In order to signal, the ligandless ErbB2 must heterodimerize with a ligand-binding member of the ErbB family. The pYtag strategy enables measurements of ErbB2’s activity despite the co-activation of epidermal growth factor receptor (EGFR). (B) Representative images of NIH3T3 cells treated with EGF (100 ng/mL). Scale bar, 20 µm. (C) Mean ± SD clearance of cytosolic ZtSH2 after treatment with EGF (100 ng/mL). n = 3 independent experiments.

Figure 4—figure supplement 1

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pYtag biosensors of additional receptor tyrosine kinases (RTKs).

(A) Images of NIH3T3 cells co-expressing iRFP-ZtSH2 with either FGFR1-FusionRed or FGFR1-CD3ε-FusionRed, treated with FGF4 (100 ng/mL). Scale bars, 20 µm. (B) Mean ± SD clearance of cytosolic ZtSH2 in cells expressing FGFR1-FusionRed (ctrl) or FGFR1-CD3ε-FusionRed (CD3ε) from (A). n = 3 independent experiments. (C) Clearance of ZtSH2 from the cytosol 10 min after treatment with FGF4 in (B). (D) Images of NIH3T3 cells co-expressing iRFP-ZtSH2 with either PDGFRβ-FusionRed or PDGFRβ-CD3ε-FusionRed, treated with PDGF-BB (100 ng/mL). Scale bars, 20 µm. (E) Mean ± SD clearance of cytosolic ZtSH2 in cells expressing PDGFRβ-FusionRed (ctrl) or PDGFRβ-CD3ε-FusionRed (CD3ε) from (D). n = 3 independent experiments. (F) Clearance of ZtSH2 from the cytosol 10 min after treatment with PDGF-BB in (E). (G) Images of NIH3T3 cells co-expressing iRFP-ZtSH2 with either VEGFR3-FusionRed or VEGFR3-CD3ε-FusionRed, treated with VEGF-C (500 ng/mL). Scale bars, 20 µm. (H) Mean ± SD clearance of cytosolic ZtSH2 in cells expressing VEGFR3-FusionRed (ctrl) or VEGFR3-CD3ε-FusionRed (CD3ε) from (G). n = 3 independent experiments. (I) Clearance of ZtSH2 from the cytosol 10 min after treatment with VEGF-C in (H). Lines denote mean values, boxes denote 25–75th percentiles, and whiskers denote minima and maxima. For each condition, n > 38 cells from three independent experiments. ***p<0.001 by Kolmogorov–Smirnov test.

Figure 5 with 3 supplements

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Orthogonal pYtags enable multiplexed receptor tyrosine kinase (RTK) biosensing.

(A) To assess the performance of the VISH2/SLP76 system as a pYtag-based biosensor, VISH2 and ZtSH2 reporters were co-expressed in NIH3T3 cells along with either SLP76- or CD3ε-labeled epidermal growth factor receptor (EGFR). (B) NIH3T3 cells co-expressing VISH2 and ZtSH2 reporters before and 3 min after treatment with EGF (100 ng/mL). Scale bars, 20 µm. (C) Mean ± SD clearances of VISH2 and ZtSH2 from the cytosol, expressed with either SLP76- or CD3ε-labeled EGFR and stimulated with EGF (100 ng/mL). n = 3 independent experiments. (D) Response of VISH2 and ZtSH2 reporters 10 min after EGF treatment in (C). n > 30 cells from three independent experiments. (E) Orthogonal pYtags can be multiplexed to monitor the activity of multiple RTKs in the same cell. (F) Images of cells expressing orthogonal reporters for EGFR and ErbB2, treated with EGF (100 ng/mL). Scale bar, 20 µm. (G) Mean ± SD trajectories for EGFR and ErbB2 activity using multiplexed pYtags. For each reporter, the mean response was normalized to its minimum and maximum measured values. n = 3 independent experiments. (H) Time to half maximal response for individual cells from (G). Lines denote mean values, boxes denote 25–75th percentiles, and whiskers denote minima and maxima. n > 30 cells from three independent experiments. ***p<0.001 by Kolmogorov–Smirnov test. See also Figure 5—video 1 and Figure 5—video 2.

Figure 5—figure supplement 1

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Comparison of epidermal growth factor receptor (EGFR) and ErbB2 responses across experiments.

(A) Mean responses of EGFR and ErbB2 to EGF (100 ng/mL) were collected from experiments with or without multiplexed biosensors (see legend at bottom right of figure) and (B) normalized to their minimum and maximum responses for comparison. (C) Time to half maximal response from (B). Lines denote mean values, boxes denote 25–75th percentiles, and whiskers denote minima and maxima. For each condition, n ≥ 30 cells from three independent experiments.

Figure 5—video 1

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Figure 5—video 2

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Figure 6 with 1 supplement

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pYtags can be used to monitor the activity of endogenous receptor tyrosine kinases (RTKs).

(A) Schematic of *EGFR* locus containing the C-terminus of epidermal growth factor receptor (EGFR), where CRISPR/Cas9 was used to label the receptor with CD3ε-mNeonGreen via homology-directed repair. (B) PCR of genomic DNA from parental or knock-in HEK293T cells. Validation primers targeting homology regions upstream and downstream of the CD3ε-mNeonGreen insert are labeled by black arrows in (A). (C) Immunoblots of EGFR in parental or knock-in HEK293T cells. (D) Images of parental or knock-in HEK293T cells treated with EGF (100 ng/mL). mScarlet-ZtSH2 images show averages of two successive frames to decrease background noise; full raw movie is included as Figure 6—video 1. Scale bar, 10 µm. (E) Mean ± SD clearance of ZtSH2 from the cytosol following treatment with EGF (100 ng/mL). parental HEK293T, n = 3 independent experiments; knock-in HEK293T, n = 4 independent experiments. (F) Clearance of ZtSH2 from the cytosol 1 min after treatment with EGF in (E). Lines denote mean values, boxes denote 25–75th percentiles, and whiskers denote minima and maxima. parental HEK293T, n = 23 cells from three independent experiments; knock-in HEK293T, n = 46 cells from four independent experiments. ***p<0.001 by Kolmogorov–Smirnov test. See also Figure 6—video 1.

Figure 6—source data 1 Uncropped gel for Figure 6B.: https://cdn.elifesciences.org/articles/82863/elife-82863-fig6-data1-v1.zip
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Figure 6—source data 2 Uncropped gel for Figure 6C.: https://cdn.elifesciences.org/articles/82863/elife-82863-fig6-data2-v1.zip
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Figure 6—video 1

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Tables

Appendix 1—key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Cell line (human)	MCF10A-5E	Janes et al., 2010	RRID:CVCL_0598
Cell line (human)	HEK293T LX	ClonTech Laboratories	Cat # 632180
Cell line (mouse)	NIH3T3	ATCC	Cat # CRL-1658
Cell line (mouse)	SYF mouse embryonic fibroblasts (MEFs)	ATCC	Cat # CRL-2459
Cell line (Escherichia coli)	Stellar chemically competent cells	ClonTech Laboratories	Cat # 636763
Recombinant DNA reagent	pCMV-dR8.91 lentivirus packaging plasmid	Gift from Prof. Didier Trono, EPFL	Addgene # 12263
Recombinant DNA reagent	pMD2.G lenti helper plasmid	Gift from Prof. Didier Trono, EPFL	Addgene # 12259
Recombinant DNA reagent	pHR EGFR-FusionRed	Yang et al., 2021	Addgene # 179263
Recombinant DNA reagent	pHR EGFR-CD3ζ1-FusionRed	This paper	N/A	EGFR-ITAM construct
Recombinant DNA reagent	pHR EGFR-CD3ζ2-FusionRed	This paper	N/A	EGFR-ITAM construct
Recombinant DNA reagent	pHR EGFR-CD3ζ3-FusionRed	This paper	N/A	EGFR-ITAM construct
Recombinant DNA reagent	pHR EGFR-CD3γ-FusionRed	This paper	N/A	EGFR-ITAM construct
Recombinant DNA reagent	pHR EGFR-CD3δ-FusionRed	This paper	N/A	EGFR-ITAM construct
Recombinant DNA reagent	pHR EGFR-CD3ε-FusionRed	This paper	Addgene # 188626	EGFR-ITAM construct
Recombinant DNA reagent	pHR EGFR- CD3ε-mNeonGreen	This paper	N/A	EGFR-ITAM construct
Recombinant DNA reagent	pHR iRFP-ZtSH2	This paper	Addgene # 188627	ZtSH2 biosensor
Recombinant DNA reagent	pHR mScarlet-ZtSH2	This paper	N/A	ZtSH2 biosensor
Recombinant DNA reagent	pHR EGFR(R84K)-CD3ε-FusionRed	This paper	N/A	GBM-mutant EGFR construct (Figure 3)
Recombinant DNA reagent	pHR EGFR(A265V)-CD3ε-FusionRed	This paper	N/A	GBM-mutant EGFR construct (Figure 3)
Recombinant DNA reagent	pHR ErbB2-FusionRed	This paper	N/A	ITAM-less ErbB2 construct (Figure 4)
Recombinant DNA reagent	pHR ErbB2- CD3ε-FusionRed	This paper	Addgene # 188628	ITAM-tagged ErbB2 (Figure 4)
Recombinant DNA reagent	pHR EGFR-Citrine	This paper	N/A	Fluorescent EGFR construct (Figure 4)
Recombinant DNA reagent	pHR Clover-VISH2	This paper	Addgene # 188629	tSH2 biosensor (Figure 5)
Recombinant DNA reagent	pHR EGFR-CD3ε-TagBFP	This paper	N/A	ITAM-tagged EGFR (Figure 5)
Recombinant DNA reagent	pHR EGFR-SLP76-TagBFP	This paper	Addgene # 188630	ITAM-tagged EGFR (Figure 5)
Recombinant DNA reagent	pHR Grb2-TagBFP	This paper	Addgene # 188631	Grb2-based biosensor (Figure 1)
Recombinant DNA reagent	pHR FGFR1-CD3ε-FusionRed	This paper	Addgene # 188632	ITAM-tagged FGFR1 (Figure 4)
Recombinant DNA reagent	pHR FGFR1-FusionRed	This paper	N/A	ITAM-less FGFR1 (Figure 4)
Recombinant DNA reagent	pHR PDGFRβ-CD3ε-FusionRed	This paper	N/A	ITAM-tagged PDGFR (Figure 4)
Recombinant DNA reagent	pHR PDGFRβ-FusionRed	This paper	N/A	ITAM-less PDGFR (Figure 4)
Recombinant DNA reagent	pHR VEGFR3-CD3ε-FusionRed	This paper	N/A	ITAM-tagged VEGFR (Figure 4)
Recombinant DNA reagent	pHR VEGFR3-FusionRed	This paper	N/A	ITAM-less VEGFR (Figure 4)
Recombinant DNA reagent	pHR ErkKTR-TagBFP	9	N/A
Recombinant DNA reagent	pX330 EGFR-sgRNA	This paper, using a plasmid from Feng Zhang, MIT	Addgene # 188633	EGFR-targeting gRNA (Figure 6)
Recombinant DNA reagent	pUC19 EGFRup-CD3ε-mNeonGreen-EGFRdown	This paper	Addgene # 188634	CRISPR plasmid for EGFR modification (Figure 6)
Sequence-based reagent	PEF122 forward primer	This paper	5′- TTCTTTTGCAGCAACAGCAAGAGGGCCCTCCC-3′	Used to verify CRISPR tagging; see ‘Methods’
Sequence-based reagent	PEF123 reverse primer	This paper	5’- TCCGTTTCTTCTTTGCCCAGGAAGGGACAGAGTGGCTTATCC-3’	Used to verify CRISPR tagging; see ‘Methods’
Antibody	Anti-EGFR antibody (rabbit monoclonal)	Cell Signaling Technology	Cat # 4267	Used at 1:1000 for western blotting
Antibody	Anti-pEGFR antibody (rabbit monoclonal)	Cell Signaling Technology	Cat # 3777	Used at 1:1000 for western blotting
Antibody	Anti-β-actin antibody (mouse monoclonal)	Cell Signaling Technology	Cat # 3700	Used at 1:1000 for western blotting
Antibody	Anti-pAkt antibody (rabbit polyclonal)	Cell Signaling Technology	Cat # 9271	Used at 1:1000 for western blotting
Antibody	Anti-ppErk antibody (rabbit polyclonal)	Cell Signaling Technology	Cat # 9101	Used at 1:1000 for western blotting
Antibody	Anti-EEA1 antibody (mouse monoclonal)	Cell Signaling Technology	Cat # 48453	Used at 1:100 for immunostaining
Antibody	Anti-ZAP70 antibody (rabbit monoclonal)	Cell Signaling Technology	Cat # 3165	Used at 1:1000 for western blotting
Antibody	Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (goat polyclonal)	Invitrogen	Cat # A-11001	Used at 1:500 for immunostaining
Antibody	Goat anti-Rabbit IgG (Heavy chain), Superclonal Recombinant Secondary Antibody, Alexa Fluor 647 (goat polyclonal)	Invitrogen	Cat # A27040	Used at 1:500 for immunostaining
Antibody	Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (goat polyclonal)	Invitrogen	Cat # A-21236	Used at 1:500 for immunostaining
Antibody	IRDye 680RD Goat anti-Mouse IgG antibody (goat polyclonal)	LI-COR	Cat # 926-68070	Used at 1:10,000 for western blotting
Antibody	IRDye 800CW Goat anti-Rabbit IgG antibody (goat polyclonal)	LI-COR	Cat # 926-32211	Used at 1:10,000 for western blotting
Peptide, recombinant protein	Bovine serum albumin	Sigma-Aldrich	Cat # 12659
Peptide, recombinant protein	Fibronectin	Corning	Cat # CB-40008A	Cell adhesion coating
Peptide, recombinant protein	ClonAmp HiFi PCR polymerase	ClonTech Laboratories	Cat # 639298	Polymerase
Peptide, recombinant protein	Insulin	Sigma-Aldrich	Cat # I6634
Peptide, recombinant protein	Cholera toxin	Sigma-Aldrich	Cat # C8052
Peptide, recombinant protein	L-glutamine	Gibco	Cat # 25030-081
Peptide, recombinant protein	EGF	R&D Systems	Cat # 236-EG-200
Peptide, recombinant protein	Epiregulin	R&D Systems	Cat # 1195-EP-025
Peptide, recombinant protein	Epigen	R&D Systems	Cat # 6629-EP-025
Peptide, recombinant protein	FGF4	R&D Systems	Cat # 235-F4-025
Peptide, recombinant protein	PDGF-BB	Millipore Sigma	Cat # P3201
Peptide, recombinant protein	VEGF-C	R&D Systems	Cat # 9199-VC-025
Chemical compound, drug	Gefitinib	Cell Signaling Technology	Cat # 4765
Chemical compound, drug	Hydrocortisone	Sigma-Aldrich	Cat # H0888
Chemical compound, drug	Penicillin/ streptomycin	Gibco	Cat # 15140–122
Chemical compound, drug	TrypLE Express	Gibco	Cat # 12605-028
Chemical compound, drug	FuGENE HD	Promega	Cat # E2311
Chemical compound, drug	Lipofectamine 3000	Thermo Fisher Scientific	Cat # L3000015
Chemical compound, drug	Aminopropyl trimethoxysilane	Sigma-Aldrich	Cat # 281778
Chemical compound, drug	Glutaraldehyde	Sigma-Aldrich	Cat # 340855
Chemical compound, drug	40% acrylamide solution	Bio-Rad	Cat # 1610140
Chemical compound, drug	2% bis-acrylamide solution	Bio-Rad	Cat # 161-0142
Chemical compound, drug	N,N,N’,N’-Tetramethyl ethylenediamine (TEMED)	Sigma-Aldrich	Cat # T9281
Chemical compound, drug	Ammonium persulfate (APS)	Sigma-Aldrich	Cat # A3678
Commercial assay or kit	inFusion HD cloning kit	ClonTech Laboratories	Cat # 638911	Cloning kit
Other	DMEM/F12	Gibco	Cat # 11320033	Culture media
Other	Horse serum	Gibco	Cat # 16050122	Serum for culture media
Other	DMEM	Gibco	Cat # 11995-065	Culture media
Other	Fetal bovine serum	R&D Systems	Cat # S11150	Serum for culture media
Software, algorithm	FIJI	Schindelin et al., 2012	http://fiji.sc; RRID:SCR_00228
Software, algorithm	Python code for computational model; analysis code for raw data	This paper	https://github.com/toettchlab/Farahani2022/ (copy archived at toettchlab, 2023)
Software, algorithm	R Studio 1.1.456	RStudio	rstudio.com; RRID:SCR_000432

Appendix 1—table 1

Equations used in the mathematical model.

L-EGFR: ligand-bound EGFR; EGFR:EGFR: EGFR in dimeric form; EGFR: EGFR bound to ZtSH2.

Species	Notation	Equation
Soluble ligand (L)	N₁	$\frac{d N_{1}}{d t} = 0$ (A1)
Unbound ZtSH2 (*)	N₂	$\frac{d N_{2}}{d t} = - k_{8} N_{2} * (2 N_{6} + 2 N_{7} + N_{9} + N_{10} + N_{11}) + k_{9} * (N_{8} + N_{9} + N_{10} + N_{11} + 2 N_{12} + 2 N_{13} + 2 N_{14} + N_{15} + N_{16})$ (A2)
EGFR	N₃	$\frac{d N_{3}}{d t} = - k_{1} N_{3} N_{1} + k_{2} N_{4} - 2 k_{5} N_{3}^{2} - k_{5} N_{3} N_{4} - k_{5} N_{3} N_{15} - k_{5} N_{3} N_{16} + 2 k_{6} N_{5} + k_{6} N_{8} + k_{7} N_{6} + k_{7} N_{9} + k_{9} N_{15}$ (A3)
L-EGFR	N₄	$\frac{d N_{4}}{d t} = k_{1} N_{3} N_{1} - k_{2} N_{4} - k_{5} N_{3} N_{4} - 2 k_{5} N_{4}^{2} - k_{5} N_{4} N_{15} - k_{5} N_{4} N_{16} + k_{7} N_{6} + k_{7} N_{10} + 2 k_{7} N_{7} + k_{7} N_{11} + k_{9} N_{16}$ (A4)
EGFR:EGFR	N₅	$\frac{d N_{5}}{d t} = {- 2 k}_{1} N_{5} N_{1} + k_{3} N_{6} + k_{5} N_{3}^{2} - k_{6} N_{5} + k_{9} N_{8}$ (A5)
L-EGFR:EGFR	N₆	$\frac{d N_{6}}{d t} = - k_{1} N_{6} N_{1} + 2 k_{4} N_{7} + 2 k_{1} N_{5} N_{1} - k_{3} N_{6} + k_{5} N_{3} N_{4} - k_{7} N_{6} - 2 k_{8} N_{6} N_{2} + k_{9} N_{9} + k_{9} N_{10}$ (A6)
L-EGFR:L-EGFR	N₇	$\frac{d N_{7}}{d t} = k_{5} N_{6} N_{1} {- 2 k}_{4} N_{7} + k_{5} N_{4}^{2} {- k}_{7} N_{7} - {2 k}_{8} N_{7} N_{2} + k_{9} N_{11}$ (A7)
EGFR*:EGFR	N₈	$\frac{d N_{8}}{d t} = - 2 k_{1} N_{8} N_{1} + k_{3} N_{9} + k_{3} N_{10} + k_{5} N_{3} N_{15} - k_{6} N_{8} - k_{9} N_{8} + {2 k}_{9} N_{12}$ (A8)
L-EGFR*:EGFR	N₉	$\frac{d N_{9}}{d t} = k_{1} N_{8} N_{1} - k_{1} N_{9} N_{1} - k_{3} N_{9} + k_{4} N_{11} + k_{5} N_{3} N_{16} - k_{7} N_{9} + k_{8} N_{6} N_{2} - k_{8} N_{9} N_{2} - k_{9} N_{9} + k_{9} N_{13}$ (A9)
L-EGFR:EGFR*	N₁₀	$\frac{d N_{10}}{d t} = k_{1} N_{8} N_{1} - k_{3} N_{10} - k_{1} N_{10} N_{1} + k_{4} N_{11} + k_{5} N_{4} N_{15} - k_{7} N_{10} + k_{8} N_{6} N_{2} - k_{9} N_{10} - k_{8} N_{10} N_{2} + k_{9} N_{13}$ (A10)
L-EGFR*:L-EGFR	N₁₁	$\frac{d N_{11}}{d t} = k_{1} N_{9} N_{1} - {2 k}_{4} N_{11} + k_{1} N_{10} N_{1} + k_{5} {N_{4} N}_{16} - k_{7} N_{11} + {2 k}_{8} N_{7} N_{2} - k_{9} N_{11} - k_{8} N_{11} N_{2} + {2 k}_{9} N_{14}$ (A11)
EGFR:EGFR	N₁₂	$\frac{d N_{12}}{d t} = - {2 k}_{1} N_{12} N_{1} + k_{3} N_{13} + k_{5} N_{15}^{2} - k_{6} N_{12} - {2 k}_{9} N_{12}$ (A12)
L-EGFR:EGFR	N₁₃	$\frac{d N_{13}}{d t} = {2 k}_{1} N_{12} N_{1} - k_{3} N_{13} - k_{1} N_{13} N_{1} + {2 k}_{4} N_{14} + k_{5} N_{15} N_{16} - k_{7} N_{13} + k_{8} N_{9} N_{2} - 2 k_{9} N_{13} + k_{8} N_{10} N_{2}$ (A13)
L-EGFR:L-EGFR	N₁₄	$\frac{d N_{14}}{d t} = k_{1} N_{13} N_{1} - {2 k}_{4} N_{14} + k_{5} N_{16}^{2} - k_{7} N_{14} + k_{8} N_{11} N_{2} - {2 k}_{9} N_{14}$ (A14)
EGFR*	N₁₅	$\frac{d N_{15}}{d t} = {- k}_{1} N_{15} N_{1} + k_{2} N_{16} - 2 k_{5} N_{15}^{2} + 2 k_{6} N_{12} - k_{5} {N_{3} N}_{15} + k_{6} N_{8} - k_{5} N_{15} N_{16} + k_{7} N_{13} - k_{5} {N_{4} N}_{15} + k_{7} N_{10} - k_{9} N_{15}$ (A15)
L-EGFR*	N₁₆	$\frac{d N_{16}}{d t} = k_{1} N_{15} N_{1} - k_{2} N_{16} - 2 k_{5} N_{16}^{2} + 2 k_{7} N_{14} - k_{5} N_{15} N_{16} + k_{7} N_{13} - k_{5} N_{16} N_{3} + k_{7} N_{9} - k_{5} {N_{4} N}_{16} + k_{7} N_{11} - k_{9} N_{16}$ (A16)

Appendix 1—table 2

Parameters used in the mathematical model.

Parameter	Notation	Value	Units	Notes
Receptor–ligand binding	k₁	0.03	nM^–1 s^–1	Schoeberl et al., 2002
Ligand dissociating from L-EGFR (β = 1 corresponds to EGF)	k₂	β*6.6e-3	s^–1	Macdonald and Pike, 2008
Ligand dissociating from L-EGFR:EGFR (β = 1 corresponds to EGF)	k₃	β*5.7e-3	s^–1	Macdonald and Pike, 2008
Ligand dissociating from L-EGFR:L-EGFR	k₄	0.087	s^–1	Macdonald and Pike, 2008
Receptor dimerization and activation	k₅	1e-5	nM^–1 s^–1	Estimated
EGFR:EGFR dissociation	k₆	5e-3; variable values in Figure 3—figure supplement 2.	s^–1	Estimated
L-EGFR:EGFR dissociation (γ = 1 corresponds to EGF)	k₇	γ*1e-4	s^–1	Estimated
ZtSH2 binding to receptor	k₈	5	nM^–1 s^–1	Kd from Ottinger et al., 1998; kinetics set to be ~10 s based on our experimental measurements of ZtSH2 translocation
ZtSH2 dissociating from receptor	k₉	16.67	s^–1	Kd from Ottinger et al., 1998; kinetics set to be ~10 s based on our experimental measurements of ZtSH2 translocation
Scaling parameter for ligand–receptor binding	β	1 for EGF; 50 for low-affinity ligands; variable values in Figure 3C	Unitless	Freed et al., 2017
Scaling parameter for dimerization of ligand-bound receptors	γ	1 for EGF; 100 for low-affinity ligands; variable values in Figure 3C	Unitless	Freed et al., 2017; Hu et al., 2022

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Payam E Farahani
Xiaoyu Yang
Emily V Mesev
Kaylan A Fomby
Ellen H Brumbaugh-Reed
Caleb J Bashor
Celeste M Nelson
Jared E Toettcher

(2023)

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

eLife 12:e82863.

https://doi.org/10.7554/eLife.82863

Figures

pYtags: a biosensing strategy to monitor receptor tyrosine kinase (RTK) activity in living cells.

Figure 1—source data 1

Grb2 fails to discriminate between immunoreceptor tyrosine-based activation motif (ITAM)-labeled and unlabeled RTKs.

Effects of the expression levels of pYtag components in NIH3T3 cells.

Effects of the expression levels of pYtag components in HEK293T cells.

Mathematical modeling of the effects of pYtag component expression levels on biosensor readout.

Timelapse of iRFP-ZtSH2 in NIH3T3 cells co-expressing iRFP-ZtSH2 and EGFR-CD3ε-FusionRed.

Monitoring epidermal growth factor receptor (EGFR) signaling at subcellular and multicellular length scales.

Analysis of epidermal growth factor receptor (EGFR) and ZtSH2 internalization.

Maximum intensity projection timelapse images of MCF10A cells co-expressing iRFP-ZtSH2 (left panel), EGFR-CD3ε-FusionRed (middle panel), and ErkKTR-BFP (right panel), cultured on soft substrata and treated with EGF (100 ng/mL).

Epidermal growth factor receptor (EGFR) pYtag reveals dose- and ligand-dependent signaling dynamics.

ZtSH2 and epidermal growth factor receptor (EGFR) localization in response to different ligands.

Ligand-free dimers in mathematical model recapitulate biphasic signaling response of epidermal growth factor receptor (EGFR).

Monitoring distinct receptor tyrosine kinases (RTKs) in heterodimeric complexes.

pYtag biosensors of additional receptor tyrosine kinases (RTKs).

Orthogonal pYtags enable multiplexed receptor tyrosine kinase (RTK) biosensing.

Comparison of epidermal growth factor receptor (EGFR) and ErbB2 responses across experiments.

Timelapse of NIH3T3 cells co-expressing VISH2 and ZtSH2 reporters, and either SLP76- or CD3ε-labeled epidermal growth factor receptor (EGFR), treated with EGF (100 ng/mL).

Timelapse of NIH3T3 cells co-expressing pYtag biosensors for epidermal growth factor receptor (EGFR) and ErbB2, treated with EGF (100 ng/mL).

pYtags can be used to monitor the activity of endogenous receptor tyrosine kinases (RTKs).

Figure 6—source data 1

Figure 6—source data 2

Timelapse of HEK293T cells expressing mScarlet-ZtSH2 and an endogenously labeled EGFR-CD3ε-mNeonGreen, treated with EGF (100 ng/mL).

Tables

Equations used in the mathematical model.

Parameters used in the mathematical model.

Additional files

MDAR checklist

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Downloads (link to download the article as PDF)

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pYtags: a biosensing strategy to monitor receptor tyrosine kinase (RTK) activity in living cells.

Figure 1—source data 1

Grb2 fails to discriminate between immunoreceptor tyrosine-based activation motif (ITAM)-labeled and unlabeled RTKs.

Effects of the expression levels of pYtag components in NIH3T3 cells.

Effects of the expression levels of pYtag components in HEK293T cells.

Mathematical modeling of the effects of pYtag component expression levels on biosensor readout.

Timelapse of iRFP-ZtSH2 in NIH3T3 cells co-expressing iRFP-ZtSH2 and EGFR-CD3ε-FusionRed.

Monitoring epidermal growth factor receptor (EGFR) signaling at subcellular and multicellular length scales.

Analysis of epidermal growth factor receptor (EGFR) and ZtSH2 internalization.

Maximum intensity projection timelapse images of MCF10A cells co-expressing iRFP-ZtSH2 (left panel), EGFR-CD3ε-FusionRed (middle panel), and ErkKTR-BFP (right panel), cultured on soft substrata and treated with EGF (100 ng/mL).

Epidermal growth factor receptor (EGFR) pYtag reveals dose- and ligand-dependent signaling dynamics.

ZtSH2 and epidermal growth factor receptor (EGFR) localization in response to different ligands.

Ligand-free dimers in mathematical model recapitulate biphasic signaling response of epidermal growth factor receptor (EGFR).

Monitoring distinct receptor tyrosine kinases (RTKs) in heterodimeric complexes.

pYtag biosensors of additional receptor tyrosine kinases (RTKs).

Orthogonal pYtags enable multiplexed receptor tyrosine kinase (RTK) biosensing.

Comparison of epidermal growth factor receptor (EGFR) and ErbB2 responses across experiments.

Timelapse of NIH3T3 cells co-expressing VISH2 and ZtSH2 reporters, and either SLP76- or CD3ε-labeled epidermal growth factor receptor (EGFR), treated with EGF (100 ng/mL).

Timelapse of NIH3T3 cells co-expressing pYtag biosensors for epidermal growth factor receptor (EGFR) and ErbB2, treated with EGF (100 ng/mL).

pYtags can be used to monitor the activity of endogenous receptor tyrosine kinases (RTKs).

Figure 6—source data 1

Figure 6—source data 2

Timelapse of HEK293T cells expressing mScarlet-ZtSH2 and an endogenously labeled EGFR-CD3ε-mNeonGreen, treated with EGF (100 ng/mL).

Equations used in the mathematical model.

Parameters used in the mathematical model.

MDAR checklist

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)