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Design and evaluation of engineered protein biosensors for live-cell imaging of EGFR phosphorylation

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Science Signaling  04 Jun 2019:
Vol. 12, Issue 584, eaap7584
DOI: 10.1126/scisignal.aap7584
  • Fig. 1 The tSH2-WT biosensor lacks specificity and does not faithfully track EGFR phosphorylation kinetics.

    (A) Normalized membrane recruitment of tSH2-WT in response to stimulation of NR6 parental cells and EGFR-expressing NR6 cells with EGF, as measured by TIRF microscopy (n = 5 cells from five independent experiments). (B) Normalized membrane recruitment of tSH2-WT in NR6 parental cells treated with sodium orthovanadate (n = 7 cells from seven independent experiments). For (A) and (B), error bars indicating 95% confidence intervals are shaded. (C) Far-Western blotting analysis of NR6 parental cells (P) and NR6 cells expressing wild-type EGFR (W) stimulated with sodium orthovanadate (V) for 60 min or EGF (E) for 10 min. Cell lysates were blotted and probed with tSH2-WT. Blot is representative of n = 3 independent experiments. Rightmost lane shows molecular weight ladder. (D) Kinetics of pEGFR predicted by the mathematical model. (E) Predicted effect of binding affinity on the interaction between an idealized biosensor and the target on biosensor readout. The desired koff is higher (lower affinity), and therefore, biosensor binding does not markedly perturb receptor dynamics. (F) Internalization of labeled EGF measured by flow cytometry in EGFR-expressing NR6 cells transfected with tSH2-WT, compared to untransfected cells (UC). *P < 0.05 for comparison with untransfected cells (n = 3 independent experiments).

  • Fig. 2 A mutant SH2 domain (mSH2) biosensor closely tracks the expected kinetics of EGFR phosphorylation and is more specific than cSH2 for the EGFR pTyr992.

    (A) Far-Western blotting analysis of NR6 parental cells (P) and NR6 cells expressing wild-type EGFR (W) stimulated with sodium orthovanadate (V) for 60 min or EGF (E) for 10 min. Leftmost lane shows molecular weight ladder. Cell lysates were blotted and probed with cSH2. Blot is representative of n = 3 independent experiments. (B) Normalized membrane recruitment of cSH2, measured by TIRF microscopy, in response to stimulation of EGFR-expressing NR6 cells with EGF (n = 21 cells analyzed from five independent experiments) or NR6 parental cells with sodium orthovanadate (n = 21 cells analyzed from three biological experiments). (C) Yeast cells displaying mSH2 on the cell surface were labeled with biotinylated synthetic peptides corresponding to the pTyr992 (pY992), pTyr1068 (pY1068), or pTyr1148 (pY1148) sites in EGFR, followed by secondary labeling with streptavidin-phycoerythrin (SA-PE). A control sample of yeast cells labeled only with SA-PE is also shown (n = 3 independent experiments). (D) Normalized membrane recruitment of mSH2, measured by TIRF microscopy, in response to EGF stimulation of NR6 parental cells (n = 7 cells analyzed from seven independent experiments) or EGFR-expressing NR6 cells (n = 18 cells analyzed from five independent experiments). (E) Normalized membrane recruitment of mSH2, measured by TIRF microscopy, in response to sodium orthovanadate treatment of NR6 parental cells (n = 7 cells from seven independent experiments). (F and G) Normalized expression of either the c′1000 EGFR truncation mutant (F) (n = 5 cells from five independent experiments) or the c′1000F EGFR mutant with the Y992F mutation (G) (n = 6 cells from six independent experiments). The 95% confidence intervals for normalized recruitment are shaded in each figure.

  • Fig. 3 mSH2 displays affinity for pTyr1021 of PDGFR in addition to pTyr992 of EGFR.

    (A) Far-Western blotting analysis of NR6 parental cells (P) and NR6 cells expressing wild-type EGFR (W) stimulated with sodium orthovanadate (V) or EGF (E). Cell lysates were blotted and probed with mSH2. Blot is representative of n = 3 independent experiments. Rightmost lane shows molecular weight ladder. (B) Yeast cells displaying mSH2 or tSH2-WT on the cell surface were labeled with a biotinylated synthetic peptide corresponding to the pTyr1021 site in PDGFR, followed by secondary labeling with SA-PE. A control sample of yeast cells labeled with only SA-PE is also shown (n = 3 independent experiments). (C) Normalized membrane recruitment of mSH2 (n = 27 cells from seven independent experiments) and tSH2-WT (n = 4 cells from four independent experiments) in response to PDGF stimulation of NR6 parental cells. The 95% confidence intervals are shaded.

  • Fig. 4 The SPY992 biosensor closely tracks the expected kinetics of EGFR phosphorylation and displays increased specificity for the EGFR pTyr992 site of EGFR in live-cell imaging.

    (A) Yeast cells displaying SPY992 on the cell surface were labeled with biotinylated synthetic peptides corresponding to the pTyr992 or Tyr992 site in EGFR and the pTyr1021 site in PDGFR, followed by secondary labeling with SA-PE. A control sample of yeast cells labeled with only SA-PE is also shown (n = 3 independent experiments). (B) Normalized membrane recruitment of SPY992 in response to EGF stimulation of EGFR-expressing NR6 cells pretreated with gefitinib (n = 27 cells from two independent experiments) or not pretreated (n = 110 cells from five independent experiments), as measured by TIRF microscopy. P < 0.0001 by two-way analysis of variance (ANOVA). (C) Normalized membrane recruitment of SPY992 in response to EGF stimulation of NR6 cells expressing the c′1000 EGFR truncation mutant (n = 20 cells from four independent experiments) and NR6 cells expressing the c′1000F EGFR mutant with the Y992F mutation (n = 23 cells from five independent experiments). P < 0.0001 by two-way ANOVA. (D) Normalized membrane recruitment of SPY992 in response to PDGF stimulation of NR6 parental cells (n = 21 cells from four independent experiments). (E) Normalized membrane recruitment of SPY992 in response to EGF stimulation of MDA-MB-231 cells pretreated with gefitinib for 5 min (n = 97 cells from four independent experiments) or not pretreated (n = 52 cells from six independent experiments). P < 0.0001 by two-way ANOVA. (F) Normalized membrane recruitment of SPY992 in EGFR-expressing NR6 cells in response to stimulation with a subthreshold concentration of EGF (300 pM). Data from cells with an active response (n = 105 cells from five independent experiments) and no response (n = 95 cells from five independent experiments) are shown. P < 0.0001 by two-way ANOVA. The 95% confidence intervals are shaded (B to F).

  • Fig. 5 The SPY1148 biosensor responds to EGFR phosphorylation and displays increased specificity for the pTyr1148 site of EGFR in live-cell imaging experiments.

    (A) Yeast cells displaying SPY1148 on the cell surface were labeled with biotinylated synthetic peptides corresponding to the pTyr1148 or Tyr1148 site in EGFR, followed by secondary labeling with SA-PE. A control sample of yeast cells labeled with only SA-PE is also shown. (B to D) Normalized membrane recruitment of SPY1148 in response to EGF stimulation of NR6 cells expressing wild-type EGFR (B) (n = 213 cells from eight independent experiments), the c′1000 EGFR truncation mutant (C) (n = 144 cells from five independent experiments), or the c′1000F EGFR mutant with the Y992F mutation (D) (n = 144 cells from five independent experiments). EGFR compared to c′1000 and c′1000f is significantly different, P < 0.0001 by two-way ANOVA, and 95% confidence intervals are shaded in (B) to (D).

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/584/eaap7584/DC1

    Fig. S1. Analysis of intermediate cell populations during library screening.

    Fig. S2. Normalized recruitment of tSH2-WT and mSH2 biosensors.

    Fig. S3. Effect of changing binding affinity or biosensor concentration on the readout from the biosensor.

    Fig. S4. Comparison of biosensor responses.

    Table S1. Protein sequences of mSH2, SPY992, and SPY1148.

    Table S2. Primer sequences.

    Table S3. Peptides used for library screening.

  • This PDF file includes:

    • Fig. S1. Analysis of intermediate cell populations during library screening.
    • Fig. S2. Normalized recruitment of tSH2-WT and mSH2 biosensors.
    • Fig. S3. Effect of changing binding affinity or biosensor concentration on the readout from the biosensor.
    • Fig. S4. Comparison of biosensor responses.
    • Table S1. Protein sequences of mSH2, SPY992, and SPY1148.
    • Table S2. Primer sequences.
    • Table S3. Peptides used for library screening.

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