Fluorescent tags allow live monitoring of growth factor signaling proteins inside living cells RiceUniversity
. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jonathan Cooper as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Alex J B Kreutzberger .
2. Discrepancy between apparent sensitivity of the approach , with the inability to detect ERBB2 activation by EGF unless EGFR is co-over-expressed. Use of supra-physiological concentrations of ligand is also a concern. The sensitivity of the system should be tested with lower EGF concentrations. Overall, it is felt that, as written, the manuscript provides a proof-of-concept rather than making a strong case for others to adapt this assay to study their receptors of interest.1. On page 9, the authors state:"Notably, we observed that pYtag-expressing cells stimulated for at least 30 min with EGF contained internalized vesicles that were positive for both total EGFR and ZtSH2 .
There are several other concerns regarding the technology, especially its apparent lack of sensitivity. Maximal biological response to activated EGFR is typically achieved with only a couple thousand occupied receptors per cell . Is this detectable? The results of the HEK293 experiments suggest that this is the case. However, instead of editing those cells, it would be informative to try MCF10A cells, which have hundreds of thousands of endogenous receptors.
In summary, we have addressed the Editor’s concerns by providing new experimental evidence and computational results showing that sensitive biosensor responses can be achieved by scaling ZtSH2 expression to EGFR expression ; showing that the kinetics of biosensor activation are relatively robust to biosensor expression level; and performing comparisons in the same parental cell line between endogenous receptor levels and overexpression, which reveal more rapid and pronounced receptor clearance...
Use of supra-physiological concentrations of ligand is also a concern. The sensitivity of the system should be tested with lower EGF concentrations. – Rate of access to the probe : These are very interesting questions, but we are unsure what experiments the editor and reviewers might have in mind to further address them. We have extensive experience over a decade with recruiting proteins from cytosol to membrane and a timescale of 10-20 sec for membrane recruitment is very typical across many contexts, from mammalian cell lines toembryos . We observe membrane translocation on the same timescale here .
We would argue strongly in favor of the model’s inclusion because it was instrumental in interpreting the stark differences in EGFR dynamics that we observe in response to different ligands! It also helped us design a validation experiment, where we alter EGFR-EGFR dimerization affinity and shift the dynamics of EREG to a more EGF-like response.
We thank the reviewers and editors for this insightful comment and have now tested whether endocytosis is different for our pYtagged EGFR compared to a control construct. We have added a new supplementary figure quantifying EGFR membrane intensity and colocalization of EGFR with EEA1, an early endosomal marker, over time after EGF stimulation. We performed this analysis for cells expressing our EGFR-ITAM system versus an ITAM-less EGFR construct expressed at the same level.
We appreciate the reviewer’s comments, which we have addressed in two ways. First, we have added new data demonstrating that EGFR and ZtSH2 indeed colocalize with the early endosome marker EEA1 after EGF stimulation in both NIH3T3 and HEK293T cells , as well as for an ITAM-less EGFR control . 3. The experiments over-expressing the pYtags were mainly performed in NIH3T3 cells, whereas the CRISPR-Cas9 knock-in was done in HEK 293T. It would be useful to check whether the same differences would be also seen in HEK 293T over-expressing the tag. After assessing this, further characterization of the signal emitted by the cell line generated by CRISPR-Cas9 would add some key concepts to RTK activity dynamics .
While we agree that three replicates are an important standard for virtually all experiments, we would argue that different standards apply to screen designed to generate a lead candidate for further study. For example, a drug screen or directed evolution campaign is often carried out just once, but of course the major hits are characterized in detail in many subsequent triplicate experiments.
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