1. This review reflects comments and contributions by Joachim Goedhart, Arthur Molines, Attya Omer, Sónia Gomes Pereira, Bhatia Sonam, Rajan Thakur. Review synthesized by Sree Rama Chaitanya Sridhara.

    NOTCH1-mediated cellular signaling determines the fate of developing cells during embryogenesis and also helps maintain tissue homeostasis (e.g., blood vessel formation, nervous system plasticity, and bone regulation). NOTCH1 performs a plethora of biological functions by maneuvering gene expression patterns. Furthermore, anomalies in NOTCH1 signaling cause pathological conditions in humans. For example, in breast cancer, NOTCH1 signaling regulates cancer stem cells, proliferation rates, metastasis, and chemosensitivity. Hence, it is important to understand the spatio-temporal regulation of NOTCH1 signaling in mammalian cells. Therefore, the authors of the current preprint engineered a light-sensitive NOTCH1 in mammalian cells to investigate the NOTCH1-mediated gene expression program and its functional impact on breast cancer cells.

    The preprint reports the major findings clearly, we lay out some suggestions that can improve the clarity and robustness of the reported findings.

    Major comments

    1.‘To facilitate the expression of both proteins in cells, we joined them through a P2A (porcine teschovirus-1 2A) “self-cleaving” sequence so that both could be expressed within one reading frame (Results, Fig 1B).’ - An excess of ZDK-NICD could lead to a constant activation. It’s advisable to look at the expression level of each of these proteins (e.g., immunoblots).

    2.‘The P2A is a self-cleaving peptide, which enables the formation of two separate proteins after translation (Fig 1C). In the dark, LOV2 and Zdk1-NICD are located at the cell membrane. Upon release from the cell membrane, the hN1ICD translocate to the nucleus where it acts as a transcriptional activator together with CSL and MAML (Fig 1A).’ - These findings may require further experimental evidence or additional discussion. Since this is a proof-of-concept study, there should be sufficient details that the system is working as planned. This includes: testing the membrane localization of the receptor and the levels of the two peptides separated by the P2A, and the translocation of the NCID domain. It may also be relevant to check if the two separate proteins are actually synthesized (using immunoblots), to test the optimal cleavage. While some functional activation of the NOTCH pathway is reported by over-expression of HES1, HEY1 (presuming that the NICD is translocated) it would be helpful for the readers to visualize it.

    1. While the initial experiments were performed in HEK293 cells, the latter was done in breast cancer cells. It is relevant to repeat the same initial experiments in breast cancer cells to make sure the reporter system is working in breast cancer cells.

    4.‘The γ-secretase inhibitor DAPT (10 μM) was used to rule out an effect on the growth of spheroids by endogenous Notch activity.’- Please report the data for this statement. The following conditions could be tested to gauge the full effect of the system: untreated control, DMSO control, 10uM DAPT, Light, and Dark.

    5.Figure 3 -There does not seem to be a clear difference between light and dark exposed spheroids. Also, the Y-axis is different between the graphs. It might be helpful to keep the Y-axis constant. Additionally, performing more experiments and imaging at higher magnifications could increase the robustness of the data.

    6.‘Notch activity was blocked by a γ-secretase inhibitor (DAPT). Using optoNotch we can modulate receptor activation to discriminate between different modes of NOTCH1 activation, as suggested by differential binding and force used by each of the ligands.’ - It is advisable to report data supporting this statement.

    7.The authors have shown that 1hr is the timepoint that induces the highest changes to gene expression (Fig. 1). However, some of the experiments were performed with light pulses for 3hrs (Fig. 2). Please clarify if there is a reason for using a different duration across experiments.

    1. ‘The total content of N1ICD in both of these stable cell lines was substantially higher than in WT cells (where there is some endogenous N1ICD) (Fig 2B-C).’ Please provide quantification for this data. Also, is there a way to differentiate between the WT N1ICD and the engineered one? If so, could an explanation be provided.

    Minor comments

    1.Abstract ‘Here, we circumvent this step by regulating Notch activity by light. To achieve this, we have engineered a membrane-bound optogenetic NOTCH1 receptor (optoNotch) to control the activation of NOTCH1 intracellular domain (N1ICD) and its downstream transcriptional activities’ - For clarity, this fragment could be rewritten as follows: ‘Here, we report on a system that allows the activation of Notch signaling by light. To this end, we used the heterodimerization between the photosensitive protein LOV and Zdk. The interaction between the membrane located LOV protein and the Zdk fused to N1ICD is lost upon illumination with blue light. As a consequence, the N1ICDactivates downstream transcriptional activities.’ Strictly speaking, the engineered optoNotch is not a membrane bound receptor. The intracellular activating domain has been tethered to the plasma membrane using a heterodimerizing system that is sensitive to light. Light-induced dissociation releases the intracellular domain and triggers downstream activities.

    2.Recommend spelling out abbreviations e.g., ICD, CSL, MAML.

    3.'avoids' instead of ‘allows avoiding’ (Introduction).

    4.Results ’via the Myristoylation-targeting sequence (MTS)’ -The consensus sequence is MG. Is that used here, or is the Myristoylation-targeting sequence (MTS) a longer peptide (as it is usually used to increase membrane labeling efficiency)?

    5.Recommend checking that the figures have sufficient resolution after conversion of the document to PDF.

    6.Consider illustrating a cartoon of the natural NOTCH receptor domain structure in Fig. 1 (for readers unfamiliar with NOTCH signaling).

    7.Results ‘This reaction resulted in a very low luciferase signal after photoactivation of the optoNotch system no matter the length of activation (0.05-second pulses for 1, 3, or 12 hours) (Fig. 1E).’ - Before moving to the results, the text could include an explanation for the engineered target gene CSL-LUC that is used as a read-out here. Also, consider illustrating it in Fig 1.

    8.Include the remnant of the P2A peptide in Fig 1C. The P2A leaves a 'scar' and it is important to include this information.

    9.Keep the axes and scale consistent in Fig 1 E-H.

    10.Data related to Fig 1H – was this measured in cells expressing WT LOV2 or the LOV2 V416L?

    11.‘All three genes showed increased expression in light-activated cells.’- Is it known, and can it be discussed how this compares to the natural activation of the NOTCH receptor. This would be useful to mention even if unknown.

    12.‘To confirm the influence of the optoNotch system on breast cancer development under physiological-mimicking conditions.’ - As the tests measure growth properties rather than development, recommend reframing the sentence.

    13.Please state what the error bars represent in Fig 3A-D?

    14.Molecular cloning - could the full sequence of the optoNotch system be reported (for instance as supplemental).

    References

    Please kindly cite relevant references:

    • receptor upon mechanical pulling by any of the ligands’ (Introduction).
    • LOVTRAP’ (Results).
    • pTriEx-NTOM20-LOV plasmid, Zdark-1 (Zdk1) sequence from pTriEX-mCherry-Zdk1 (Methods, Molecular cloning) - Please cite the sources or cat.no.
    • to the original protocol’ (Methods, Molecular cloning).
    • MCF7 and MDA-MB-468 stable cell lines’ (Methods, Immunostaining and Flow Cytometry) - Please cite sources if not generated by the lab.
    Read the original source
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