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An improved Erk biosensor reveals oscillatory Erk dynamics driven by mitotic erasure during early development

Scott G. Wilcockson, Luca Guglielmi, Pablo Araguas Rodriguez, Marc Amoyel, Caroline S. Hill

Preprint posted on 4 November 2022 https://www.biorxiv.org/content/10.1101/2022.11.03.515001v1

Want Erk-cellent representation of Erk signalling dynamics? Wilcockson and authors explore an improved biosensor that faithfully reports Erk activity without being impacted by Cdk1

Selected by Sakurako Kobayashi

Background

Extracellular signal regulated kinase (Erk) signalling is a central signalling axis involved in regulating cellular growth, proliferation and fate specification (Lavoie et al., 2020). In order to understand the regulation of Erk-dependent processes, it is essential to develop sensitive, but accurate, biosensors that will capture Erk signalling dynamics in vivo. Despite the need to detect a dynamic range of signalling, current methods can only weakly discriminate between high and low Erk activity.

Since the development of kinase translocation reporters (KTRs), a number of new KTR biosensors have been applied to in vivo systems (Regot et al., 2014). KTRs, including Erk-KTR, report kinase activity by converting phosphorylating events into nucleo-cytoplasmic shuttling events that can be measured by confocal microscopy. This allows for rapid and quantifiable measurements of ERK activity based upon subcellular localisation of a fluorescent fusion protein (Regot et al., 2014). The ability of KTRs to dynamically report kinase activity at single-cell resolution, without the need of expensive and time-consuming imaging platforms, has made it an attractive tool for live-cell based imaging.

Visualising Erk signalling dynamics in the early embryo is essential for understanding how early fate decisions are made. Activated Erk, or phosphorylated-Erk (P-Erk), is induced by fibroblast growth factor (Fgf) signalling at the blastula embryonic margin. From as early as 4.3 hours post-fertilisation (hpf) in the zebrafish embryo, Fgf ligands are concentrated in the presumptive dorsal organiser, as a result of Nodal signalling (Mathieu et al., 2004). As development progresses, Nodal continues to induce Fgf expression in the most marginal cells, leading to a gradient of Fgf/Erk signalling, and ultimately, patterning mesodermal and endodermal lineages (Fürthauer et al., 2004).

The preprint I have chosen to highlight introduces a modified version of the Erk-KTR biosensor, which was created in order to distinguish between real Erk signalling from Cdk1 activity. It was used here to study the cause of Fgf/Erk signalling heterogeneity during embryonic development and subsequent patterning of the blastula at the embryonic margin.


Key findings

An improved Erk-specific biosensor

In this present study, Wilcockson and authors observed Erk-KTR to display off-target activity, particularly during cell cycle remodelling at the mid-blastula transition. Transgenic Erk-KTR zebrafish embryos treated with an Erk inhibitor (MEKi), as well as a Cyclin Dependent Kinase 1 (CDK1) inhibitor, resulted in maximal nuclear localisation (low Erk activity) of the biosensor, suggesting that Erk-KTR is regulated by a combination of Erk and Cdk1.  To circumvent this, the authors introduced a point mutation in the putative cyclin-docking site of the Erk-docking domain, in hopes to reduce Cdk1 responsiveness. An additional Erk docking site was also introduced to ensure that this change would not reduce Erk binding.

This modified Erk-KTR biosensor (modErk-KTR), compared to the original version, showed high Erk activity (cytoplasm>nuclear expression) from as early as 3.6 hpf, and was restricted to the presumptive dorsal organiser. Progressively, this reporter showed expansion throughout the embryonic margin, with very few animal pole cells showing Erk activity (cytoplasm<nuclear expression). Again, ­embryos were treated with Erk and CDK1 inhibitors and it was found that CDK1 inihibition alone did not affect Erk biosensor activity, while Erk inhibition, as well as combined Erk and CDK1 inhibition, reduced its activity.

The authors next tested the functionality of modErk-KTR in a number of systems where rapidly dividing cells can be found. In the Drosophila larvae, for example, modErk-KTR, compared to Erk-KTR, accurately reported high Erk activity at the poles of the animal and low Erk activity at the mediolateral regions. Furthermore, modErk-KTR localisation was shown not to be influenced by cell cycle in the imaginal disc eye, contrary to what was found using Erk-KTR. Overall, in combination with additional functional studies not further discussed here (refer to: Figure 4E, F; Figure S1B; Figure S2B, C for further reading), the authors could show that 1) modERK-KTR displays improved sensitivity to Erk signalling and, 2) modERK-KTR is not sensitive to Cdk1 expression.

modERK-KTR reads out Fgf/Erk signalling gradient formation in real time

The authors next utilised this modified Erk biosensor to characterise Fgf/Erk signalling dynamics in the zebrafish embryo. By taking images every 5 minutes, the authors established an Erk signalling gradient from the margin. Interestingly, a high level of Erk signalling variability was noted in these cells, and at every time point, indicating Erk signalling heterogeneity at the margin. In addition, treatment with an Erk signalling inhibitor showed that the cells closest to the margin were more sensitive to Erk inhibition. However, this sensitivity was found to be significantly delayed for modERK-KTR cells situated in the same region, compared to endogenous P-Erk, despite displaying the highest Erk activity. The authors suggested that this difference, between P-Erk and modERK-KTR, was due to the modErk-KTR reporter only reporting cumulative Erk expression in this region. Nevertheless, these findings address an important question surrounding the heterogeneity of Erk signalling at the embryonic margin, and also the nature of Erk dynamics during this period of development.

Mitotic erasure induces oscillatory Fgf/Erk signalling dynamics in the presumptive mesendoderm

Finally, the authors explored cell cycle dynamics with modErk-KTR activity in the early zebrafish embryo. Interestingly, the authors describe a rapid decrease in Erk activity 2-3 minutes before the cells enter mitosis, and then a re-activation again post-mitosis. Although there was no correlation in Erk dynamics post-mitosis between mother and daughter cells, sister cells displayed similar Erk oscillations, reflecting the temporal regulation of Erk signalling. Spatially, the authors found that cells closest to the embryonic margin had the fastest activation rate and higher levels of Erk activity compared to those that were further away. This was positively correlated with the availability of Fgf8a ligands in the extracellular compartment at the embryonic margin, suggesting that this was likely to be the cause of the rapid Erk re-activation.

Indeed, the further the cells were away from the margin, the more variable in Erk re-activation time they were. This points towards an interesting mechanism that describes Fgf/Erk patterning during development. In addition, this work highlights the potential of asynchronous cell divisions and how this, as well as other sources, can induce heterogeneity in some of the early cell fate decisions that are made in the developing embryo.


What I like about the preprint

Methods for studying Erk signalling dynamics have mainly focussed on using biochemical assays or tissue staining on fixed materials. Wilcockson and colleagues highlight a new biosensor that directly studies Erk dynamics in cells that pattern the developing blastula.

The reason I like this preprint is that it takes an existing tool and simply asks: “how can we improve on this?” The approach of modifying potential interaction domains to accommodate for undesirable traits, and then subjecting the modifications to multiple routes of validation, is deeply valuable and should be acknowledged. In addition, the authors have directly addressed concerns about using biosensors in densely packed, non-uniform, or highly proliferative cells, which significantly improves the utility of modErk-KTR.

Besides of the findings themselves, I think this preprint benchmarks the level of validation that is necessary before providing a resource to the community. The modERK-KTR system was verified in multiple in vitro and in vivo contexts, including settings of low/high Erk signalling and low/high cell divisions. I also liked that the authors clearly laid out the limitations of the tool, and potential causes of these; another aspect that is important when providing a new resource.

 

References

Fürthauer, M., Van Celst, J., Thisse, C., and Thisse, B. (2004). Fgf signalling controls the dorsoventral patterning of the zebrafish embryo. Development 131, 2853-2864.

Lavoie, H., Gagnon, J., and Therrien, M. (2020). ERK signalling: a master regulator of cell behaviour, life and fate. Nat Rev Mol Cell Biol 21, 607-632.

Mathieu, J., Griffin, K., Herbomel, P., Dickmeis, T., Strähle, U., Kimelman, D., Rosa, F.M., and Peyriéras, N. (2004). Nodal and Fgf pathways interact through a positive regulatory loop and synergize to maintain mesodermal cell populations. Development 131, 629-641.

Regot, S., Hughey, J.J., Bajar, B.T., Carrasco, S., and Covert, M.W. (2014). High-sensitivity measurements of multiple kinase activities in live single cells. Cell 157, 1724-1734.


Future directions and questions for the authors

Q1. Do the authors have any future plans to make any other modifications to ERK-KTR or modErk-KTR?

Q2. Did the authors notice asymmetry of Erk signalling at the embryonic margin?

Q3. How early does the nuclear membrane break down before mitosis? Can this potentially influence Erk biosensor activity?

Q4. Did the introduction of the additional Erk docking site increase the specificity of the biosensor independently of changing the cyclin-dependent site?

Q5. Would you expect the rapid decrease in Erk activity (prior to mitosis) to be rescued in cells that are treated with anti-mitotic agents?

Tags: biosensor, development, drosophila, erk, fgf signalling, kinase translocation reporter, mitosis, zebrafish

Posted on: 21 December 2022

doi: https://doi.org/10.1242/prelights.33398

Read preprint (No Ratings Yet)

Author's response

Scott G. Wilcockson shared

Q1. Do the authors have any future plans to make any other modifications to ERK-KTR or modErk-KTR?

We are now happy with the Erk specificity of modErk-KTR and do not have any plans to tinker with it any further for now. The modular nature of the KTR system does make it easy to modify and so it might be interesting in the future to test how Erk binding sites from different proteins, or additional regulatory elements, might improve/affect the biosensor. We do plan utilise different fluorophores for use in zebrafish and mammalian systems to enable multiplexing with other reporter systems. In addition, we will apply the polycistronic nuclear marker system to zebrafish (used here in the Drosophila transgenics) as this would be useful when using the reporter in densely packed tissues.

 

Q2. Did the authors notice asymmetry of Erk signalling at the embryonic margin?

We do observe some asymmetry in the postmitotic reactivation of Erk signalling between sister cells. If we follow sister cells over time, we only observe a weak correlation in relative Erk activity levels, based on KTR localisation, suggesting that one sister reactivates and/or achieve higher levels of Erk signalling, at least 30 mins post-mitosis. In a small number of cases this is likely due to the movement of sisters away or toward the embryonic margin. However, it is unclear what else might be driving this. It does appear that sisters closest to the margin are more likely to be more synchronised, but we will need to look into this more. It will be interesting to see if there are any differences in cell size or the inheritance of signalling molecules that could account for any asymmetry in post-mitotic reactivation.

 

Q3. How early does the nuclear membrane break down before mitosis? Can this potentially influence Erk biosensor activity?

The rapid loss of Erk signalling we observe (rapid shuttling of the KTR into the nucleus) occurs just prior to the onset of prophase (DNA condensation and nuclear envelope breakdown). Indeed, we note that upon DNA condensation, the KTR becomes evenly dispersed throughout the cytoplasm and remains so throughout mitosis. Post-mitosis we observe that upon DNA decondensation (and presumably nuclear envelope reformation), the KTR rapidly accumulates in the nucleus once more and cells gradually reactivate signalling over time. Therefore, due to the absence of the nuclear envelope during mitosis the KTR cannot function as a read-out of Erk activity, however, based on the pre- and post-mitotic levels of Erk activity (as well as P-Erk immunostains) it is clear that Erk is inactive during mitosis in the early zebrafish embryo. It would be interesting to use a nuclear envelope marker, as well as other cell cycle reporters, to precisely probe the timing of Erk signalling dynamics across the entire cell cycle. Interestingly, in the Drosophila blastoderm the nuclear envelope is only partially disassembled during mitosis. We have indeed noticed that the KTR remains nuclear throughout mitosis suggesting that Erk activity may also be low or absent during mitosis in the Drosophila blastoderm.

 

Q4. Did the introduction of the additional Erk docking site increase the specificity of the biosensor independently of changing the cyclin-dependent site?

We did not test this, but I would predict that only adding an additional Erk docking site would not improve the specificity of the reporter. There are examples in the literature illustrating the Erk-dependent response of the original Erk-KTR in many different contexts, including in zebrafish and Drosophila. We see the problem as not that the KTR was inefficient at interacting with Erk, but that it also binds cyclin-CDK complexes, as well as the fact that the target threonines that are phosphorylated are optimal for both Erk and Cdk1. Therefore, to improve Erk specificity we needed to block this additional cyclin-CDK interaction. So, we focused on the putative cyclin binding site as we were unable to change the target threonines or surrounding amino acids, as has previously been done for the Erk-FRET reporter (Ponsioen et al. 2021), as they are required for NLS function.

 

Q5. Would you expect the rapid decrease in Erk activity (prior to mitosis) to be rescued in cells that are treated with anti-mitotic agents?

Great question! This is exactly the kind of thing we are excited to start exploring now. How is Fgf/Erk signalling and downstream outputs influenced by proliferation? Indeed, we might hypothesise that if we stop cells dividing then we will lose these periods of Erk inactivity. If so, we could then start interrogating whether there is any functional consequences to the signalling dynamics we have observed in early embryonic development.

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