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Collective ERK/Akt activity waves orchestrate epithelial homeostasis by driving apoptosis-induced survival

Paolo Armando Gagliardi, Maciej Dobrzyński, Marc-Antoine Jacques, Coralie Dessauges, Robert M Hughes, Olivier Pertz

Preprint posted on 11 June 2020 https://www.biorxiv.org/content/10.1101/2020.06.11.145573v1

Article now published in Developmental Cell at http://dx.doi.org/10.1016/j.devcel.2021.05.007

Staying alive! - and the role of apoptosis in neighbour communication.

Selected by Mariana De Niz

Categories: cell biology

Background

The epithelium is a self-organizing tissue that coordinates cell division and cell death to maintain its barrier function (i.e. epithelial homeostasis). Cell death events continuously challenge epithelial barrier function, yet are crucial to eliminate old or critically damaged cells. Apoptotic cells can influence the fate of neighboring cells in different ways including inducing proliferation, resulting in wound-repair processes inducing further apoptosis during developmental processes requiring collective apoptosis; or triggering survival fates. These different processes imply coordination of signaling pathways that regulate proliferation, survival or apoptosis fates. However, how these signaling pathways are regulated at the single-cell level, and spatio-temporally integrated at the population level remains poorly understood.

Previous work has shown that mitogen-activated protein kinase (MAPK)/ERK-phosphoinositide-3 kinase (PI3K)/Akt signaling networks are crucial for regulation of cell fate, and play a role both at population, and single cell levels. In their work, Gagliardi et al (1) explore the roles of EGF and ERK/Akt in regulating additional single-cell fate decisions, and how the latter are integrated at the cell population level to ensure epithelial homeostasis.

Figure 1. ERK/Akt waves involve EGFR and MMP signaling. Cartoon shows the propagation mechanism of the ERK/Akt activity waves (from Ref.1).

 

Key findings and developments

How are ERK/Akt pulses propagated from apoptotic cells to neighbouring healthy cells?

The authors generated an epithelial cell line with  various reporters for Histone 2B (H2B), ERK-KTR and FoxO3a to investigate single-cell ERK/Akt activity dynamics in epithelia (the latter two biosensors report single-cell ERK and Akt activity by displaying reversible nuclear-to-cytosol translocation upon phosphorylation), and an automated image analysis pipeline to segment and track nuclei (based on H2B), and extract cytosolic to nuclear fluorescence intensities to quantify ERK/Akt activities.

They began by observing ERK/Akt pulses in starved cells, and showed that starved monolayers display spatially and temporally coordinated ERK/Akt activity pulses, resulting in collective signaling events. Such events were triggered by apoptosis resulting from the starvation-induced stress. In such case, the wave of ERK/Akt activity pulses originated from an apoptotic cell, and radiated sequentially to the subsequent layers of healthy neighbouring cells. Notably, the proportion of neighboring cells with ERK/Akt pulses sharply decreases across the layers (i.e. 90% of cells are activated in the first, 30% in the second, and 10% in the third layer).

When during the apoptosis process are ERK/Akt waves initiated?

The morphological events occurring in apoptosis include nuclear shrinkage, plasma membrane blebbing, chromatin condensation, extrusion of the apoptotic cell, nuclear fragmentation, and disaggregation into apoptotic bodies. Based on this, the authors went on to determine at what point during the sequence of apoptotic events, ERK/Akt waves were initiated. They found that the onset of ERK/Akt waves coincided with nuclear shrinkage. Moreover, they used OptoBAX, an optogenetic apoptosis actuator, to selectively induce cell death with single-cell resolution. They observed that OptoBAX-induced apoptosis triggered an ERK wave identical to the one caused by spontaneous apoptosis. To determine whether a correlation existed between ERK/Akt waves and caspase activity, they treated cells with a pan-caspase inhibitor. They found that this treatment did not prevent ERK/Akt waves neither in spontaneous events, not in OptoBAX-triggered apoptosis.

Which signaling pathways are involved in ERK/Act wave triggering?

Specific ERK or Akt inhibition resulted in abrogation of ERK or Akt, but not both, suggesting that both pathways are activated by an upstream signaling node. Based on this, the authors went on to explore upstream actuators. They inhibited EGFR catalytic activity, and showed that this completely abrogated apoptosis-triggered ERK/Akt waves, both in spontaneous events and in OptoBax-triggered apoptosis. Interestingly they showed equal effects upon antibody-based inhibition of EGFR or drug-mediated inhibition of MMP. Altogether, this suggests that ERK/Akt waves are triggered by EGFR and MMP signaling, and that they likely depend on MMP-mediated cleavage of pro-EGF ligands.

Do ERK/Akt waves regulate cell extrusion?

As a next step, the functional significance of apoptosis-triggered ERK/Akt waves was investigated. This included testing the hypothesis that ERK/Akt waves regulate the cytoskeletal process and mechanical forces participating in extrusion and/or closing of the epithelial gap. Drug-based inhibition of EGFR-dependent ERK/Akt waves, however, did not block the process of extrusion.

Do apoptosis-triggered ERK/Akt waves generate signals that regulate fate decisions?

The authors began by testing the hypothesis that ERK and Akt signaling waves provide local survival signals. For this, they generated computational approaches to analyse ERK signaling, and trained a convolutional neural network (CNN) to separate apoptotic from non-apoptotic cells based on ERK activity. First, they explored whether ERK activity dynamics differed between apoptotic cells before nuclear shrinkage, and non-apoptotic cells within the same time period. The results, based on the frequency of ERK pulses, suggest that the apoptosis/survival fates in starved monolayers depend on the ERK pulse frequency, whereby trajectories without any pulses correspond to apoptotic cells in the majority of cases, while in most trajectories with 2-5 pulses or more, cell do not undergo apoptosis. The next step was to test if different signaling dynamics in apoptotic and non-apoptotic cells specifically correlated with collective signaling events. Using computational approaches they found that collective ERK signaling events occurred mostly in non-apoptotic cells. Finally, they explored whether an ERK pulse within an apoptosis-triggered signaling wave locally promotes survival. They found that within a 4 hours time-window, secondary apoptosis is significantly less likely to occur in cells that experienced an ERK pulse induced by the primary event, suggesting that an ERK activity pulse within an apoptosis-triggered signaling wave induces survival within this time limit.

How is survival fate modulated?

Given that the results suggest that a critical pulse frequency of ERK and perhaps also of Akt are required to promote the survival fate, the authors went on to determine whether specific dynamic signaling frequencies regulate the survival fate. For this, they used two optogenetic systems to evoke synthetic signaling pulse regimes with different frequencies. One of the systems activates both ERK and Akt signaling, while the other selectively controls ERK activity only. The authors used different stimulation regimes– varying in time frequency, and evaluated apoptosis. Protection against apoptosis was observed when ERK pulses were triggered within short time regimes (3 hour intervals), but were lost within longer regimes (6 and 12 hour intervals). ERK or Akt inhibition abrogated the protection granted by optogenetic stimulation. Altogether, the results suggests that ERK signaling is sufficient to exert the pro-survival effects.

What are the physiological effects of apoptosis-induced survival?

The authors then explored whether ERK/Akt wave-mediated local survival in the vicinity of apoptotic sites contributes to endothelial homeostasis and tissue integrity in response to stress. They observed that starvation resulted in a transient peak of apoptosis that began 2-3 hours after starvation, and lasted another 2-3 until a steady-state apoptosis rate was reached. ERK/Akt activities immediately decreased with starvation and were transiently re-activated with kinetics that were slightly delayed with respect to the apoptotic rate. This suggests that the monolayer can adapt to starvation-induced stress by regulating survival to maintain homeostasis and tissue integrity.

The authors built a mathematical model consisting of two components (cells undergoing apoptosis and protection from cell death (population averaged ERK/Akt activity triggered by apoptosis) to capture the dynamic relationship between apoptosis and survival. They then explored 3 scenarios: the first scenario, considered a low rate of apoptosis and a high rate of protection (mimicking the starvation experiment) – in this case, the model agreed with the experiment, in that an episode of increased apoptosis is followed by a delayed induction of protection. In the second scenario, the rate of apoptosis was increased. The model predicted a higher protection level and apoptosis rate after the initial apoptotic event. Experimental approaches were in line with these predictions. Finally, in the third scenario, the protection rate was lowered. In this case, the apoptosis rate was higher. The experimental approaches were also in line with these predictions.

Importantly, the authors show that changes in apoptosis impact tissue integrity, as measured by holes observed in the epithelium following drug-mediated damage. These results suggest that single-cell level apoptosis-induced survival- signaling contributes to population-level tissue integrity by its ability to react to insults of different intensities, and by spatially distancing the apoptotic sites.

What I like about this preprint

I think the topic is very relevant to various fields of study within cell biology and pathology. Moreover, I found the way of approaching the questions very thorough, and the range of methods used is extraordinary – with many generated by the authors themselves. I like microscopy and image analysis, and rarely had I seen this range of techniques used in a single paper in the context of cell biology. Moreover, I enjoyed reading the work- the questions follow a very logical order, making this an enjoyable read.

References

  1. Gagliardi PA et al, Collective ERK/Act activity waves orchestrate epithelial homeostasis by driving apoptosis-induced survival, bioRxiv, 2020.

 

Posted on: 13 July 2020

doi: https://doi.org/10.1242/prelights.23041

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Author's response

Paolo Gagliardi shared

Open questions 

1.I enjoyed reading your work a lot. My first question is whether there is anything known in vivo in homeostasis and disease, about apoptosis-induced survival, and in general about ERK/Akt signaling?

Thank you for your appreciation on our work. We think that apoptosis-induced survival (AiS) can be relevant in vivo as well. In 2014, Bilak A et al (doi:10.1371/journal.pgen.1004220) found that induction of apoptosis in wing imaginal discs of Drosophila larva makes the neighbouring cells more resistant to apoptosis induced by ionizing radiations. Such a protection mechanism requires the activation of the anti-apoptotic microRNA Bantam and the tyrosine kinase receptor tie. More recently, Valon L et al (doi.org/10.1101/2020.03.17.994921) have found that in the Drosophila pupal notum there is a wave of ERK activity that propagates from apoptotic cells in the neighbouring ones. Such a wave is fundamental to protect the neighbours from further apoptosis, avoiding multiple apoptotic events and loss of the barrier function. So far, this mechanism has never been shown in vivo in vertebrates in a physiological context. However, we hypothesize that tumor cells could highjack this mechanism to boost their survival. For instance, in human ovarian cancer cell lines the pro-apoptotic drug doxorubicin induces the activation of the HER3‐PI3K‐AKT signalling pathway that makes cells more resistant to further apoptosis (doi.org/10.1016/j.molonc.2012.07.001).

2.As you mentioned in your work, apoptosis is crucial to eliminate senescent or damaged cells, and this likely happens at different rates in different tissues where exposure to insult is more or less likely, and senescence rates vary vastly. Do you envisage that in each tissue, apoptosis induced survival and maintenance of epithelial homeostasis follows the observations you saw in your 3 mathematical scenarios?

So far, we have proven that apoptosis induces AiS, but we do not have any evidence about senescence. If we consider only apoptosis, it is very likely that different tissues vary for the apoptotic rates, for the extent of propagation of the ERK/Akt wave and for the intensity of the protection effect. So, depending on these properties it is possible to think a large variety of scenarios depending on the tissue type or the physiological/pathological conditions. Our computational model allows us to explore a large space of protection activation and apoptosis rate constants where we can identify other scenarios corresponding to other tissue types or treatments.

3.There are diseases whereby apoptosis of damaged/senescent/infected cells is compromised as part of the pathology. While lack of excision of damaged cells might compromise tissue homeostasis and barrier effect, how do you envisage, based on your findings, that this will affect the rest of the tissue in a spatio-temporal manner?

Based on what we know, extrusion and AiS are independently regulated. Indeed, whereas extrusion is a process that requires mechanical forces to be executed, AiS relies on paracrine signalling.  It is possible to think a condition in which extrusion is inhibited, but the mechanism responsible for the ERK/Akt wave propagation is still working. This is the case of caspases inhibition with zVAD-FMK (Figures 2K and L) that completely inhibits the process of extrusion, but it is still able to produce the ERK/Akt activity wave. However, failed extrusions might be in the long term detrimental for the epithelial barrier function despite AiS is still working. Dead cell bodies and debris might accumulate in the space between cells causing a leak in the epithelial barrier. We think that it is an interesting topic to be explored. For instance, it is interesting to understand if cell death bodies inside the epithelium can have a different effect on AiS compared to properly executed epithelial extrusion.

4.While apoptosis is one of the mechanisms of cell death and of ensuring tissue homeostasis, there are other forms of cell death, like autophagy or necrosis. How do you think death by these different mechanisms would influence -induced survival responses?

AiS is a general principle, based on very simple rules: the dying cell must have the ability to deliver a message to the neighbours and the neighbours in turn must be able to encode such a message in the survival phenotype. In this article, we explored only apoptosis and we describe a mechanism that involves EGFR-ligand release by metallo-proteases and the binding of mature EGFR-ligands to the EGFR receptor. But in principle any paracrine signalling combined with a proper transduction pathway in the neighbours could generate an effect like AiS. Further studies are needed to understand if a similar AiS is present also with the other types of programmed cell death.

5.You explored in your work, how protection remains for a certain amount of time following apoptosis and ERK/Akt waves. In the case of consistent stimuli in a tissue (such as inflammation) or ongoing apoptosis, how do you envisage this would affect the survival of neighbouring cells? Namely, is there a threshold of stimulus, whereby protection in neighbouring cells would be lost?

This is a very good point. It is well known that at high ligand concentration, transmembrane receptors can head to a degradation pathway instead of being recycled back to the plasma membrane. This causes the reduction of the overall number of exposed receptors and consequent partial desensitization of the cell to constant stimuli. This mechanism is well studied for the case of EGFR, that is indeed the receptor that mediates AiS in our model.In addition, during propagation of the ERK/Akt activity waves, we see that where the waves stop there are neighbouring cells that do not show any pulse of ERK/Akt activity. This suggest that there is a threshold below which neighbouring cells fail to respond to the EGFR ligand. Such a threshold effect is very important to define the propagation size of the ERK/Akt activity wave.

Combining these two observations, we can hypothesize that if there is a constant background ligand concentration cells could adapt to be responsive to stimulation coming from the apoptotic cell. Further studies are needed to understand if it is the case.

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