Collective MAPK Signaling Dynamics Coordinates Epithelial Homeostasis

Abstract

12Epithelial tissues are constantly challenged by individual cell fate decisions while 13 maintaining barrier function. During oncogenesis, mutant and normal cells also differ in their 14 signaling states and cellular behaviors creating competitive interactions that are poorly 15 understood. Here we show that the temporal patterns of MAPK activity are decoded by the 16 ADAM17-EGFR paracrine signaling axis to coordinate migration of neighboring cells and promote 17 extrusion of aberrantly-signaling cells. Concurrently, neighboring cells increase proliferation to 18 maintain cell density while oncogene expressing cells undergo cell cycle arrest. Moreover, the 19 stress MAPK p38 elicits the same paracrine signaling and extrusion response, suggesting that 20 the ADAM17-EGFR pathway constitutes a quality control mechanism to eliminate and replace 21 unfit cells from epithelial tissues. Overall, we show that the temporal patterns of MAPK activity 22 coordinates both single and collective cell behaviors to maintain tissue homeostasis. 23 24 27 cancers 1 . In normal conditions the ERK pathway promotes proliferation, differentiation, survival 28 and cell migration 2 . Conversely, during oncogenesis mutations or amplification of ERK pathway 29 components can also promote oncogene-induced senescence 3 (OIS) or cellular extrusion from 30 epithelial monolayers 4,5 . The mechanisms underlying dose dependent effects of ERK signaling 31 have been intensely studied using bulk cell population assays. However, the advent of single cell 32 analysis has shown that single cells often behave qualitatively different than bulk populations. In 33 fact, in vivo and in vitro studies have now shown that pulsatile or sustained ERK activity have 34 different effects on cell fate 6-12 . Whether oncogenic perturbations also have different functional 35 outcomes depending on downstream signaling dynamics remains unknown. To address this 36 question, an isogenic single-cell approach with temporal control of oncogene expression is 37 needed. 38 39 Recent in vivo studies revealed that oncogene expression can trigger cell-cell competition and 40 tissue level responses involving normal neighboring cells 13-16 . During these events, the 41 mechanistic basis of competition and the signaling events involved in recognition between normal 42 and diseased cells are poorly understood. Coincidentally, propagating ERK signaling waves that 43 depend on the sheddase ADAM17 have been observed in mouse epidermis and intestinal 44 organoids, but the physiological role of these collective signaling events in homeostasis remains 45 unclear 7,8,17 . Understanding of the context-dependent mechanisms of paracrine signaling and cell-46 cell competition upon oncogene expression holds the key to unlocking new therapeutic strategies. 47 48 Here we combine live cell imaging of signaling biosensors with inducible expression of oncogenes 49 to study the cell autonomous and non-cell autonomous effects of oncogene expression in 50 epithelial monolayers. Our data shows that pulsa...

Spatio-temporal Control of ERK Pulse Frequency Coordinates Fate Decisions during Mammary Acinar Morphogenesis

“…We then imaged single-cell ERK dynamics in stage 1 acini using 3D confocal spinning disk microscopy of both the H2B and ERK KTR channels with time resolutions of 3 -5 minutes, until acini started to suffer from phototoxicity (which was observed after 10 -23 hours of imaging). Visual inspection of micrographs of stage 1 acini revealed that different cells displayed asynchronous ERK pulses ( Figure 2A) as previously observed in 2D cultures (Aikin et al, 2020;Albeck et al, 2013;Gagliardi et al, 2020). To quantify single-cell ERK activity trajectories, we used a custom-modified version of the opensource LEVER software (Wait et al, 2014;Winter et al, 2016) to segment and track nuclei based on their H2B signal; and to extract ERK activity trajectories by calculating the ratio of the ERK KTR intensities in the nucleus over a voxel mask around the nucleus ( Figure 2B).…”

Cell Extrusion: Crowd Pushing and Sticky Neighbours

“…Moreover, it is very difficult at this stage to connect the perinuclear relocalisation of actin with the convergent movement of cells. Interestingly, these waves share many similarities with a recent description of ERK waves observed in vicinity of apoptotic cells and BRAF transformed cells [19]. ERK waves also trigger collective movements toward the transformed cells which drive their apical extrusion.…”

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

“…3C, S4A-I). These results suggest that ERK/Akt waves depend on MMP-mediated cleavage of pro-EGF ligands (Dong et al 1999;Aikin et al 2019) (summarized in Fig. 3D).…”