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

Abstract

Cell death events continuously challenge epithelial barrier function, yet are crucial to eliminate old or critically damaged cells. How such apoptotic events are spatio-temporally organized to maintain epithelial homeostasis remains unclear. We observe waves of Extracellular Signal-Regulated Kinase (ERK) and AKT serine/threonine kinase (Akt) activity pulses that originate from apoptotic cells and propagate radially to healthy surrounding cells. Such a propagation requires Epidermal Growth Factor Receptor (EGFR) and matrix metalloproteinase (MMP) signaling. At the single-cell level, ERK/Akt waves act as spatial survival signals that locally protect cells in the vicinity of the epithelial injury from apoptosis for a period of 3-4 hours. At the cell population level, ERK/Akt waves maintain epithelial homeostasis (EH) in response to mild or intense insults. Disruption of this spatial signaling system results in the inability of a model epithelial tissue to ensure barrier function in response to cellular stress.

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

“…Outer cells displayed ERK frequencies of typically one pulse every 2 hours on average, while inner cells pulsed rarer and often did not exhibit ERK pulses at all ( Figure 4A). This is consistent with a similar phenomenon in serumstarved 2D MCF10A cultures in which dying cells evoke local waves of single-cell ERK pulses that each induce survival for approximately 4 hours (Gagliardi et al, 2020).…”

“…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).…”

“…In non-tumorigenic MCF10A mammary epithelial cells cultured in 2D, fate decisions are controlled by the average frequency of asynchronous, singlecell ERK activity pulses of constant amplitude and duration, that we refer to as ERK frequency from now on. Low ERK frequency predisposes cells to apoptosis, whereas high ERK frequency drives survival and cell cycle progression (Albeck et al, 2013; Gagliardi et al, 2020). Single-cell ERK dynamics have, however, never been measured in 3D mammary acini.…”

CODEX, a neural network approach to explore signaling dynamics landscapes

“…H2B-miRFP703, ERK-KTR-mTurquoise2 and FoxO3a-mNeonGreen constructs were generated and subcloned in the piggy PiggyBac vectors pMP-PB, pSB-HPB and pPB3.0.Blast as previously described 17 . Upon transfection of these plasmids with FuGene (Promega), cells were treated with 2.5 μg/ml Puromycin, 25 μg/ml Hygromycin and 5 μg/ml Blasticidin to select stably expressing cells.…”

“…To obtain single-cell bivariate signalling trajectories of ERK and Akt activities we used a dedicated image analysis pipeline, as previously described 17 . First, we trained a random forest classifier based on different pixel features (Ilastik 18 ) to separate H2B-miRFP703 fluorescence from background signal.…”