Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia

Abstract

For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

Figure 1. Both apoptotic and non-apoptotic epithelial cells extrude at locations of high crowding during homeostasis and development

Non-apoptotic (caspase-negative) and apoptotic (caspase-positive) extrusions at the surface of human colon epithelia (a, b), zebrafish epidermis (d, e), and confluent MDCK monolayers (g, h). Extrusions typically occur at sites of high crowding (indicated by yellow cells in c, f, and i). Arrows indicate direction of force from mitoses and migration. Scale bars=10 μm.

Figure 2. Characterisation of experimental overcrowding

(a), Experimental design for overcrowding monolayers grown on stretched matrix. Only cells in the grey area (right) were analysed to ensure a homogeneous strain field. (b–e) Images of the nuclei of control unstretched monolayers, and monolayers 0.5–6 hours post-overcrowding were quantified in (f). Mean number of nuclei per field is plotted, error bars represent s.e.m. (g–i) Monolayers at 0.5 and 2 hours post-release show that adherens and tight junctions maintain integrity after overcrowding, as compared to control. (j), Monolayer stained for actin and DNA at 2 hours after release on Cy5-labeled fibronectin shows that contacts to substratum are maintained. Scale bars = 10 μm. ***=p<0.0005, **=p<0.005, *=p<0.05.

Figure 3. Overcrowding MDCK monolayers promotes extrusion of non-apoptotic cells

(a), Quantification of live and apoptotic extruding cells 2 hrs post-overcrowding. (b), Live cell extrusion requires ROCK, Sphingosine Kinase, and S1P₂ (JTE-013). (c), Cell viability 2 or 24 hrs after extrusion. (d), Growth of extruded cells collected at 2 hrs post crowding after 24hrs and 5 days. Gd³⁺ inhibits both overcrowding-induced extrusion (e) and homeostatic extrusion (g) whereas Bcl-2 over-expression and JNK inhibition only affect UV-C-induced apoptotic extrusion (f). n≤7 independent experiments, mean ± s.e.m. Scale bars=100 μm. ***=p<0.0005, **=p<0.005, *=p<0.05.

Figure 4. Disrupting the stretch-activated channel Piezo1 in vivo blocks live cell extrusion and causes epithelial mass formation

(a) Proliferation and extrusion rates during zebrafish tail fin development. (b) Gd³⁺-treatment and piezo1 photo-MO block extrusion (n=15–17 embryos from 3 independent experiments, mean ± s.e.m). Brightfield and confocal projections of untreated (c,d), Gd³⁺-treated (f,g), and piezo1 photo-MO knockdown (i, j) in 60hpf developing zebrafish. (e, h), Tracks from red circle mark the movement of individual epithelial cells in control and Gd³⁺-treated Tg(cldnb:lynGFP) developing zebrafish. (k) Epidermal cell mass from a piezo1 photo-MO knockdown in Kaede zebrafish, where yellow cells are photo-converted. (l), Model of induced cell death versus homeostatic cell death. Arrowheads denote cell accumulations and asterisk an extrusion. Scale bars in c, f, & i = 100 μm, in d, g, & j =10 μm. ***=p<0.0005, **=p<0.005, *=p<0.05.