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# Epithelial cell extrusion at a glance

Aline Grata, Romain Levayer

► To cite this version:

Aline Grata, Romain Levayer. Epithelial cell extrusion at a glance. Journal of Cell Science, 2025, 138 (8), pp.jcs263786. 10.1242/jcs.263786. pasteur-05295651

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CELL SCIENCE AT A GLANCE

# Epithelial cell extrusion at a glance

Aline Grata and Romain Levayer*

## ABSTRACT

The robustness and plasticity of epithelial tissues rely on the capacity of such tissues to eliminate cells without affecting their sealing. This is achieved by epithelial cell extrusion – a well-orchestrated series of remodelling steps involving the eliminated cell and its neighbours – which ensures the constant maintenance of mechanical and chemical barrier properties while allowing cell expulsion. In this Cell Science at a Glance and the accompanying poster, we describe the remodelling steps that take place within dying or extruding cells, as well as neighbouring cells, outlining the commonalities and variations

between tissues and organisms. These steps include reorganization of the cytoskeleton and remodelling of cell-cell junctions that alters their contribution to mechanical coupling and mechanotransduction. We also discuss larger-scale coordination between cells and the contribution of cell extrusion to tissue morphogenesis, epithelial surveillance mechanisms, and pathologies such as cancer and chronic inflammation. Altogether, we outline the complexity and plasticity of this minimalist morphogenetic process.

**KEY WORDS:** Epithelium, Cell extrusion, Apoptosis, Cell-cell adhesion, Junctions, Actomyosin, Caspases

Department of Developmental and Stem Cell Biology, Institut Pasteur, Université de Paris Cité, CNRS UMR 3738, 25 rue du Dr. Roux, 75015 Paris, France.

*Author for correspondence (romain.levayer@pasteur.fr)

R.L., 0000-0001-6769-1465

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

## Introduction

Epithelia are tissues composed of polarised cells interconnected through various junctions, forming protective yet very plastic chemical and mechanical barriers. During embryogenesis, epithelial cells divide, die, migrate and differentiate to shape tissues and organs. In adults, epithelia with high cell turnover, such as in the intestine or the skin, undergo constant cycles of cell division and death to maintain

<figure><img src="image_3.png" /></figure>

See supplementary information for a high-resolution version of the poster.
their size and function (Pellettieri and Sanchez Alvarado, 2007). In the adult human gut, billions of cells are eliminated daily without impacting its sealing (Blander, 2016). Epithelial tissue integrity is maintained by tightly orchestrated cell removal, in which both the expelled cell and its neighbours undergo coordinated deformations to prevent formation of gaps in the tissue. This process, called epithelial cell extrusion, is essential for the maintenance of tissue integrity (Rosenblatt et al., 2001). Cell extrusion was first described in the context of elimination of apoptotic cells, namely caspase-dependent cell extrusion (Rosenblatt et al., 2001), in which caspases directly target a number of core regulators of cell architecture in the dying cell. In other contexts, live cells can be extruded independently of apoptosis induction. Live-cell extrusion can expel epithelial cells that carry oncogenic mutations (Tanimura and Fujita, 2020), which is not necessarily associated with subsequent cell death, or can occur when cells become too crowded within the tissue, through the activation of the stretch-sensitive ion channel Piezo (herein referring to Piezo1) (Eisenhoffer et al., 2012). Cells expelled from the tissue eventually undergo cell death through anoikis, in which apoptosis is triggered upon cell detachment from the substrate (Frisch and Francis, 1994). We will distinguish these two types of extrusion, when possible, throughout the text and poster, focusing on the processes associated with ‘caspase-dependent extrusion’ and the regulatory steps specific to ‘live-cell extrusion’, which are independent of caspases.

In this Cell Science at a Glance article and the accompanying poster, we delineate the specificities and commonalities of these modes of extrusion. Cell extrusion is conserved throughout metazoans and is consubstantial with the presence of epithelia, from *Trichoplax* (Fortunato et al., 2021), sponges (De Goeij et al., 2009) and *Planaria* (Lee et al., 2024) to insects (Levayer et al., 2016) and vertebrates (Gu et al., 2011). The vast majority of cell extrusion in vertebrates is associated with expulsion of cells from the apical surface, whereas in insects most extrusion is directed to the basal side (Nanavati et al., 2020). However, there are many exceptions to this rule, including apical extrusion of gut enterocytes in *Drosophila* (Martin et al., 2018), extrusion without clear direction in the *Drosophila* ovarium (Torres et al., 2017) and the co-existence of apical and basal extrusions in the *Xenopus laevis* embryo (Ventrella et al., 2023).

Here, we provide a broad overview of the regulation and functions of cell extrusion, focusing on events of cell extrusion leading to cell death and excluding delamination events associated with cell differentiation. We first outline the structural rearrangements and signalling pathways involved in extrusion at the cellular level, within the extruding cell as well as in its immediate neighbours. We then discuss the roles of mechanotransduction, cell-cell junctions, and the larger-scale collective dynamics and tissue-scale parameters that affect extrusion. Lastly, we describe the physiological functions of cell extrusion and its pathological relevance, concluding with a list of important open questions.

## Regulation of cell extrusion at the cellular level

Cell extrusion entails several stages of deformation and cellular rearrangement, both in the extruding cell and in its neighbours. In this section, we discuss the cytoskeletal and subcellular rearrangements that drive these shape changes.

### Cell-autonomous structural changes

The first morphological change associated with extrusion is apical constriction of the extruding cell, driven by a cell-autonomous F-actin and non-muscle myosin II (MyoII) ring at the cell apex (Kuipers et al., 2014; Rosenblatt et al., 2001) (see poster). Increased RhoA activity

precedes the assembly of the actomyosin ring, and inhibition of RhoA in the extruding cell partially blocks apical constriction (Kuipers et al., 2014). The mechanisms driving actomyosin ring assembly in the extruding cell remain unclear, especially in the context of live-cell extrusion, but are slightly more established during caspase-dependent extrusion, in which active caspases in the dying cell directly target cytoskeleton regulators. In MCF10A mammary epithelial cells and Madin-Darby canine kidney (MDCK) cells, MyoII activation and actomyosin ring assembly is driven by caspase-mediated cleavage of the kinase MRCKα (also known as CDC42BPA), which renders it constitutively active and promotes MyoII light chain phosphorylation (Gagliardi et al., 2017) (see poster). Similarly, caspase cleavage generates constitutively active ROCK1, another kinase that targets MyoII (Coleman et al., 2001; Sebbagh et al., 2001), promoting cell extrusion in Caco2 colorectal adenocarcinoma cells (Gagliardi et al., 2017) and MDCK cells (Lubkov and Bar-Sagi, 2014). Alternatively, in the *Drosophila* pupal notum, a single-layer epithelium, effector caspases promote apical constriction through the disassembly of an apical microtubule mesh, which increases cell deformability and compressibility independently of MyoII regulation (Villars et al., 2022) (see poster). Finally, during the last phase of apical constriction, the extruding cell releases localised extracellular membrane vesicles to facilitate cell expulsion (Kira et al., 2023). Thus, the extruding cell displays various cytoskeletal and structural rearrangements that contribute to apical constriction. These changes are synchronised with neighbouring cells, which play an active role slightly later in the extrusion process.

### Cytoskeletal changes in neighbouring cells

While contractility increases within the extruding cell, a supracellular actomyosin ring also assembles in the neighbouring cells surrounding the extruding cell (Rosenblatt et al., 2001). This supracellular ring appears typically 20–40 min after the formation of the first intracellular actomyosin ring in an extruding MDCK cell (Gagliardi et al., 2017; Kuipers et al., 2014) and is essential for the completion of extrusion (Rosenblatt et al., 2001); however, this timescale varies between tissues, from a few minutes in zebrafish (Duszycz et al., 2021) to several hours in *Drosophila* gut (Martin et al., 2018). This ring progressively slides towards the basal surface while constricting, thereby exerting forces that push the extruding cell out apically (Kuipers et al., 2014; Le et al., 2021). Three-dimensional vertex models, simulating epithelial shape by modelling the dynamics of cell vertices (Alt et al., 2017), suggest that the relative tension between apical and basal areas of the extruding cell set the extrusion direction (Okuda and Fujimoto, 2020; Staneva and Levayer, 2023). As such, the direction in which the supracellular contractile ring slides should determine extrusion direction. Since basal focal adhesions (FAs) are mechanically connected to the supracellular actomyosin ring through radial actin cables (Le et al., 2021), the direction of extrusion could also be modulated by the strength of anchoring of the supracellular ring to FAs.

The formation of the supracellular actomyosin ring in the neighbours is thought to be driven by local delivery of the Rho guanine-nucleotide-exchange factor p115RhoGEF (also known as ARHGEF1) via rearrangement of microtubules, which direct their plus ends towards the basal side of the junctions shared with the extruding cell (Slattum et al., 2009). Local RhoA activation is also regulated by the bioactive lipid sphingosine-1-phosphate (S1P), which is released by the extruding cell and bound by its G-protein-coupled receptor S1P2 (also known as S1PR2) on the neighbouring cells, thereby promoting RhoA activation and supracellular actomyosin ring formation (Gu et al., 2011) (see poster). S1P signalling is essential for
caspase-dependent extrusion (Gu et al., 2011), crowding-induced live-cell extrusion (Eisenhoffer et al., 2012) and oncogenic cell extrusion (Yamamoto et al., 2016). However, recent experiments have shown that S1P primarily has a global permissive function for extrusion rather than a local instructive role (Atieh et al., 2021; Duszyc et al., 2021; Yamamoto et al., 2016), as discussed further below. Alternatively, RhoA activation in the neighbouring cells also depends on mechanotransduction and the contractility of the extruding cell (Duszyc et al., 2021), as discussed in the next section. Neighbouring cells also extend lamellipodia via actin polymerisation basal to the extruding cell (Duszyc et al., 2021; Kocgozlu et al., 2016; Kuipers et al., 2014) (see poster), which likely exerts pushing forces on the basal side and weakens the adhesion of the extruding cell to the extracellular matrix (ECM). Previously, the contribution of lamellipodia was mostly linked to extrusions at low epithelial density, whereas the supracellular actomyosin ring was thought to be a dominant factor in extrusions at high cell density (Kocgozlu et al., 2016). This binary distinction has recently been nuanced by data showing co-existence of the two mechanisms, with a dominant role of basal protrusion and discontinuous or faint recruitment of actomyosin in neighbours occurring upon weakening of cell-cell adhesion, compared to fewer basal protrusions and stronger contributions from a more continuous supracellular actomyosin ring associated with low substrate adhesion, ensuring the robustness of extrusion despite variation in cell-cell and cell-ECM adhesion (Le et al., 2021).

To conclude, the neighbours of the extruding cell play crucial roles in the extrusion process: first, by generating contractility through the formation of a supracellular actomyosin cable, and second, by extending basal lamellipodia below the extruding cell. Importantly, throughout these changes, the tissue remains sealed, with junctions enabling extensive mechanical coupling between cells.

## Junctional remodelling and mechanotransduction

In this section, we describe the remodelling of cell-cell junctions during extrusion, highlighting their essential roles for anchoring mechanical forces, mediating mechanotransduction and coordinating the remodelling steps between the extruding cell and its neighbours (see poster).

### Mechanotransduction at adherens junctions

Adherens junctions (AJs) are central regulators of epithelial cell-cell adhesion mostly mediated by the transmembrane adherence protein E-cadherin (E-cad; also known as CDH1 in vertebrates, Shotgun in *Drosophila*) coupled intracellularly to actin through α-catenins and β-catenin (also known as CTNNB1 in vertebrates, Armadillo in *Drosophila*) (Lecuit and Yap, 2015). During extrusion, shrinking of the apical area of the extruding cell correlates with a radial elongation of the neighbours, likely driven by passive stretching of the neighbours via constriction of the cell-autonomous actomyosin ring in the extruding cell (Lubkov and Bar-Sagi, 2014; Rosenblatt et al., 2001). This elongation requires mechanical coupling through AJs between the extruding cell and its neighbours (Kuipers et al., 2014; Lubkov and Bar-Sagi, 2014). Accordingly, knockdown of E-cad perturbs neighbour elongation, leading to extrusion failure and gap formation (Lubkov and Bar-Sagi, 2014). Force transmission is therefore essential for promoting contractility and the progression of cell extrusion. Indeed, AJs coordinate the assembly of the supracellular actomyosin ring through mechanotransduction (Duszyc et al., 2021; Michael et al., 2016; Teo et al., 2020a): tension-dependent recruitment of the mechanosensitive protein myosin VI (MYO6) in the neighbouring

cells facilitates relocalisation of the RhoA activators p114RhoGEF (also known as ARHGEF18) and p115RhoGEF to the junctions shared with the extruding cell (Duszyc et al., 2021), and E-cad also facilitates supracellular ring assembly through the tension-dependent recruitment of coronin1B at AJs in the neighbouring cells, which promotes MyoII association through F-actin realignment (Michael et al., 2016) (see poster).

### E-cadherin dynamics during cell extrusion

E-cad perdurance at AJs seems essential for orchestrating cell extrusion, but other studies have outlined permissive roles of E-cad remodelling and AJ disassembly for extrusion progression. E-cad dynamics during cell extrusion are remarkably context dependent. E-cad accumulates during apical constriction of extruding cells in the *Drosophila* pupal notum (Villars et al., 2022), whereas it is fully disassembled prior to the termination of apical constriction during larval epidermal cell extrusion in the pupal abdomen (Teng et al., 2017). During caspase-dependent extrusion, caspase-mediated cleavage of β-catenin in the dying cell destabilises E-cad, promoting its removal from the cell membrane and facilitating cell detachment (Kessler and Muller, 2009) (see poster). Alternatively, decreased E-cad levels promote oncogenic cell extrusion, and induction of E-cad extracellular domain cleavage by tobacco etch virus protease is sufficient to promote cell extrusion in MDCK cells (Grieve and Rabouille, 2014). Endocytosis of E-cad also promotes mutant Ras<sup>V12</sup> MDCK cell apical extrusion (Saitoh et al., 2017), and upregulation of E-cad endocytosis during late morphogenesis of the *Drosophila* pupal abdomen promotes larval epidermal cell extrusion (Michel and Dahmann, 2020). Interestingly extrusion is redirected toward the basal side upon E-cad depletion in MDCK cells cultured on thick collagen gel (Balasubramaniam et al., 2025), suggesting a potential role of E-cad in extrusion direction. However, in the multiple contexts described above, no unequivocal correlation has been observed between E-cad dynamics during extrusion and extrusion direction, suggesting that other factors must regulate direction.

### Desmosomal junctions

In vertebrates, desmosomal junctions, which are linked to keratin filaments and located basally to AJs, are also essential for cell-cell adhesion and force transmission (Green et al., 2019). During cell extrusion, ‘old’ desmosomes between the extruding cells and neighbours co-exist with basal desmosomes that are newly formed between neighbours, ensuring the continuity of tissue mechanical integrity (Thomas et al., 2020). Desmosomes contribute to rearrangement of the cytoskeleton in neighbouring cells by aligning cytokeratin-18 (KRT18) and forming an actomyosin cable at the junctional sites (see poster). Disruption of desmosomal components impairs actomyosin contractility and extrusion, suggesting that anchoring of forces through desmosomes might help to coordinate contraction (Thomas et al., 2020).

### Other junctions

The role of other cell junctions in cell extrusion is relatively poorly characterised. Tight junctions (septate junctions in invertebrates) are essential for regulating the sealing properties of epithelia (Citi et al., 2024). Therefore, their regulation during extrusion should be highly relevant. Tight junctions are located apical to AJs, whereas invertebrate septate junctions are more basal. Surprisingly, the dynamics of tight and septate junctions during cell extrusion are very underexplored, although preliminary results suggest that they are maintained throughout extrusion of larval epidermal cells in the
*Drosophila* pupal abdomen (Teng et al., 2017), and early work suggested that depletion of the tight junction component occludin in MDCK cells reduces the rate of apoptotic cell extrusion (Yu et al., 2005). Similarly, recent work has outlined the role of MyoII-dependent recruitment of tight junction components in the formation of new tight junctions between neighbours, which are required to maintain sealing properties during extrusion (Cho et al., 2024). Describing the dynamics of tight and septate junctions will be extremely important for understanding how sealing is ensured throughout extrusion. Finally, the roles and dynamics of basal junctions and ECM attachments via FAs in cell extrusion also remain poorly understood. Similar to AJs, initial maintenance of FAs might help to anchor contractile forces to the basal side and direct the basal movement of the supracellular contractile ring (Le et al., 2021). Accordingly, talin – an FA component that couples integrins to actin – has been shown to remain in FAs during basal extrusion in the *Drosophila* pupal leg, and its depletion has been found to affect apical-to-basal force transmission (Ambrosini et al., 2019).

In summary, cell extrusion induces remodelling in the neighbouring cells through mechanical coupling via cell–cell junctions and mechanotransduction, promoting contractility in the neighbouring cells. The contribution of E-cad dynamics to extrusion is highly context dependent: whereas persistence of E-cad is required for efficient extrusion and force transmission, its subsequent cleavage or downregulation promotes extrusion in specific contexts. Similar anchoring functions have been described for desmosomes; however, the dynamics and roles of tight and septate junctions as well as FAs in extrusion remain poorly characterised. These paradoxical functions of junctions, which need to be preserved to transmit forces and maintain sealing but also need to be dismantled by the end of extrusion, can only be resolved by a very tight regulation of junctional dynamics during extrusion.

## Regulation of extrusion at the tissue scale

Cell extrusion is also tuned through tissue-scale parameters. In this section, we outline behaviours that promote extrusion observed in more distant neighbouring cells, as well as the permissive role of the mechanical state of the tissue (see poster).

## Collective migration

Extracellular signal-regulated kinase [ERK; herein referring to ERK1 and ERK2 in vertebrates (also known as MAPK3 and MAPK1, respectively), Rolled in *Drosophila*, a core regulator of cell growth and survival, coordinates collective cell migration during wound healing and morphogenesis through propagation of waves of ERK signalling and long-range cell polarity alignment (Boocock et al., 2021; Hino et al., 2020; Ishii et al., 2021). In oncogenic cell extrusion, induction of active Ras and Raf GTPases (which act upstream of ERK) in a subset of cells also generates spontaneous ERK waves that propagate several cell rows away and trigger collective migration toward the oncogenic cell, which promotes its live extrusion (Aikin et al., 2020). Similarly, in MDCK cells and zebrafish fin, $Ca^{2+}$ waves propagate from apoptotic cells or cells with active Ras, promoting actin rearrangements and coordinated movements toward the extruding cell (Takeuchi et al., 2020) (see poster). Finally, collective migration also promotes spontaneous caspase activation and cell extrusion through generation of local cell misalignment and topological defects that locally increase pressure (Saw et al., 2017). Thus, cells beyond the first layer of neighbours promote extrusion by collective migration toward the extruding cell, most likely due to the build-up of local pressure squeezing out the cell.

## Global tissue tension

The global mechanical state of the epithelial tissue can also modulate the rate of extrusion. Intuitively, the constriction of the extruding cell must overcome the resisting forces from the neighbours and pre-existing tissue tension. Accordingly, increasing tissue tension through expression of a constitutively active form of myosin regulatory light chain or treatment with tumour necrosis factor alpha (TNF$\alpha$, also known as TNF) inhibits extrusion downstream of apoptosis induction in Caco2 cells, whereas tension release through global MyoII inhibition upregulates extrusion (Mann et al., 2024). This is in good agreement with findings in the *Drosophila* pupal notum, where tension and MyoII fluctuations, promoted by low Rho-associated kinase (Rok) activity or dampened by Rok hyperactivation, are permissive for junction remodelling and cell extrusion (Curran et al., 2017). Furthermore, recent studies suggest that the main function of S1P is to globally prime the tissue for extrusion (Atieh et al., 2021; Duszyc et al., 2021), rather than its earlier identified instructive function in promoting local Rho GTPase activation (Gu et al., 2011). Accordingly, inducing global damage in zebrafish fin through DNA crosslinking leads to ubiquitous caspase activation and S1P accumulation, which promotes cell extrusion through a global reduction of tissue tension and stiffness (Atieh et al., 2021). Alternatively, Src kinase-dependant local reduction of tension in the junctions of neighbouring cells orthogonal to the extruding cell (i.e. those sharing a single vertex with the dying cell) facilitates extrusion progression (Teo et al., 2020b).

Taken together, these insights emphasise that a mechanically permissive tissue environment is essential for the extrusion process. Cell extrusion is therefore not just a very locally regulated event but is also closely linked to distant neighbours and global tissue properties. ERK and $Ca^{2+}$ waves drive extrusion by coordinating collective convergent movements, pushing the extruding cell out of the tissue. The tissue tensional state, notably influenced by S1P signalling, also plays a key role in extrusion efficiency.

## Physiological and pathological relevance of cell extrusion

In this section, we outline the physiological functions of cell extrusion, including its roles in morphogenesis and removal of oncogenic cells. We then discuss the pathological outcomes associated with misregulation of cell extrusion.

### Cell extrusion as a force generator

Cell extrusion not only influences local cytoarchitecture but actively drives movements that shape the tissue. Three types of tissue deformation can be generated by cell extrusion (see poster). Firstly, cell extrusion can promote tissue fusion through the generation of in-plane pulling forces between the two fusing tissues, which might result from the contraction of the dying cells and/or the supracellular actomyosin ring, as shown by the active role of amnioserosa cell apoptosis in fusing the two apposed epidermal sheets during *Drosophila* dorsal closure (Toyama et al., 2008) or the contribution of larval epidermal cell apoptosis to the fusion of the two neighbouring wing disc nota in the *Drosophila* pupal thorax (Athilingam et al., 2022). Secondly, extrusion can generate out-of-plane orthogonal forces through the generation of actomyosin cables along the apicobasal axis (Ambrosini et al., 2019; Monier et al., 2015), which initiates epithelial folding in the *Drosophila* pupal leg (Monier et al., 2015), as well as local bending of the neural tube in the chick embryo (Roellig et al., 2022). Thirdly, cell extrusion can also generate rotational forces (or torque), promoting tissue rotation or twisting. For instance, the 360° looping of the *Drosophila* genitalia at pupal stage is promoted by the upregulation
of apoptosis between two rotating rings of cells, which plays a permissive role by releasing friction and fluidifying the interface between the rotating rings (Suzanne et al., 2010) and/or an active role by generating polarised pulling forces through an unknown mechanism (Kuranaga et al., 2011). Finally, cell extrusion might also play a permissive role in morphogenesis by reducing the global tissue viscosity (Ranft et al., 2010). Therefore, local cytoskeletal remodelling caused by cell extrusion can drive larger epithelial movements, such as fusion, bending and rotation, that altogether contribute to organ morphogenesis.

## Pro- and anti-tumoural functions

Epithelial defence against cancer is a process by which transformed or oncogenic cells (for example, cells with active Ras or active Src) are selectively removed from an epithelium via apical live-cell extrusion (Tanimura and Fujita, 2020). This mechanism ensures that oncogenic cells do not invade other tissues in the organism by expelling them into the lumen. Interestingly, in other contexts, oncogenic Ras<sup>V12</sup> mutations can drive tissue invasion through aberrant basal extrusion as a result of S1P degradation via elevated autophagy (Slattum and Rosenblatt, 2014). Consistently, Ras<sup>V12</sup> clones in zebrafish larval epidermis were shown to undergo single-cell basal delamination followed by cell dedifferentiation and invasion through blood circulation, reminiscent of the early phase of metastasis (Fadul et al., 2021). These findings highlight how cell extrusion can be the first line of defence against cancer, but also how oncogenic mutations can sometimes highjack extrusion mechanisms by changing the direction of expulsion, contributing to tumour initiation, progression and metastasis (see poster).

## Epithelial barrier function and pathogen invasion

Epithelial layers defend the body and organs against invading pathogens. Although extrusion can be used to selectively remove infected cells, some bacteria exploit or interfere with epithelial extrusion to breach this barrier. For instance, MDCK cells infected by *Listeria monocytogenes* are collectively expelled apically from the epithelial layer through the activation of NF-κB, reduction in stiffness of the infected cells relative to neighbouring cells, and the pressure induced by migration of neighbour cells (Bastounis et al., 2021). In contrast, certain variants of *Neisseria gonorrhoeae* actively inhibit cell extrusion in the mucosa by promoting ECM adhesion through the activation of β1 integrins, thereby increasing perdurance of infected cells (Muenzner et al., 2010, 2005).

## Excessive or aberrant extrusion and inflammation

Finally, misregulation of extrusion or excessive cell extrusion can affect the barrier function of epithelia and promote inflammation (see poster). Blocking apical extrusion leads to the perdurance of apoptotic cells within the epithelium, which stimulates secretion of the pro-inflammatory signal interleukin-8 (IL-8) through release of extracellular ATP from the dying cells and activation of the ATP receptor P2Y11 (also known as P2RY11) in neighbours (Duszyc et al., 2023). Excessive extrusion can also promote inflammation, which eventually disrupts epithelial barrier function, as reported in inflammatory bowel disease (Blander, 2016; Levayer, 2024). In the *Drosophila* pupal notum (Valon et al., 2021) and in MCF10A and MDCK cells (Gagliardi et al., 2021), excessive extrusion and cell death is reported to be limited by transient ERK activation in neighbours of extruding cells. ERK pulses or waves transiently inhibit cell death, thereby buffering down the rate of extrusion (Gagliardi et al., 2021) and preventing the formation of clusters of extruding cells, which are detrimental for epithelial sealing (Valon

et al., 2021). Finally, excessive extrusion has more recently been associated with asthma, an inflammatory airway pathology (Bagley et al., 2024). During an asthma attack, airway narrowing causes epithelial crowding and triggers a high rate of crowding-induced extrusion through activation of Piezo and S1P signalling, which leads to epithelial detachment and inflammation (Bagley et al., 2024).

## Conclusions and outstanding questions

Epithelial extrusion plays a crucial role in tissue homeostasis and morphogenesis by facilitating the removal of apoptotic and aberrant cells while ensuring barrier integrity. Here, we have discussed the various remodelling steps that take place in the extruding and neighbouring cells, as well as key molecular regulators, including targets of caspases, the S1P pathway and mechanotransduction at AJs. We have also outlined larger-scale contributions to extrusion, such as collective migration and global tissue mechanical properties. It is important to stress that cell extrusion is a highly plastic process that can vary according to the tissue, organism and mechanical environment. Because of this versatility and complexity, many aspects of cell extrusion remain not fully understood (see poster). For instance, it remains unknown what factors set the direction of extrusion – namely, apical or basal. Similarly, the mechanisms that temporally regulate the maintenance and later disassembly of AJs remain unclear. Globally, the quantitative phenomenology of cell junctions during extrusion is largely incomplete. It also remains unclear how the chronology of the different remodelling steps is orchestrated. Lastly, we have a very limited understanding of how caspases promote cell extrusion, mostly due to the very limited number of caspase substrates that have been identified to be relevant for extrusion. Given the prevalence of extrusion, its multiple physiological functions and pathological relevance, these questions will surely stimulate intensive research in the near future.

## Acknowledgements

We thank members of the Cell Death and Epithelial Homeostasis group for constructive comments on the manuscript.

## Competing interests

The authors declare no competing or financial interests.

## Funding

Work in the lab of R.L. is supported by the Institut Pasteur and the European Union (European Research Council; PrApEDoC, 101085444), the Agence Nationale de la Recherche (ANR) projet de recherche collaborative (PRC) CoECECa, the ANR PRC MAPEFLU, and by ANR-10-LABX-0073 and the Centre National de la Recherche Scientifique (CNRS; UMR 3738). Views and opinions expressed are those of the authors only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them. Open Access funding provided by the Agence Nationale de la Recherche (PRC CoECECa). Deposited in PMC for immediate release.

## High-resolution poster and poster panels

A high-resolution version of the poster and individual poster panels are available for downloading at https://journals.biologists.com/jcs/article-lookup/doi/10.1242/jcs.263786#supplementary-data

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