Crk mediates Csk-Hippo signaling independently of Yap tyrosine phosphorylation to induce cell extrusion.

Src family kinases (SFKs), including Src, Fyn and Yes, play important roles in development and cancer. Despite being first discovered as the Yes-associated protein, the regulation of Yap by SFKs remains poorly understood. Here, through single-cell analysis and genetic lineage tracing, we show that the pan-epithelial ablation of C-terminal Src kinase (Csk) in the lacrimal gland unleashes broad Src signaling but specifically causes extrusion and apoptosis of acinar progenitors at a time when they are shielded by myoepithelial cells from the basement membrane. Csk mutants can be phenocopied by constitutively active Yap and rescued by deleting Yap or Taz, indicating a significant functional overlap between Src and Yap signaling. Although Src-induced tyrosine phosphorylation has long been believed to regulate Yap activity, we find that mutating these tyrosine residues in both Yap and Taz fails to perturb mouse development or alleviate the Csk lacrimal gland phenotype. In contrast, Yap loses Hippo signaling-dependent serine phosphorylation and translocates into the nucleus in Csk mutants. Further chemical genetics studies demonstrate that acute inhibition of Csk enhances Crk/CrkL phosphorylation and Rac1 activity, whereas removing Crk/CrkL or Rac1/Rap1 ameliorates the Csk mutant phenotype. These results show that Src controls Hippo-Yap signaling through the Crk/CrkL-Rac/Rap axis to promote cell extrusion.

Cell extrusion - a novel mechanism driving neural crest cell delamination.

Neural crest cells (NCC) comprise a heterogeneous population of cells with variable potency, that contribute to nearly every tissue and organ system throughout the body. Considered unique to vertebrates, NCC are transiently generated within the dorsolateral region of the neural plate or neural tube, during neurulation. Their delamination and migration are crucial events in embryo development as the differentiation of NCC is heavily influenced by their final resting locations. Previous work in avian and aquatic species has shown that NCC delaminate via an epithelial-mesenchymal transition (EMT), which transforms these stem and progenitor cells from static polarized epithelial cells into migratory mesenchymal cells with fluid front and back polarity. However, the cellular and molecular drivers facilitating NCC delamination in mammals are poorly understood. We performed live timelapse imaging of NCC delamination in mouse embryos and discovered a group of cells that exit the neuroepithelium as isolated round cells, which then halt for a short period prior to acquiring the mesenchymal migratory morphology classically associated with most delaminating NCC. High magnification imaging and protein localization analyses of the cytoskeleton, together with measurements of pressure and tension of delaminating NCC and neighboring neuroepithelial cells, revealed these round NCC are extruded from the neuroepithelium prior to completion of EMT. Furthermore, we demonstrate that cranial NCC are extruded through activation of the mechanosensitive ion channel, PIEZO1, a key regulator of the live cell extrusion pathway, revealing a new role for PIEZO1 in neural crest cell development. Our results elucidating the cellular and molecular dynamics orchestrating NCC delamination support a model in which high pressure and tension in the neuroepithelium results in activation of the live cell extrusion pathway and delamination of a subpopulation of NCC in parallel with EMT. This model has broad implications for our understanding of cell delamination in development and disease.

Assembly, dynamics and remodeling of epithelial cell junctions throughout development.

Cell junctions play key roles in epithelial integrity. During development, when epithelia undergo extensive morphogenesis, these junctions must be remodeled in order to maintain mechanochemical barriers and ensure the cohesion of the tissue. In this Review, we present a comprehensive and integrated description of junctional remodeling mechanisms in epithelial cells during development, from embryonic to adult epithelia. We largely focus on Drosophila, as quantitative analyses in this organism have provided a detailed characterization of the molecular mechanisms governing cell topologies, and discuss the conservation of these mechanisms across metazoans. We consider how changes at the molecular level translate to tissue-scale irreversible deformations, exploring the composition and assembly of cellular interfaces to unveil how junctions are remodeled to preserve tissue homeostasis during cell division, intercalation, invagination, ingression and extrusion.

Toward a predictive understanding of epithelial cell death.

Epithelial cell death is highly prevalent during development and tissue homeostasis. While we have a rather good understanding of the molecular regulators of programmed cell death, especially for apoptosis, we still fail to predict when, where, how many and which specific cells will die in a tissue. This likely relies on the much more complex picture of apoptosis regulation in a tissular and epithelial context, which entails cell autonomous but also non-cell autonomous factors, diverse feedback and multiple layers of regulation of the commitment to apoptosis. In this review, we illustrate this complexity of epithelial apoptosis regulation by describing these different layers of control, all demonstrating that local cell death probability is a complex emerging feature. We first focus on non-cell autonomous factors that can locally modulate the rate of cell death, including cell competition, mechanical input and geometry as well as systemic effects. We then describe the multiple feedback mechanisms generated by cell death itself. We also outline the multiple layers of regulation of epithelial cell death, including the coordination of extrusion and regulation occurring downstream of effector caspases. Eventually, we propose a roadmap to reach a more predictive understanding of cell death regulation in an epithelial context.

Assessing mechanical agency during apical apoptotic cell extrusion.

Epithelial tissues maintain homeostasis through the continual addition and removal of cells. Homeostasis is necessary for epithelia to maintain barrier function and prevent the accumulation of defective cells. Unfit, excess, and dying cells can be removed from epithelia by the process of extrusion. Controlled cell death and extrusion in the epithelium of the larval zebrafish tail fin coincides with oscillation of cell area, both in the extruding cell and its neighbors. Both cell-autonomous and non-autonomous factors have been proposed to contribute to extrusion but have been challenging to test by experimental approaches. Here we develop a dynamic cell-based biophysical model that recapitulates the process of oscillatory cell extrusion to test and compare the relative contributions of these factors. Our model incorporates the mechanical properties of individual epithelial cells in a two-dimensional simulation as repelling active particles. The area of cells destined to extrude oscillates with varying durations or amplitudes, decreasing their mechanical contribution to the epithelium and surrendering their space to surrounding cells. Quantitative variations in cell shape and size during extrusion are visualized by a hybrid weighted Voronoi tessellation technique that renders individual cell mechanical properties directly into an epithelial sheet. To explore the role of autonomous and non-autonomous mechanics, we vary the biophysical properties and behaviors of extruding cells and neighbors such as the period and amplitude of repulsive forces, cell density, and tissue viscosity. Our data suggest that cell autonomous processes are major contributors to the dynamics of extrusion, with the mechanical microenvironment providing a less pronounced contribution. Our computational model based on in vivo data serves as a tool to provide insights into the cellular dynamics and localized changes in mechanics that promote elimination of unwanted cells from epithelia during homeostatic tissue maintenance.

Tug-of-war via ERK signaling pathway for tissue organization - ERK activation to force generation.

Extracellular signal-regulated kinase (ERK) plays a crucial role in regulating collective cell behaviors observed in diverse biological phenomena. Emerging studies have shed light on the involvement of the ERK signaling pathway in the reception and generation of mechanical forces, thereby governing local mechanical interactions within multicellular tissues. Although limited in number, studies have provided insights into how ERK-mediated mechanical interactions contribute to multicellular organization. Here we explore the impact of ERK-mediated mechanical interactions on tissue morphogenesis, cell extrusion in homeostasis, and their interplay with the physical microenvironments of the extracellular matrix. We conclude that the coupling system of ERK activity with mechanical forces offers a promising avenue to unravel the emergent collective dynamics underlying tissue organization.

Deciphering the Role of p60AmotL2 in Epithelial Extrusion and Cell Detachment.

Preserving an accurate cell count is crucial for maintaining homeostasis. Apical extrusion, a process in which redundant cells are eliminated by neighboring cells, plays a key role in this regard. Recent studies have revealed that apical extrusion can also be triggered in cells transformed by oncogenes, suggesting it may be a mechanism through which tumor cells escape their microenvironment. In previous work, we demonstrated that p60AmotL2 modulates the E-cadherin function by inhibiting its connection to radial actin filaments. This isoform of AmotL2 is expressed in invasive breast and colon tumors and promotes invasion in vitro and in vivo. Transcriptionally regulated by c-Fos, p60AmotL2 is induced by local stress signals such as severe hypoxia. In this study, we investigated the normal role of p60AmotL2 in epithelial tissues. We found that this isoform is predominantly expressed in the gut, where cells experience rapid turnover. Through time-lapse imaging, we present evidence that cells expressing p60AmotL2 are extruded by their normal neighboring cells. Based on these findings, we hypothesize that tumor cells exploit this pathway to detach from normal epithelia and invade surrounding tissues.

Bidirectional multiciliated cell extrusion is controlled by Notch-driven basal extrusion and Piezo1-driven apical extrusion.

Xenopus embryos are covered with a complex epithelium containing numerous multiciliated cells (MCCs). During late-stage development, there is a dramatic remodeling of the epithelium that involves the complete loss of MCCs. Cell extrusion is a well-characterized process for driving cell loss while maintaining epithelial barrier function. Normal cell extrusion is typically unidirectional, whereas bidirectional extrusion is often associated with disease (e.g. cancer). We describe two distinct mechanisms for MCC extrusion, a basal extrusion driven by Notch signaling and an apical extrusion driven by Piezo1. Early in the process there is a strong bias towards basal extrusion, but as development continues there is a shift towards apical extrusion. Importantly, response to the Notch signal is age dependent and governed by the maintenance of the MCC transcriptional program such that extension of this program is protective against cell loss. In contrast, later apical extrusion is regulated by Piezo1, such that premature activation of Piezo1 leads to early extrusion while blocking Piezo1 leads to MCC maintenance. Distinct mechanisms for MCC loss underlie the importance of their removal during epithelial remodeling.

Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion.

Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells. However, the underlying mechanisms of cell delamination from the cell layer are unclear. Here, we report a conserved execution mechanism of apoptotic cell extrusion. We found extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells.

Cell polarity and extrusion: How to polarize extrusion and extrude misspolarized cells?

The barrier function of epithelia is one of the cornerstones of the body plan organization of metazoans. It relies on the polarity of epithelial cells which organizes along the apico-basal axis the mechanical properties, signaling as well as transport. This barrier function is however constantly challenged by the fast turnover of epithelia occurring during morphogenesis or adult tissue homeostasis. Yet, the sealing property of the tissue can be maintained thanks to cell extrusion: a series of remodeling steps involving the dying cell and its neighbors leading to seamless cell expulsion. Alternatively, the tissue architecture can also be challenged by local damages or the emergence of mutant cells that may alter its organization. This includes mutants of the polarity complexes which can generate neoplastic overgrowths or be eliminated by cell competition when surrounded by wild type cells. In this review, we will provide an overview of the regulation of cell extrusion in various tissues focusing on the relationship between cell polarity, cell organization and the direction of cell expulsion. We will then describe how local perturbations of polarity can also trigger cell elimination either by apoptosis or by cell exclusion, focusing specifically on how polarity defects can be directly causal to cell elimination. Overall, we propose a general framework connecting the influence of polarity on cell extrusion and its contribution to aberrant cell elimination.

Mechanical basis and topological routes to cell elimination.

Cell layers eliminate unwanted cells through the extrusion process, which underlines healthy versus flawed tissue behaviors. Although several biochemical pathways have been identified, the underlying mechanical basis including the forces involved in cellular extrusion remains largely unexplored. Utilizing a phase-field model of a three-dimensional cell layer, we study the interplay of cell extrusion with cell-cell and cell-substrate interactions in a flat monolayer. Independent tuning of cell-cell versus cell-substrate adhesion forces reveals that extrusion events can be distinctly linked to defects in nematic and hexatic orders associated with cellular arrangements. Specifically, we show that by increasing relative cell-cell adhesion forces the cell monolayer can switch between the collective tendency towards fivefold, hexatic, disclinations relative to half-integer, nematic, defects for extruding a cell. We unify our findings by accessing three-dimensional mechanical stress fields to show that an extrusion event acts as a mechanism to relieve localized stress concentration.

ECM-transmitted shear stress induces apoptotic cell extrusion in early breast gland development.

Epithelial cells of human breast glands are exposed to various mechanical ECM stresses that regulate tissue development and homeostasis. Mechanoadaptation of breast gland tissue to ECM-transmitted shear stress remained poorly investigated due to the lack of valid experimental approaches. Therefore, we created a magnetic shear strain device that enabled, for the first time, to analyze the instant shear strain response of human breast gland cells. MCF10A-derived breast acini with basement membranes (BM) of defined maturation state and basoapical polarization were used to resemble breast gland morphogenesis in vitro. The novel biophysical tool was used to apply cyclic shear strain with defined amplitudes (≤15%, 0.2 Hz) over 22 h on living spheroids embedded in an ultrasoft matrix (<60 Pa). We demonstrated that breast spheroids gain resistance to shear strain, which increased with BM maturation and basoapical polarization. Most intriguingly, poorly developed spheroids were prone to cyclic strain-induced extrusion of apoptotic cells from the spheroid body. In contrast, matured spheroids were insensitive to this mechanoresponse-indicating changing mechanosensing or mechanotransduction mechanisms during breast tissue morphogenesis. Together, we introduced a versatile tool to study cyclic shear stress responses of 3D cell culture models. It can be used to strain, in principle, all kinds of cell clusters, even those that grow only in ultrasoft hydrogels. We believe that this approach opens new doors to gain new insights into dynamic shear strain-induced mechanobiological regulation circuits between cells and their ECM.

A Stiff Extracellular Matrix Favors the Mechanical Cell Competition that Leads to Extrusion of Bacterially-Infected Epithelial Cells.

Cell competition refers to the mechanism whereby less fit cells ("losers") are sensed and eliminated by more fit neighboring cells ("winners") and arises during many processes including intracellular bacterial infection. Extracellular matrix (ECM) stiffness can regulate important cellular functions, such as motility, by modulating the physical forces that cells transduce and could thus modulate the output of cellular competitions. Herein, we employ a computational model to investigate the previously overlooked role of ECM stiffness in modulating the forceful extrusion of infected "loser" cells by uninfected "winner" cells. We find that increasing ECM stiffness promotes the collective squeezing and subsequent extrusion of infected cells due to differential cell displacements and cellular force generation. Moreover, we discover that an increase in the ratio of uninfected to infected cell stiffness as well as a smaller infection focus size, independently promote squeezing of infected cells, and this phenomenon is more prominent on stiffer compared to softer matrices. Our experimental findings validate the computational predictions by demonstrating increased collective cell extrusion on stiff matrices and glass as opposed to softer matrices, which is associated with decreased bacterial spread in the basal cell monolayer in vitro. Collectively, our results suggest that ECM stiffness plays a major role in modulating the competition between infected and uninfected cells, with stiffer matrices promoting this battle through differential modulation of cell mechanics between the two cell populations.

A unique form of collective epithelial migration is crucial for tissue fusion in the secondary palate and can overcome loss of epithelial apoptosis.

Tissue fusion frequently requires the removal of an epithelium that intervenes distinct primordia to form one continuous structure. In the mammalian secondary palate, a midline epithelial seam (MES) forms between two palatal shelves and must be removed to allow mesenchymal confluence. Abundant apoptosis and cell extrusion support their importance in MES removal. However, genetically disrupting the intrinsic apoptotic regulators BAX and BAK within the MES results in complete loss of cell death and cell extrusion, but successful removal of the MES. Novel static- and live-imaging approaches reveal that the MES is removed through streaming migration of epithelial trails and islands to reach the oral and nasal epithelial surfaces. Epithelial trail cells that express the basal epithelial marker ΔNp63 begin to express periderm markers, suggesting that migration is concomitant with differentiation. Live imaging reveals anisotropic actomyosin contractility within epithelial trails, and genetic ablation of actomyosin contractility results in dispersion of epithelial collectives and failure of normal MES migration. These findings demonstrate redundancy between cellular mechanisms of morphogenesis, and reveal a crucial and unique form of collective epithelial migration during tissue fusion.

Impact of Epithelial Cell Shedding on Intestinal Homeostasis.

The gut barrier acts as a first line of defense in the body, and plays a vital role in nutrition and immunoregulation. A layer of epithelial cells bound together via intercellular junction proteins maintains intestinal barrier integrity. Based on a tight equilibrium between cell extrusion and cell restitution, the renewal of the epithelium (epithelial turnover) permits the preservation of cell numbers. As the last step within the epithelial turnover, cell shedding occurs due to the pressure of cell division and migration from the base of the crypt. During this process, redistribution of tight junction proteins enables the sealing of the epithelial gap left by the extruded cell, and thereby maintains barrier function. Disturbance in cell shedding can create transient gaps (leaky gut) or cell accumulation in the epithelial layer. In fact, numerous studies have described the association between dysregulated cell shedding and infection, inflammation, and cancer; thus epithelial cell extrusion is considered a key defense mechanism. In the gastrointestinal tract, altered cell shedding has been observed in mouse models of intestinal inflammation and appears as a potential cause of barrier loss in human inflammatory bowel disease (IBD). Despite the relevance of this process, there are many unanswered questions regarding cell shedding. The investigation of those mechanisms controlling cell extrusion in the gut will definitely contribute to our understanding of intestinal homeostasis. In this review, we summarized the current knowledge about intestinal cell shedding under both physiological and pathological circumstances.

Calcium wave propagation during cell extrusion.

Oncogenically transformed or apoptotic cells are removed from epithelial sheets by cell-cell communication between the transformed/apoptotic cells (extruding cells) and the nearest neighboring cells. Cell extrusion is driven by actomyosin contraction and lamellipodial crawling of the nearest neighboring cells. Recent studies have found that distal cell communication also plays a role in cell extrusion. Specifically, distal cells located 3-16 cells away from the extruding cell are coordinated by calcium waves and collectively migrate toward the extruding cell to initiate cell extrusion. Here, I describe how calcium waves are generated and contribute to the extrusion of cells in mammals and zebrafish.

Extracellular ATP facilitates cell extrusion from epithelial layers mediated by cell competition or apoptosis.

For the maintenance of epithelial homeostasis, various aberrant or dysfunctional cells are actively eliminated from epithelial layers. This cell extrusion process mainly falls into two modes: cell-competition-mediated extrusion and apoptotic extrusion. However, it is not clearly understood whether and how these processes are governed by common molecular mechanisms. In this study, we demonstrate that the reactive oxygen species (ROS) levels are elevated within a wide range of epithelial layers around extruding transformed or apoptotic cells. The downregulation of ROS suppresses the extrusion process. Furthermore, ATP is extracellularly secreted from extruding cells, which promotes the ROS level and cell extrusion. Moreover, the extracellular ATP and ROS pathways positively regulate the polarized movements of surrounding cells toward extruding cells in both cell-competition-mediated and apoptotic extrusion. Hence, extracellular ATP acts as an "extrude me" signal and plays a prevalent role in cell extrusion, thereby sustaining epithelial homeostasis and preventing pathological conditions or disorders.

Rapid lamellipodial responses by neighbor cells drive epithelial sealing in response to pyroptotic cell death.

Cell injury poses a substantial challenge for epithelia homeostasis. Several cellular processes preserve epithelial barriers in response to apoptosis, but less is known about other forms of cell death, such as pyroptosis. Here we use an inducible caspase-1 system to analyze how colon epithelial monolayers respond to pyroptosis. We confirm that sporadic pyroptotic cells are physically eliminated from confluent monolayers by apical extrusion. This is accompanied by a transient defect in barrier function at the site of the pyroptotic cells. By visualizing cell shape changes and traction patterns in combination with cytoskeletal inhibitors, we show that rapid lamellipodial responses in the neighbor cells are responsible for correcting the leakage and resealing the barrier. Cell contractility is not required for this resealing response, in contrast to the response to apoptosis. Therefore, pyroptosis elicits a distinct homeostatic response from the epithelium that is driven by the stimulation of lamellipodia in neighbor cells.

γδ Intraepithelial Lymphocytes Facilitate Pathological Epithelial Cell Shedding Via CD103-Mediated Granzyme Release.

BACKGROUND & AIMS: Excessive shedding of apoptotic enterocytes into the intestinal lumen is observed in inflammatory bowel disease and is correlated with disease relapse. Based on their cytolytic capacity and surveillance behavior, we investigated whether intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IELs) are actively involved in the shedding of enterocytes into the lumen. METHODS: Intravital microscopy was performed on GFP γδ T cell reporter mice treated with intraperitoneal lipopolysaccharide (10 mg/kg) for 90 minutes to induce tumor necrosis factor-mediated apoptosis. Cell shedding in various knockout or transgenic mice in the presence or absence of blocking antibody was quantified by immunostaining for ZO-1 funnels and cleaved caspase-3 (CC3). Granzyme A and granzyme B release from ex vivo-stimulated γδ IELs was quantified by enzyme-linked immunosorbent assay. Immunostaining for γδ T cell receptor and CC3 was performed on duodenal and ileal biopsies from controls and patients with Crohn's disease. RESULTS: Intravital microscopy of lipopolysaccharide-treated mice revealed that γδ IELs make extended contact with shedding enterocytes. These prolonged interactions require CD103 engagement by E-cadherin, and CD103 knockout or blockade significantly reduced lipopolysaccharide-induced shedding. Furthermore, we found that granzymes A and B, but not perforin, are required for cell shedding. These extracellular granzymes are released by γδ IELs both constitutively and after CD103/E-cadherin ligation. Moreover, we found that the frequency of γδ IEL localization to CC3-positive enterocytes is increased in Crohn's disease biopsies compared with healthy controls. CONCLUSIONS: Our results uncover a previously unrecognized role for γδ IELs in facilitating tumor necrosis factor-mediated shedding of apoptotic enterocytes via CD103-mediated extracellular granzyme release.

Yersinia pseudotuberculosis YopE prevents uptake by M cells and instigates M cell extrusion in human ileal enteroid-derived monolayers.

Many pathogens use M cells to access the underlying Peyer's patches and spread to systemic sites via the lymph as demonstrated by ligated loop murine intestinal models. However, the study of interactions between M cells and microbial pathogens has stalled due to the lack of cell culture systems. To overcome this obstacle, we use human ileal enteroid-derived monolayers containing five intestinal cell types including M cells to study the interactions between the enteric pathogen, Yersinia pseudotuberculosis (Yptb), and M cells. The Yptb type three secretion system (T3SS) effector Yops inhibit host defenses including phagocytosis and are critical for colonization of the intestine and Peyer's patches. Therefore, it is not understood how Yptb traverses through M cells to breach the epithelium. By growing Yptb under two physiological conditions that mimic the early infectious stage (low T3SS-expression) or host-adapted stage (high T3SS-expression), we found that large numbers of Yptb specifically associated with M cells, recapitulating murine studies. Transcytosis through M cells was significantly higher by Yptb expressing low levels of T3SS, because YopE and YopH prevented Yptb uptake. YopE also caused M cells to extrude from the epithelium without inducing cell-death or disrupting monolayer integrity. Sequential infection with early infectious stage Yptb reduced host-adapted Yptb association with M cells. These data underscore the strength of enteroids as a model by discovering that Yops impede M cell function, indicating that early infectious stage Yptb more effectively penetrates M cells while the host may defend against M cell penetration of host-adapted Yptb.