Epithelial Apicobasal Polarity Research Articles

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell–cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Diamond controls epithelial polarity through the dynactin-dynein complex.

Epithelial polarity is critical for proper functions of epithelial tissues, tumorigenesis, and metastasis. The evolutionarily conserved transmembrane protein Crumbs (Crb) is a key regulator of epithelial polarity. Both Crb protein and its transcripts are apically localized in epithelial cells. However, it remains not fully understood how they are targeted to the apical domain. Here, using Drosophila ovarian follicular epithelia as a model, we show that epithelial polarity is lost and Crb protein is absent in the apical domain in follicular cells (FCs) in the absence of Diamond (Dind). Interestingly, Dind is found to associate with different components of the dynactin-dynein complex through co-IP-MS analysis. Dind stabilizes dynactin and depletion of dynactin results in almost identical defects as those observed in dind-defective FCs. Finally, both Dind and dynactin are also required for the apical localization of crb transcripts in FCs. Thus our data illustrate that Dind functions through dynactin/dynein-mediated transport of both Crb protein and its transcripts to the apical domain to control epithelial apico-basal (A/B) polarity.

GPR125 (ADGRA3) is an autocleavable adhesion GPCR that traffics with Dlg1 to the basolateral membrane and regulates epithelial apicobasal polarity

The adhesion family of G protein–coupled receptors (GPCRs) is defined by an N-terminal large extracellular region that contains various adhesion-related domains and a highly-conserved GPCR-autoproteolysis-inducing (GAIN) domain, the latter of which is located immediately before a canonical seven-transmembrane domain. These receptors are expressed widely and involved in various functions including development, angiogenesis, synapse formation, and tumorigenesis. GPR125 (ADGRA3), an orphan adhesion GPCR, has been shown to modulate planar cell polarity in gastrulating zebrafish, but its biochemical properties and role in mammalian cells have remained largely unknown. Here, we show that human GPR125 likely undergoes cis-autoproteolysis when expressed in canine kidney epithelial MDCK cells and human embryonic kidney HEK293 cells. The cleavage appears to occur at an atypical GPCR proteolysis site within the GAIN domain during an early stage of receptor biosynthesis. The products, i.e., the N-terminal and C-terminal fragments, seem to remain associated after self-proteolysis, as observed in other adhesion GPCRs. Furthermore, in polarized MDCK cells, GPR125 is exclusively recruited to the basolateral domain of the plasma membrane. The recruitment likely requires the C-terminal PDZ-domain–binding motif of GPR125 and its interaction with the cell polarity protein Dlg1. Knockdown of GPR125 as well as that of Dlg1 results in formation of aberrant cysts with multiple lumens in Matrigel 3D culture of MDCK cells. Consistent with the multilumen phenotype, mitotic spindles are incorrectly oriented during cystogenesis in GPR125-KO MDCK cells. Thus, the basolateral protein GPR125, an autocleavable adhesion GPCR, appears to play a crucial role in apicobasal polarization in epithelial cells.

Insights Into Mechanisms of Oriented Division From Studies in 3D Cellular Models.

In multicellular organisms, epithelial cells are key elements of tissue organization. In developing tissues, cellular proliferation and differentiation are under the tight regulation of morphogenetic programs, that ensure the correct organ formation and functioning. In these processes, mitotic rates and division orientation are crucial in regulating the velocity and the timing of the forming tissue. Division orientation, specified by mitotic spindle placement with respect to epithelial apico-basal polarity, controls not only the partitioning of cellular components but also the positioning of the daughter cells within the tissue, and hence the contacts that daughter cells retain with the surrounding microenvironment. Daughter cells positioning is important to determine signal sensing and fate, and therefore the final function of the developing organ. In this review, we will discuss recent discoveries regarding the mechanistics of planar divisions in mammalian epithelial cells, summarizing technologies and model systems used to study oriented cell divisions in vitro such as three-dimensional cysts of immortalized cells and intestinal organoids. We also highlight how misorientation is corrected in vivo and in vitro, and how it might contribute to the onset of pathological conditions.

Expression of Neighbor of Punc E11 (NOPE) in early stage esophageal adenocarcinoma is associated with reduced survival

Current recommendations suggest neoadjuvant treatment in node-positive esophageal cancer or tumors staged T3 and upwards but some T2 N0 patients might benefit from neoadjuvant therapy. It is of clinical relevance to identify this subgroup. Loss of epithelial apicobasal polarity is a key factor in the development of invasive capabilities of carcinoma. The oncofetal stem/progenitor cell marker NOPE is expressed in adult depolarized murine hepatocytes and in murine/human hepatocellular carcinoma. We analyzed NOPE expression in 363 patients with esophageal adenocarcinoma using an RNA Scope Assay on a tissue microarray and correlated results with clinical data. Median follow-up was 57.7 months with a 5-year survival rate of 26.6%. NOPE was detectable in 32 patients (8.8%). In pT1/2 stages, NOPE expression was associated with a significantly reduced median OS of 6.3 months (95% CI 1.2–19.4 months), the median OS is not reached in the NOPE-negative group (calculated mean OS 117.1 months) (P = 0.012). In advanced tumor stages, a NOPE dependent survival difference was not detected. This is the first report of NOPE expression demonstrating a prognostic value in esophageal cancer. Early stage, NOPE positive patients are at a high risk of tumor progression and may benefit from neoadjuvant treatment analogous to advanced stage cancer.

Metabolic regulation of cell competition

Cell Competition is a selective process by which viable cells are eliminated from developing or adult tissues by interactions with their neighbors. In many cases, the eliminated cells (losers) display reduced fitness, yet they would be able to sustain tissue growth or maintenance in a homotypic environment, and are only eliminated when confronted with surrounding wild type cells (winners). In addition, cells with oncogenic mutations that do not show reduced fitness can also be eliminated from tissues when surrounded by wild type cells. Depending on the context, transformed cells can also become supercompetitors and eliminate surrounding wild type cells, thereby promoting tumor formation. Several factors have been shown to play essential roles in Cell Competition, including genes relevant in developmental growth, tumor formation and epithelial apico-basal polarity. Recent discoveries, however, suggest that energy metabolism plays a central role in very different models of cell competition. Here we review the involvement of mitochondrial dynamics and metabolism, autophagy and nutritional status in cell competition and discuss the possible implications of this emerging field.

Zebrafish Crb1, Localizing Uniquely to the Cell Membranes around Cone Photoreceptor Axonemes, Alleviates Light Damage to Photoreceptors and Modulates Cones' Light Responsiveness.

The crumbs (crb) apical polarity genes are essential for the development and functions of epithelia. Adult zebrafish retinal neuroepithelium expresses three crb genes (crb1, crb2a, and crb2b); however, it is unknown whether and how Crb1 differs from other Crb proteins in expression, localization, and functions. Here, we show that, unlike zebrafish Crb2a and Crb2b as well as mammalian Crb1 and Crb2, zebrafish Crb1 does not localize to the subapical regions of photoreceptors and Müller glial cells; rather, it localizes to a small region of cone outer segments: the cell membranes surrounding the axonemes. Moreover, zebrafish Crb1 is not required for retinal morphogenesis and photoreceptor patterning. Interestingly, Crb1 promotes rod survival under strong white light irradiation in a previously unreported non--cell-autonomous fashion; in addition, Crb1 delays UV and blue cones' chromatin condensation caused by UV light irradiation. Finally, Crb1 plays a role in cones' responsiveness to light through an arrestin-translocation-independent mechanism. The localization of Crb1 and its functions do not differ between male and female fish. We conclude that zebrafish Crb1 has diverged from other vertebrate Crb proteins, representing a neofunctionalization in Crb biology during evolution.SIGNIFICANCE STATEMENT Apicobasal polarity of epithelia is an important property that underlies the morphogenesis and functions of epithelial tissues. Epithelial apicobasal polarity is controlled by many polarity genes, including the crb genes. In vertebrates, multiple crb genes have been identified, but the differences in their expression patterns and functions are not fully understood. Here, we report a novel subcellular localization of zebrafish Crb1 in retinal cone photoreceptors and evidence for its new functions in photoreceptor maintenance and light responsiveness. This study expands our understanding of the biology of the crb genes in epithelia, including retinal neuroepithelium.

The Mammalian Crumbs Complex Defines a Distinct Polarity Domain Apical of Epithelial Tight Junctions

Epithelial apico-basal polarity is established through the asymmetric cortical distribution of the Par, Crumbs and Scribble polarity modules. Apical (Par and Crumbs) and basolateral (Scribble) polarity modules overlap at the apical-lateral border, which, in mammals, is defined by the apical junctional complex (AJC). The AJC is composed of tight junctions (TJ) and adherens junctions (AJ) and plays fundamental roles in epithelial morphogenesis and plasticity. However, the molecular composition and precise sub-junctional organization of the AJC and its associated polarity regulators are not well defined. Here, we used the peroxidase APEX2 for quantitative proximity proteomics (QPP) and electron microscopy (EM) imaging to dissect the architecture of the AJC in fully polarized MDCK-II cells. We present a high-confidence proteome of the apical-lateral border in which TJ and AJ components and apical and lateral compartment markers are spatially resolved. We further demonstrate that the Crumbs complex (Pals1, PatJ, Lin7c, and Crumbs3) defines a hitherto unidentified membrane compartment apical of TJ, which we coin the vertebrate marginal zone (VMZ). QPP, imaging, and immunoprecipitation assays showed that the HOMER scaffolding proteins, PKN2 and PTPN13, and the membrane-proximal HIPPO pathway proteins ARHGAP29 and STXBP4 are recruited to the VMZ via the PDZ domains of PatJ. Taken together, our work defines the spatial and molecular organization of the apical-lateral border in mammalian epithelial cells, reveals an intriguing molecular and spatial conservation of invertebrate and vertebrate cell polarity protein domains, and identifies a VMZ-associated protein network implicated in HIPPO signaling and the control of the cortical actin cytoskeleton.

KLF4 Coordinates Corneal Epithelial Apical-Basal Polarity and Plane of Cell Division and Is Downregulated in Ocular Surface Squamous Neoplasia.

PurposePreviously, we demonstrated that Krüppel-like factor 4 (KLF4) promotes corneal epithelial (CE) homeostasis by suppressing epithelial-mesenchymal transition (EMT) and TGF-β signaling. As TGF-β affects epithelial apicobasal polarity (ABP) and plane of division, we investigated the role of KLF4 in these processes.Methods Klf4 was ablated in adult ternary transgenic Klf4Δ/ΔCE (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mouse CE using doxycycline chow. ABP and plane of division markers’ expression in Klf4Δ/ΔCE and human ocular surface squamous neoplasia (OSSN) tissues relative to controls was evaluated by quantitative PCR, immunoblots, and/or immunofluorescent staining.Results Klf4Δ/ΔCE CE cells displayed downregulation of apical Pals1 and Crumbs1, apicolateral Par3, and basolateral Scribble, as well as upregulation of Rho family GTPase Cdc42, suggesting disruption of ABP. Phalloidin staining revealed that the Klf4Δ/ΔCE CE actin cytoskeleton is disrupted. Klf4Δ/ΔCE cells favored vertical plane of division within 67.5° to 90° of the CE basement membrane (39% and 47% of the dividing cells relative to 23% and 26% in the control based on phospho-histone-H3 and survivin, respectively), resulting in more dividing cells within the Klf4Δ/ΔCE CE as reported previously. KLF4 was downregulated in human OSSN tissues that displayed EMT and downregulation of PAR3, PALS1, and SCRIB, consistent with a protective role for KLF4.ConclusionsBy demonstrating that Klf4 ablation affects CE expression of ABP markers and Cdc42, cytoskeletal actin organization, and the plane of cell division and that KLF4 is downregulated in OSSN tissues that display EMT and lack ABP, these results elucidate the key integrative role of KLF4 in coordinating CE cell polarity and plane of division, loss of which results in OSSN.

Scribble, Erbin, and Lano redundantly regulate epithelial polarity and apical adhesion complex.

The basolateral protein Scribble (Scrib), a member of the LAP protein family, is essential for epithelial apicobasal polarity (ABP) in Drosophila However, a conserved function for this protein in mammals is unclear. Here we show that the crucial role for Scrib in ABP has remained obscure due to the compensatory function of two other LAP proteins, Erbin and Lano. A combined Scrib/Erbin/Lano knockout disorganizes the cell-cell junctions and the cytoskeleton. It also results in mislocalization of several apical (Par6, aPKC, and Pals1) and basolateral (Llgl1 and Llgl2) identity proteins. These defects can be rescued by the conserved "LU" region of these LAP proteins. Structure-function analysis of this region determined that the so-called LAPSDb domain is essential for basolateral targeting of these proteins, while the LAPSDa domain is essential for supporting the membrane basolateral identity and binding to Llgl. In contrast to the key role in Drosophila, mislocalization of Llgl proteins does not appear to be critical in the scrib ABP phenotype.

Perturbation of epithelial apicobasal polarity by rhomboid family‐1 gene overexpression

The human rhomboid family (RHBDF)1 gene is highly expressed in breast cancer under clinical conditions but not in normal mammary gland tissues. Silencing the RHBDF1 gene in breast cancer xenograft tumors leads to inhibition of tumor growth. We show in this study that artificially raising RHBDF1 protein levels in the mammary epithelial cells MCF-10A results in severe perturbations of the ability of the cells to form lumen-containing acini, either in 3-dimensional cell cultures or implanted in mouse mammary fat pads. Knocking down RHBDF1 with short hairpin (sh)RNA leads to restoration of acinus formation. Consistently, RHBDF1 overexpression gives rise to disordered distribution of polarity markers GM130 and laminin-5, which otherwise are located in apical and basal positions, respectively, in the acini. Further investigations reveal that RHBDF1 directly binds to Par6a, a component of a protein complex consisting of partitioning-defective scaffold protein (Par)6, Par3, renin-angiotensin system-related C3 botulinum toxin substrate (Rac)1, and cell-division cycle (Cdc)42, which is structurally critical to the formation of apicobasal polarity. RHBDF1 binding to Par6a results in collapse of the protein complex and thus disruption of polarity formation. Since early stages of breast cancer are characterized by the loss of mammary gland epithelial cell polarity, our findings indicate that perturbations of apicobasal polarity by high levels of RHBDF1 is a significant attribute in the development of breast neoplasia.-Peng, X.-M., Gao, S., Deng, H.-T., Cai, H.-X., Zhou, Z., Xiang, R., Zhang, Q.-Z., Li, L.-Y. Perturbation of epithelial apicobasal polarity by rhomboid family-1 gene overexpression.

Biallelic Mutation of ARHGEF18, Involved in the Determination of Epithelial Apicobasal Polarity, Causes Adult-Onset Retinal Degeneration

Mutations in more than 250 genes are implicated in inherited retinal dystrophy; the encoded proteins are involved in a broad spectrum of pathways. The presence of unsolved families after highly parallel sequencing strategies suggests that further genes remain to be identified. Whole-exome and -genome sequencing studies employed here in large cohorts of affected individuals revealed biallelic mutations in ARHGEF18 in three such individuals. ARHGEF18 encodes ARHGEF18, a guanine nucleotide exchange factor that activates RHOA, a small GTPase protein that is a key component of tight junctions and adherens junctions. This biological pathway is known to be important for retinal development and function, as mutation of CRB1, encoding another component, causes retinal dystrophy. The retinal structure in individuals with ARHGEF18 mutations resembled that seen in subjects with CRB1 mutations. Five mutations were found on six alleles in the three individuals: c.808A>G (p.Thr270Ala), c.1617+5G>A (p.Asp540Glyfs∗63), c.1996C>T (p.Arg666∗), c.2632G>T (p.Glu878∗), and c.2738_2761del (p.Arg913_Glu920del). Functional tests suggest that each disease genotype might retain some ARHGEF18 activity, such that the phenotype described here is not the consequence of nullizygosity. In particular, the p.Thr270Ala missense variant affects a highly conserved residue in the DBL homology domain, which is required for the interaction and activation of RHOA. Previously, knock-out of Arhgef18 in the medaka fish has been shown to cause larval lethality which is preceded by retinal defects that resemble those seen in zebrafish Crumbs complex knock-outs. The findings described here emphasize the peculiar sensitivity of the retina to perturbations of this pathway, which is highlighted as a target for potential therapeutic strategies.

Quantification of Confocal Images Using LabVIEW for Tissue Engineering Applications.

Quantifying confocal images to enable location of specific proteins of interest in three-dimensional (3D) is important for many tissue engineering (TE) applications. Quantification of protein localization is essential for evaluation of specific scaffold constructs for cell growth and differentiation for application in TE and tissue regeneration strategies. Although obtaining information regarding protein expression levels is important, the location of proteins within cells grown on scaffolds is often the key to evaluating scaffold efficacy. Functional epithelial cell monolayers must be organized with apicobasal polarity with proteins specifically localized to the apical or basolateral regions of cells in many organs. In this work, a customized program was developed using the LabVIEW platform to quantify protein positions in Z-stacks of confocal images of epithelial cell monolayers. The program's functionality is demonstrated through salivary gland TE, since functional salivary epithelial cells must correctly orient many proteins on the apical and basolateral membranes. Bio-LabVIEW Image Matrix Evaluation (Bio-LIME) takes 3D information collected from confocal Z-stack images and processes the fluorescence at each pixel to determine cell heights, nuclei heights, nuclei widths, protein localization, and cell count. As a demonstration of its utility, Bio-LIME was used to quantify the 3D location of the Zonula occludens-1 protein contained within tight junctions and its change in 3D position in response to chemical modification of the scaffold with laminin. Additionally, Bio-LIME was used to demonstrate that there is no advantage of sub-100 nm poly lactic-co-glycolic acid nanofibers over 250 nm fibers for epithelial apicobasal polarization. Bio-LIME will be broadly applicable for quantification of proteins in 3D that are grown in many different contexts.

Cell Competition: Mechanisms and Physiological Roles.

Cell-competitive interactions are widespread in nature and determine the outcome of a vast variety of biological processes. A particular class of competitive interactions takes place when alterations in intrinsic cellular properties are sensed nonautonomously by comparison between neighboring cells, resulting in the selective elimination of one cell population. This type of cell competition was first described four decades ago in developing epithelia of Drosophila. In the last 15 years, further molecular and cellular analyses have provided essential knowledge about the mechanisms, universality, and physiological relevance of cell competition. The two main phenomena triggering cell competition are alterations in cellular metabolic status and alterations in epithelial apico-basal polarity, while other reported pathways are less characterized. Cell competition plays essential roles in quality control, homeostasis, and repair of developing and adult tissues, and depending on the context, it may function as a tumor-suppressing or tumor-promoting mechanism.

Rab35 GTPase couples cell division with initiation of epithelial apico-basal polarity and lumen opening.

Establishment and maintenance of apico-basal polarity in epithelial organs must be tightly coupled with cell division, but the underlying molecular mechanisms are largely unknown. Using 3D cultures of renal MDCK cells (cysts), we found that the Rab35 GTPase plays a crucial role in polarity initiation and apical lumen positioning during the first cell division of cyst development. At the molecular level, Rab35 physically couples cytokinesis with the initiation of apico-basal polarity by tethering intracellular vesicles containing key apical determinants at the cleavage site. These vesicles transport aPKC, Cdc42, Crumbs3 and the lumen-promoting factor Podocalyxin, and are tethered through a direct interaction between Rab35 and the cytoplasmic tail of Podocalyxin. Consequently, Rab35 inactivation leads to complete inversion of apico-basal polarity in 3D cysts. This novel and unconventional mode of Rab-dependent vesicle targeting provides a simple mechanism for triggering both initiation of apico-basal polarity and lumen opening at the centre of cysts.

Regulation of epithelial cell polarity by PAR-3 depends on Girdin transcription and Girdin-Gαi3 signaling.

Epithelial apicobasal polarity has fundamental roles in epithelial physiology and morphogenesis. The PAR complex, comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC), is involved in determining cell polarity in various biological contexts, including in epithelial cells. However, it is not fully understood how the PAR complex induces apicobasal polarity. In this study, we found that PAR-3 regulates the protein expression of Girdin (also known as GIV or CCDC88A), a guanine-nucleotide-exchange factor (GEF) for heterotrimeric Gαi subunits, at the transcriptional level by cooperating with the AP-2 transcription factor. In addition, we confirmed that PAR-3 physically interacts with Girdin, and show that Girdin, together with the Gαi3 (also known as GNAI3), controls tight junction formation, apical domain development and actin organization downstream of PAR-3. Taken together, our findings suggest that transcriptional upregulation of Girdin expression and Girdin-Gαi3 signaling play crucial roles in regulating epithelial apicobasal polarity through the PAR complex.

Genes Regulating Epithelial Polarity Are Critical Suppressors of Esophageal Oncogenesis.

Esophageal cancer is an aggressive disease featured by early lymphatic and hematogenous dissemination, and is the sixth leading cause of cancer-related deaths worldwide. The proper formation of apicobasal polarity is essential for normal epithelium physiology and tissue homeostasis, while loss of polarity is a hallmark of cancer development including esophageal oncogenesis. In this review, we summarized the stages of esophageal cancer development associated with the loss or deregulation of epithelial cell apicobasal polarity. Loss of epithelial apicobasal polarity exerts an indispensable role in the initiation of esophageal oncogenesis, tumor progression, and the advancement of tumors from benign to malignant. In particular, we reviewed the involvement of several critical genes, including Lkb1, claudin-4, claudin-7, Par3, Lgl1, E-cadherin, and the Scnn1 gene family. Understanding the role of apicobasal regulators may lead to new paradigms for treatment of esophageal tumors, including improvement of prognostication, early diagnosis, and individually tailored therapeutic interventions in esophageal oncology.

The BTB-zinc Finger Transcription Factor Abrupt Acts as an Epithelial Oncogene in Drosophila melanogaster through Maintaining a Progenitor-like Cell State

The capacity of tumour cells to maintain continual overgrowth potential has been linked to the commandeering of normal self-renewal pathways. Using an epithelial cancer model in Drosophila melanogaster, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, scribbled (scrib), and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with scrib loss of function, Abrupt promotes the formation of massive tumours in the eye/antennal disc. The steroid hormone receptor coactivator, Taiman (a homologue of SRC3/AIB1), is known to associate with Abrupt, and Taiman overexpression also drives tumour formation in cooperation with the loss of Scrib. Expression arrays and ChIP-Seq indicates that Abrupt overexpression represses a large number of genes, including steroid hormone-response genes and multiple cell fate regulators, thereby maintaining cells within an epithelial progenitor-like state. The progenitor-like state is characterised by the failure to express the conserved Eyes absent/Dachshund regulatory complex in the eye disc, and in the antennal disc by the failure to express cell fate regulators that define the temporal elaboration of the appendage along the proximo-distal axis downstream of Distalless. Loss of scrib promotes cooperation with Abrupt through impaired Hippo signalling, which is required and sufficient for cooperative overgrowth with Abrupt, and JNK (Jun kinase) signalling, which is required for tumour cell migration/invasion but not overgrowth. These results thus identify a novel cooperating oncogene, identify mammalian family members of which are also known oncogenes, and demonstrate that epithelial tumours in Drosophila can be characterised by the maintenance of a progenitor-like state.

A functional role of the extracellular domain of Crumbs in cell architecture and apicobasal polarity

Regulated cell shape changes in epithelial cells, which contribute to most organs and tissues, are at the basis of morphogenesis. Crumbs (Crb) is an essential apical determinant controlling epithelial apicobasal polarity. Here we provide evidence for a novel role of Crb apical localisation and stabilisation in controlling cell shape through apical domain organisation and adherens junction positioning. We find that Crb apical stabilisation requires the extracellular domain. In vivo results from Drosophila suggest that the extracellular domain assists Crb apical stabilisation by mediating Crb-Crb interactions at opposing cell membranes. We further confirm Crb-Crb extracellular interactions by showing that the extracellular domain of Crb is sufficient to promote cell aggregation in vitro. Furthermore, we report that Crb apical stabilisation mediated by the extracellular domain is also required for maintenance of Crb apicobasal polarity. Our results provide new insights into the mechanisms of apicobasal polarity and the cellular mechanisms of tissue architecture.