The First Defined Null Allele of the Notch Regulator, a Suppressor of Deltex: Uncovering Its Novel Roles in Drosophila melanogaster Oogenesis.

Suppressor of deltex (Su(dx)) is a Drosophila melanogaster member of the NEDD4 family of the HECT domain E3 ubiquitin ligases. Su(dx) acts as a regulator of Notch endocytic trafficking, promoting Notch lysosomal degradation and the down-regulation of both ligand-dependent and ligand-independent signalling, the latter involving trafficking through the endocytic pathway and activation of the endo/lysosomal membrane. Mutations of Su(dx) result in developmental phenotypes in the Drosophila wing that reflect increased Notch signalling, leading to gaps in the specification of the wing veins, and Su(dx) functions to provide the developmental robustness of Notch activity to environmental temperature shifts. The full developmental functions of Su(dx) are unclear; however, this is due to a lack of a clearly defined null allele. Here we report the first defined null mutation of Su(dx), generated by P-element excision, which removes the complete open reading frame. We show that the mutation is recessive-viable, with the Notch gain of function phenotypes affecting wing vein and leg development. We further uncover new roles for Su(dx) in Drosophila oogenesis, where it regulates interfollicular stalk formation, egg chamber separation and germline cyst enwrapment by the follicle stem cells. Interestingly, while the null allele exhibited a gain in Notch activity during oogenesis, the previously described Su(dx)SP allele, which carries a seven amino acid in-frame deletion, displayed a Notch loss of function phenotypes and an increase in follicle stem cell turnover. This is despite both alleles displaying similar Notch gain of function in wing development. We attribute this unexpected context-dependent outcome of Su(dx)sp being due to the partial retention of function by the intact C2 and WW domain regions of the protein. Our results extend our understanding of the developmental role of Su(dx) in the tissue renewal and homeostasis of the Drosophila ovary and illustrate the importance of examining an allelic series of mutations to fully understand developmental functions.

Alternative mechanisms of Notch activation by partitioning into distinct endosomal domains.

Different membrane microdomain compositions provide unique environments that can regulate signaling receptor function. We identify microdomains on the endosome membrane of Drosophila endosomes, enriched in lipid-raft or clathrin/ESCRT-0, which are associated with Notch activation by distinct, ligand-independent mechanisms. Transfer of Notch between microdomains is regulated by Deltex and Suppressor of deltex ubiquitin ligases and is limited by a gate-keeper role for ESCRT complexes. Ubiquitination of Notch by Deltex recruits it to the clathrin/ESCRT-0 microdomain and enhances Notch activation by an ADAM10-independent/TRPML-dependent mechanism. This requirement for Deltex is bypassed by the downregulation of ESCRT-III. In contrast, while ESCRT-I depletion also activates Notch, it does so by an ADAM10-dependent/TRPML-independent mechanism and Notch is retained in the lipid raft-like microdomain. In the absence of such endosomal perturbation, different activating Notch mutations also localize to different microdomains and are activated by different mechanisms. Our findings demonstrate the interplay between Notch regulators, endosomal trafficking components, and Notch genetics, which defines membrane locations and activation mechanisms.

Efficiency calibrations of radiation detectors for source localization and characterization at long source-detector distances for gamma and neutron sources.

Deployment of radiation detectors under field conditions for the purposes of security, safety or response has increased in recent years. Effective use of such instruments in the field necessitates careful consideration of the efficiency of the detector - both peak and total - at distances which may extend beyond 100 m. Difficulties in addressing the determination of both peak and total efficiencies across the energy range of interest and at long distances reduces the utility of such systems in effectively characterising radiation sources in the field. Empirical approaches to such calibrations are difficult. Approaches such as Monte Carlo simulations can become challenging with respect to time and computational requirements as source-detector distances become greater and in consideration of total efficiency. This paper presents a computationally efficient method of calculating peak efficiency at distances more than 300 m using efficiency transfer from a parallel beam geometry to point sources at extended distances. The relationship between total and peak efficiency at extended distances is explored and means of estimating the total efficiency from the peak efficiency are discussed. The ratio of the total efficiency to the peak efficiency increases as a function of the source-detector distance. The relationship is linear at distances longer than 50 m and is independent of photon energy. Usefulness of the efficiency calibration as a function of the source-detector distance was demonstrated in a field experiment. Total efficiency calibration measurements were performed for a neutron counter. An AmBe source was then successfully localized and characterised using four measurements at arbitrary locations far away from the unknown source. This kind of capability is useful for the authorities responding to nuclear accidents or security events. It has important operational implications, including the safety of the personnel involved.

Ubiquitylation is required for the incorporation of the Notch receptor into intraluminal vesicles to prevent prolonged and ligand-independent activation of the pathway.

BACKGROUND: Ubiquitylation of the ligands and the receptor plays an important part in the regulation of the activity of the evolutionary conserved Notch signalling pathway. However, its function for activation of Notch is not completely understood, despite the identification of several E3 ligases devoted to the receptor. RESULTS: Here we analysed a variant of the Notch receptor where all lysines in its intracellular domain are replaced by arginines. Our analysis of this variant revealed that ubiquitylation of Notch is not essential for its endocytosis. We identified two functions for ubiquitylation of lysines in the Notch receptor. First, it is required for the degradation of free Notch intracellular domain (NICD) in the nucleus, which prevents a prolonged activation of the pathway. More importantly, it is also required for the incorporation of Notch into intraluminal vesicles of maturing endosomes to prevent ligand-independent activation of the pathway from late endosomal compartments. CONCLUSIONS: The findings clarify the role of lysine-dependent ubiquitylation of the Notch receptor and indicate that Notch is endocytosed by several independent operating mechanisms.

E3 Ubiquitin Ligase Regulators of Notch Receptor Endocytosis: From Flies to Humans.

Notch is a developmental receptor, conserved in the evolution of the metazoa, which regulates cell fate proliferation and survival in numerous developmental contexts, and also regulates tissue renewal and repair in adult organisms. Notch is activated by proteolytic removal of its extracellular domain and the subsequent release of its intracellular domain, which then acts in the nucleus as part of a transcription factor complex. Numerous regulatory mechanisms exist to tune the amplitude, duration and spatial patterning of this core signalling mechanism. In Drosophila, Deltex (Dx) and Suppressor of dx (Su(dx)) are E3 ubiquitin ligases which interact with the Notch intracellular domain to regulate its endocytic trafficking, with impacts on both ligand-dependent and ligand-independent signal activation. Homologues of Dx and Su(dx) have been shown to also interact with one or more of the four mammalian Notch proteins and other target substrates. Studies have shown similarities, specialisations and diversifications of the roles of these Notch regulators. This review collates together current research on vertebrate Dx and Su(dx)-related proteins, provides an overview of their various roles, and discusses their contributions to cell fate regulation and disease.

Inhibition of Wnt signalling by Notch via two distinct mechanisms.

Notch and Wnt are two essential signalling pathways that help to shape animals during development and to sustain adult tissue homeostasis. Although they are often active at the same time within a tissue, they typically have opposing effects on cell fate decisions. In fact, crosstalk between the two pathways is important in generating the great diversity of cell types that we find in metazoans. Several different mechanisms have been proposed that allow Notch to limit Wnt signalling, driving a Notch-ON/Wnt-OFF state. Here we explore these different mechanisms in human cells and demonstrate two distinct mechanisms by which Notch itself, can limit the transcriptional activity of ß-catenin. At the membrane, independently of DSL ligands, Notch1 can antagonise ß-catenin activity through an endocytic mechanism that requires its interaction with Deltex and sequesters ß-catenin into the membrane fraction. Within the nucleus, the intracellular domain of Notch1 can also limit ß-catenin induced transcription through the formation of a complex that requires its interaction with RBPjκ. We believe these mechanisms contribute to the robustness of cell-fate decisions by sharpening the distinction between opposing Notch/Wnt responses.

Notch3 in Development, Health and Disease.

Notch3 is one of four mammalian Notch proteins, which act as signalling receptors to control cell fate in many developmental and adult tissue contexts. Notch signalling continues to be important in the adult organism for tissue maintenance and renewal and mis-regulation of Notch is involved in many diseases. Genetic studies have shown that Notch3 gene knockouts are viable and have limited developmental defects, focussed mostly on defects in the arterial smooth muscle cell lineage. Additional studies have revealed overlapping roles for Notch3 with other Notch proteins, which widen the range of developmental functions. In the adult, Notch3, in collaboration with other Notch proteins, is involved in stem cell regulation in different tissues in stem cell regulation in different tissues, and it also controls the plasticity of the vascular smooth muscle phenotype involved in arterial vessel remodelling. Overexpression, gene amplification and mis-activation of Notch3 are associated with different cancers, in particular triple negative breast cancer and ovarian cancer. Mutations of Notch3 are associated with a dominantly inherited disease CADASIL (cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy), and there is further evidence linking Notch3 misregulation to hypertensive disease. Here we discuss the distinctive roles of Notch3 in development, health and disease, different views as to the underlying mechanisms of its activation and misregulation in different contexts and potential for therapeutic intervention.

Whole Exome Sequencing Reveals the Major Genetic Contributors to Nonsyndromic Tetralogy of Fallot.

RATIONALE: Familial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, nonsyndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease phenotype. Rare genetic variants have been identified as important contributors to the risk of congenital heart disease, but relatively small numbers of TOF cases have been studied to date. OBJECTIVE: We used whole exome sequencing to assess the prevalence of unique, deleterious variants in the largest cohort of nonsyndromic TOF patients reported to date. METHODS AND RESULTS: Eight hundred twenty-nine TOF patients underwent whole exome sequencing. The presence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database and a scaled combined annotation-dependent depletion score of ≥20. The clustering of variants in 2 genes, NOTCH1 and FLT4, surpassed thresholds for genome-wide significance (assigned as P<5×10-8) after correction for multiple comparisons. NOTCH1 was most frequently found to harbor unique, deleterious variants. Thirty-one changes were observed in 37 probands (4.5%; 95% CI, 3.2%-6.1%) and included 7 loss-of-function variants 22 missense variants and 2 in-frame indels. Sanger sequencing of the unaffected parents of 7 cases identified 5 de novo variants. Three NOTCH1 variants (p.G200R, p.C607Y, and p.N1875S) were subjected to functional evaluation, and 2 showed a reduction in Jagged1-induced NOTCH signaling. FLT4 variants were found in 2.4% (95% CI, 1.6%-3.8%) of TOF patients, with 21 patients harboring 22 unique, deleterious variants. The variants identified were distinct to those that cause the congenital lymphoedema syndrome Milroy disease. In addition to NOTCH1, FLT4 and the well-established TOF gene, TBX1, we identified potential association with variants in several other candidates, including RYR1, ZFPM1, CAMTA2, DLX6, and PCM1. CONCLUSIONS: The NOTCH1 locus is the most frequent site of genetic variants predisposing to nonsyndromic TOF, followed by FLT4. Together, variants in these genes are found in almost 7% of TOF patients.

The Drosophila ZO-1 protein Polychaetoid suppresses Deltex-regulated Notch activity to modulate germline stem cell niche formation.

The developmental signalling protein Notch can be proteolytically activated following ligand-interaction at the cell surface, or can be activated independently of its ligands, following Deltex (Dx)-induced Notch endocytosis and trafficking to the lysosomal membrane. The means by which different pools of Notch are directed towards these alternative outcomes remains poorly understood. We found that the Drosophila ZO-1 protein Polychaetoid (Pyd) suppresses specifically the Dx-induced form of Notch activation both in vivo and in cell culture assays. In vivo we confirmed the physiological relevance and direction of the Pyd/Dx interaction by showing that the expanded ovary stem cell niche phenotypes of pyd mutants require the presence of functional Dx and other components that are specific to the Dx-induced Notch activation mechanism. In S2 cells we found that Pyd can form a complex with Dx and Notch at the cell surface and reduce Dx-induced Notch endocytosis. Similar to other known activities of ZO-1 family proteins, the action of Pyd on Dx-induced endocytosis and signalling was found to be cell density dependent. Thus, together, our results suggest an alternative means by which external cues can tune Notch signalling through Pyd regulation of Dx-induced Notch trafficking.

Combining genetic and biophysical approaches to probe the structure and function relationships of the notch receptor.

Notch is a conserved cell signalling receptor regulating many aspects of development and tissue homeostasis. Notch is activated by ligand-induced proteolytic cleavages that release the Notch intracellular domain, which relocates to the nucleus to regulate gene transcription. Proteolytic activation first requires mechanical force to be applied to the Notch extracellular domain through an endocytic pulling mechanism transmitted through the ligand/receptor interface. This exposes the proteolytic cleavage site allowing the signal to be initiated following removal of the Notch extracellular domain. Ligands can also act, when expressed in the same cell, through non-productive cis-interactions to inhibit Notch activity. Furthermore, ligand selectivity and Notch activation are regulated by numerous post-translational modifications of the extracellular domain. Additional non-canonical trans and cis interactions with other regulatory proteins may modulate alternative mechanisms of Notch activation that depend on endocytic trafficking of the full-length receptor and proteolytic release of the intracellular domain from endo-lysosomal surface. Mutations of Notch, located in different regions of the protein, are associated with a spectrum of different loss and gain of function phenotypes and offer the possibility to dissect distinct regulatory interactions and mechanisms, particularly when combined with detailed structural analysis of Notch in complex with various regulatory partners.

DisGeNET: a discovery platform for the dynamical exploration of human diseases and their genes.

DisGeNET is a comprehensive discovery platform designed to address a variety of questions concerning the genetic underpinning of human diseases. DisGeNET contains over 380,000 associations between >16,000 genes and 13,000 diseases, which makes it one of the largest repositories currently available of its kind. DisGeNET integrates expert-curated databases with text-mined data, covers information on Mendelian and complex diseases, and includes data from animal disease models. It features a score based on the supporting evidence to prioritize gene-disease associations. It is an open access resource available through a web interface, a Cytoscape plugin and as a Semantic Web resource. The web interface supports user-friendly data exploration and navigation. DisGeNET data can also be analysed via the DisGeNET Cytoscape plugin, and enriched with the annotations of other plugins of this popular network analysis software suite. Finally, the information contained in DisGeNET can be expanded and complemented using Semantic Web technologies and linked to a variety of resources already present in the Linked Data cloud. Hence, DisGeNET offers one of the most comprehensive collections of human gene-disease associations and a valuable set of tools for investigating the molecular mechanisms underlying diseases of genetic origin, designed to fulfill the needs of different user profiles, including bioinformaticians, biologists and health-care practitioners. Database URL: http://www.disgenet.org/

Reversible regulation of stem cell niche size associated with dietary control of Notch signalling.

BACKGROUND: Stem cells can respond to environmental and physiological inputs to adaptively remodel tissues. Little is known about whether stem cell niches are similarly responsive. The Drosophila ovary germline stem cell (GSC) niche is a well-studied model, which is comprised of cap cells that provide anchorage and maintenance signals for GSCs to maintain oogenesis. Previous studies have shown a strong link between diet and the regulation of oogenesis, making this a useful model system in which to investigate dietary regulation of the niche and its associated stem cells. RESULTS: We show that the Drosophila ovary GSC cap cell niche is a dynamic structure, which can contract and expand in fluctuating dietary conditions. Cap cells are lost when adult flies are shifted to nutrient poor diet and are restored after returning flies to nutrient-rich medium. Notch signalling in cap and escort cells is similarly reduced and restored by dietary shifts to nutrient poor and rich media. In old flies decreased Notch signalling is associated with decreased robustness of the niche to dietary changes. We demonstrated using a Notch temperature sensitive allele that removal and restoration of Notch signalling also leads to a reduction and re-expansion of the niche. Changes in niche size were not associated with apoptosis or cell division. We identified two distinct roles for Notch in the adult germarium. Notch can act in cap cells to prevent their loss while activation of Notch in the flanking escort cells results in expansion of the niche. CONCLUSIONS: We provide evidence that dietary changes alone are sufficient to alter Notch signalling and reversibly change niche size in the adult in wild type flies. We show Notch acts in different cells to maintain and re-expand the niche and propose a model in which cell fate transitions between cap cells and flanking somatic cells accounts for niche dynamics. These findings reveal an unexpected reversible plasticity of the GSC niche whose responses provide an integrated read out of the physiological status of the fly that is modulated by diet and age.

Compensatory flux changes within an endocytic trafficking network maintain thermal robustness of Notch signaling.

Developmental signaling is remarkably robust to environmental variation, including temperature. For example, in ectothermic animals such as Drosophila, Notch signaling is maintained within functional limits across a wide temperature range. We combine experimental and computational approaches to show that temperature compensation of Notch signaling is achieved by an unexpected variety of endocytic-dependent routes to Notch activation which, when superimposed on ligand-induced activation, act as a robustness module. Thermal compensation arises through an altered balance of fluxes within competing trafficking routes, coupled with temperature-dependent ubiquitination of Notch. This flexible ensemble of trafficking routes supports Notch signaling at low temperature but can be switched to restrain Notch signaling at high temperature and thus compensates for the inherent temperature sensitivity of ligand-induced activation. The outcome is to extend the physiological range over which normal development can occur. Similar mechanisms may provide thermal robustness for other developmental signals.

Norrish Type I surface photochemistry for butyrophenone on TiO2(110).

The photofragmentation of butyrophenone yields benzoate and a propyl radical on oxidized TiO2(110). Oxygen dissociates in native oxygen vacancies to produce reactive oxygen adatoms which react with butyrophenone to create photoactive butyrophenone-O complexes that are sensitive to hole oxidation created upon UV illumination. The same O adatoms also trap one of the primary photoproducts, phenyl-CO, to produce benzoate. The reaction proceeds via a Norrish Type I like process involving α-CC cleavage on the surface, in contrast to the gas phase where a Norrish Type II pathway predominates. The mechanism is probed using mass spectrometry and, for the first time, scanning tunneling microscopy (STM). Our STM experiments show that there is a 1-to-1 correspondence between the immobile butyrophenone-O complex and formation of a benzoate on the surface. We also demonstrate that the benzoate species is in close proximity to the original butyrophenone complex, indicating that benzoate is produced on a time scale more rapid than diffusion of the photoproducts. While the photoproducts of butyrophenone decomposition are similar to ketone oxidation reported previously, butyrophenone reacts via a different starting ground state, based on STM and density functional theory studies. Specifically, butyrophenone does not produce a dioxyalkylene species, which has been proposed to be the photoactive state for other ketones. Based on a combination of STM experiments and density functional theory, we propose that a peroxy-like configuration where the oxygen adatom stabilizes the butyrophenone through its carbonyl oxygen is the surface intermediate that photodecomposes. These results demonstrate the importance of the excited state in determining the photochemistry of ketones on surfaces.

Molecular basis for Jagged-1/Serrate ligand recognition by the Notch receptor.

We have mapped a Jagged/Serrate-binding site to specific residues within the 12th EGF domain of human and Drosophila Notch. Two critical residues, involved in a hydrophobic interaction, provide a ligand-binding platform and are adjacent to a Fringe-sensitive residue that modulates Notch activity. Our data suggest that small variations within the binding site fine-tune ligand specificity, which may explain the observed sequence heterogeneity in mammalian Notch paralogues, and should allow the development of paralogue-specific ligand-blocking antibodies. As a proof of principle, we have generated a Notch-1-specific monoclonal antibody that blocks binding, thus paving the way for antibody tools for research and therapeutic applications.

Sequential photo-oxidation of methanol to methyl formate on TiO2(110).

Methyl formate is produced from the photo-oxidation of methanol on preoxidized TiO(2)(110). We demonstrate that two consecutive photo-oxidation steps lead to methyl formate using mass spectrometry and scanning tunneling microscopy. The first step in methanol oxidation is formation of methoxy by the thermal dissociation of the O-H bond to yield adsorbed CH(3)O and water. Formaldehyde is produced via hole-mediated oxidation of adsorbed methoxy in the first photochemical step. Next, transient HCO is made photochemically from formaldehyde. The HCO couples with residual methoxy on the surface to yield methyl formate. Exposure of the titania surface to O(2) is required for these photo-oxidation steps in order to heal surface and near-surface defects that can serve as hole traps. Notably, residual O adatoms are not required for photochemical production of methyl formate or formaldehyde. All O adatoms react thermally with methanol to form methoxy and gaseous water at rt, leaving a surface devoid of O adatoms. The mechanism provides insight into the photochemistry of TiO(2) and suggests general synthetic pathways that are the result of the ability to activate both alkoxides and aldehydes using photons.

Endocytic routes to Notch activation.

It is well established that Notch signalling is activated in response to ligand binding through a series of proteolytic cleavages that release the Notch intracellular domain, allowing it to translocate to the nucleus to regulate downstream target gene expression. However there is still much to learn about the mechanisms that can bring about these proteolytic events in the numerous physiological contexts in which signal activation occurs. A number of studies have suggested that endocytosis of Notch contributes to the signal activation process, but the molecular details are unclear and controversial. There is conflicting data as to whether endocytosis of the receptor is essential for ligand-induced signalling or supplements it. Other studies have revealed that Notch can be activated in the endosomal pathway, independently of its ligands, through the activity of Deltex, a Ring-domain Ubiquitin ligase that binds to the Notch intracellular domain. However, it is unclear how the Deltex-activation mechanism relates to that of ligand-induced signalling, or to ectopic Notch signalling brought about by disruption of ESCRT complexes that affect multivesicular body formation. This review will address these issues and argue that the data are best reconciled by proposing distinct activation mechanisms in different cellular locations that contribute to the cellular pool of the soluble Notch intracellular domain. The resulting signalling network may provide developmental robustness to environmental and genetic variation.

Formation of one-dimensional electronic states along the step edges of CeO2(111).

Scanning tunneling microscopy (STM) combined with density functional theory (DFT) are used to analyze the structural and electronic properties of step edges on the surface of CeO(2)(111) films grown on Ru(0001). Depending on the preparation conditions, 211 or 110-oriented steps develop on the surface, which results in the formation of ceria ad-islands with hexagonal or triangular shapes. STM conductance spectroscopy reveals pronounced differences in the electronic properties of the step edges, as reflected in different onset positions of the ceria conduction band. The band shifts are related to the development of distinct edge electronic states that split-off from the ceria conduction band, as shown with DFT calculations. The separation of the edge states from the main band is governed by the atom-coordination and local charge-distribution along the edge, the latter giving rise to the development of electrostatic dipoles. We expect that the observed edge morphologies determine not only the electronic properties but also the adsorption behavior of step edges on the CeO(2)(111) surface.

Roles of Drosophila deltex in Notch receptor endocytic trafficking and activation.

Cell signaling mediated by the Notch receptor (N) regulates many cell-fate decisions and is partly controlled by the endocytic trafficking of N. Drosophila deltex (dx) encodes an evolutionarily conserved regulator of N signaling, an E3-ubiquitin ligase, which ubiquitinates N's intracellular domain. Although Dx was shown to function in N endocytosis in studies of dx over-expression, the roles of endogenous Dx have remained hidden. Here, we investigated N endocytosis in a dx-null Drosophila mutant and found that endogenous Dx is required for at least two steps of N trafficking: the incorporation of N into endocytic vesicles from the plasma membrane and the transport of N from early endosomes to lysosomes. In the absence of Dx functions, N was stabilized in unknown endocytic compartments, where it was probably insulated from transport to lysosomes. We also found that canonical N signaling and Dx-mediated N signaling are activated in two different endocytic compartments, before N is incorporated into multivesicular body (MVB) interluminal vesicles and after N is transported from MVBs, respectively. The endocytic compartment in which Dx-mediated N signaling is activated appears to coincide with the activity of endogenous Dx in N trafficking. These findings extend our understanding of how N's trafficking and activation are correlated.

Su(dx) E3 ubiquitin ligase-dependent and -independent functions of polychaetoid, the Drosophila ZO-1 homologue.

Zona occludens (ZO) proteins are molecular scaffolds localized to cell junctions, which regulate epithelial integrity in mammals. Using newly generated null alleles, we demonstrate that polychaetoid (pyd), the unique Drosophila melanogaster ZO homologue, regulates accumulation of adherens junction-localized receptors, such as Notch, although it is dispensable for epithelial polarization. Pyd positively regulates Notch signaling during sensory organ development but acts negatively on Notch to restrict the ovary germline stem cell niche. In both contexts, we identify a core antagonistic interaction between Pyd and the WW domain E3 ubiquitin ligase Su(dx). Pyd binds Su(dx) directly, in part through a noncanonical WW-binding motif. Pyd also restricts epithelial wing cell numbers to control adult wing shape, a function associated with the FERM protein Expanded and independent of Su(dx). As both Su(dx) and Expanded regulate trafficking, we propose that a conserved role of ZO proteins is to coordinate receptor trafficking and signaling with junctional organization.