Clathrin Adaptor AP-1 Research Articles

Role of adaptor protein complexes in generating functionally distinct synaptic vesicle pools.

The synaptic vesicle (SV) cycle ensures the release of neurotransmitters and the replenishment of SVs to sustain neuronal activity. Multiple endocytosis and sorting pathways contribute to the recapture of the SV membrane and proteins after fusion. Adaptor protein (AP) complexes are among the critical components of the SV retrieval machinery. The canonical clathrin adaptor AP2 ensures the replenishment of most SVs across many neuronal populations. An alternative AP1/AP3-dependent process mediates the formation of a subset of SVs that differ from AP2 vesicles in molecular composition and respond preferentially during higher frequency firing. Furthermore, recent studies show that vesicular transporters for different neurotransmitters depend to a different extent on the AP3 pathway and this affects the release properties of the respective neurotransmitters. This review focuses on the current understanding of the AP-dependent molecular and functional diversity among SVs. We also discuss the contribution of these pathways to the regulation of neurotransmitter release across neuronal populations.

Endocytosis blocks the vesicular secretion of exosome marker proteins.

Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.

Lipid nanodiscs as a template for high-resolution cryo-EM structures of peripheral membrane proteins

Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient binding to the membrane has a profound impact on protein function, serving to induce conformational changes and alter biochemical and biophysical parameters by increasing the local concentration of factors and restricting diffusion to two dimensions. Despite the centrality of the membrane in serving as a template for cell biology, there are few reported high-resolution structures of peripheral membrane proteins bound to the membrane. We analyzed the utility of lipid nanodiscs to serve as a template for cryo-EM analysis of peripheral membrane proteins. We tested a variety of nanodiscs and we report a 3.3 Å structure of the AP2 clathrin adaptor complex bound to a 17-nm nanodisc, with sufficient resolution to visualize a bound lipid head group. Our data demonstrate that lipid nanodiscs are amenable to high-resolution structure determination of peripheral membrane proteins and provide a framework for extending this analysis to other systems.

Immunofluorescence as a Method to Study Golgi Organization in Larval Salivary Glands of Drosophila melanogaster.

Immunofluorescence is an important research tool in cell biology that reveals structural organization of subcellular organelles by detecting their associated constituents. Here, we describe an antibody staining method to detect Golgi-associated proteins in Drosophila larval salivary glands, using the cis-Golgi protein Lava lamp and the clathrin adaptor AP-1 as a suitable example. Golgi bodies immunostained using this protocol can be visualized using confocal or structured illumination microscopy.

Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization.

Clathrin-mediated endocytosis (CME) is the main route of internalization from the plasma membrane. It is known that the heterotetrameric AP2 clathrin adaptor must open to simultaneously engage membrane and endocytic cargo, yet it is unclear how transmembrane cargos are captured to catalyze CME. Using cryogenic-electron microscopy, we discover a new way in which mouse AP2 can reorganize to expose membrane- and cargo-binding pockets, which is not observed in clathrin-coated structures. Instead, it is stimulated by endocytic pioneer proteins called muniscins, which do not enter vesicles. Muniscin-engaged AP2 is primed to rearrange into the vesicle-competent conformation on binding the tyrosine cargo internalization motif (YxxΦ). We propose adaptor priming as a checkpoint to ensure cargo internalization.

Retrograde transport of CDMPR depends on several machineries as analyzed by sulfatable nanobodies.

Retrograde protein transport from the cell surface and endosomes to the TGN is essential for membrane homeostasis in general and for the recycling of mannose-6-phosphate receptors (MPRs) for sorting of lysosomal hydrolases in particular. We used a nanobody-based sulfation tool to more directly determine transport kinetics from the plasma membrane to the TGN for the cation-dependent MPR (CDMPR) with and without rapid or gradual inactivation of candidate machinery proteins. Although knockdown of retromer (Vps26), epsinR, or Rab9a reduced CDMPR arrival to the TGN, no effect was observed upon silencing of TIP47. Strikingly, when retrograde transport was analyzed by rapamycin-induced rapid depletion (knocksideways) or long-term depletion by knockdown of the clathrin adaptor AP-1 or of the GGA machinery, distinct phenotypes in sulfation kinetics were observed, suggesting a potential role of GGA adaptors in retrograde and anterograde transport. Our study illustrates the usefulness of derivatized, sulfation-competent nanobodies, reveals novel insights into CDMPR trafficking biology, and further outlines that the selection of machinery inactivation is critical for phenotype analysis.

Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses.

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/adaptor protein complex 2 (AP-2)-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in mouse hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a revised model for the endocytosis of SV membranes at mammalian central synapses.

Reconstituting and Purifying Assembly Intermediates of Clathrin Adaptors AP1 and AP2.

Clathrin-coated vesicles mediate membrane cargo transportation from the plasma membrane, the trans-Golgi network, the endosome, and the lysosome. Heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) are bridges that link cargo-loaded membranes to clathrin coats. Assembly of AP2 was previously considered to be spontaneous; however, a recent study found AP2 assembly is a highly orchestrated process controlled by alpha and gamma adaptin binding protein (AAGAB). Evidence shows that AAGAB controls AP1 assembly in a similar way. Insights into the orchestrated assembly process and three-dimensional structures of assembly intermediates are only emerging. Here, we describe a protocol for reconstitution and purification of the complexes containing AAGAB and AP1 or AP2 subunits, known as AP1 and AP2 hemicomplexes. Our purification routinely yields milligrams of pure complexes suitable for structural analysis by X-ray crystallography and electron microscopy.

Clathrin adaptors mediate two sequential pathways of intra-Golgi recycling.

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5-dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5-dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.

AAGAB is an assembly chaperone regulating AP1 and AP2 clathrin adaptors.

Multimeric cargo adaptors such as AP2 play central roles in intracellular membrane trafficking. We recently discovered that the assembly of the AP2 adaptor complex, a key player in clathrin-mediated endocytosis, is a highly organized process controlled by alpha- and gamma-adaptin-binding protein (AAGAB, also known as p34). In this study, we demonstrate that besides AP2, AAGAB also regulates the assembly of AP1, a cargo adaptor involved in clathrin-mediated transport between the trans-Golgi network and the endosome. However, AAGAB is not involved in the formation of other adaptor complexes, including AP3. AAGAB promotes AP1 assembly by binding and stabilizing the γ and σ subunits of AP1, and its mutation abolishes AP1 assembly and disrupts AP1-mediated cargo trafficking. Comparative proteomic analyses indicate that AAGAB mutation massively alters surface protein homeostasis, and its loss-of-function phenotypes reflect the synergistic effects of AP1 and AP2 deficiency. Taken together, these findings establish AAGAB as an assembly chaperone for both AP1 and AP2 adaptors and pave the way for understanding the pathogenesis of AAGAB-linked diseases.

NEW GENES AND DISEASES: EP.301 The alpha2-subunit of the AP2 clathrin adaptor as the causal gene in an atypical myopathy with granulofilamentous inclusions

Clathrin-mediated endocytosis is the best characterized process for the entry of proteins and lipids at the plasma membrane of eukaryotic cells. We have previously shown that at costameres, muscle adhesion and force transmission sites, clathrin recruited by the AP2 adaptor forms flat plaques required for actin and desmin intermediate filament organization. Here, we describe a patient presenting a progressive muscular atrophy with loss of ambulation, complete tetraplegia, ophthalmoplegia, severe scoliosis and lower limb contractures and no cardiac involvement. Electron microscopy of muscle biopsy revealed inclusions composed of filamentous material, vacuoles, honeycomb structures and thin granular material surcharge. This case was classified as a myofibrillar myopathy with granulofilamentous inclusion bodies despite no mutation in DES, FHL1, CRYAB and other usual suspect genes. Whole exome sequencing identified two compound heterozygote single-nucleotide variants in the AP2A2 gene encoding the AP2 a2-subunit. Immunofluorescent labeling on muscle sections displayed intracellular aggregates positive for AP2, clathrin and desmin. Depletion of total AP2 in mouse muscle induced a dystrophic phenotype with severe force loss, subsarcolemmal disorganization, vacuoles and neuromuscular junction defects while specifically depleting AP2 a2-subunit induced small subsarcolemmal disorganization and light neuromuscular junction defects. Lastly, transferrin uptake assays of endocytosis function in the patient fibroblasts showed a decrease in endocytosis rates compared to fibroblasts from his healthy parents. Altogether, our results suggest that AP2A2 could be the causal gene in this family and AP2A2 mutations leading to a severe but atypical myopathy. Mutations in AP2A2 could represent the first case in a wider family of atypical myopathies with desmin inclusions caused by mutations in genes encoding proteins of the endocytosis machinery.

Phosphorylation of the AMPA receptor subunit GluA1 regulates clathrin-mediated receptor internalization.

Synaptic strength is altered during synaptic plasticity by controlling the number of AMPA receptors (AMPARs) at excitatory synapses. During long-term potentiation and synaptic upscaling, AMPARs are accumulated at synapses to increase synaptic strength. Neuronal activity leads to phosphorylation of AMPAR subunit GluA1 (also known as GRIA1) and subsequent elevation of GluA1 surface expression, either by an increase in receptor forward trafficking to the synaptic membrane or a decrease in receptor internalization. However, the molecular pathways underlying GluA1 phosphorylation-induced elevation of surface AMPAR expression are not completely understood. Here, we employ fluorescence recovery after photobleaching (FRAP) to reveal that phosphorylation of GluA1 serine 845 (S845) predominantly plays a role in receptor internalization, rather than forward trafficking, during synaptic plasticity. Notably, internalization of AMPARs depends upon the clathrin adaptor AP2, which recruits cargo proteins into endocytic clathrin-coated pits. In fact, we further reveal that an increase in GluA1 S845 phosphorylation upon two distinct forms of synaptic plasticity diminishes the binding of the AP2 adaptor, reducing internalization and resulting in elevation of GluA1 surface expression. We thus demonstrate a mechanism of GluA1 phosphorylation-regulated clathrin-mediated internalization of AMPARs.

Mechanism of p38 MAPK-induced EGFR endocytosis and its crosstalk with ligand-induced pathways.

Ligand binding triggers clathrin-mediated and, at high ligand concentrations, clathrin-independent endocytosis of EGFR. Clathrin-mediated endocytosis (CME) of EGFR is also induced by stimuli activating p38 MAPK. Mechanisms of both ligand- and p38-induced endocytosis are not fully understood, and how these pathways intermingle when concurrently activated remains unknown. Here we dissect the mechanisms of p38-induced endocytosis using a pH-sensitive model of endogenous EGFR, which is extracellularly tagged with a fluorogen-activating protein, and propose a unifying model of the crosstalk between multiple EGFR endocytosis pathways. We found that a new locus of p38-dependent phosphorylation in EGFR is essential for the receptor dileucine motif interaction with the σ2 subunit of clathrin adaptor AP2 and concomitant receptor internalization. p38-dependent endocytosis of EGFR induced by cytokines was additive to CME induced by picomolar EGF concentrations but constrained to internalizing ligand-free EGFRs due to Grb2 recruitment by ligand-activated EGFRs. Nanomolar EGF concentrations rerouted EGFR from CME to clathrin-independent endocytosis, primarily by diminishing p38-dependent endocytosis.

Spatially resolved cell polarity proteomics of a human epiblast model.

Critical early steps in human embryonic development include polarization of the inner cell mass, followed by formation of an expanded lumen that will become the epiblast cavity. Recently described three-dimensional (3D) human pluripotent stem cell-derived cyst (hPSC-cyst) structures can replicate these processes. To gain mechanistic insights into the poorly understood machinery involved in epiblast cavity formation, we interrogated the proteomes of apical and basolateral membrane territories in 3D human hPSC-cysts. APEX2-based proximity bioinylation, followed by quantitative mass spectrometry, revealed a variety of proteins without previous annotation to specific membrane subdomains. Functional experiments validated the requirement for several apically enriched proteins in cyst morphogenesis. In particular, we found a key role for the AP-1 clathrin adaptor complex in expanding the apical membrane domains during lumen establishment. These findings highlight the robust power of this proximity labeling approach for discovering novel regulators of epithelial morphogenesis in 3D stem cell-based models.

The Effects of Apelin and Elabela Ligands on Apelin Receptor Distinct Signaling Profiles.

Apelin and Elabela are endogenous peptide ligands for Apelin receptor (APJ), a widely expressed G protein-coupled receptor. They constitute a spatiotemporal dual ligand system to control APJ signal transduction and function. We investigated the effects of Apelin-13, pGlu1-apelin-13, Apelin-17, Apelin-36, Elabela-21 and Elabela-32 peptides on APJ signal transduction. Whether different ligands are biased to different APJ mediated signal transduction pathways was studied. We observed the different changes of G protein dependent and β-arrestin dependent signaling pathways after APJ was activated by six peptide ligands. We demonstrated that stimulation with APJ ligands resulted in dose-dependent increases in both G protein dependent [cyclic AMP (cAMP), Ca2+ mobilization, and the early phase extracellular related kinase (ERK) activation] and β-arrestin dependent [GRKs, β-arrestin 1, β-arrestin 2, and β2 subunit of the clathrin adaptor AP2] signaling pathways. However, the ligands exhibited distinct signaling profiles. Elabela-32 showed a >1000-fold bias to the β-statin-dependent signaling pathway. These data provide that Apelin-17 was biased toward β-arrestin dependent signaling. Eabela-21 and pGlu1-Apelin-13 exhibited very distinct activities on the G protein dependent pathway. The activity profiles of these ligands could be valuable for the development of drugs with high selectivity for specific APJ downstream signaling pathways.

Unconventional endocytosis and trafficking of transferrin receptor induced by iron.

The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH4Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.

Interferon Induction By HbS, SERINC5 Upregulation and Reduction of HIV-1 Nef and AP2 Contribute to HIV-1 Restriction in Sickle Cell Disease

Concerted action of numerous positively acting cellular factors is essential for Human immunodeficiency virus type 1 (HIV-1) replication but in turn is challenged by anti-viral restriction factors. Previously we showed that ex vivo one round HIV-1 replication and replication of fully competent T-tropic HIV-1(IIIB) is significantly reduced in peripheral blood mononuclear cells (PBMCs) obtained from patients with Sickle Cell Disease (SCD). Further, we identified and confirmed CDKN1A (p21) and CH25H as host restriction factors expressed in SCD PBMCs that may contribute to the HIV-1 inhibition, in addition to the previously reported SAMHD1 and IKBα. Since CH25H is an interferon stimulated gene (ISG), we analyzed IRFs and interferon expression in SCD PBMCs. Higher levels of IRF7 and IFNβ mRNA were observed in SCD PBMCs compared to controls. We probed further to ascertain if hemin or sickle Hb was responsible for interferon response. We found upregulation of IFNβ in THP-1 - derived macrophages treated with lysates of HbSS RBCs or purified HbS as compared to untreated or HbA treated controls. HbSS RBCs lysates and purified HbS inhibited HIV-1 gag mRNA expression in monocyte-derived macrophages infected with HIV-1(Ba-L). Recent clinical study showed increased levels of CD4 in HIV-1 infected SCD patients in Africa. Thus we analyzed CD4 levels in HIV-1 IIIB infected SCD PBMCs, and found them to be higher compared to controls. Levels of HIV-1 nef mRNA, that controls CD4 expression was lower in HIV-1 IIIB infected SCD PBMCs. As Nef counteracts SERINC3/5 restriction factor, we analyzed its expression as well as the expression of AP2 clathrin adaptor that is required for Nef mediated internalization of CD4. AP2 expression was lower and SERINC5 expression was higher in SCD PBMCs. CONCLUSIONS: SCD PBMCs could resist HIV-1 infection because of the increased IFNβ production by macrophages exposed to HbSS or sickle cell RBCs. SCD PBMC have increased levels of SERNIC5 and lower levels of HIV-1 Nef and host AP2 expression that, culumlatively, can increased CD4 levels and lead to the overall improved immunological health of SCD patients. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 1SC1HL150685, 5U54MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures No relevant conflicts of interest to declare.

Endosome-Localized Clathrin Adaptors AP-1 and GGA2 Support Cell Surface Expression of a Subset of RTKs for Cell Growth

The role of Golgi/endosome-localized clathrin adaptors in the maintenance of steady-state cell surface receptor tyrosine kinases (RTKs) is not well known. Here, we show that the epidermal growth factor receptor (EGFR), an RTK, associates preferentially with both AP-1 and GGA2 in vitro. AP-1 depletion reduces the expression of EGFR protein by promoting its lysosomal degradation. Proximity ligation assays demonstrated that the interaction of EGFR with AP-1 or GGA2 occurs in Rab11-positive recycling endosomes. Western blot analysis revealed that the depletion of AP-1 or GGA2 decreased surface expression of additional RTKs, such as Erb-B4 and MET. AP-1 depletion suppressed the growth of H1975 cancer cells and normal ARPE-19 cells. AP-1 was expressed in endosomes at high levels in several cancer tissues. Collectively, these results indicate that AP-1 and GGA2 function to retrieve a subset of RTKs in recycling endosomes, thereby sustaining their cell surface expression and, consequently, cancer cell growth.

Nef Dimerization is Required for Antagonism of the Host Cell Restriction Factor, SERINC5

HIV‐1 Nef is a virulence factor that is essential for immune escape of HIV‐infected cells, viral pathogenesis and AIDS progression. Nef lacks intrinsic biochemical activity, functioning instead by interacting with host cell effector proteins to hijack multiple signaling and endocytic pathways. Many Nef functions depend on dimerization of the protein, including enhancement of viral infectivity, replication, and immune cell receptor downregulation. Work in our lab, through both X‐ray crystallography and virological assays, has shown that distinct hydrophobic residues (L112, Y115, F121) are key in Nef dimer formation, and disruption of these residues significantly impairs many Nef functions. Pharmacologic disruption of the Nef homodimer may therefore represent a novel approach to antiretroviral therapy.Here we investigated whether Nef dimerization is required for antagonism of the host cell restriction factor SERINC5 (S5). S5 is a potent suppressor of HIV‐1 infectivity that is antagonized by Nef via binding and downregulating S5 from the surface of infected cells, preventing its incorporation into nascent virions. For our studies, HIV‐1 virions were produced in 293T cells transfected with the HIV‐1 provirus and S5, Jurkat WT and S5‐knockout cells, or donor PBMCs (which naturally express high levels of S5). Viral supernatants were harvested from these “producer” cells and normalized for p24 AlphaLISA. Infectivity of producer‐cell viruses was assessed using TZM‐bl reporter cells, which couple the HIV‐1 LTR to expression of luciferase. Mutation of conserved Nef residues essential for homodimerization (L112A/D, Y115D, F121A) reduced the infectivity of HIV‐1 produced from all three producer cell lines in a S5‐dependent manner. S5 incorporation into virions was increased in the presence of Nef dimer interface mutants, suggesting that dimerization is important in its ability to exclude S5 from newly synthesized virions. Recruitment, downregulation, and endocytic trafficking of S5 by wild type and dimerization‐defective Nef mutants were also examined via confocal microscopy. Nef dimer interface mutants also failed to induce colocalization of S5 with Rab7+ late endosomes, suggesting that Nef dimerization is essential for S5 internalization and degradation via the lyso‐endosomal pathway. By contrast, Nef mutants retained their ability to interact with both S5 and subunits of the AP‐2 clathrin adaptor protein essential for S5 endocytosis, as shown via bimolecular fluorescence complementation. Taken together, these results demonstrate that downregulation of S5 and subsequent enhancement of viral infectivity requires Nef homodimerization, providing further support for pharmacological targeting of Nef quaternary structure in HIV‐1 cure strategies.

The Clathrin adaptor AP-1 and Stratum act in parallel pathways to control Notch activation in Drosophila sensory organ precursors cells.

ABSTRACTDrosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells, the identity of which relies on activation of Notch at cytokinesis. Although Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, the basal pool of Notch is reported to be the main contributor for Notch activation in the pIIa cell. Intra-lineage signalling requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. We have previously reported that AP-1 and Stratum regulate the trafficking of Notch and Sanpodo from the trans-Golgi network to the basolateral membrane. Loss of AP-1 or Stratum caused mild Notch gain-of-function phenotypes. Here, we report that their concomitant loss results in a penetrant Notch gain-of-function phenotype, indicating that they control parallel pathways. Although unequal partitioning of cell fate determinants and cell polarity were unaffected, we observed increased amounts of signalling-competent Notch as well as Delta and Sanpodo at the apical pIIa-pIIb interface, at the expense of the basal pool of Notch. We propose that AP-1 and Stratum operate in parallel pathways to localize Notch and control where receptor activation takes place.