Phosphatidic acid increases Notch signalling by affecting Sanpodo trafficking during Drosophila sensory organ development.

Organ cell diversity depends on binary cell-fate decisions mediated by the Notch signalling pathway during development and tissue homeostasis. A clear example is the series of binary cell-fate decisions that take place during asymmetric cell divisions that give rise to the sensory organs of Drosophila melanogaster. The regulated trafficking of Sanpodo, a transmembrane protein that potentiates receptor activity, plays a pivotal role in this process. Membrane lipids can regulate many signalling pathways by affecting receptor and ligand trafficking. It remains unknown, however, whether phosphatidic acid regulates Notch-mediated binary cell-fate decisions during asymmetric cell divisions, and what are the cellular mechanisms involved. Here we show that increased phosphatidic acid derived from Phospholipase D leads to defects in binary cell-fate decisions that are compatible with ectopic Notch activation in precursor cells, where it is normally inactive. Null mutants of numb or the α-subunit of Adaptor Protein complex-2 enhance dominantly this phenotype while removing a copy of Notch or sanpodo suppresses it. In vivo analyses show that Sanpodo localization decreases at acidic compartments, associated with increased internalization of Notch. We propose that Phospholipase D-derived phosphatidic acid promotes ectopic Notch signalling by increasing receptor endocytosis and inhibiting Sanpodo trafficking towards acidic endosomes.

Dynamin Regulates Autophagy by Modulating Lysosomal Function.

Autophagy is a central lysosomal degradation pathway required for maintaining cellular homeostasis and its dysfunction is associated with numerous human diseases. To identify players in autophagy, we tested ∼1200 chemically induced mutations on the X chromosome in Drosophila fat body clones and discovered that shibire (shi) plays an essential role in starvation-induced autophagy. shi encodes a dynamin protein required for fission of clathrin-coated vesicles from the plasma membrane during endocytosis. We showed that Shi is dispensable for autophagy initiation and autophagosome-lysosome fusion, but required for lysosomal/autolysosomal acidification. We also showed that other endocytic core machinery components like clathrin and AP2 play similar but not identical roles in regulating autophagy and lysosomal function as dynamin. Previous studies suggested that dynamin directly regulates autophagosome formation and autophagic lysosome reformation (ALR) through its excision activity. Here, we provide evidence that dynamin also regulates autophagy indirectly by regulating lysosomal function.

New Insights to Clathrin and Adaptor Protein 2 for the Design and Development of Therapeutic Strategies.

The Amyloid Precursor Protein (APP) has been extensively studied for its role as the precursor of the ß-amyloid protein (Aß) in Alzheimer's disease (AD). However, our understanding of the normal function of APP is still patchy. Emerging evidence indicates that a dysfunction in APP trafficking and degradation can be responsible for neuronal deficits and progressive degeneration in humans. We recently reported that the Y682 mutation in the 682YENPTY687 domain of APP affects its binding to specific adaptor proteins and leads to its anomalous trafficking, to defects in the autophagy machinery and to neuronal degeneration. In order to identify adaptors that influence APP function, we performed pull-down experiments followed by quantitative mass spectrometry (MS) on hippocampal tissue extracts of three month-old mice incubated with either the 682YENPTY687 peptide, its mutated form, 682GENPTY687 or its phosphorylated form, 682pYENPTY687. Our experiments resulted in the identification of two proteins involved in APP internalization and trafficking: Clathrin heavy chain (hc) and its Adaptor Protein 2 (AP-2). Overall our results consolidate and refine the importance of Y682 in APP normal functions from an animal model of premature aging and dementia. Additionally, they open the perspective to consider Clathrin hc and AP-2 as potential targets for the design and development of new therapeutic strategies.

Clathrin/AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses.

Neurotransmission depends on presynaptic membrane retrieval and local reformation of synaptic vesicles (SVs) at nerve terminals. The mechanisms involved in these processes are highly controversial with evidence being presented for SV membranes being retrieved exclusively via clathrin-mediated endocytosis (CME) from the plasma membrane or via ultrafast endocytosis independent of clathrin. Here we show that clathrin and its major adaptor protein 2 (AP-2) in addition to the plasma membrane operate at internal endosome-like vacuoles to regenerate SVs but are not essential for membrane retrieval. Depletion of clathrin or conditional knockout of AP-2 result in defects in SV reformation and an accumulation of endosome-like vacuoles generated by clathrin-independent endocytosis (CIE) via dynamin 1/3 and endophilin. These results together with theoretical modeling provide a conceptual framework for how synapses capitalize on clathrin-independent membrane retrieval and clathrin/AP-2-mediated SV reformation from endosome-like vacuoles to maintain excitability over a broad range of stimulation frequencies.

TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy.

The RabGAP protein TBC1D5 controls cellular endomembrane trafficking processes and binds the retromer subunit VPS29 and the ubiquitin-like protein ATG8 (LC3). Here, we describe that TBC1D5 also associates with ATG9 and the active ULK1 complex during autophagy. Moreover, ATG9 and TBC1D5 interact with clathrin and the AP2 complex. Depletion of TBC1D5 leads to missorting of ATG9 to late endosomes upon activation of autophagy, whereas inhibition of clathrin-mediated endocytosis or AP2 depletion alters ATG9 trafficking and its association with TBC1D5. Taken together, our data show that TBC1D5 and the AP2 complex are important novel regulators of the rerouting of ATG9-containing vesicular carriers toward sites of autophagosome formation.

Adaptor protein2 (AP2) orchestrates CXCR2-mediated cell migration.

The chemokine receptor CXCR2 is vital for inflammation, wound healing, angiogenesis, cancer progression and metastasis. Adaptor protein 2 (AP2), a clathrin binding heterotetrameric protein comprised of α, ß2, µ2 and σ2 subunits, facilitates clathrin-mediated endocytosis. Mutation of the LLKIL motif in the CXCR2 carboxyl-terminal domain (CTD) results in loss of AP2 binding to the receptor and loss of ligand-mediated receptor internalization and chemotaxis. AP2 knockdown also results in diminished ligand-mediated CXCR2 internalization, polarization and chemotaxis. Using knockdown/rescue approaches with AP2-µ2 mutants, the binding domains were characterized in reference to CXCR2 internalization and chemotaxis. When in an open conformation, µ2 Patch 1 and Patch 2 domains bind tightly to membrane PIP2 phospholipids. When AP2-µ2, is replaced with µ2 mutated in Patch 1 and/or Patch 2 domains, ligand-mediated receptor binding and internalization are not lost. However, chemotaxis requires AP2-µ2 Patch 1, but not Patch 2. AP2-σ2 has been demonstrated to bind dileucine motifs to facilitate internalization. Expression of AP2-σ2 V88D and V98S dominant negative mutants resulted in loss of CXCR2 mediated chemotaxis. Thus, AP2 binding to both membrane phosphatidylinositol phospholipids and dileucine motifs is crucial for directional migration or chemotaxis. Moreover, AP2-mediated receptor internalization can be dissociated from AP2-mediated chemotaxis.

The endocytosis of cellulose synthase in Arabidopsis is dependent on µ2, a clathrin-mediated endocytosis adaptin.

Clathrin-mediated endocytosis (CME) is the best-characterized type of endocytosis in eukaryotic cells. Plants appear to possess all of the molecular components necessary to carry out CME; however, functional characterization of the components is still in its infancy. A yeast two-hybrid screen identified µ2 as a putative interaction partner of CELLULOSE SYNTHASE6 (CESA6). Arabidopsis (Arabidopsis thaliana) µ2 is homologous to the medium subunit 2 of the mammalian ADAPTOR PROTEIN COMPLEX2 (AP2). In mammals, the AP2 complex acts as the central hub of CME by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis. We confirmed that µ2 interacts with multiple CESA proteins through the µ-homology domain of µ2, which is involved in specific interactions with endocytic cargo proteins in mammals. Consistent with its role in mediating the endocytosis of cargos at the plasma membrane, µ2-YELLOW FLUORESCENT PROTEIN localized to transient foci at the plasma membrane, and loss of µ2 resulted in defects in bulk endocytosis. Furthermore, loss of µ2 led to increased accumulation of YELLOW FLUORESCENT PROTEIN-CESA6 particles at the plasma membrane. Our results suggest that CESA represents a new class of CME cargo proteins and that plant cells might regulate cellulose synthesis by controlling the abundance of active CESA complexes at the plasma membrane through CME.

Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint.

Numerous endocytic accessory proteins (EAPs) mediate assembly and maturation of clathrin-coated pits (CCPs) into cargo-containing vesicles. Analysis of EAP function through bulk measurement of cargo uptake has been hampered due to potential redundancy among EAPs and, as we show here, the plasticity and resilience of clathrin-mediated endocytosis (CME). Instead, EAP function is best studied by uncovering the correlation between variations in EAP association to individual CCPs and the resulting variations in maturation. However, most EAPs bind to CCPs in low numbers, making the measurement of EAP association via fused fluorescent reporters highly susceptible to detection errors. Here, we present a framework for unbiased measurement of EAP recruitment to CCPs and their direct effects on CCP dynamics. We identify dynamin and the EAP-binding α-adaptin appendage domain of the AP2 adaptor as switches in a regulated, multistep maturation process and provide direct evidence for a molecular checkpoint in CME.

Otoferlin couples to clathrin-mediated endocytosis in mature cochlear inner hair cells.

The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.

Clathrin and AP2 are required for PtdIns(4,5)P2-mediated formation of LRP6 signalosomes.

Canonical Wnt signaling is initiated by the binding of Wnt proteins to their receptors, low-density lipoprotein-related protein 5 and 6 (LRP5/6) and frizzled proteins, leading to phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P(2)) production, signalosome formation, and LRP phosphorylation. However, the mechanism by which PtdIns(4,5)P(2) regulates the signalosome formation remains unclear. Here we show that clathrin and adaptor protein 2 (AP2) were part of the LRP6 signalosomes. The presence of clathrin and AP2 in the LRP6 signalosomes depended on PtdIns(4,5)P(2), and both clathrin and AP2 were required for the formation of LRP6 signalosomes. In addition, WNT3A-induced LRP6 signalosomes were primarily localized at cell surfaces, and WNT3A did not induce marked LRP6 internalization. However, rapid PtdIns(4,5)P(2) hydrolysis induced artificially after WNT3A stimulation could lead to marked LRP6 internalization. Moreover, we observed WNT3A-induced LRP6 and clathrin clustering at cell surfaces using super-resolution fluorescence microscopy. Therefore, we conclude that PtdIns(4,5)P(2) promotes the assembly of LRP6 signalosomes via the recruitment of AP2 and clathrin and that LRP6 internalization may not be a prerequisite for Wnt signaling to ß-catenin stabilization.

Adaptor protein complex 2-mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis.

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real-time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP-2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin-dependent endocytosis, thus implying the likelihood of specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also upregulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H(+) transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H(+) transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage-sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor-regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation.

Fishing for clathrin-coated pit nucleators.

The membrane-curvature-inducing protein Fcho was proposed to be part of a ubiquitous nucleation mechanism for clathrin-coated pits. However, studies in developing zebrafish embryos now indicate a role for Fcho as a receptor-specific adaptor in bone morphogenetic protein (BMP) signalling, rather than a global coated-pit nucleator.

Distinct and separable activities of the endocytic clathrin-coat components Fcho1/2 and AP-2 in developmental patterning.

Clathrin-mediated endocytosis occurs at multiple independent import sites on the plasma membrane, but how these positions are selected and how different cargo is simultaneously recognized is obscure. FCHO1 and FCHO2 are early-arriving proteins at surface clathrin assemblies and are speculated to act as compulsory coat nucleators, preceding the core clathrin adaptor AP-2. Here, we show that the µ-homology domain of FCHO1/2 represents an endocytic interaction hub. Translational silencing of fcho1 in zebrafish embryos causes strong dorsoventral patterning defects analogous to Bmp signal failure. The Fcho1 µ-homology domain interacts with the Bmp receptor Alk8, uncovering an endocytic component that positively modulates Bmp signal transmission. Still, the fcho1 morphant phenotype is distinct from severe embryonic defects apparent when AP-2 is depleted. Our data thus challenge the primacy of FCHO1/2 in coat initiation.

AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function.

UNLABELLED: The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of α- and µ2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3×FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. CONCLUSION: AP2 mediates the internalization and subsequent degradation of CM-resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis.

Eps15 and Epsin1 are crucial for enteropathogenic Escherichia coli pedestal formation despite the absence of adaptor protein 2.

Enteropathogenic Escherichia coli (EPEC) are primarily extracellular pathogens that generate actin-rich structures known as pedestals during their pathogenesis. Surprising evidence has demonstrated that despite maintaining an extracellular location, EPEC require the endocytic protein, clathrin, for pedestal formation. To evaluate the strategies EPEC use to usurp endocytic machinery, we investigated the roles of a number of clathrin-coated pits components, adaptor protein 2 (AP-2), Eps15 and epsin1, during EPEC infections. We demonstrated that in conjunction with clathrin, pedestal formation also required the recruitment of Eps15 and epsin1 but not AP-2. Because AP-2 orchestrates the recruitment of clathrin, Eps15, and epsin1, as well as other adaptors, during assembly of clathrin-coated pits at the plasma membrane, our findings reveal a novel internalization subversion strategy employed by EPEC. These results further emphasize the recent paradigm that endocytic proteins are important for EPEC-mediated disease.

Molecular mechanism and physiological functions of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is the endocytic portal into cells through which cargo is packaged into vesicles with the aid of a clathrin coat. It is fundamental to neurotransmission, signal transduction and the regulation of many plasma membrane activities and is thus essential to higher eukaryotic life. Morphological stages of vesicle formation are mirrored by progression through various protein modules (complexes). The process involves the formation of a putative FCH domain only (FCHO) initiation complex, which matures through adaptor protein 2 (AP2)-dependent cargo selection, and subsequent coat building, dynamin-mediated scission and finally auxilin- and heat shock cognate 70 (HSC70)-dependent uncoating. Some modules can be used in other pathways, and additions or substitutions confer cell specificity and adaptability.

Viral factors involved in adapter-related protein complex 2 alpha 1 subunit-mediated regulation of human immunodeficiency virus type 1 replication.

The presence of siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2alpha) enhances human immunodeficiency virus type 1 (HIV-1) replication by up-regulating nuclear transport of viral genome. In this report, we examined possible viral factors involved in AP2alpha-mediated regulation of HIV-1 replication, namely, Gag matrix protein (MA), integrase (IN) and Vpr. Replication of mutant viruses lacking the nucleophilic property of one of these viral proteins was significantly enhanced by treating cells with AP2alpha siRNA, indicating that Gag MA, IN or Vpr is not specifically involved in AP2alpha-mediated enhancement of viral replication. In contrast, AP2alpha siRNA showed no effect on the level of gene transduction mediated by HIV-1-derived lentiviral vector (LV). Although virus-like LV particle and parental HIV-1 particle are composed of almost equivalent viral structural proteins, LV particles lack three accessory proteins, Vif, Vpr and Vpu, and a large portion of the HIV-1 genome. Vif, Vpr and Vpu were dispensable for AP2alpha siRNA-mediated enhancement of HIV-1 replication, indicating that a particular part of the HIV-1 genomic fragment deleted in the LV genome might be required for the enhancing effect of AP2alpha siRNA on viral replication. Taken together, these results suggest that an as yet undetermined gene fragment of the HIV-1 genome is involved in AP2alpha-mediated regulation of HIV-1 replication.

Endocytic internalization routes required for delta/notch signaling.

The internalization of transmembrane receptors from the cell surface plays a central role in signal regulation. Receptor internalization can occur through different routes; however, because of the difficulty in selectively blocking these routes in vivo, their roles in signaling are poorly understood. Here we use null mutations in Drosophila dynamin, clathrin, and AP-2 adaptor subunits to analyze internalization requirements for the Delta ligand and its receptor, Notch. Bulk Notch is internalized via AP-2-dependent endocytosis, but signaling by Notch requires AP-2-independent clathrin-dependent endocytosis, highlighting a distinction between Notch endocytic routes required for degradation versus signaling activation. Signaling by Delta requires dynamin, but whether this generates a pulling force of Delta on Notch or allows for Delta entry into a recycling pathway to gain signaling competence is widely debated. Surprisingly, we show that signaling by Delta in germline cells can occur by clathrin-independent endocytosis, when endosomal entry is blocked, and when activity of Rab11 or its effectors is reduced, suggesting that Delta need not pass through a recognized recycling pathway to achieve signaling competence. The absolute requirement for dynamin-dependent endocytosis but not endosomal entry or Rab11 activity supports "pulling force" rather than "recycling" models for Delta activation.