Apical secretion of Wnt1 in polarized epithelial cells is regulated by exocyst-mediated trafficking.

Wnts are glycosylated proteins secreted from various cell types including mesenchymal, hematopoietic and epithelial cells. Directional secretion of Wnts in polarized epithelial cells is unique; Wnt11 is secreted apically, whereas Wnt5a and Wnt3a are secreted basolaterally. Here, we found that Wnt1 is equivalently secreted both apically and basolaterally in MDCK cells. Wnt1 was modified with a complex- or hybrid-type glycan at Asn29 and Asn359 and the high-mannose- or hybrid-type glycan at Asn316. Although glycosylation of Wnt11 at the N-terminal site was shown to be essential for its apical secretion, glycosylation of Asn29 of Wnt1 was not required. Instead, the apical secretion of Wnt1 was inhibited by knockdown of Sec6 and Sec8, suggesting that Wnt1 is secreted apically via exocyst-mediated transport. Basolateral secretion of Wnt1 was mediated by clathrin and AP-1, in mechanism similar to that used by Wnt5a and Wnt3a. Although Wingless was reported to be transcytosed to the basolateral region in the Drosophila wing disc, transcytosis was not involved in the basolateral secretion of Wnt1. Thus, the polarized secretion of Wnt1 is regulated by different mechanisms than other Wnts.

Alternative Splicing in Ca(V)2.2 Regulates Neuronal Trafficking via Adaptor Protein Complex-1 Adaptor Protein Motifs.

N-type voltage-gated calcium (Ca(V)2.2) channels are expressed in neurons and targeted to the plasma membrane of presynaptic terminals, facilitating neurotransmitter release. Here, we find that the adaptor protein complex-1 (AP-1) mediates trafficking of Ca(V)2.2 from the trans-Golgi network to the cell surface. Examination of splice variants of Ca(V)2.2, containing either exon 37a (selectively expressed in nociceptors) or 37b in the proximal C terminus, reveal that canonical AP-1 binding motifs, YxxΦ and [DE]xxxL[LI], present only in exon 37a, enhance intracellular trafficking of exon 37a-containing Ca(V)2.2 to the axons and plasma membrane of rat DRG neurons. Finally, we identify differential effects of dopamine-2 receptor (D2R) and its agonist-induced activation on trafficking of Ca(V)2.2 isoforms. D2R slowed the endocytosis of Ca(V)2.2 containing exon 37b, but not exon 37a, and activation by the agonist quinpirole reversed the effect of the D2R. Our work thus reveals key mechanisms involved in the trafficking of N-type calcium channels.

Analyzing the role of AP-1B in polarized sorting from recycling endosomes in epithelial cells.

Epithelial cells polarize their plasma membrane into apical and basolateral domains where the apical membrane faces the luminal side of an organ and the basolateral membrane is in contact with neighboring cells and the basement membrane. To maintain this polarity, newly synthesized and internalized cargos must be sorted to their correct target domain. Over the last ten years, recycling endosomes have emerged as an important sorting station at which proteins destined for the apical membrane are segregated from those destined for the basolateral membrane. Essential for basolateral sorting from recycling endosomes is the tissue-specific adaptor complex AP-1B. This chapter describes experimental protocols to analyze the AP-1B function in epithelial cells including the analysis of protein sorting in LLC-PK1 cells lines, immunoprecipitation of cargo proteins after chemical crosslinking to AP-1B, and radioactive pulse-chase experiments in MDCK cells depleted of the AP-1B subunit µ1B.

A role for adaptor protein complex 1 in protein targeting to rhoptry organelles in Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum possesses sophisticated systems of protein secretion to modulate host cell invasion and remodeling. In the present study, we provide insights into the function of the AP-1 complex in P. falciparum. We utilized GFP fusion constructs for live cell imaging, as well as fixed parasites in immunofluorescence analysis, to study adaptor protein mu1 (Pfµ1) mediated protein trafficking in P. falciparum. In trophozoites Pfµ1 showed similar dynamic localization to that of several Golgi/ER markers, indicating Golgi/ER localization. Treatment of transgenic parasites with Brefeldin A altered the localization of Golgi-associated Pfµ1, supporting the localization studies. Co-localization studies showed considerable overlap of Pfµ1 with the resident rhoptry proteins, rhoptry associated protein 1 (RAP1) and Cytoadherence linked asexual gene 3.1 (Clag3.1) in schizont stage. Immunoprecipitation experiments with Pfµ1 and PfRAP1 revealed an interaction, which may be mediated through an intermediate transmembrane cargo receptor. A specific role for Pfµ1 in trafficking was suggested by treatment with AlF4, which resulted in a shift to a predominantly ER-associated compartment and consequent decrease in co-localization with the Golgi marker GRASP. Together, these results suggest a role for the AP-1 complex in rhoptry protein trafficking in P. falciparum.

Basolateral sorting of the Mg²âº transporter CNNM4 requires interaction with AP-1A and AP-1B.

Ancient conserved domain protein/cyclin M (CNNM) 4 is an evolutionarily conserved Mg(2+) transporter that localizes at the basolateral membrane of the intestinal epithelia. Here, we show the complementary importance of clathrin adaptor protein (AP) complexes AP-1A and AP-1B in basolateral sorting of CNNM4. We first confirmed the basolateral localization of both endogenous and ectopically expressed CNNM4 in Madin-Darby Canine Kidney cells, which form highly polarized epithelia in culture. Single knockdown of µ1B, a cargo-recognition subunit of AP-1B, did not affect basolateral localization, but simultaneous knockdown of the µ1A subunit of AP-1A abrogated localization. Mutational analyses showed the importance of three conserved dileucine motifs in CNNM4 for both basolateral sorting and interaction with µ1A and µ1B. These results imply that CNNM4 is sorted to the basolateral membrane by the complementary function of AP-1A and AP-1B.

Adaptor protein complexes-1 and 3 are involved at distinct stages of flavivirus life-cycle.

Intracellular protein trafficking pathways are hijacked by viruses at various stages of viral life-cycle. Heterotetrameric adaptor protein complexes (APs) mediate vesicular trafficking at distinct intracellular sites and are essential for maintaining the organellar homeostasis. In the present study, we studied the effect of AP-1 and AP-3 deficiency on flavivirus infection in cells functionally lacking these proteins. We show that AP-1 and AP-3 participate in flavivirus life-cycle at distinct stages. AP-3-deficient cells showed delay in initiation of Japanese encephalitis virus and dengue virus RNA replication, which resulted in reduction of infectious virus production. AP-3 was found to colocalize with RNA replication compartments in infected wild-type cells. AP-1 deficiency affected later stages of dengue virus infection where increased intracellular accumulation of infectious virus was observed. Therefore, our results propose a novel role for AP-1 and AP-3 at distinct stages of infection of some of the RNA viruses.

AP-1B-mediated protein sorting regulates polarity and proliferation of intestinal epithelial cells in mice.

BACKGROUND & AIMS: In epithelial cells, protein sorting mechanisms regulate localization of plasma membrane proteins that generate and maintain cell polarity. The clathrin-adaptor protein (AP) complex AP-1B is expressed specifically in polarized epithelial cells, where it regulates basolateral sorting of membrane proteins. However, little is known about its physiological significance. METHODS: We analyzed the intestinal epithelia of mice deficient in Ap1m2 (Ap1m2(-/-) mice), which encodes the AP-1B µ1B subunit, and compared it with 129/B6/CD1 littermates (controls). Notch signaling was inhibited by intraperitoneal injection of dibenzazepine, and ß-catenin signaling was inhibited by injection of IWR1. Intestinal tissue samples were collected and analyzed by immunofluorescence analysis. RESULTS: Ap1m2(-/-) mice developed intestinal epithelial cell hyperplasia. The polarity of intestinal epithelial cells was disrupted, as indicated by the appearance of ectopic microvilli-like structures on the lateral plasma membrane and mislocalization of basolateral membrane proteins, including the low-density lipoprotein receptor and E-cadherin. The E-cadherin-ß-catenin complex therefore was disrupted at the adherens junction, resulting in nuclear translocation of ß-catenin. This resulted in up-regulation of genes regulated by ß-catenin/transcription factor 4 (Tcf4) complex, and increased the proliferation of intestinal epithelial cells. CONCLUSIONS: AP-1B is required for protein sorting and polarization of intestinal cells in mice. Loss of AP-1B in the intestinal epithelia results in mislocalization of E-cadherin, activation of ß-catenin/Tcf4 complex, proliferation, and hyperplasia.

AP-1 is required for the maintenance of apico-basal polarity in the C. elegans intestine.

Epithelial tubes perform functions that are essential for the survival of multicellular organisms. Understanding how their polarised features are maintained is therefore crucial. By analysing the function of the clathrin adaptor AP-1 in the C. elegans intestine, we found that AP-1 is required for epithelial polarity maintenance. Depletion of AP-1 subunits does not affect epithelial polarity establishment or the formation of the intestinal lumen. However, the loss of AP-1 affects the polarised distribution of both apical and basolateral transmembrane proteins. Moreover, it triggers de novo formation of ectopic apical lumens between intestinal cells along the lateral membranes later during embryogenesis. We also found that AP-1 is specifically required for the apical localisation of the small GTPase CDC-42 and the polarity determinant PAR-6. Our results demonstrate that AP-1 controls an apical trafficking pathway required for the maintenance of epithelial polarity in vivo in a tubular epithelium.

Clathrin and AP-1 regulate apical polarity and lumen formation during C. elegans tubulogenesis.

Clathrin coats vesicles in all eukaryotic cells and has a well-defined role in endocytosis, moving molecules away from the plasma membrane. Its function on routes towards the plasma membrane was only recently appreciated and is thought to be limited to basolateral transport. Here, an unbiased RNAi-based tubulogenesis screen identifies a role of clathrin (CHC-1) and its AP-1 adaptor in apical polarity during de novo lumenal membrane biogenesis in the C. elegans intestine. We show that CHC-1/AP-1-mediated polarized transport intersects with a sphingolipid-dependent apical sorting process. Depleting each presumed trafficking component mislocalizes the same set of apical membrane molecules basolaterally, including the polarity regulator PAR-6, and generates ectopic lateral lumens. GFP::CHC-1 and BODIPY-ceramide vesicles associate perinuclearly and assemble asymmetrically at polarized plasma membrane domains in a co-dependent and AP-1-dependent manner. Based on these findings, we propose a trafficking pathway for apical membrane polarity and lumen morphogenesis that implies: (1) a clathrin/AP-1 function on an apically directed transport route; and (2) the convergence of this route with a sphingolipid-dependent apical trafficking path.

AP1B plays an important role in intestinal tumorigenesis with the truncating mutation of an APC gene.

Recent evidence has suggested that carcinoma is accompanied by the loss of cell polarity. An epithelial cell-specific form of the AP-1 clathrin adaptor complex, AP1B, is involved in the polarized transport of membrane proteins to the basolateral surface of epithelial cells. In our study, we investigated whether AP1B is involved in intestinal tumorigenesis. The cellular polarity of intestinal tumor cells was examined using APC(Min/+) mice as an in vivo model and SW480 cells with a truncating mutation in the adenomatous polyposis coli (APC) gene as an in vitro model by confocal microscopy. Next, the expression of AP1B in intestinal tumor cells was examined by real-time polymerase chain reaction (PCR) and Western blotting. The localization of ß-catenin and the expression of AP1B in the tumor tissue of patients with colorectal cancer were evaluated by confocal microscopy and real-time PCR, respectively, and the relationships among cell polarity, AP1B expression and intestinal tumorigenesis were examined. Cellular polarity was lost in intestinal tumor cells, and the expression of AP1B was downregulated. In addition, the reduction in the expression level of AP1B correlated with the nuclear localization of ß-catenin in human colorectal cancer. Our study indicates the close associations between AP1B, intestinal tumorigenesis and mutations in the APC gene. This is the first report to reveal the relationships among AP1B, cellular polarity and intestinal tumorigenesis, and achieving a detailed understanding of AP1B will hopefully lead to discovery of therapeutic targets and novel biomarkers for intestinal cancer.

Characterization of Trex1 induction by IFN-γ in murine macrophages.

3' Repair exonuclease (Trex1) is the most abundant mammalian 3' → 5' DNA exonuclease with specificity for ssDNA. Trex1 deficiency has been linked to the development of autoimmune disease in mice and humans, causing Aicardi-Goutières syndrome in the latter. In addition, polymorphisms in Trex1 are associated with systemic lupus erythematosus. On the basis of all these observations, it has been hypothesized that Trex1 acts by digesting an endogenous DNA substrate. In this study, we report that Trex1 is regulated by IFN-γ during the activation of primary macrophages. IFN-γ upregulates Trex1 with the time course of an early gene, and this induction occurs at the transcription level. The half-life of mRNA is relatively short (half-life of 70 min). The coding sequence of Trex1 has only one exon and an intron of 260 bp in the promoter in the nontranslated mRNA. Three transcription start sites were detected, the one at -580 bp being the most important. In transient transfection experiments using the Trex1 promoter, we have found that two IFN-γ activation site boxes, as well as an adaptor protein complex 1 box, were required for the IFN-γ-dependent induction. By using EMSA assays and chromatin immune precipitation assays, we determined that STAT1 binds to the IFN-γ activation site boxes. The requirement of STAT1 for Trex1 induction was confirmed using macrophages from Stat1 knockout mice. We also establish that c-Jun protein, but not c-Fos, jun-B, or CREB, bound to the adaptor protein complex 1 box. Therefore, our results indicate that IFN-γ induces the expression of the Trex1 exonuclease through STAT1 and c-Jun.

The AP-1 clathrin adaptor facilitates cilium formation and functions with RAB-8 in C. elegans ciliary membrane transport.

Clathrin adaptor (AP) complexes facilitate membrane trafficking between subcellular compartments. One such compartment is the cilium, whose dysfunction underlies disorders classified as ciliopathies. Although AP-1mu subunit (UNC-101) is linked to cilium formation and targeting of transmembrane proteins (ODR-10) to nematode sensory cilia at distal dendrite tips, these functions remain poorly understood. Here, using Caenorhabditis elegans sensory neurons and mammalian cell culture models, we find conservation of AP-1 function in facilitating cilium morphology, positioning and orientation, and microtubule stability and acetylation. These defects appear to be independent of IFT, because AP-1-depleted cells possess normal IFT protein localisation and motility. By contrast, disruption of chc-1 (clathrin) or rab-8 phenocopies unc-101 worms, preventing ODR-10 vesicle formation and causing misrouting of ODR-10 to all plasma membrane destinations. Finally, ODR-10 colocalises with RAB-8 in cell soma and they cotranslocate along dendrites, whereas ODR-10 and UNC-101 signals do not overlap. Together, these data implicate conserved roles for metazoan AP-1 in facilitating cilium structure and function, and suggest cooperation with RAB-8 to coordinate distinct early steps in neuronal ciliary membrane sorting and trafficking.

Human immunodeficiency virus type-1 gag and host vesicular trafficking pathways.

The Gag protein of HIV-1 directs the particle assembly process. Gag recruits components of the cellular vesicular trafficking machinery in order to traverse the cytoplasm of the cell and reach the particle assembly site. The plasma membrane is the primary site of particle assembly in most cell types, while in macrophages an unusual intracellular membrane-bound compartment bearing markers of late endosomes and the plasma membrane is the predominant assembly site. Plasma membrane specificity of assembly may be directed by components of lipid rafts and the cytoplasmic leaflet component PI(4,5)P(2). Recent work has highlighted the role of adaptor protein complexes, protein sorting and recycling pathways, components of the multivesicular body, and cellular motor proteins in facilitating HIV assembly and budding. This review presents an overview of the relevant vesicular trafficking pathways and describes the individual components implicated in interactions with Gag.

Induction of asymmetrical cell division to analyze spindle-dependent organelle partitioning using correlative microscopy techniques.

This protocol describes an assay for the induction of asymmetrical cell division where the entire spindle is segregated into only one of the daughter cells. The procedure consists of four stages: (i) generation of asymmetrical monoasters by arresting cells in early mitosis with a kinesin Eg5 inhibitor; (ii) induction of cell division by microinjection of recombinant Mad1 protein or by the addition of a Cdk1 inhibitor; (iii) monitoring the division process by phase-contrast time-lapse microscopy; and (iv) processing for correlative immunofluorescence or correlative electron microscopy. This approach can be applied to determine the requirement for the mitotic spindle in organelle partitioning as well as to investigate the role of the monopolar spindle in cytokinesis. Moreover, the generated nucleus-lacking cytoplast provides an ideal environment to test the feasibility and activity of biological processes in the absence of genomic influence. The protocol takes 2-4 d to complete.

Synaptic vesicle recycling at CNS snapses without AP-2.

Synaptic vesicles (SVs) are composed of approximately 10 types of transmembrane proteins that must be recycled after exocytosis of neurotransmitter. The mechanisms for resorting these proteins into synaptic vesicles once incorporated into the plasma membrane after exocytosis are poorly understood. The adaptor complex AP-2 is the major clathrin-associated adaptor for cargo recognition at the plasma membrane. Here, we have investigated its role in synaptic vesicle endocytosis. shRNA-mediated knockdown of the AP-2 complex results in an approximately 96% reduction of this protein complex in primary neurons. We used simultaneous expression of shRNA and pHluorin-tagged vesicle components to show that the absence of AP-2 significantly slows but does not prevent the endocytosis of four of the major synaptic vesicle transmembrane proteins. We show that in the absence of AP-2, the AP-1 adaptor complex appears to functionally substitute for AP-2 but results in complex internalization kinetics that are now sensitive to the guanine-nucleotide exchange factor for ADP-ribosylation factor GTPase (ARF-GEF) inhibitor brefeldin-A (BFA). Simultaneous removal of both AP-2 and AP-1 prevents this compensatory substitution and results in slowed but functional endocytosis. These results demonstrate that in the absence of AP-2, SV proteins still become endocytosed, and synaptic vesicle recycling remains operational.

Adaptor heat shock protein complex formation regulates trafficking of the asialoglycoprotein receptor.

In the asialoglycoprotein receptor (ASGPR) endocytic pathway, internalized receptors pass through early, recycling, and sorting endosomal compartments before returning to the cell surface. Sorting motifs in the cytoplasmic domain (CD) and protein interactions with these sequences presumably direct receptor trafficking. Previous studies have shown that association of a potential sorting heat shock protein (HSP) heterocomplex with the ASGPR-CD was regulated by casein kinase 2 (CK2)-mediated phosphorylation. Mass spectrometry and immunoblot analyses identified five of these ASGPR-CD-associated proteins as the molecular chaperones glycoprotein 96, HSP70, HSP90, cyclophilin A, and FK 506 binding protein. The present study was undertaken to determine whether any of the adaptor protein complexes (AP1, AP2, or AP3) were selectivity associated with the ASGPR-CD. In conjunction with molecular chaperones, AP2 and AP1 were recovered from a CK2 phosphorylated agarose-GSH-GST-ASGPR-CD matrix. Binding of AP3 was independent of the phosphorylation status of the CD matrix. Inhibition of CK2-mediated phosphorylation with tetrabromobenzotriazole prevented AP recovery within an immunoadsorbed ASGPR complex. Rapamycin, which dissociates the HSP heterocomplex from ASGPR-CD, thereby altering receptor trafficking also, inhibited AP association. Similar results were obtained with an inhibitor of HSP90 heterocomplex formation, geldanmycin. The data presented provide evidence that recruitment of AP1 and AP2, which is necessary for appropriate receptor trafficking, is mediated by the interaction of AP with the ASGPR-CD-bound HSP complex.

Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes.

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.