[This corrects the article DOI: 10.1371/journal.pone.0181724.].

Members of the Dickkopf (Dkk) family of Wnt antagonists interrupt Wnt-induced receptor assembly and participate in axial patterning and cell fate determination. One family member, DKK3, does not block Wnt receptor activation. Loss of Dkk3 expression in cancer is associated with hyperproliferation and dysregulated ß-catenin signaling, and ectopic expression of Dkk3 halts cancer growth. The molecular events mediating the DKK3-dependent arrest of ß-catenin-driven cell proliferation in cancer cells are unknown. Here we report the identification of a new intracellular gene product originating from the Dkk3 locus. This Dkk3b transcript originates from a second transcriptional start site located in intron 2 of the Dkk3 gene. It is essential for early mouse development and is a newly recognized regulator of ß-catenin signaling and cell proliferation. Dkk3b interrupts nuclear translocation ß-catenin by capturing cytoplasmic, unphosphorylated ß-catenin in an extra-nuclear complex with ß-TrCP. These data reveal a new regulator of one of the most studied signal transduction pathways in metazoans and provides a novel, completely untapped therapeutic target for silencing the aberrant ß-catenin signaling that drives hyperproliferation in many cancers.

Sorting of EGF and transferrin at the plasma membrane and by cargo-specific signaling to EEA1-enriched endosomes" (2008).

Background: We report a 6.5 year-old female with a homozygous missense mutation in ZFYVE20, encoding Rabenosyn-5 (Rbsn-5), a highly conserved multi-domain protein implicated in receptor-mediated endocytosis. The clinical presentation includes intractable seizures, developmental delay, microcephaly, dysostosis, osteopenia, craniofacial dysmorphism, macrocytosis and megaloblastoid erythropoiesis. Biochemical findings include transient cobalamin deficiency, severe hypertriglyceridemia upon ketogenic diet, microalbuminuria and partial cathepsin D deficiency.Methods and results: Whole exome sequencing followed by Sanger sequencing confirmed a rare (frequency:0.003987) homozygous missense mutation, g. 15,116,371 G > A (c.1273G > A), in ZFYVE20 resulting in an amino acid change from Glycine to Arginine at position 425 of the Rbsn protein (p.Gly425Arg), as the only mutation segregating with disease in the family. Studies in fibroblasts revealed expression and localization of Rbsn-5G425R in wild-type manner, but a 50% decrease in transferrin accumulation, which is corrected by wild-type allele transfection. Furthermore, the patient's fibroblasts displayed an impaired proliferation rate, cytoskeletal and lysosomal abnormalities.Conclusion: These results are consistent with a functional defect in the early endocytic pathway resulting from mutation in Rbsn-5, which secondarily disrupts multiple cellular functions dependent on endocytosis, leading to a severe multi-organ disorder.

Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic.

Antibody-mediated intracellular delivery of therapeutic agents has been considered for treatment of a variety of diseases. These approaches involve targeting cell-surface receptor proteins expressed by tumors or viral proteins expressed on infected cells. We examined the intracellular trafficking of a viral cell-surface-expressed protein, rabies G, with or without binding a specific antibody, ARG1. We found that antibody binding shifts the native intracellular trafficking pathway of rabies G in an Fc-independent manner. Kinetic studies indicate that the ARG1/rabies G complex progressively co-localized with clathrin, early endosomes, late endosomes, and lysosomes after addition to cells. This pathway was different from that taken by rabies G without addition of antibody, which localized with recycling endosomes. Findings were recapitulated using a cellular receptor with a well-defined endogenous recycling pathway. We conclude that antibody binding to cell-surface proteins induces redirection of intracellular trafficking of unbound or ligand bound receptors to a specific degradation pathway. These findings have broad implications for future developments of antibody-based therapeutics.

Recent work has led to the identification of novel endocytic compartments with functional roles in both protein trafficking and growth factor signal transduction. The phosphatidylinositol 3-phosphate binding, FYVE domain-containing protein WDFY2 is localized to a distinct subset of early endosomes, which are localized close to the plasma membrane. Here, we find that the serine/threonine kinase Akt interacts with these endosomes in an isoform-specific manner. Using quantitative fluorescence microscopy we demonstrate specific co-localization of WDFY2 with endogenous Akt2, but not Akt1. Moreover, depletion of WDFY2 leads to impaired phosphorylation of Akt in response to insulin due to isoform specific reduction of Akt2, but not Akt1, protein levels, and to a marked reduction in the insulin-stimulated phosphorylation of numerous Akt substrates. This is accompanied by an impairment in insulin-stimulated glucose transport and, after prolonged silencing, a reduction in the level of expression of adipogenic genes. We propose that WDFY2-enriched endosomes serve as a scaffold that enables specificity of insulin signaling through Akt2.

The biological function of receptors is determined by their appropriate trafficking through the endosomal pathway. Following internalization, the transferrin (Tf) receptor quantitatively recycles to the plasma membrane, whereas the epidermal growth factor (EGF) receptor undergoes degradation. To determine how Tf and EGF engage these two different pathways we imaged their binding and early endocytic pathway in live cells using total internal reflection fluorescence microscopy (TIRF-M). We find that EGF and Tf bind to distinct plasma membrane regions and are incorporated into different endocytic vesicles. After internalization, both EGF-enriched and Tf-enriched vesicles interact with endosomes containing early endosome antigen 1 (EEA1). EGF is incorporated and retained in these endosomes, while Tf-containing vesicles rapidly dissociate and move to a juxtanuclear compartment. Endocytic vesicles carrying EGF recruit more Rab5 GTPase than those carrying Tf, which, by strengthening their association with EEA1-enriched endosomes, may provide a mechanism for the observed cargo-specific sorting. These results reveal pre-endocytic sorting of Tf and EGF, a specialized role for EEA1-enriched endosomes in EGF trafficking, and a potential mechanism for cargo-specified sorting of endocytic vesicles by these endosomes.

The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12-13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include proteins that have not been characterized in any species. One of these, WDFY2, appears to play an important role in early endocytosis and was revealed in a RNAi (RNA interference) screen in C. elegans. Interestingly, some proteins contain FYVE-like domains in C. elegans and D. melanogaster, but have lost this domain during evolution. One of these is the homologue of Rabatin-5, a protein that, in mammalian cells, binds both Rab5 and Rabex-5, a guanine-nucleotide exchange factor for Rab5. Thus the Rabatin-5 homologue suggests that mechanisms to link PtdIns3P and Rab5 activation developed in evolution. In mammalian cells, these mechanisms are apparent in the existence of proteins that bind PtdIns3P and Rab GTPases, such as EEA1, Rabenosyn-5 and Rabip4'. Despite the comparable ability to bind to PtdIns3P in vitro, FYVE domains display widely variable abilities to interact with endosomes in intact cells. This variation is due to three distinct properties of FYVE domains conferred by residues that are not involved in PtdIns3P head group recognition: These properties are: (i) the propensity to oligomerize, (ii) the ability to insert into the membrane bilayer, and (iii) differing electrostatic interactions with the bilayer surface. The different binding properties are likely to regulate the extent and duration of the interaction of specific FYVE domain-containing proteins with early endosomes, and thereby their biological function.

Type I deiodinase is the best characterized member of a small family of seleno enzymes catalyzing the bioactivation and disposal of thyroid hormone. This enzyme is an integral membrane protein composed of two 27-kDa subunits that assemble into a functional enzyme after translation using a highly conserved sequence of 16 amino acids in the C-terminal half of the polypeptide, (148)DFLXXYIXEAHXXDGW(163). In this study, we used alanine scanning mutagenesis to identify the key residues in this domain required for holoenzyme assembly. Overexpression of sequential alanine-substituted mutants of a dimerization domain-green fluorescent protein fusion showed that sequence (IYI154)-I-152 was required for type I enzyme assembly and that a catalytically active monomer was generated by a single I152A substitution. Overexpression of the sequential alanine-substituted dimerization domain mutants in type II selenodeiodinase-expressing cells showed that five residues ((FLIVY157)-F-153) at the beginning and three residues ((164)SDG(166)) at the end of this region were required for the assembly of the type II enzyme. In vitro binding analysis revealed a free energy of association of -60 +/- 5kJ/mol for the noncovalent interaction between dimerization domain monomers. These data identify and characterize the essential residues in the dimerization domain that are responsible for the post-translational assembly of selenodeiodinases.

The FYVE domain binds with high specificity and avidity to phosphatidylinositol 3-phosphate. It is present in ≈30 proteins in humans, some of which have been implicated in functions ranging from early endosome fusion to signal transduction through the TGF-β receptor. To develop a further understanding of the biological roles of this protein family, we turned to the nematode Caenorhabditis elegans , which contains only 12 genes predicted to encode for phosphatidylinositol 3-phosphate binding, FYVE domain-containing proteins, all of which have homologs in the human genome. Each of these proteins was targeted individually by RNA interference. One protein, WDFY2, produced a strong inhibition of endocytosis when silenced. WDFY2 contains WD40 motifs and a FYVE domain, is highly conserved between species, and localizes to a set of small endosomes that reside within 100 nm from the plasma membrane. These endosomes are involved in transferrin uptake but lack the classical endosomal markers Rab5 and EEA1. Silencing of WDFY2 by siRNA in mammalian cells impaired transferrin endocytosis. These studies reveal the important, conserved role of WDFY2 in endocytosis, and the existence of a subset of early endosomes, closely associated with the plasma membrane, that may constitute the first stage of endocytic processing of internalized cargo.

Clathrin assembly at the plasma membrane is a fundamental process required for endocytosis. In cultured cells, most of the clathrin is localized to large patches that display little lateral mobility. The functional role of these regions is not clear, and it has been thought that they may represent artifacts of cell adhesion of cultured cells. Here we have analyzed clathrin organization in primary adipose cells isolated from mice, which are nonadherent and fully differentiated. The majority of clathrin on the plasma membrane of these cells (> 60%) was found in large clathrin patches that displayed virtually no lateral mobility and persisted for many minutes, and a smaller amount was found in small spots that appeared and disappeared rapidly. Direct visualization of transferrin revealed that it bound onto large arrays of clathrin, internalizing through vesicles that emerge from these domains. High resolution imaging (50 images/s) revealed fluorescence intensity fluctuations consistent with the formation and detachment of coated vesicles from within large patches. These results reveal that large clathrin assemblies are active regions of endocytosis in mammalian cells and highlight the importance of understanding the mechanistic basis for this organization.

Endocytosis is important for the regulation of signal transduction and for the movement of essential cellular components from outside the cell to their appropriate intracellular compartment(s). Two established mechanisms of endocytosis are clathrinmediated (CME) and clathrin-independent endocytosis, and they are responsible for internalization of different ligands. In this study, the newly established technique of total internal reflection fluorescent microscopy (TIRF-M) was used, along with standard biochemical and molecular biological tools, to systematically study the sorting and early trafficking of two established ligands of endocytosis, transferrin (Tf) and epidermal growth factor (EGF). TIRF-M studies revealed that Tf binds its receptor that is located in large clathrin arrays positioned just below the surface of the cell and that these large clathrin platforms serves as the major site of CME at the plasma membrane. EGF endocytosis is very different and occurs as follows 1) the liganded EGFR recruits Rab5 to the plasma membrane, 2) Rab5 concentrates around vesicles containing liganded EGFR and 3) these vesicles co-localize with EEA1 enriched endosomes. EEA1 was shown to play a pivotal role in EGF endocytosis, establishing a new role for EEA1 in vesicle trafficking in addition to its role in tethering and fusion. Finally, WDFY2, a new FYVE domain protein was shown to decorate a specific subset of vesicles, upstream of the EEA1 vesicle pool that appear to participate in Tf endocytosis. These studies establish new functions and components of endocytosis that enhances our understanding of this complex process.