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The primary role of endosomal system is endocytic trafficking – to sort out internalized macromolecules and proteins to their destined cellular localizations. Incorporation of sorted cargos into multivesicular bodies (MVBs) confers specificities and determines their fates. This central point of the endosomal trafficking separates MVBs in two directions. The MVB populations fuse either with lysosomes to initiate autophagy or with plasma membrane to release small extracellular vesicles. Factors contributing to the selection of cargo and direction of trafficking incorporate the cells’ metabolic status and stress level. In this review, we discuss the molecular cues responsible for differential cargo sorting into MVBs and trafficking directions of MVBs in the endosomal network. Keywords: Exosomes; degradative MVB; secretory MVB; physiological stress; endocytic machinery; lysosome

Extracellular vesicles are membrane particles secreted from cells into all body fluids. They convey signals that induce long-term changes in cellular behavior. During development, EV secretion and the specific loading of signalling factors in EVs contributes to organ development and tissue differentiation. Different biomolecules such as proteins, lipids and nucleic acids transmit these signals and the content, size, and membrane composition of EVs are highly dynamic and depend on the cellular source, state, and environmental conditions.Here we review current in vivo studies in different model organisms regarding extracellular vesicles and their physiological role in the formation of cell types, tissues and refined body structures.

WNT signaling is a key developmental pathway in tissue organization. A recent focus of research is the secretion of WNT proteins from source cells. Research over the past decade on how WNTs are produced and released into the extracellular space has unravelled very specific control mechanisms in the early secretory pathway, specialized trafficking routes, and redundant forms of packaging for delivery to target cells. In this review I discuss the findings that WNT proteins have been found on extracellular vesicles (EVs) such as exosomes and possible functional implications. There is an ongoing debate in the WNT signaling field whether EV are relevant in vivo and can fulfill specific functions, also fueled by the general preconception of EV secretion as cellular garbage disposal. As part of the EV research community, I want to give an overview of what we know and don't know about WNT secretion on EVs and offer a more unifying model that can explain current discrepancies in observations regarding WNT secretion.

Secretory Wnt trafficking can be studied in the polarized epithelial monolayer of Drosophila wing imaginal discs (WID). In this tissue, Wg (Drosophila Wnt-I) is presented on the apical surface of its source cells before being internalized into the endosomal pathway. Long-range Wg secretion and spread depend on secondary secretion from endosomal compartments, but the exact post-endocytic fate of Wg is poorly understood. Here, we summarize and present three protocols for the immunofluorescence-based visualization and quantitation of different pools of intracellular and extracellular Wg in WID: (1) steady-state extracellular Wg; (2) dynamic Wg trafficking inside endosomal compartments; and (3) dynamic Wg release to the cell surface. Using a genetic driver system for gene manipulation specifically at the posterior part of the WID (EnGal4) provides a robust internal control that allows for direct comparison of signal intensities of control and manipulated compartments of the same WID. Therefore, it also circumvents the high degree of staining variability usually associated with whole-tissue samples. In combination with the genetic manipulation of Wg pathway components that is easily feasible in Drosophila, these methods provide a tool-set for the dissection of secretory Wg trafficking and can help us to understand how Wnt proteins travel along endosomal compartments for short- and long-range signal secretion. Graphic abstract: Figure 1. Visualization of extracellular and intracellular Wg trafficking in Drosophila wing imaginal discs. While staining of extracellular Wg without permeabilization exclusively visualizes Wg bound to the extracellular surface (left), Wg uptake and endosomal trafficking can be visualized using an antibody uptake assay (middle). Dynamic Wg release can be visualized by performing a non-permeabilizing staining at a permissive temperature that sustains secretory Wg transport (right).

Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. We identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, we found that phosphorylation regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. We propose that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes.

Morphogens are important signalling molecules for tissue development and their secretion requires tight regulation. In the wing imaginal disc of flies, the morphogen Wnt/Wingless is apically presented by the secreting cell and re-internalized before final long-range secretion. Why Wnt molecules undergo these trafficking steps and the nature of the regulatory control within the endosomal compartment remain unclear. Here, we have investigated how Wnts are sorted at the level of endosomes by the versatile v-SNARE Ykt6. Using in vivo genetics, proximity-dependent proteomics and in vitro biochemical analyses, we show that most Ykt6 is present in the cytosol, but can be recruited to de-acidified compartments and recycle Wnts to the plasma membrane via Rab4-positive recycling endosomes. Thus, we propose a molecular mechanism by which producing cells integrate and leverage endocytosis and recycling via Ykt6 to coordinate extracellular Wnt levels., Summary: SNARE-dependent secondary secretion via Rab4 recycling endosomes plays an important role in coordinating long-range extracellular levels of the hydrophobic morphogen Wnt/Wingless.

Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion. However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6-RNAi mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. The latter accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies. Taken together, we show that a regulatory switch in Ykt6 fine-tunes sorting of Wnts in endosomes.

Autocrine Wnt10b signaling ensures proper mitotic microtubule polymerization and faithful chromosome segregation., Wnt signaling is crucial for proper development, tissue homeostasis and cell cycle regulation. A key role of Wnt signaling is the GSK3β-mediated stabilization of β-catenin, which mediates many of the critical roles of Wnt signaling. In addition, it was recently revealed that Wnt signaling can also act independently of β-catenin. In fact, Wnt mediated stabilization of proteins (Wnt/STOP) that involves an LRP6-DVL–dependent signaling cascade is required for proper regulation of mitosis and for faithful chromosome segregation in human somatic cells. We show that inhibition of Wnt/LRP6 signaling causes whole chromosome missegregation and aneuploidy by triggering abnormally increased microtubule growth rates in mitotic spindles, and this is mediated by increased GSK3β activity. We demonstrate that proper mitosis and maintenance of numerical chromosome stability requires continuous basal autocrine Wnt signaling that involves secretion of Wnts. Importantly, we identified Wnt10b as a Wnt ligand required for the maintenance of normal mitotic microtubule dynamics and for proper chromosome segregation. Thus, a self-maintaining Wnt10b-GSK3β–driven cellular machinery ensures the proper execution of mitosis and karyotype stability in human somatic cells.

In prostate cancer and other malignancies sensitive and robust biomarkers are lacking or have relevant limitations. Prostate specific antigen (PSA), the only biomarker widely used in prostate cancer, is suffering from low specificity. Exosomes offer new perspectives in the discovery of blood-based biomarkers. Here we present a proof-of principle study for a proteomics-based identification pipeline, implementing existing data sources, to exemplarily identify exosome-based biomarker candidates in prostate cancer. Exosomes from malignant PC3 and benign PNT1A cells and from FBS-containing medium were isolated using sequential ultracentrifugation. Exosome and control samples were analyzed on an LTQ-Orbitrap XL mass spectrometer. Proteomic data is available via ProteomeXchange with identifier PXD003651. We developed a scoring scheme to rank 64 proteins exclusively found in PC3 exosomes, integrating data from four public databases and published mass spectrometry data sets. Among the top candidates, we focused on the tight junction protein claudin 3. Retests under serum-free conditions using immunoblotting and immunogold labeling confirmed the presence of claudin 3 on PC3 exosomes. Claudin 3 levels were determined in the blood plasma of patients with localized (n = 58; 42 with Gleason score 6–7, 16 with Gleason score ≥8) and metastatic prostate cancer (n = 11) compared with patients with benign prostatic hyperplasia (n = 15) and healthy individuals (n = 15) using ELISA, without prior laborious exosome isolation. ANOVA showed different CLDN3 plasma levels in these groups (p = 0.004). CLDN3 levels were higher in patients with Gleason ≥8 tumors compared with patients with benign prostatic hyperplasia (p = 0.012) and Gleason 6–7 tumors (p = 0.029). In patients with localized tumors CLDN3 levels predicted a Gleason score ≥ 8 (AUC = 0.705; p = 0.016) and did not correlate with serum PSA. By using the described workflow claudin 3 was identified and validated as a potential blood-based biomarker in prostate cancer. Furthermore this workflow could serve as a template to be used in other cancer entities.

// Kerstin Menck 1,* , Florian Klemm 1,* , Julia Christina Gross 2 , Tobias Pukrop 1 , Dirk Wenzel 3 , Claudia Binder 1 1 Dept of Hematology/Oncology, University Medicine, Gottingen, Germany 2 Div. Signaling and Functional Genomics, German Cancer Research Center, Heidelberg, Germany 3 Max Planck Institute for Biophysical Chemistry, Gottingen, Germany * These authors contributed equally to this work. Correspondence: Claudia Binder, email: // Keywords : Breast cancer, exosomes, Evi, microvesicles, macrophages, Wnt 5a Received : August 27, 2013 Accepted : October 19, 2013 Published : October 21, 2013 Abstract Recently, we have shown that macrophage (MΦ)-induced invasion of breast cancer cells requires upregulation of Wnt 5a in MΦ leading to activation of β-Catenin-independent Wnt signaling in the tumor cells. However, it remained unclear, how malignant cells induce Wnt 5a in MΦ and how it is transferred back to the cancer cells. Here we identify two types of extracellular particles as essential for this intercellular interaction in both directions. Plasma membrane-derived microvesicles (MV) as well as exosomes from breast cancer cells, although biologically distinct populations, both induce Wnt 5a in MΦ. In contrast, the particle-free supernatant and vesicles from benign cells, such as platelets, have no such effect. Induction is antagonized by the Wnt inhibitor Dickkopf-1. Subsequently, Wnt 5a is shuttled via responding MΦ-MV and exosomes to the tumor cells enhancing their invasion. Wnt 5a export on both vesicle fractions depends at least partially on the cargo protein Evenness interrupted (Evi). Its knockdown leads to Wnt 5a depletion of both particle populations and reduced vesicle-mediated invasion. In conclusion, MV and exosomes are critical for MΦ-induced invasion of cancer cells since they are responsible for upregulation of MΦ-Wnt 5a as well as for its delivery to the recipient cells via a reciprocal loop. Although of different biogenesis, both populations share common features regarding function and Evi-dependent secretion of non-canonical Wnts.

Cryptochromes are a ubiquitous group of blue-light absorbing flavoproteins that in the mammalian retina have an important role in the circadian clock. In birds, cryptochrome 1a (Cry1a), localized in the UV/violet-sensitive S1 cone photoreceptors, is proposed to be the retinal receptor molecule of the light-dependent magnetic compass. The retinal localization of mammalian Cry1, homologue to avian Cry1a, is unknown and it is open whether mammalian Cry1 is also involved in magnetic field sensing. To constrain the possible role of retinal Cry1, we immunohistochemically analysed 90 mammalian species across 48 families in 16 orders, using an antiserum against the Cry1 C-terminus that in birds labels only the photo-activated conformation. In the Carnivora families Canidae, Mustelidae and Ursidae and in some Primates, Cry1 was consistently labeled in the outer segment of the shortwave-sensitive S1 cones. This finding would be compatible with a magnetoreceptive function of Cry1 in these taxa. In all other taxa, Cry1 was not detected by the antiserum that likely also in mammals labels the photo-activated conformation, although Western blots showed Cry1 in mouse retinal cell nuclei. We speculate that in the mouse and the other negative-tested mammals Cry1 is involved in circadian functions as a non-light-responsive protein.