Abstract
Accumulating evidence suggests that exosomes are heterogeneous in molecular composition and physical properties. Here we examined whether epithelial cells secrete a heterogeneous population of exosomes, and if that is the case, whether epithelial cell polarity affects release of different populations of exosomes, especially that of those carrying Wnt. Sucrose-density ultracentrifugation and molecular marker analysis revealed that different populations of exosomes or exosome-like vesicles were released from MDCK cells depending on the cell polarity. Wnt3a associated with these vesicles were detectable in culture media collected from both apical and basolateral sides of the cells. Basolaterally secreted Wnt3a were co-fractionated with a typical exosomal protein TSG101 in fractions having typical exosome densities. In contrast, most of apically secreted Wnt3a, as well as Wnt11, were co-fractionated with CD63 and Hsp70, which are also common to the most exosomes, but recovered in higher density fractions. Wnt3a exhibiting similar floatation behavior to the apically secreted ones were also detectable in the culture media of Wnt3a-expressing L and HEK293 cells. The lipidation of Wnt3a was required for its basolateral secretion in exosomes but was dispensable for the apical one. Thus, epithelial cells release Wnt via distinct populations of vesicles differing in secretion polarity and lipidation dependency.
Highlights
Formation in multivesicular bodies (MVBs), an ESCRT-independent mechanism has been described[13,14,15]
Proteome analysis of 2 distinct populations of exosomes released from cells of a human colon carcinoma cell line revealed that molecules trafficking to the apical or basolateral side are differently enriched in these 2 exosome populations, suggesting that epithelial cells release different populations of exosomes from their apical and basolateral surfaces[19]
We found that heterogeneous populations of exosome-like extracellular vesicles were secreted in a polarity-dependent manner and that the secreted Wnt proteins were differently packaged into distinct populations of vesicles depending on the cell polarity
Summary
Formation in MVBs, an ESCRT-independent mechanism has been described[13,14,15]. These findings raise the question as to whether the heterogeneity of exosomes is relevant to the regulation of intercellular communications via exosomes. In Drosophila embryos, the mRNA and proteins of Wingless (Wg), the main Drosophila Wnt, accumulate in the apical region of epithelial cells[29,30,31,32] In contrast to this cytoplasmic localization, Wg is extracellularly concentrated on the basolateral surface of its producing cells in imaginal discs[29,32]. It still remains to be elucidated whether Wnt-associated exosomes are secreted in an apico-basal polarity-dependent manner and, if this is the case, how its polarized distribution is regulated in the biogenesis of exosomes To answer these questions, we examined exosome-mediated secretion of Wnt proteins by utilizing Madin-Darby canine kidney (MDCK) cells, which can generate a properly polarized epithelial sheet in culture[37]. We found that heterogeneous populations of exosome-like extracellular vesicles were secreted in a polarity-dependent manner and that the secreted Wnt proteins were differently packaged into distinct populations of vesicles depending on the cell polarity
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