WNT5a export onto extracellular vesicles studied at single‐molecule and single‐vesicle resolution

• DOI: 10.1111/febs.70074
• Publication Date: 2025-04-02T04:00:48Z
• Authors: Antonia Schubert; Ajaree Mongkolsit...; Andrei Kobitski; Matthias Schulz; Oksana Voloshanenko; Meike Schaffrinski; Nadine Winkler; Michelle Neßling; Karsten Richter; Dominique Kranz; Karin Nienhaus; Dirk Jäger; Lorenz Trümper; Judith Büntzel; Claudia Binder; Gerd Ulrich Nienhaus; Michael Boutros
• Keywords: Exosome; Extracellular vesicles; Cell Signaling

WNT signaling governs development, homeostasis, and aging of cells tissues, is frequently dysregulated in pathophysiological processes such as cancer. proteins are hydrophobic traverse the intercellular space between secreting receiving on various carriers, including extracellular vesicles (EVs). Here, we address relevance different EV fractions other vehicles for WNT5a protein, a non‐canonical ligand that signals independently beta‐catenin. Its highly context‐dependent roles cancer (either tumor‐suppressive or tumor‐promoting) have been attributed to two distinct isoforms, Short (WNT5aS) Long (WNT5aL), resulting from signal peptide cleavage sites. To explore possible differences secretion transport, developed fusion constructs with fluorescent (FPs) mScarlet mOxNeonGreen. Functional reporter assays revealed both isoforms inhibit canonical signaling, EVs produced by WNT5a‐bearing tumor cells, carrying either induced invasiveness luminal A breast cell line MCF7. We used fluorescence intensity distribution analysis (FIDA) correlation spectroscopy (FCS) characterize at single‐molecule sensitivity WNT5aL‐bearing entities secreted HEK293T cells. Importantly, found most WNT5aL remained monomeric supernatant after ultracentrifugation; only minor fraction was EV‐bound. further determined average sizes number per EV. Our detailed biophysical physical nature populations an important step toward understanding cargo loading future studies.