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WNT5a export onto extracellular vesicles studied at single-molecule and single-vesicle resolution
Antonia Schubert1,2,3,4,5, Ajaree Mongkolsittisilp6, Andrei Kobitski6
1Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany.
The FEBS Journal
|April 1, 2025
Summary
WNT5a protein, crucial in cancer, travels via extracellular vesicles (EVs). This study reveals most WNT5a remains free, not EV-bound, impacting cancer cell invasiveness.
Area of Science:
- Cell biology
- Molecular biology
- Biophysics
Background:
- WNT signaling is vital for cell and tissue function, but its dysregulation drives diseases like cancer.
- WNT proteins, including WNT5a, are hydrophobic and utilize carriers like extracellular vesicles (EVs) for intercellular transport.
- WNT5a isoforms (WNT5aS and WNT5aL) exhibit context-dependent roles in cancer, potentially due to differential signaling or transport.
Purpose of the Study:
- To investigate the secretion and extracellular transport mechanisms of WNT5a isoforms.
- To determine the association of WNT5a with different extracellular vesicle (EV) fractions and other secreted entities.
- To analyze the biophysical properties of WNT5a-carrying EVs and their impact on cancer cell behavior.
Main Methods:
- Development of fluorescent protein (FP) fusion constructs for WNT5a isoforms (WNT5aS, WNT5aL).
- Functional reporter assays to assess WNT signaling inhibition and cancer cell invasiveness.
- Advanced biophysical techniques: Fluorescence Intensity Distribution Analysis (FIDA) and Fluorescence Correlation Spectroscopy (FCS) for single-molecule analysis.
- Ultracentrifugation to separate EV fractions and characterization of EV size and WNT5a loading.
Main Results:
- Both WNT5a isoforms were found to inhibit canonical WNT signaling.
- EVs carrying WNT5a isoforms induced invasiveness in MCF7 breast cancer cells.
- Single-molecule analysis revealed that the majority of WNT5aL remained monomeric and free in the supernatant, with only a minor fraction associated with EVs.
- Characterization of EV size distribution and WNT5a loading capacity per EV.
Conclusions:
- WNT5a isoforms can be transported via EVs, influencing cancer cell invasiveness.
- A significant portion of WNT5a exists independently of EVs in the extracellular environment.
- Detailed biophysical characterization of WNT5a-associated entities is crucial for understanding its cargo loading and context-dependent signaling in disease.
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