An experimental approach to investigate extracellular vesicle-mediated transfer of lipids between fungal cells

Extracellular vesicles (EVs) are key mediators of communication in fungal populations, but the mechanisms underlying lipid exchange through EVs remain poorly characterized. Here, we describe an experimental approach to visualize lipid transfer between fungal cells using the lipophilic dye FM 1-43. This styryl dye efficiently stained membranous structures in multiple fungal species, including Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus, Saccharomyces cerevisiae, and Sporothrix brasiliensis. Staining of the acapsular C. neoformans strain Cap67 with FM 1-43 generated fluorescent EVs, as shown by nano flow cytometry. Supernatants from FM 1-43-stained Cap67 cultures successfully transferred fluorescence to previously unstained cells, suggesting EV-mediated lipid exchange. The removal of EVs through ultracentrifugation of the supernatants effectively nullified this observation. A transwell system separating stained donor and unstained acceptor cells by a 0.4 µm membrane demonstrated that fluorescence transfer occurred without direct cell contact, with stronger signals observed when acapsular cells were used as donors. Using this model and acapsular C. neoformans cells as lipid acceptors, interspecies lipid transfer was detected when Cryptococcus deuterogattii and C. albicans were used as donor cells, although fluorescence levels were lower compared to intraspecies exchanges. Together, these results establish a tractable framework to monitor EV-mediated lipid trafficking among fungi. This model offers new experimental opportunities to dissect fungal communication mediated by EVs.IMPORTANCEFungal cells release extracellular vesicles (EVs) that mediate intercellular communication, but the mechanisms and biological consequences of this process remain underexplored. Here, we provide experimental evidence that lipids can be exchanged between fungal cells via EVs, visualized using the lipophilic dye FM 1-43. This approach allows tracking of lipid trafficking between cells of the same or different species, even in the absence of direct contact. Understanding EV-mediated lipid exchange may offer insights into fungal physiology, signaling, and adaptation. These findings establish a model that can be broadly applied to study vesicle biology and intercellular communication in other microorganisms.