Abstract
Extracellular vesicles (EVs) encompass a variety of vesicles secreted into the extracellular space. EVs have been implicated in promoting tumor metastasis, but the molecular composition of tumor-derived EV sub-types and the mechanisms by which molecules are sorted into EVs remain mostly unknown. We report the separation of two small EV sub-populations from a metastatic breast cancer cell line, with biochemical features consistent with different sub-cellular origins. These EV sub-types use different mechanisms of miRNA sorting (selective and non-selective), suggesting that sorting occurs via fundamentally distinct processes, possibly dependent on EV origin. Using biochemical and genetic tools, we identified the Lupus La protein as mediating sorting of selectively packaged miRNAs. We found that two motifs embedded in miR-122 are responsible for high-affinity binding to Lupus La and sorting into vesicles formed in a cell-free reaction. Thus, tumor cells can simultaneously deploy multiple EV species using distinct sorting mechanisms that may enable diverse functions in normal and cancer biology.
Highlights
Extracellular vesicles (EVs) are membranous compartments that consist of a lipid bilayer with a unique set of transmembrane proteins enclosing soluble contents that include nucleic acids and proteins (Colombo et al, 2014)
With the aid of a cell-free reaction that recapitulates the sorting of miRNAs into vesicles formed in vitro (Shurtleff et al, 2016), we found that the Lupus La protein (La protein) is required for the selective sorting of miRNAs found in the high buoyant density small EVs (sEVs) species, a conclusion that was confirmed in cells repressed for the expression of the La protein
We developed an EV purification strategy involving differential ultracentrifugation followed by buoyant density flotation on a linear iodixanol gradient (Figure 1a and Figure 1—figure supplement 1)
Summary
Extracellular vesicles (EVs) are membranous compartments that consist of a lipid bilayer with a unique set of transmembrane proteins enclosing soluble contents that include nucleic acids and proteins (Colombo et al, 2014). Cells release an array of EV sub-populations, which can be broadly classified into two categories: exosomes and shedding vesicles, distinguished by the cell membrane of origin. Exosomes are 30–150 nm vesicles that originate in the endocytic pathway. Their secretion to the extracellular space occurs upon multivesicular body (MVB) fusion with the plasma membrane resulting in the release of intraluminal vesicles (ILVs) to the extracellular space (Harding et al, 1983; Pan et al, 1985). Shedding vesicles and exosomes are termed as large and small EVs respectively. Their isolation is generally achieved by differential ultracentrifugation.
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