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W Dismuke, Brian McKay, Aaditya Khatri, Kristin Perkumas, W Daniel Stamer; Tracking protein endocytosis and trafficking to exosomes released from trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2003.
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© ARVO (1962-2015); The Authors (2016-present)
Aqueous humor and conditioned media from cultured human trabecular meshwork (TM) cells contain exosomes, extracellular vesicles with an unclear physiological role. Exosomes have been shown to act as signal transducers and vehicles for delivery of small RNAs and enzymes to adjacent cells. However, regulation of exosomal cargo loading is poorly understood. Here we test the hypothesis that plasma membrane proteins from human TM cells are trafficked to exosomes in response to external stimuli.
To monitor plasma membrane protein trafficking to exosomes, we utilized a novel experimental paradigm. Cultured human TM cells were serum starved and biotinylated on their cell surface. Cells were then incubated with or without fetal bovine serum (10% v:v) for various lengths of time. Following serum stimulation, media was collected over time and exosomes were isolated by serial ultracentrifugations and characterized by established techniques. Exosome proteins were separated by SDS-PAGE and biotinylated proteins were detected with strepavadin conjugated HRP.
Proteins accessible to the extracellular surface of human trabecular meshwork cells were exclusively labeled with biotin, as indicated by lack of cytoplasmic actin biotinylation . As early as 4 hours after media change, biotinylated proteins were detected on exosomes released from human TM cells, regardless of the presence of serum. Notably, we observed the appearance of several unique biotinylated proteins in exosomes from cells that were stimulated with serum. In particular, a ~65kDa, biotinylated exosome protein was reproducibly observed. Treatment of cells with the Ca++ ionophore, ionomycin (10μM, 10 minutes), at the end of the 4 hour serum stimulation increased the abundance of the observed biotinylated exosome proteins.
Our data demonstrates that plasma membrane proteins from human TM cells are endocytosed, trafficked to, incorporated in and are constitutively released with exosomes. Additionally, we detected a unique set of proteins trafficked to exosomes upon ligand stimulation. These findings suggest that the endocytic pathway is very active in TM cells and that exosomal trafficking responds to environmental cues. Both are potentially involved in the regulation receptor signaling, outflow facility and thus intraocular pressure.
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