The isolation, proteomic analysis and function of Dictyoselium exosomes

Authors

• PW Kriebel
• L Jenkins
• K Narayan
• L Liu
• CA Parent

Abstract

In Dictyostelium, binding of the chemoattractant cAMP to its G protein coupled receptor activates various effectors including the adenylyl cyclase, ACA, which converts ATP into cAMP. A large portion of synthesized cAMP is secreted to relay signals to neighboring cells causing the cells to align in a head to tail fashion (streaming) during aggregation. We have previously shown that ACA is present in intraluminal vesicles of multivesicular-like bodies, which collect at the back of cells and are released as exosomes in trails behind the cells. We have also previously shown that the collection and release of ACA exosomes is essential for cell streaming and chemotactic signal relay during Dictyostelium cell aggregation. To isolate exosomes from cell culture supernatant we used differential centrifugations and sucrose density gradients, which yielded exosomes 80 to 150nm in diameter, with a density near 1.19mg/ml and containing ACA. Mass spectrophotometry revealed robust representation of canonical exosomal proteins and other proteins identified as orthologues of proteins also found in mammalian exosomes. Mass spectrophotometry also identified several Abc transporters that may be involved in the release of cAMP. We now show that exosomes contain cAMP, that exosomes release cAMP and that exosomes attract cells in a cAMP dependent manner. Finally we used Abc transporter inhibitors to demonstrate that cAMP is actively transported outside the exosome. These results demonstrate that exosomes released by Dictyostelium cells are involved in relaying the cAMP signal so that cells stream to form an aggregate.

Scientific Focus Area: Cell Biology

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