The isolation and proteomic analysis of Dictyostelium exosomes

Tuesday, October 25, 2011 — Poster Session II
Noon – 2:00 p.m.	Natcher Conference Center	NCI	PROTEOM-3

Authors

• P Kriebel
• L Jenkins
• G Zhang
• C 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 during the formation of aggregates. We have previously shown that vesicles containing ACA are specifically trafficked to the back of migrating Dictyostelium cells, and are essential for cells to align in a head to tail fashion during chemotaxis. This pool of ACA containing vesicles provides a compartment from which cAMP is locally released. Electron microscope analysis revealed ACA enrichment on multi-vesicular bodies that coalesce at the back of cells and fuse with the plasma membrane to release their vesicular content (exosomes) containing ACA, to the outside in trails. We have developed methods to isolate these ACA containing exosomes from cell culture supernatant, employing differential centrifugations, sucrose density gradients and LTQ mass spectrophotometry. These results indicate that exosomes may be a conserved organelle in both Dictyostelium and mammals. By exploring the protein and lipid composition of Dictyostelium exosomes we wish to gain insight into the mechanisms that regulate cAMP secretion and the role of exosomes in cell-cell signaling during chemotaxis.

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