title: Mechanism of evenness interrupted (Evi)-exosome release at synaptic boutons.
abstract: Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release.
authors.affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. kate.koles@umassmed.edu
authors.forename: Kate
authors.initials: K
authors.lastname: Koles
authors.forename: John
authors.initials: J
authors.lastname: Nunnari
authors.forename: Ceren
authors.initials: C
authors.lastname: Korkut
authors.forename: Romina
authors.initials: R
authors.lastname: Barria
authors.forename: Cassandra
authors.initials: C
authors.lastname: Brewer
authors.forename: Yihang
authors.initials: Y
authors.lastname: Li
authors.forename: John
authors.initials: J
authors.lastname: Leszyk
authors.forename: Bing
authors.initials: B
authors.lastname: Zhang
authors.forename: Vivian
authors.initials: V
authors.lastname: Budnik
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000502:physiology; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D055354:Exosomes / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D024701:Myosin Type V / Q000235:genetics / Q000378:metabolism; D009469:Neuromuscular Junction / Q000235:genetics / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology*; D013572:Synaptic Vesicles / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
chemical_list: D029721:Drosophila Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D024701:Myosin Type V; D020691:rab GTP-Binding Proteins; C511046:Wls protein, Drosophila; C559123:Rab11 protein, Drosophila; D000099193:rab11 GTP-Binding Proteins