ABSTRACT
Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. Here we show that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling.
Subject(s)
Drosophila Proteins/metabolism , Exosomes/metabolism , Presynaptic Terminals/metabolism , Signal Transduction/physiology , Synaptic Potentials/physiology , Synaptotagmins/metabolism , Animals , Animals, Genetically Modified , Drosophila , Exosomes/chemistry , Neuromuscular Junction/chemistry , Neuromuscular Junction/metabolism , Presynaptic Terminals/chemistry , Synapses/chemistry , Synapses/metabolismABSTRACT
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.
Subject(s)
Drosophila Proteins/metabolism , Exosomes/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Neuromuscular Junction/metabolism , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Animals , Biological Transport/physiology , Drosophila Proteins/genetics , Drosophila melanogaster , Exosomes/genetics , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Myosin Type V/genetics , Myosin Type V/metabolism , Neuromuscular Junction/genetics , Synaptic Vesicles/genetics , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
Wnts play pivotal roles during development and in the mature nervous system. However, the mechanism by which Wnts traffic between cells has remained elusive. Here we demonstrate a mechanism of Wnt transmission through release of exosome-like vesicles containing the Wnt-binding protein Evenness Interrupted/Wntless/Sprinter (Evi/Wls/Srt). We show that at the Drosophila larval neuromuscular junction (NMJ), presynaptic vesicular release of Evi is required for the secretion of the Wnt, Wingless (Wg). We also show that Evi acts cell-autonomously in the postsynaptic Wnt-receiving cell to target dGRIP, a Wg-receptor-interacting protein, to postsynaptic sites. Upon Evi loss of function, dGRIP is not properly targeted to synaptic sites, interfering with postsynaptic Wnt signal transduction. These findings uncover a previously unknown cellular mechanism by which a secreted Wnt is transported across synapses by Evi-containing vesicles and reveal trafficking functions of Evi in both the Wnt-producing and the Wnt-receiving cells. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.
Subject(s)
Drosophila Proteins/metabolism , Drosophila/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Signal Transduction , Synaptic Vesicles/metabolism , Wnt1 Protein/metabolism , Animals , Carrier Proteins/metabolism , Frizzled Receptors/metabolism , Membrane Proteins , Motor Neurons/metabolism , Nerve Tissue Proteins/metabolism , Neuromuscular Junction , Protein Transport , Receptors, G-Protein-Coupled/metabolism , SynapsesABSTRACT
Although WNTs have been long thought of as regulators of cell fate, recent studies highlight their involvement in crucial aspects of synaptic development in the nervous system. Particularly compelling are recent studies of the neuromuscular junction in nematodes, insects, fish and mammals. These studies place WNTs as major determinants of synapse differentiation and neurotransmitter receptor clustering.
