Study of synaptic vesicles for membrane fusion and extracellular vesicles for cell signaling using single-molecule imaging technique

Abstract

Vesicle transport is very crucial mechanism which occurs in every cells mediating many biological events and by which cells communicate each other and then maintain physiology and homeostasis. However, sometimes oncogenic cells also transfer abnormal signals by emitting ‘extracellular vesicles (EVs)’ which, therefore, are recently emerging therapeutic opportunities. Here, we ask how distinct types of EVs differently represent their parental cells from an oncoprotein-delivery perspective. By measuring protein-protein interactions (PPIs) between EGFR and representative downstream effector proteins (called “probes”) in EVs produced by NSCLC cells, we test which type of EVs better represents the status of its cell of origin. While the phosphotyrosine level on EGFR in EVs has been investigated and others observed the activation of signaling proteins downstream of EGFR, direct measurement of PPIs between signaling proteins from EVs has not been reported so far. Our data show that microvesicles closely reflect the cellular EGFR in their downstream signaling potential while exosomes generally display upregulated PPIs. Using drug-resistant cells and EVs derived from them, we also study how EVs adjust their PPI landscape when it has drug resistance. Ultimately, we hope EVs thus characterized could be utilized in clinical diagnosis and prognosis with clearer understanding of their implications: microvesicles as reflectors of parent cells and exosomes as predictors of drug sensitivity. Another important vesicle trafficking is synaptic vesicle fusion, which deals with neurotransmitter release in synapse, at the end of neuron. It has been known that intracellular Ca regulates transmitter release, and synaptotagmin is its regulator, that was observed direct control of $Ca^{2+}$ concentration at a large terminal, in vivo experiments. However, a mechanism of the fusion has not been fully understood and contribution of syt to the membrane fusion is also controversial whether fusogen or not. Therefore, many in vitro studies have been struggling to mimic fast $Ca^{2+}$-triggered fusion, but there still is a room for a reliable and conserved result. By developing ability to detect content mixing in single vesicle fusion events, we here show that such dynamic $Ca^{2+}$-dependent Syt1 activity is also observed at the content mixing level, not only lipid mixing. Deactivation of SNAREs interferes stimulation of syt so that we infer its role of $Ca^{2+}$ regulator operates only when SNAREs are active as a fusion machinery.