Over the last decade, Extracellular Vesicles (EVs), including Exosomes, emerged as an important vector of intercellular communication. EVs have been proposed to transfer cargoes such as lipids, nucleic acids and proteins from donor to acceptor tissues or cells. EVs have been associated with several physiological functions and diseases. Today, EV biology is an intense and high impact research topic, which promises to revolutionize translational research through the development of EV-mimetics designed for targeted delivery of therapeutics. Tremendous progresses have been made to better understand the mechanisms that regulate EV secretion. However, our knowledge of EV uptake and content delivery within the acceptor cells is still very limited. In this project, we propose a combination of cell biology experiments coupled with in vitro studies and structural replicate electron microscopy to study how EVs fuse with their target membranes. In a first aim, we will capitalize on our previously published cell-based assays and imaging methods (optical-and electron-microscopy) to 1) further characterize EV uptake and content delivery 2) identify new proteins involved in the fusion process through a candidate approach, 3) determine basic parameters such as cargo size/ type that condition EV delivery through fusion. We will test several proteins candidates that are suspected to control EV uptake and delivery. We will focus our work on a family of proteins name hEnvs, derived from ancestral viral envelop proteins that have been integrated in human genome, and also IFITM1 and 3 proteins. We suspect that hEnv might be involved in pH-dependent EV delivery whereas IFITM1 and 3 would inhibit EV-content delivery. The major actors of endocytosis pathways (clathrin, dynamin, caveolin) will also be tested. Our bulk assays will be complemented with morphological analysis (fluorescent microcopy and classical electron microscopy), to analyze the distribution of EV markers on cells In a second aim, we plan to develop novel fluorescent-based assays to image EV membrane fusion in real time using fluorescent microscopy and correlative EM to capture all the intermediates. Our main goal is to formally establish that membrane fusion is the mechanism responsible for EV content delivery. This mechanism has never been proved so far. Our work will provide an important breakthrough in understanding and proving this delivery content. We will adapt a cell-free assay that used acceptor Plasma Membrane (PM) sheets in suspension coupled with single particle tracking diffusion. Using fluorescent microscopy, we will follow in real-time and on single EV the diffusion of EV, the docking and the fusion reactions. In addition, the same in vitro assay will be used to directly visualize the fusion process at the ultrastructural level. We propose to image the samples through platinum-replica electron microscopy (PREM). EVs will be loaded on the top of the deposited PM sheets and fixed. Initially we expect to dissect the fusion reaction and capture all the fusion-intermediates (including the fusion pore itself) by rapidly fixing the samples at different time points under different conditions Note that all candidates mentioned in Aim1 can be quickly tested within this cell-free imaging assay to directly demonstrate if cell phenotypes observed in Aim 1 correspond to perturbation of the fusion reaction. In other term we will establish causality and not just correlation.