Sec1/Munc18 (SM) proteins are required for every step of intracellular membrane fusion, but their molecular mechanism of action has been unclear. In this work, we demonstrate a fundamental role of the SM protein: to act as a stimulatory subunit of its cognate SNARE fusion machinery. In a reconstituted system, mammalian SNARE pairs assemble between bilayers to drive a basal fusion reaction. Munc18-1/nSec1, a synaptic SM protein required for neurotransmitter release, strongly accelerates this reaction through direct contact with both t- and v-SNAREs. Munc18-1 accelerates fusion only for the cognate SNAREs for exocytosis, therefore enhancing fusion specificity.

Membrane fusion occurs when SNAREpins fold up between lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy-versus-distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (approximately 2-4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k(B)T) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers (40-50 k(B)T).

Extracellular vesicles (EVs) are recognized as nature's own carriers to transport macromolecules throughout the body. Hijacking this endogenous communication system represents an attractive strategy for advanced drug delivery. However, efficient and reproducible loading of EVs with therapeutic or imaging agents still represents a bottleneck for their use as a drug delivery system. Here, we developed a method for modifying cell-derived EVs through their fusion with liposomes containing both membrane and soluble cargoes. The fusion of EVs with functionalized liposomes was triggered by polyethylene glycol (PEG) to create smart biosynthetic hybrid vectors. This versatile method proved to be efficient to enrich EVs with exogenous lipophilic or hydrophilic compounds, while preserving their intrinsic content and biological properties. Hybrid EVs improved cellular delivery efficiency of a chemotherapeutic compound by a factor of 3-4, as compared to the free drug or the drug-loaded liposome precursor. On one side, this method allows the biocamouflage of liposomes by enriching their lipid bilayer and inner compartment with biogenic molecules. On the other side, the proposed fusion strategy enables efficient EV loading, and the pharmaceutical development of EVs with adaptable activity and drug delivery property.