En route to dynamic life processes by SNARE-mediated fusion of polymer and hybrid membranes.

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes. Here, we show that synthetic amphiphile membranes also undergo fusion, mediated by the protein machinery for synaptic secretion. We integrated fusogenic SNAREs in polymer and hybrid vesicles and observed efficient membrane and content mixing. We determined bending rigidity and pore edge tension as key parameters for fusion and described its plausible progression through cryo-EM snapshots. These findings demonstrate that dynamic membrane phenomena can be reconstituted in synthetic materials, thereby providing new tools for the assembly of synthetic protocells.

Tight docking of membranes before fusion represents a metastable state with unique properties.

Membrane fusion is fundamental to biological processes as diverse as membrane trafficking or viral infection. Proteins catalyzing membrane fusion need to overcome energy barriers to induce intermediate steps in which the integrity of bilayers is lost. Here, we investigate the structural features of tightly docked intermediates preceding hemifusion. Using lipid vesicles in which progression to hemifusion is arrested, we show that the metastable intermediate does not require but is enhanced by divalent cations and is characterized by the absence of proteins and local membrane thickening. Molecular dynamics simulations reveal that thickening is due to profound lipid rearrangements induced by dehydration of the membrane surface.

Differential Diffusional Properties in Loose and Tight Docking Prior to Membrane Fusion.

Fusion of biological membranes, although mediated by divergent proteins, is believed to follow a common pathway. It proceeds through distinct steps, including docking, merger of proximal leaflets (stalk formation), and formation of a fusion pore. However, the structure of these intermediates is difficult to study because of their short lifetime. Previously, we observed a loosely and tightly docked state preceding leaflet merger using arresting point mutations in SNARE proteins, but the nature of these states remained elusive. Here, we used interferometric scattering (iSCAT) microscopy to monitor diffusion of single vesicles across the surface of giant unilamellar vesicles (GUVs). We observed that the diffusion coefficients of arrested vesicles decreased during progression through the intermediate states. Modeling allowed for predicting the number of tethering SNARE complexes upon loose docking and the size of the interacting membrane patches upon tight docking. These results shed new light on the nature of membrane-membrane interactions immediately before fusion.

Local externalization of phosphatidylserine mediates developmental synaptic pruning by microglia.

Neuronal circuit assembly requires the fine balance between synapse formation and elimination. Microglia, through the elimination of supernumerary synapses, have an established role in this process. While the microglial receptor TREM2 and the soluble complement proteins C1q and C3 are recognized as key players, the neuronal molecular components that specify synapses to be eliminated are still undefined. Here, we show that exposed phosphatidylserine (PS) represents a neuronal "eat-me" signal involved in microglial-mediated pruning. In hippocampal neuron and microglia co-cultures, synapse elimination can be partially prevented by blocking accessibility of exposed PS using Annexin V or through microglial loss of TREM2. In vivo, PS exposure at both hippocampal and retinogeniculate synapses and engulfment of PS-labeled material by microglia occurs during established developmental periods of microglial-mediated synapse elimination. Mice deficient in C1q, which fail to properly refine retinogeniculate connections, have elevated presynaptic PS exposure and reduced PS engulfment by microglia. These data provide mechanistic insight into microglial-mediated synapse pruning and identify a novel role of developmentally regulated neuronal PS exposure that is common among developing brain structures.

A convenient protocol for generating giant unilamellar vesicles containing SNARE proteins using electroformation.

Reconstitution of membrane proteins in artificial membranes is an essential prerequisite for functional studies that depend on the context of an intact membrane. While straight-forward protocols for reconstituting proteins in small unilamellar vesicles were developed many years ago, it is much more difficult to prepare large membranes containing membrane proteins at biologically relevant concentrations. Giant unilamellar vesicles (GUVs) represent a model system that is characterised by low curvature, controllable tension, and large surface that can be easily visualised with microscopy, but protein insertion is notoriously difficult. Here we describe a convenient method for efficient generation of GUVs containing functionally active SNARE proteins that govern exocytosis of synaptic vesicles. Preparation of proteo-GUVs requires a simple, in-house-built device, standard and inexpensive electronic equipment, and employs a straight-forward protocol that largely avoids damage of the proteins. The procedure allows upscaling and multiplexing, thus providing a platform for establishing and optimizing preparation of GUVs containing membrane proteins for a diverse array of applications.

Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.

Rapid SNARE-Mediated Fusion of Liposomes and Chromaffin Granules with Giant Unilamellar Vesicles.

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins are the main catalysts for membrane fusion in the secretory pathway of eukaryotic cells. In vitro, SNAREs are sufficient to mediate effective fusion of both native and artificial membranes. Here we have established, to our knowledge, a new platform for monitoring SNARE-mediated docking and fusion between giant unilamellar vesicles (GUVs) and smaller liposomes or purified secretory granules with high temporal and spatial resolution. Analysis of fusion is restricted to the free-standing part of the GUV-membrane exhibiting low curvature and a lack of surface contact, thus avoiding adhesion-mediated interference with the fusion reaction as in fusion with supported bilayers or surface-immobilized small vesicles. Our results show that liposomes and chromaffin granules fuse with GUVs containing activated SNAREs with only few milliseconds delay between docking and fusion. We conclude that after initial contact in trans, SNAREs alone can complete fusion at a rate close to fast neuronal exocytosis.

Computed tomography analysis of guinea pig bone: architecture, bone thickness and dimensions throughout development.

The domestic guinea pig, Cavia aperea f. porcellus, belongs to the Caviidae family of rodents. It is an important species as a pet, a source of food and in medical research. Adult weight is achieved at 8-12 months and life expectancy is ∼5-6 years. Our aim was to map bone local thickness, structure and dimensions across developmental stages in the normal animal. Guinea pigs (n = 23) that had died of natural causes were collected and the bones manually extracted and cleaned. Institutional ethical permission was given under the UK Home Office guidelines and the Veterinary Surgeons Act. X-ray Micro Computed Tomography (microCT) was undertaken on the left and right scapula, humerus and femur from each animal to ascertain bone local thickness. Images were also used to undertake manual and automated bone measurements, volumes and surface areas, identify and describe nutrient, supratrochlear and supracondylar foramina. Statistical analysis between groups was carried out using ANOVA with post-hoc testing. Our data mapped a number of dimensions, and mean and maximum bone thickness of the scapula, humerus and femur in guinea pigs aged 0-1 month, 1-3 months, 3-6 months, 6 months-1 year and 1-4 years. Bone dimensions, growth rates and local bone thicknesses differed between ages and between the scapula, humerus and femur. The microCT and imaging software technology showed very distinct differences between the relative local bone thickness across the structure of the bones. Only one bone showed a singular nutrient foramen, every other bone had between 2 and 5, and every nutrient canal ran in an oblique direction. In contrast to other species, a supratrochlear foramen was observed in every humerus whereas the supracondylar foramen was always absent. Our data showed the bone local thickness, bone structure and measurements of guinea pig bones from birth to 4 years old. Importantly it showed that bone development continued after 1 year, the point at which most guinea pigs have reached full weight. This study is the first to show the high abundance (100% in this study) of the supratrochlear foramen within the guinea pig humerus and the complete absence of a supracondylar foramen, which is different to many other species and may also affect potential fracture points and frequencies. Understanding bone morphology and growth is essential in not only understanding the requirements of the healthy guinea pig, but also necessary in order to investigate disease states.

Synaptic PI(3,4,5)P3 is required for Syntaxin1A clustering and neurotransmitter release.

PI(3,4,5)P3 is a low-abundance lipid thought to play a role in the regulation of synaptic activity; however, the mechanism remains obscure. We have constructed novel split Venus-based probes and used superresolution imaging to localize PI(3,4,5)P3 at Drosophila larval neuromuscular synapses. We find the lipid in membrane domains at neurotransmitter release sites, where it concentrates with Syntaxin1A, a protein essential for vesicle fusion. Reducing PI(3,4,5)P3 availability disperses Syntaxin1A clusters and increasing PI(3,4,5)P3 levels rescues this defect. In artificial giant unilamellar vesicles, PI(3,4,5)P3 also induces Syntaxin1A domain formation and this clustering, in vitro and in vivo, is dependent on positively charged residues in the Syntaxin1A-juxtamembrane domain. Functionally, reduced PI(3,4,5)P3 causes temperature-sensitive paralysis and reduced neurotransmitter release, a phenotype also seen in animals expressing a Syntaxin1A with a mutated juxtamembrane domain. Thus, our data indicate that PI(3,4,5)P3, based on electrostatic interactions, clusters Syntaxin1A at release sites to regulate neurotransmitter release.

Characterization of a photonic crystal fiber mode converter using low coherence interferometry.

The relative group delay of the different modes present in an all-fiber LP11 mode converter at a central wavelength of 750 nm is observed using low coherence interferometric imaging. We have simultaneously measured the relative group delay and computed the intensity and the phase distribution of the modes emitted from the mode converter end face using a Fourier technique, providing unequivocal identification of the modes involved.