Witkowska, A. | bioRxiv Science

bioRxiv2020

Local externalization of phosphatidylserine mediates developmental synaptic pruning by microglia

Neuronal circuits assembly requires the fine equilibrium 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 key players in this process, the neuronal molecular components that tag synapses to be eliminated are still undefined. Here we show that exposed phosphatidylserine (PS) represents a neuronal eat-me signal enabling microglial-mediated synapse pruning. In hippocampal neuron and microglia co-cultures, synapse elimination can be prevented by blocking accessibility of exposed PS using Annexin V or through microglial loss of TREM2. In vivo, exposed PS is detectable at both hippocampal and retinogeniculate synapses, where exposure coincides with the onset of synapse elimination and increased PS engulfment by microglia. Mice deficient in C1q, which fail to properly refine retinogeniculate connections, display elevated exposed PS and reduced PS engulfment by microglia. These data provide mechanistic insight into microglial-mediated synapse pruning and identify a novel role of developmentally regulated PS exposure that is common among developing brain structures.

neuroscience↗

bioRxiv2020

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 due to 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.

biophysics↗