Differential diffusional properties in loose and tight docking prior to membrane fusion

Abstract

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. membrane fusion intermediates | iSCAT microscopy | SNARE proteins | particle tracking | tethered diffusion | GUV Correspondence: rjahn@gwdg.de (R.J.), vahid.sandoghdar@mpl.mpg.de (V.S.), and agata.witkowska@wp.eu (A.W.)

Kinetic analysis of neuronal SNARE protein interactions

“…The joining of the two lipid bilayers is influenced by factors such as charge, curvature, lipid composition, and by specific proteins present on the membrane surface. There are several recognized stages that characterize fusion: (i) docking; (ii) dehydration; (iii) hemifusion stalk formation; (iv) pore opening and expansion (Hernández and Podbilewicz, 2017;Witkowska et al, 2020Witkowska et al, , 2021Yavuz et al, 2018). These stages are accompanied by corresponding energy barriers that need to be overcome in order for fusion to occur.…”

“…of SNARE complex formation in different stages of fusion (Hernandez et al, 2012;Witkowska et al, 2020;Yavuz et al, 2018).…”