SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Abstract

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo. Video LinkThe video component of this article can be found at

FisB relies on homo-oligomerization and lipid-binding to catalyze membrane fission in bacteria

“…1 ml of cells was washed twice with PBS containing 50 μg/ml fosfomycin and mounted on a PBS agar pad also containing fosfomycin. Cells were imaged using a Olympus IX81 microscope with a home-built polarized TIRF setup 92,93 . Exposure times were 50 ms for BDR2061 and 100 ms for BMB014.…”

Fusion Pore Formation Observed during SNARE-Mediated Vesicle Fusion with Pore-Spanning Membranes

“…This observation implies a very rapid formation of a fusion pore concomitant with the release of the content dye and collapse of the vesicle into the PSM. The majority of fusion assays on the single vesicle level rely on lipid mixing to observe merging of the two membranes (16,29,31,32,42,43). Assays that make use of the release of an entrapped dye in the vesicle lumen to gather information about fusion pore formation are mainly based on planar membranes supported on glass surfaces lacking a second aqueous compartment (20,22,21,44).…”

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

“…Exocytosis is unique among all biological fusion reactions in that pore dynamics can be observed with sub-millisecond temporal resolution under native conditions using high resolution electrophysiological and electrochemical methods (Travis and Wightman, 1998 ; Lindau, 2012 ). In addition, high temporal resolution of single-pore measurements was recently achieved in biochemically defined systems, promising to illuminate many mechanistic questions (Nikolaus and Karatekin, 2016 ; Stratton et al, 2016 ; Wu et al, 2016 , 2017 ). Finally, atomistic (Blanchard et al, 2014 ; Han et al, 2016b ) and coarse-grained (CG) simulations (Risselada et al, 2011 ; Han et al, 2015 ; Mostafavi et al, 2017 ) of fusogen protein-membrane systems have provided important insights into the role of TMDs in fusion pore regulation.…”