Optimizing Excitation Polarization to Probe Fusion Pore Dynamics using TIRF Microscopy

Abstract

During hormone or neurotransmitter release via exocytosis, fusion pores may open and close (flicker) repeatedly before resealing (“kiss & run” fusion) or dilating (full fusion) irreversibly. Pore dynamics regulate the amount and kinetics of cargo release, and determine the mode of recycling, but mechanisms that govern pore dynamics are not understood. This is in large part due to a lack of reconstituted assays with single-pore sensitivity and millisecond time resolution. We recently described a polarized total internal reflection (pTIRF) microscopy assay to monitor fusion of proteoliposomes to planar lipid bilayers supported on a soft polymer cushion with single molecule sensitivity and ∼15 ms temporal resolution. Fluorescently labeled small unilamellar vesicles, reconstituted with exocytotic/neuronal v-SNAREs (v-SUVs), fuse with a supported bilayer containing the cognate t-SNAREs (t-SBL). Each fusion event is accompanied by changes in the total fluorescence intensity surrounding the fusion site, as the lipid-linked labels diffuse from the liposome into the supported bilayer through the fusion pore. Analysis of the intensity changes, combined with a mathematical model, provides information on pore dynamics (Stratton et al. Biophys. J. 2016). In principle, three factors can contribute to intensity changes upon fusion: 1) dequenching of fluorophores, 2) evanescent field decay and 3) a change in the average orientation of the fluorophore dipole moments with respect to the excitation polarization field, as the fluorophores move from the round liposome into the flat bilayer. Here, we systematically varied the polarization of the excitation field and quantified its contribution to intensity changes for different lipid-linked fluorophores. Large increases facilitate detection of fusion events and quantification of lipid release kinetics.