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

Joerg Nikolaus; Erdem Karatekin

doi:10.3791/54349

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Method Article

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

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DOI:

10.3791/54349

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August 24th, 2016

Joerg Nikolaus¹^,² , Erdem Karatekin¹^,²^,³^,⁴

¹Department of Cellular and Molecular Physiology, Yale University School of Medicine, ²Nanobiology Institute, Yale University, ³Department of Molecular Biophysics and Biochemistry, Yale University, ⁴Laboratoire de Neurophotonique, Université Paris Descartes, Faculté des Sciences Fondamentales et Biomédicales, Centre National de la Recherche Scientifique (CNRS)

Summary

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

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.

Introduction

Membrane fusion is a universal biological process required for intracellular trafficking of lipids and proteins, secretion, fertilization, development, and enveloped virus entry into host organisms^1-3. For most intracellular fusion reactions including release of hormones and neurotransmitters via exocytosis, the energy to fuse two lipid bilayers is provided by formation of a four-helix bundle between cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, anchored in the vesicle (v-SNARE) and the target membrane (t-SNARE)⁴, respectively. Synaptic vesicle exocytosis is the most tightly regulated fus....