Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy

The fusion pore is the initial narrow connection that forms between fusing membranes. During vesicular release of hormones or neurotransmitters, the nanometer-sized fusion pore may open-close repeatedly (flicker) before resealing or dilating irreversibly, leading to kiss-and-run or full-fusion event...

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Autores principales:	Joerg Nikolaus, Kasey Hancock, Maria Tsemperouli, David Baddeley, Erdem Karatekin
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	membrane fusion SNARE-mediated membrane fusion total internal reflection fluorescence microscopy fusion pore liposome-supported bilayer fusion assay Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/01fcd20291524125bb67f74978da266e
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spelling	oai:doaj.org-article:01fcd20291524125bb67f74978da266e2021-11-10T07:40:24ZOptimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy2296-889X10.3389/fmolb.2021.740408https://doaj.org/article/01fcd20291524125bb67f74978da266e2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fmolb.2021.740408/fullhttps://doaj.org/toc/2296-889XThe fusion pore is the initial narrow connection that forms between fusing membranes. During vesicular release of hormones or neurotransmitters, the nanometer-sized fusion pore may open-close repeatedly (flicker) before resealing or dilating irreversibly, leading to kiss-and-run or full-fusion events, respectively. Pore dynamics govern vesicle cargo release and the mode of vesicle recycling, but the mechanisms are poorly understood. This is partly due to a lack of reconstituted assays that combine single-pore sensitivity and high time resolution. Total internal reflection fluorescence (TIRF) microscopy offers unique advantages for characterizing single membrane fusion events, but signals depend on effects that are difficult to disentangle, including the polarization of the excitation electric field, vesicle size, photobleaching, orientation of the excitation dipoles of the fluorophores with respect to the membrane, and the evanescent field depth. Commercial TIRF microscopes do not allow control of excitation polarization, further complicating analysis. To overcome these challenges, we built a polarization-controlled total internal reflection fluorescence (pTIRF) microscope and monitored fusion of proteoliposomes with planar lipid bilayers with single molecule sensitivity and ∼15 ms temporal resolution. Using pTIRF microscopy, we detected docking and fusion of fluorescently labeled small unilamellar vesicles, reconstituted with exocytotic/neuronal v-SNARE proteins (vSUVs), with a supported bilayer containing the cognate t-SNAREs (tSBL). By varying the excitation polarization angle, we were able to identify a dye-dependent optimal polarization at which the fluorescence increase upon fusion was maximal, facilitating event detection and analysis of lipid transfer kinetics. An improved algorithm allowed us to estimate the size of the fusing vSUV and the fusion pore openness (the fraction of time the pore is open) for every event. For most events, lipid transfer was much slower than expected for diffusion through an open pore, suggesting that fusion pore flickering limits lipid release. We find a weak correlation between fusion pore openness and vesicle area. The approach can be used to study mechanisms governing fusion pore dynamics in a wide range of membrane fusion processes.Joerg NikolausJoerg NikolausKasey HancockKasey HancockKasey HancockMaria TsemperouliMaria TsemperouliDavid BaddeleyDavid BaddeleyErdem KaratekinErdem KaratekinErdem KaratekinErdem KaratekinFrontiers Media S.A.articlemembrane fusionSNARE-mediated membrane fusiontotal internal reflection fluorescence microscopyfusion poreliposome-supported bilayer fusion assayBiology (General)QH301-705.5ENFrontiers in Molecular Biosciences, Vol 8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	membrane fusion SNARE-mediated membrane fusion total internal reflection fluorescence microscopy fusion pore liposome-supported bilayer fusion assay Biology (General) QH301-705.5
spellingShingle	membrane fusion SNARE-mediated membrane fusion total internal reflection fluorescence microscopy fusion pore liposome-supported bilayer fusion assay Biology (General) QH301-705.5 Joerg Nikolaus Joerg Nikolaus Kasey Hancock Kasey Hancock Kasey Hancock Maria Tsemperouli Maria Tsemperouli David Baddeley David Baddeley Erdem Karatekin Erdem Karatekin Erdem Karatekin Erdem Karatekin Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy
description	The fusion pore is the initial narrow connection that forms between fusing membranes. During vesicular release of hormones or neurotransmitters, the nanometer-sized fusion pore may open-close repeatedly (flicker) before resealing or dilating irreversibly, leading to kiss-and-run or full-fusion events, respectively. Pore dynamics govern vesicle cargo release and the mode of vesicle recycling, but the mechanisms are poorly understood. This is partly due to a lack of reconstituted assays that combine single-pore sensitivity and high time resolution. Total internal reflection fluorescence (TIRF) microscopy offers unique advantages for characterizing single membrane fusion events, but signals depend on effects that are difficult to disentangle, including the polarization of the excitation electric field, vesicle size, photobleaching, orientation of the excitation dipoles of the fluorophores with respect to the membrane, and the evanescent field depth. Commercial TIRF microscopes do not allow control of excitation polarization, further complicating analysis. To overcome these challenges, we built a polarization-controlled total internal reflection fluorescence (pTIRF) microscope and monitored fusion of proteoliposomes with planar lipid bilayers with single molecule sensitivity and ∼15 ms temporal resolution. Using pTIRF microscopy, we detected docking and fusion of fluorescently labeled small unilamellar vesicles, reconstituted with exocytotic/neuronal v-SNARE proteins (vSUVs), with a supported bilayer containing the cognate t-SNAREs (tSBL). By varying the excitation polarization angle, we were able to identify a dye-dependent optimal polarization at which the fluorescence increase upon fusion was maximal, facilitating event detection and analysis of lipid transfer kinetics. An improved algorithm allowed us to estimate the size of the fusing vSUV and the fusion pore openness (the fraction of time the pore is open) for every event. For most events, lipid transfer was much slower than expected for diffusion through an open pore, suggesting that fusion pore flickering limits lipid release. We find a weak correlation between fusion pore openness and vesicle area. The approach can be used to study mechanisms governing fusion pore dynamics in a wide range of membrane fusion processes.
format	article
author	Joerg Nikolaus Joerg Nikolaus Kasey Hancock Kasey Hancock Kasey Hancock Maria Tsemperouli Maria Tsemperouli David Baddeley David Baddeley Erdem Karatekin Erdem Karatekin Erdem Karatekin Erdem Karatekin
author_facet	Joerg Nikolaus Joerg Nikolaus Kasey Hancock Kasey Hancock Kasey Hancock Maria Tsemperouli Maria Tsemperouli David Baddeley David Baddeley Erdem Karatekin Erdem Karatekin Erdem Karatekin Erdem Karatekin
author_sort	Joerg Nikolaus
title	Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy
title_short	Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy
title_full	Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy
title_fullStr	Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy
title_full_unstemmed	Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy
title_sort	optimal detection of fusion pore dynamics using polarized total internal reflection fluorescence microscopy
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/01fcd20291524125bb67f74978da266e
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Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy

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