A Nanodisc-Cell Fusion Assay with Single-Pore Sensitivity and Sub-millisecond Time Resolution

Dudzinski, Natasha; Wu, Zhenyong; Karatekin, Erdem

doi:10.1007/978-1-4939-8760-3_17

Cited by 6 publications

(14 citation statements)

References 34 publications

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“…3A). To prevent exclusion of low conductance signals from analysis, we set a threshold close to the baseline and a minimum threshold-crossing duration to detect open subperiods in a burst [38,62]. Pore properties exhibit broad distributions ( Fig.…”

Section: Nanodisc-cell Fusionmentioning

confidence: 99%

“…As for the ND-cell experiments, the composition of the outer plasma membrane is very different than that of the inner leaflet which a vesicle normally encounters during exocytosis. During ND-cell fusion, we actually do find some pores that have relatively stable conductances, but many of these are excluded from analysis because their amplitude/duration is lower than the set threshold [38,62]. Channellike pores that conform to the cut-off to be included in analysis are rare and the conductances are not uniform for a given condition (Fig.…”

Section: Nanodisc-black Lipid Membrane (Blm) Fusionmentioning

confidence: 99%

“…Release of sulforhodamine B from single t‐ SNARE liposomes upon fusion with v‐ SNARE ND s has also been monitored in a dye efflux assay using surface‐tethered liposomes . (C) ND ‐cell fusion can be monitored in various manners . Top: ‘flipped’ t‐ SNARE cells expressing complementary neuronal t‐ SNARE s ectopically with the SNARE domain facing the extracellular space were fused with v‐ SNARE ND s .…”

Section: Nanodisc‐based Fusion Assays: a New Look At Fusion Poresmentioning

confidence: 99%

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Toward a unified picture of the exocytotic fusion pore

Karatekin

2018

FEBS Letters

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Neurotransmitter and hormone release involve calcium-triggered fusion of a cargo-loaded vesicle with the plasma membrane. The initial connection between the fusing membranes, called the fusion pore, can evolve in various ways, including rapid dilation to allow full cargo release, slow expansion, repeated opening-closing, and resealing. Pore dynamics determine the kinetics of cargo release and the mode of vesicle recycling, but how these processes are controlled are poorly understood. Previous reconstitutions could not monitor single pores, limiting mechanistic insight they could provide. Recently developed nanodisc-based fusion assays allow reconstitution and monitoring of single pores with unprecedented detail and hold great promise for future discoveries. They recapitulate various aspects of exocytotic fusion pores, but comparison is difficult because different approaches suggested very different exocytotic fusion pore properties, even for the same cell type. In this Review, I discuss how most of the data can be reconciled, by recognizing how different methods probe different aspects of the same fusion process. The resulting picture is that fusion pores have broadly distributed properties arising from stochastic processes which can be modulated by physical constraints imposed by proteins, lipids and membranes.

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Section: Nanodisc-cell Fusionmentioning

confidence: 99%

Section: Nanodisc-black Lipid Membrane (Blm) Fusionmentioning

confidence: 99%

Section: Nanodisc‐based Fusion Assays: a New Look At Fusion Poresmentioning

confidence: 99%

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Toward a unified picture of the exocytotic fusion pore

Karatekin

2018

FEBS Letters

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“…The zippering of the cognate vSNARE and tSNARE into SNAREpins brings the two membranes in close apposition and eventually opens up a fusion pore between the vesicle and the target compartment. The expansion of the fusion pore beyond 2 nm in diameter has been largely studied, both in vivo and in vitro ( 5 – 12 ). However, the efficiency of cargo release is likely to depend on the initial phase of the expansion process, that is, when the pore is below 2 nm.…”

mentioning

confidence: 99%

“…This scenario is only scarcely described in the literature because of technical difficulties required to accurately characterize such small pores. Recent studies based on in vitro electrophysiology approaches have shown the possibility to overcome these limitation1 using nanodisc-based fusion processes ( 7 – 11 ).…”

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confidence: 99%

Nascent fusion pore opening monitored at single-SNAREpin resolution

Heo

Coleman

Fleury

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Vesicle fusion with a target membrane is a key event in cellular trafficking and ensures cargo transport within the cell and between cells. The formation of a protein complex, called SNAREpin, provides the energy necessary for the fusion process. In a three-dimensional microfluidic chip, we monitored the fusion of small vesicles with a suspended asymmetric lipid bilayer. Adding ion channels into the vesicles, our setup allows the observation of a single fusion event by electrophysiology with 10-μs precision. Intriguingly, we identified that small transient fusion pores of discrete sizes reversibly opened with a characteristic lifetime of ∼350 ms. The distribution of their apparent diameters displayed two peaks, at 0.4 ± 0.1 nm and 0.8 ± 0.2 nm. Varying the number of SNAREpins, we demonstrated that the first peak corresponds to fusion pores induced by a single SNAREpin and the second peak is associated with pores involving two SNAREpins acting simultaneously. The pore size fluctuations provide a direct estimate of the energy landscape of the pore. By extrapolation, the energy landscape for three SNAREpins does not exhibit any thermally significant energy barrier, showing that pores larger than 1.5 nm are spontaneously produced by three or more SNAREpins acting simultaneously, and expand indefinitely. Our results quantitatively explain why one SNAREpin is sufficient to open a fusion pore and more than three SNAREpins are required for cargo release. Finally, they also explain why a machinery that synchronizes three SNAREpins, or more, is mandatory to ensure fast neurotransmitter release during synaptic transmission.

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The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

Dharan

Thiyagarajan

et al. 2019

Preprint

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All membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered vesicular release of neurotransmitters and hormones. Expansion of this small pore to release cargo molecules is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins, beyond its established role in coupling calcium influx to fusion pore opening. Fusion pore dilation by Syt1 required interactions with SNAREs, PI(4,5)P2, and calcium. Calcium-induced insertion of the tandem C2 domain (C2AB) hydrophobic loops of Syt1 into the membrane is required for pore opening. We find that pore expansion also requires loop insertion, but through a distinct mechanism. Mathematical modelling suggests that membrane insertion re-orients the C2 domains bound to the SNARE complex, rotating the SNARE complex so as to exert force on the membranes in a mechanical lever action that increases the intermembrane distance. The increased membrane separation provokes pore dilation to offset a bending energy penalty. Our results show that Syt1 can assume a critical mechanical role in calcium-dependent fusion pore dilation during neurotransmitter and hormone release, distinct from its proposed role in generating curvature required for pore opening. SIGNIFICANCE STATEMENTMembrane fusion is a fundamental biological process, required for development, infection by enveloped viruses, fertilization, intracellular trafficking, and calcium-triggered release of neurotransmitters and hormones when cargo-laden vesicles fuse with the plasma membrane. All membrane fusion reactions proceed through a an initial, nanometer-sized fusion pore which can flicker open-closed multiple times before expanding or resealing. Pore expansion is required for efficient cargo release, but underlying mechanisms are poorly understood. Using a combination of single-pore measurements and quantitative modeling, we suggest that a complex between the neuronal calcium sensor Synaptotagmin-1 and the SNARE proteins together act as a calcium-sensitive mechanical lever to force the membranes apart and enlarge the pore, distinct from the curvature-generating mechanism of Synaptotagmin-1 proposed to promote pore opening.

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A Nanodisc-Cell Fusion Assay with Single-Pore Sensitivity and Sub-millisecond Time Resolution

Cited by 6 publications

References 34 publications

Toward a unified picture of the exocytotic fusion pore

Toward a unified picture of the exocytotic fusion pore

Nascent fusion pore opening monitored at single-SNAREpin resolution

The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

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