Fusion of Cells by Flipped SNAREs

Hu, Chuan; Ahmed, Mahiuddin; Melia, Thomas J.; Söllner, Thomas H.; Mayer, Thomas; Rothman, James E.

doi:10.1126/science.1084909

Cited by 224 publications

(219 citation statements)

References 28 publications

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“…Using a cell fusion assay, previous studies showed that cells expressing v-and t-SNAREs on the cell surface fuse spontaneously, demonstrating that SNAREs are sufficient to fuse biological membranes [10]. VAMP3 binds to plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25 [22,29,30,34].…”

Section: Resultsmentioning

confidence: 99%

“…Using a cell fusion assay [10], we report here that VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23.…”

Section: Introductionmentioning

confidence: 93%

“…To generate flipped VAMP3, the flipped syntaxin1 plasmid (pCH9) [10] was digested with XbaI and ApaI to excise the coding region of syntaxin1. The vector fragment was purified from an agarose gel.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

“…The signal sequence of preprolactin (SS) was fused to the N-termini of VAMP3, syntaxin4 and SNAP-23. The signal sequence directs translocation into the lumen of endoplasmic reticulum (ER) [10]. The transmembrane domains (TMD) at the C-termini of VAMP3 and syntaxin4 are expected to terminate the translocation and anchor VAMP3 proteins and syntaxin4 proteins on the cell surface after expression and intracellular transport.…”

Section: Flipped Snare Constructsmentioning

confidence: 99%

“…The parallel arrangement of the helices with all amino termini at one end of the helix bundle should bring the membrane anchors of the SNARE proteins and thus the vesicular and target membranes into close proximity. Energy made available from the assembly of the trans-SNARE complexes is used to drive the fusion of lipid bilayers [8][9][10][11]. After membrane fusion, the adapter protein α-SNAP (soluble NSF attachment protein) and the ATPase NSF (Nethylmaleimide-sensitive factor) dissociate the cis-SNARE complexes at the expense of ATP [12,13] to free the SNAREs for the next round of membrane fusion.…”

Section: Introductionmentioning

confidence: 99%

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Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Hardee

Minnear

2007

Experimental Cell Research

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Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the Nterminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the Nterminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 93%

Section: Construction Of Plasmidsmentioning

confidence: 99%

Section: Flipped Snare Constructsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Hardee

Minnear

2007

Experimental Cell Research

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Assessing Cell Fusion and Cytokinesis Failure as Mechanisms of Clone 9 Hepatocyte Multinucleation In Vitro

et al. 2012

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In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed-color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell-impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed-color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual-labeled multinucleated cells have resulted from fusion.

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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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Fusion of Cells by Flipped SNAREs

Cited by 224 publications

References 28 publications

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Assessing Cell Fusion and Cytokinesis Failure as Mechanisms of Clone 9 Hepatocyte Multinucleation In Vitro

Toward a unified picture of the exocytotic fusion pore

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