Liposome Fusion Assay to Monitor Intracellular Membrane Fusion Machines

Scott, Brenton L.; Komen, Jeffrey S. Van; Liu, Song; Weber, Thomas; Melia, Thomas J.; McNew, James A.

doi:10.1016/s0076-6879(03)72016-3

Cited by 61 publications

(101 citation statements)

References 40 publications

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“…GLUT4 exocytic t-SNAREs, comprising the untagged syntaxin-4 and the His 6 -tagged SNAP-23, were expressed using the same procedure as described previously for synaptic t-SNAREs (34,35). The v-SNARE proteins were expressed in a similar way as VAMP2 (36) and had no extra residues left after the tags were proteolytically removed.…”

Section: Methodsmentioning

confidence: 99%

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

Rathore

Shen

2013

Journal of Biological Chemistry

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Background: Synip is a SNARE-binding regulatory factor whose molecular mechanism remains unclear. Results: Synip acts as a selective t-SNARE-binding inhibitor that arrests membrane fusion by preventing the initiation of ternary SNARE complex assembly. Conclusion: Synip function likely represents a novel regulatory mechanism of vesicle fusion. Significance: Studies of vesicle fusion regulation provide key insights into the mechanisms of vesicle transport.

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Section: Methodsmentioning

confidence: 99%

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

Rathore

Shen

2013

Journal of Biological Chemistry

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“…All proteoliposomes were prepared by detergent dilution and dialysis and used in standard fusion assays as described previously (35).…”

Section: Methodsmentioning

confidence: 99%

The Polybasic Juxtamembrane Region of Sso1p Is Required for SNARE Function In Vivo

et al. 2005

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Exocytosis in Saccharomyces cerevisiae requires the specific interaction between the plasma membrane t-SNARE complex (Sso1/2p;Sec9p) and a vesicular v-SNARE (Snc1/2p). While SNARE proteins drive membrane fusion, many aspects of SNARE assembly and regulation are ill defined. Plasma membrane syntaxin homologs (including Sso1p) contain a highly charged juxtamembrane region between the transmembrane helix and the "SNARE domain" or core complex domain. We examined this region in vitro and in vivo by targeted sequence modification, including insertions and replacements. These modified Sso1 proteins were expressed as the sole copy of Sso in S. cerevisiae and examined for viability. We found that mutant Sso1 proteins with insertions or duplications show limited function, whereas replacement of as few as three amino acids preceding the transmembrane domain resulted in a nonfunctional SNARE in vivo. Viability is also maintained when two proline residues are inserted in the juxtamembrane of Sso1p, suggesting that helical continuity between the transmembrane domain and the core coiled-coil domain is not absolutely required. Analysis of these mutations in vitro utilizing a reconstituted fusion assay illustrates that the mutant Sso1 proteins are only moderately impaired in fusion. These results suggest that the sequence of the juxtamembrane region of Sso1p is vital for function in vivo, independent of the ability of these proteins to direct membrane fusion.Biological membrane fusion is imperative for cellular survival. Membrane fusion requires an energy-dependent reaction to overcome the repulsive nature of two opposing membranes (4, 10, 11). In cells, conformational rearrangements of specific fusion proteins drive this membrane merger (9, 44). Membrane fusion reactions in the secretory pathway have been extensively studied, and much is known about the molecular machinery, and yet many aspects of this process are not well understood. SNARE proteins (Soluble NSF attachment protein receptors) constitute the core fusion machinery (38,49) and are the final arbiters of fusion specificity (23). SNAREs are operationally divided into two groups: those that are found primarily on the transport vesicle, called v-SNAREs, and those found primarily on the target membrane, called t-SNAREs. The founding members of the SNARE superfamily were identified from bovine brain and participate in synaptic transmission (38). The neuronal SNARE complex is comprised of two t-SNAREs localized to the plasma membrane, called syntaxin 1A (3) and SNAP25 (synaptosome-associated protein of 25 kDa [28]), as well as one v-SNARE located on the synaptic vesicle, known as VAMP (vesicle-associated membrane protein or synaptobrevin [40,43]). SNARE proteins also share structural and mechanistic commonalities with viral fusogens, such as the assembly of a coiledcoil bundle structure and a hemifusion transition-state intermediate (15,22,36,39,51). However, several elements that regulate membrane fusion, including proteins such as Rab GTPases and the exocyst complex, re...

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“…LP for v-SNAREs is much lower (LP ~200, 7,000 v-SNAREs per µm 2 ), close to the v-SNARE densities found on synaptic vesicles 46,47 . To ensure protein function after recombinant expression and purification of v-and t-SNAREs it is useful to perform a simple bulk fusion assay 48 before going through all the steps of the single vesicle fusion assay. 7.…”

Section: Preparation Of V-suvs Containing Both Lipid and Content Labelsmentioning

confidence: 99%

“…13. Characterize SUV sizes using dynamic light scattering 24 or electron microscopy 48 . Store SRB containing v-SUVs at 4 °C up to ~3-4 days.…”

Section: Preparation Of V-suvs Containing Both Lipid and Content Labelsmentioning

confidence: 99%

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

Nikolaus

Karatekin

2016

JoVE

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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. Video LinkThe video component of this article can be found at

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Liposome Fusion Assay to Monitor Intracellular Membrane Fusion Machines

Cited by 61 publications

References 40 publications

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

The Polybasic Juxtamembrane Region of Sso1p Is Required for SNARE Function In Vivo

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

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