High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion

Kreutzberger, Alex J. B.; Kiessling, Volker; Tamm, Lukas K.

doi:10.1016/j.bpj.2015.06.022

Cited by 58 publications

(69 citation statements)

References 79 publications

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“…Clustering of t-SNAREs has been reported to be important for promoting membrane fusion and these clusters are cholesterol-dependent (49). Consistently, the Haynes group showed that platelet membrane cholesterol directly affects cargo release from platelets by affecting fusion pore formation, dilation, and full fusion (50).…”

Section: Acylation's Effects On T-snaressupporting

confidence: 50%

Dynamic cycling of t-SNARE acylation regulates platelet exocytosis

Zhang

Huang²,

Chen

et al. 2018

Journal of Biological Chemistry

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ABSTRACT:Platelets regulate vascular integrity by secreting a host of molecules that promote hemostasis and its sequelae. Given the importance of platelet exocytosis, it is critical to understand how it is controlled. The t-SNAREs, SNAP-23 and syntaxin-11, lack classical transmembrane domains (TMDs), yet both are associated with platelet membranes and redistributed into cholesterol-dependent, lipid rafts when platelets were activated. Using metabolic labeling and hydroxylamine/HCl (HA) treatment, we showed that both contain thioester-linked acyl groups. Mass spectrometry mapping further showed that syntaxin-11 was modified on Cysteine 275, 279, 280, 282, 283 and 285, was modified on Cysteine 79, 80, 83, 85, and 87. Interestingly, metabolic labeling studies showed incorporation of [ 3 H]-palmitate into the t-SNAREs increased though the protein levels were unchanged, suggesting that acylation turns over on the two t-SNAREs in resting platelets. Exogenously added fatty acids did compete with [ 3 H]-palmitate for t-SNARE labeling. To determine the effects of acylation, we measured aggregation, ADP/ATP release as well as P-selectin exposure in platelets treated with the acyl-transferase inhibitor, cerulenin, or the thioesterase inhibitor, palmostatin B. We found that cerulenin pretreatment inhibited t-SNARE acylation and platelet function in a dose-and time-dependent manner while palmostatin B had no detectable effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin suggesting that maintaining the acylation state is important for platelet function. Thus, our work shows that t-SNARE acylation is actively cycling in platelets and suggests that the enzymes regulating protein acylation could be potential targets to control platelet exocytosis in vivo.Platelet exocytosis is critical for hemostasis; however, it is increasingly obvious that platelets secrete components with (8)(9)(10)(11)(12)(13)(14). Further studies defined specific SNARE regulators, which control when and where the v-and t-SNAREs interact. Aside from the role that I κ B Kinase-mediated phosphorylation plays in controlling SNAP-23/syntaxin-11 association (15), little is known about other post-translational modifications and their effects on the platelet exocytosis.Despite their importance to secretion, neither SNAP-23 nor syntaxin-11 contains a classical transmembrane domain (TMD), nor any identifiable CAAX motifs. Instead, both proteins contain cysteine-rich regions: human syntaxin-11 has 8 cysteines with 6 at its C-terminus, and SNAP-23 contains 6 cysteines with 5 in a central domain. These cysteine-rich regions have been shown, in some cells, to be important for membrane association and are potential sites for S-acylation (16)(17)(18)(19)(20) (27,28).Past studies have shown that acylation occurs in platelets (29). Dowal et al. characterized over 200 proteins in the platelet "palmitoylome", many of which appear to be involved in platelet signaling pathways (30). Consistently, Sim et al. showed that PAT-inhibit...

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Section: Acylation's Effects On T-snaressupporting

confidence: 50%

Dynamic cycling of t-SNARE acylation regulates platelet exocytosis

Zhang

Huang²,

Chen

et al. 2018

Journal of Biological Chemistry

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“…Indeed, such two-color experiments may provide the only conclusive assessment of the presence of hemifusion in other in vitro fusion assays 18,20 . However, hemifusion can reliably be detected in single-color SUV-SBL experiments monitoring lipid release alone 22,44 .…”

Section: Discussionmentioning

confidence: 99%

“…Others have fused v-SUVs to planar bilayers reconstituted with t-SNAREs [15][16][17][21][22][23][24][25][26][27]29 . The planar geometry of the target (t-SNARE containing) bilayer better mimics the physiological fusion process of small, highly curved vesicles with a flat plasma membrane.…”

Section: Introductionmentioning

confidence: 99%

“…The Steinem group employed pore-spanning membranes reconstituted with t-SNAREs, suspended over a porous silicon nitride substrate and detected fusion with individual v-SUVs using confocal laser scanning microscopy 23 . Others fused v-SUVs to planar bilayers reconstituted with t-SNAREs, supported on a glass substrate [15][16][17]21,22,[24][25][26][27]29 . The great advantage of using supported bilayers (SBLs) is that TIRF microscopy can be used to detect docking and fusion events with excellent signal-to-noise ratio and without interference from free v-SUVs, although using microfluidics also provides single-event resolution using standard far-field epifluorescence microscopy 24 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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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“…These intermediates include tethering and docking of the two membranes to one another, initiation of fusion where a point like protrusion occurs in one of the bilayers, hemifusion stalk formation which may progress into an extended hemifusion diaphragm and then to a fusion pore, Figure 1.1 (Martens & McMahon, 2008;Chernomordik & Kozlov, 2008). In vitro fusion assays also appear to have a dead end off pathway stable hemifusion intermediate that does not lead to the opening of a fusion pore (Diao, et al, 2012;Kreutzberger, et al, 2015;Chlanda, et al, 2016). …”

Section: Membrane Fusionmentioning

confidence: 99%

Reconstitution of SNARE Mediated Membrane Fusion

Kreutzberger

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Regulated exocytosis is a process by which cells release neurotransmitters, peptides, proteins, and small molecules in response to a stimulus. This process is necessary for cell-cell communication in multi-cellular organisms. The final step of regulated exocytosis is membrane fusion, where the membrane of a secretory vesicle merges with the plasma membrane, opening a pore releasing soluble cargo. The molecular machinery that controls fusion is coupled to respond to intracellular calcium. The proteins involved include the SNARE proteins (the fusion machinery), the calcium sensor synaptotagmin, and the regulatory proteins complexin, Munc18, Munc13, and CAPS.I will show the use of planar supported bilayers as a model system to study membrane fusion of single vesicles (Chapter 3). This assay was used to investigate the activity of different plasma membrane target SNARE complexes (Chapter 4), the effect of membrane curvature (Chapter 5) and cholesterol (Chapter 6) on the fusion process, how complexin-1 interacts with the plasma membrane SNARE proteins (Chapter 7), and how the fusion process is coupled to calcium (Chapter 8). These results have led to a new model of membrane fusion. In the absence of calcium, secretory vesicle fusion is arrested in the presence of the SNARE proteins, Munc18, and complexin, but will fuse readily in the presence of calcium. Meanwhile, the protein CAPS or Munc13 act as a kinetic factors controlling the rate of fusion in response to a calcium stimulus.ii

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High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion

Cited by 58 publications

References 79 publications

Dynamic cycling of t-SNARE acylation regulates platelet exocytosis

Dynamic cycling of t-SNARE acylation regulates platelet exocytosis

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

Reconstitution of SNARE Mediated Membrane Fusion

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