Real-time intermembrane force measurements and imaging of lipid domain morphology during hemifusion

Lee, Dong Woog; Kristiansen, Kai; Donaldson, Stephen H.; Cadirov, Nicholas; Banquy, Xavier; Israelachvili, Jacob N.

doi:10.1038/ncomms8238

Cited by 26 publications

(28 citation statements)

References 30 publications

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“…Force is known to drive destabilization of apposed artificial lipid membranes toward fusion threshold (8). We found that the average eccentricity of the docked SVs' ellipsoid shape in resting terminals was significantly greater than that of undocked SVs.…”

Section: Discussionmentioning

confidence: 77%

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Variable priming of a docked synaptic vesicle

Jung

Szule

Marshall

et al. 2016

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

119

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The priming of a docked synaptic vesicle determines the probability of its membrane (VM) fusing with the presynaptic membrane (PM) when a nerve impulse arrives. To gain insight into the nature of priming, we searched by electron tomography for structural relationships correlated with fusion probability at active zones of axon terminals at frog neuromuscular junctions. For terminals fixed at rest, the contact area between the VM of docked vesicles and PM varied >10-fold with a normal distribution. There was no merging of the membranes. For terminals fixed during repetitive evoked synaptic transmission, the normal distribution of contact areas was shifted to the left, due in part to a decreased number of large contact areas, and there was a subpopulation of large contact areas where the membranes were hemifused, an intermediate preceding complete fusion. Thus, fusion probability of a docked vesicle is related to the extent of its VM-PM contact area. For terminals fixed 1 h after activity, the distribution of contact areas recovered to that at rest, indicating the extent of a VM-PM contact area is dynamic and in equilibrium. The extent of VM-PM contact areas in resting terminals correlated with eccentricity in vesicle shape caused by force toward the PM and with shortness of active zone material macromolecules linking vesicles to PM components, some thought to include Ca 2+ channels. We propose that priming is a variable continuum of events imposing variable fusion probability on each vesicle and is regulated by force-generating shortening of active zone material macromolecules in dynamic equilibrium.synapse | hemifusion | priming | active zone material | electron tomography S ynaptic vesicles (SVs) move toward and dock on (are held in contact with) the presynaptic plasma membrane (PM) of a neuron's axon terminal before fusing with the PM and releasing their neurotransmitter into the synaptic cleft to mediate synaptic transmission (1, 2). Docking is achieved by force-generating interactions of the vesicle membrane (VM) protein synaptobrevin with the PM proteins syntaxin and SNAP25 (3, 4). These interactions, which produce force by forming a coiled coil called the SNARE core complex, are regulated by auxiliary proteins (1, 5-7). Such force on the VM-PM contact site may also play a role in their fusion (8). At typical synapses, docking and fusion take place at structurally specialized regions along the PM called active zones (9, 10). Several lines of evidence suggest that the formation of the SNARE core complex occurs in the macromolecules composing the common active zone organelle, active zone material (AZM) (2,(11)(12)(13)(14), which is positioned near Ca 2+ channels concentrated in the PM at active zones (15-17). Influx of Ca 2+ through the channels after the arrival of a nerve impulse triggers fusion of the VM of docked SVs with the PM.Priming is a step in synaptic transmission between the docking of an SV on, and fusion with, the PM and accounts for the observation that relatively few docked SVs fuse with the PM...

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Section: Discussionmentioning

confidence: 77%

“…These interactions, which produce force by forming a coiled coil called the SNARE core complex, are regulated by auxiliary proteins (1,(5)(6)(7). Such force on the VM-PM contact site may also play a role in their fusion (8). At typical synapses, docking and fusion take place at structurally specialized regions along the PM called active zones (9,10).…”

mentioning

confidence: 99%

Variable priming of a docked synaptic vesicle

Jung

Szule

Marshall

et al. 2016

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

119

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“…At concentrations lower than the CAC, 5aTHQs behave as hydrophilic molecules in aqueous solutions,s howing low/no membrane affinity.W hen the concentration is higher than the CAC, aggregate forms appear.5 aTHQs in aggregate forms behave as hydrophobic molecules,a nd can fuse with and reside in lipid membranes.Ingeneral, the most important force leading to the direct fusion of membranes is hydrophobic interactions, which supports this model. [29] Molecules in membrane structures have to flexibly move for hemifusion by decreasing steric and electrostatic repulsions. [29] Molecules in membrane structures have to flexibly move for hemifusion by decreasing steric and electrostatic repulsions.…”

Section: Resultsmentioning

confidence: 99%

Chemical Interactions of Cryptic Actinomycete Metabolite 5‐Alkyl‐1,2,3,4‐tetrahydroquinolines through Aggregate Formation

et al. 2019

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Organisms often produce secondary metabolites as amixture of biosynthetically related congeners.However,why are metabolites with minor chemical variations produced simultaneously?5 -Alkyl-1,2,3,4-tetrahydroquinolines (5aTHQs) are small, lipophilic metabolites produced by Streptomyces nigrescens HEK616 when cultured with Tsukamurella pulmonis TP-B0596. Am ixture of 5aTHQs forms aggregates that showenhanced membrane affinity and biological activity.T he ability to form aggregates and membranebinding activity is regulated by the length of the alkylc hains. Aggregates with long alkylc hains were too stable to fuse with lipid membranes.H owever,i fi nactive 5aTHQ congener was mixed with active congener,t he mixture showed increased membrane affinity,e nabling cellular entry and biological activity.Therefore,itisshown that sloppiness in abiosynthetic pathway,b yw hichm inor structural variations can be produced, is functionally rational, as the metabolites show synergistic action.

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“…In the presence of calcium but absence of SNAREs, any fusion-related events are detected mainly if the integrity of the supported bilayer was partly compromised (i.e., showing defects) (17,18). The likelihood for membranes to cross the hydration barrier but not to merge increases strongly from 9 to 15% when PEG and calcium were added to the system, attributed to lowering of the hydration barrier.…”

Section: Resultsmentioning

confidence: 99%

“…Although precise data for thermodynamics and kinetics of SNARE zippering and unzippering are now available (11,14,15), few studies address how the energy landscape of membrane fusion is shaped by participation of SNAREs. Israelachvili and coworkers (16)(17)(18) were among the first to measure fusion events in the absence of proteins as a function of external force using a surface force apparatus, and Moy and coworkers (19) measured the interaction forces between reconstituted SNARE derivatives, reporting fusion events as mechanical instabilities in the approach curve of the two membrane-coated substrates. Additional advances have been made by Brouwer et al (20) and Keidel et al (21) by using optical tweezers for studying some aspects of membrane fusion.…”

mentioning

confidence: 99%

SNARE-mediated membrane fusion trajectories derived from force-clamp experiments

Oelkers

Witt

Halder

et al. 2016

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

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Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomicforce microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed.embrane fusion plays a pivotal role in many fundamental biological processes, comprising viral infection, cell-cell fusion during fertilization, tissue formation, and intracellular transport during exo-and endocytosis (1). Among the best-studied biological examples is the Ca 2+ -regulated fusion of synaptic vesicles with the presynaptic plasma membrane in neurons and chromaffin cells (2, 3). Fusion in these cells is catalyzed by concerted action of SNARE proteins through formation of a tetrameric coiled-coil complex that releases a sufficient amount of free energy to lower the barriers for membrane merging. One major energy barrier is associated with the strong repulsive hydration forces when two smooth bilayers approach each other. Other energy-costly contributions originate from lipid splaying as the initiation of stalk formation, expansion of the stalk structure driven by the strong curvature associated with membrane destabilization, hemifusion diaphragm expansion, and, finally, pore formation (4-8). The free energy associated with complex formation and folding of SNAREs was reported to be ∼35 k B T, close to the energy required to create the highly curved transition structures (40-50 k B T) (9, 10). Albeit a part of this free energy might be dissipated as heat, this coincidence of matching energy is also supported by experimental studies showing that only very few SNARE complexes are sufficient to induce fusion in artificial systems (5,6,(11)(12)(13). Although precise data for thermodynamics and kinetics of SNARE zippering and unzippering are now available (11,14,15), few studies address how the energy landscape of membrane fusion is shaped by participation of SNAREs. were among the first to measure fusion events in the absence of proteins as a function of external force using a surface force apparatus, and Moy and coworkers (19) m...

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Real-time intermembrane force measurements and imaging of lipid domain morphology during hemifusion

Cited by 26 publications

References 30 publications

Variable priming of a docked synaptic vesicle

Variable priming of a docked synaptic vesicle

Chemical Interactions of Cryptic Actinomycete Metabolite 5‐Alkyl‐1,2,3,4‐tetrahydroquinolines through Aggregate Formation

SNARE-mediated membrane fusion trajectories derived from force-clamp experiments

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