Conical Electron Tomography of a Chemical Synapse: Vesicles Docked to the Active Zone are Hemi-Fused

Zampighi, Guido A.; Zampighi, Lorenzo; Fain, N.; Lanzavecchia, Salvatore; Simon, Sidney A.; Wright, Ernest M.

doi:10.1529/biophysj.106.084814

Cited by 81 publications

(99 citation statements)

References 50 publications

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“…S1, and Table S1; all P values <0.05), indicating that the VM and PM had been fixed during the formation of a single bilayer characteristic of hemifusion, an intermediate state of membrane fusion that precedes fusion pore formation (33). VM-PM hemifusion, as assessed visually, has been reported for CNS synapses (34,35). Extent of VM-PM contact areas.…”

Section: Vm-pm Contact Sites In Terminals Fixed During Repetitive Evokedmentioning

confidence: 91%

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: Vm-pm Contact Sites In Terminals Fixed During Repetitive Evokedmentioning

confidence: 91%

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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“…This method eliminated the bias in selecting the boundaries of the dense layers. By comparing the resolution between both methods, we have estimated that the resolution of the conical maps was ϳ3 nm (Zampighi et al, , 2006.…”

Section: Figurementioning

confidence: 99%

“…To resolve these limitations, we reconstructed chemical synapses of rat neocortex, the archetypical average synapse, by conical electron tomography and analyzed the maps using semiautomatic density segmentation Zampighi et al, 2005;Cantele et al, 2007;Salvi et al, 2008). At ϳ3 nm, the resolution of these methods is sufficiently high to identify vesicles that are hemifused with the membrane of the active zone (Zampighi et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Conical Electron Tomography of a Chemical Synapse: Polyhedral Cages Dock Vesicles to the Active Zone

Zampighi¹,

Fain²,

Zampighi³

et al. 2008

J. Neurosci.

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In this study, we tested the hypothesis that the structure of the active zone of chemical synapses has remained uncertain because of limitations of conventional electron microscopy. To resolve these limitations, we reconstructed chemical synapses of rat neocortex, the archetypical "average" synapse, by conical electron tomography, a method that exhibits an isotropic in plane resolution of ϳ3 nm and eliminates the need to impose symmetry or use averaging methods to increase signal-to-noise ratios. Analysis of 17 reconstructions by semiautomatic density segmentation indicated that the active zone was constructed of a variable number of distinct "synaptic units" comprising a polyhedral cage and a corona of approximately seven vesicles. The polyhedral cages measured ϳ60 nm in diameter, with a density of ϳ44/m 2 and were associated with vesicles at the active zone ("first tier"). Vesicles in this first-tier position represented ϳ7.5% of the total number of vesicles in the terminal and were contiguous, hemifused (ϳ4% of total), or fully fused (ϳ0.5% of total) to the plasma membrane. Our study supports the hypothesis that rat neocortical synapses are constructed of variable numbers of distinct synaptic units that facilitate the docking of vesicles to the active zone and determine the number of vesicles available for immediate release.

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“…Following the "lipidic pore hypothesis", it is now well accepted that membrane fusion involves a sequence of lipidic nonbilayer intermediates, whose formation is catalyzed and guided by a specialized protein machinery (1,2). A stage termed "hemifusion" in which the lipids of the outer leaflets of two membrane-enclosed compartments about to fuse can mix, whereas the inner leaflets and the enclosed content remain unaltered (3), has been confirmed by a variety of methods including conical electron tomography (4) and, more indirectly; e.g., by electron spin resonance (5) and fluorescence recovery after photobleaching (6). Studying the fusion of protein-free bilayers of well defined lipid composition can contribute useful insights into the physical principles governing the merger of their more complex biological counterparts and clarify the effect of individual lipid species.…”

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confidence: 99%

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

Aeffner

Reusch²,

Weinhausen³

et al. 2012

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

161

246

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We have used X-ray diffraction on the rhombohedral phospholipid phase to reconstruct stalk structures in different pure lipids and lipid mixtures with unprecedented resolution, enabling a quantitative analysis of geometry, as well as curvature and hydration energies. Electron density isosurfaces are used to study shape and curvature properties of the bent lipid monolayers. We observe that the stalk structure is highly universal in different lipid systems. The associated curvatures change in a subtle, but systematic fashion upon changes in lipid composition. In addition, we have studied the hydration interaction prior to the transition from the lamellar to the stalk phase. The results indicate that facilitating dehydration is the key to promote stalk formation, which becomes favorable at an approximately constant interbilayer separation of 9.0 AE 0.5 Å for the investigated lipid compositions.curvature energy | hydration force | lipid bilayer | E xocytosis, intracellular transport, neurotransmission, fertilization, or viral entry, require that two membranes merge into one. This event, membrane fusion, involves a complex interplay of different membrane lipids, proteins, and water molecules on length scales of few nm. Following the "lipidic pore hypothesis", it is now well accepted that membrane fusion involves a sequence of lipidic nonbilayer intermediates, whose formation is catalyzed and guided by a specialized protein machinery (1, 2). A stage termed "hemifusion" in which the lipids of the outer leaflets of two membrane-enclosed compartments about to fuse can mix, whereas the inner leaflets and the enclosed content remain unaltered (3), has been confirmed by a variety of methods including conical electron tomography (4) and, more indirectly; e.g., by electron spin resonance (5) and fluorescence recovery after photobleaching (6). Studying the fusion of protein-free bilayers of well defined lipid composition can contribute useful insights into the physical principles governing the merger of their more complex biological counterparts and clarify the effect of individual lipid species.The first connection between two lipid bilayers is the so-called stalk sketched in Fig. 1A (7). The proximal lipid monolayers have merged into one strongly curved monolayer, whereas the distal ones are still separated and intact. A persistent problem in membrane biophysics is to determine the precise structure of stalks and the free energy barrier for stalk formation. In a long series of papers (8-16), this determination has been attempted within the framework of the continuum theory of membrane elasticity (17, 18). More recently, with the advent of sufficient computational power, simulations including molecular details have become feasible [e.g. (19, 20) and references therein]. While stalk formation between lipid bilayers in close contact is generally accepted as the initial step in possibly all membrane fusion reactions (7), the subsequent stages from stalk to complete fusion are still debated and different pathways may exist (21)....

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Conical Electron Tomography of a Chemical Synapse: Vesicles Docked to the Active Zone are Hemi-Fused

Cited by 81 publications

References 50 publications

Variable priming of a docked synaptic vesicle

Variable priming of a docked synaptic vesicle

Conical Electron Tomography of a Chemical Synapse: Polyhedral Cages Dock Vesicles to the Active Zone

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

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