Temporal Separation of Vesicle Release from Vesicle Fusion during Exocytosis

Troyer, Kevin P.; Wightman, R. Mark

doi:10.1074/jbc.m202856200

Cited by 52 publications

(65 citation statements)

References 49 publications

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“…4 and 8). The likely explanation is that, under hypertonic conditions known to inhibit endocytosis (37)(38)(39)(40), components of exocytosed vesicles can migrate laterally in the plasma membrane (e.g. Ref.…”

Section: Discussionmentioning

confidence: 99%

Properties of Synaptic Vesicle Pools in Mature Central Nerve Terminals

Ashton

Ushkaryov

2005

Journal of Biological Chemistry

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Readily releasable and reserve pools of synaptic vesicles play different roles in neurotransmission, and it is important to understand their recycling and interchange in mature central synapses. Using adult rat cerebrocortical synaptosomes, we have shown that 100 mosM hypertonic sucrose caused complete exocytosis of only the readily releasable pool (RRP) of synaptic vesicles containing glutamate or ␥-aminobutyric acid. Repetitive hypertonic stimulations revealed that this pool recycled (and reloaded the neurotransmitter from the cytosol) fully in <30 s and did so independently of the reserve pool. Multiple rounds of exocytosis could occur in the constant absence of extracellular Ca 2؉ . However, although each vesicle cycle includes a Ca 2؉ -independent exocytotic step, some other stage(s) critically require an elevation of cytosolic [Ca 2؉ ], and this is supplied by intracellular stores. Repetitive recycling also requires energy, but not the activity of phosphatidylinositol 4-kinase, which maintains the normal level of phosphoinositides. By varying the length of hypertonic stimulations, we found that ϳ70% of the RRP vesicles fused completely with the plasmalemma during exocytosis and could then enter silent pools, probably outside active zones. The rest of the RRP vesicles underwent very fast local recycling (possibly by kiss-and-run) and did not leave active zones. Forcing the fully fused RRP vesicles into the silent pool enabled us to measure the transfer of reserve vesicles to the RRP and to show that this process requires intact phosphatidylinositol 4-kinase and actin microfilaments. Our findings also demonstrate that respective vesicle pools have similar characteristics and requirements in excitatory and inhibitory nerve terminals.Synaptic vesicles in a nerve terminal that undergo release/recycling consist of the readily releasable pool (RRP) 2 and the reserve pool (1, 2). These vesicle populations play distinct roles in neurotransmission, and their relative sizes change with synapse maturation (3, 4). Therefore, to understand the physiologically relevant properties of the vesicle pools, it is important to study them in mature central nerve terminals. When conducted in acute slices of adult brain, such studies are usually limited to specific regions of the hippocampus and thus do not necessarily reflect the behavior of vesicle pools in the rest of the brain. An alternative, well established system, cerebrocortical synaptosomes, is more universal and has several important advantages: (i) synchronized stimulation of all nerve terminals even by viscous solutions such as sucrose, (ii) direct comparison of terminals containing different transmitters, (iii) high-throughput pharmacological analysis, (iv) evaluation of multiple samples, and (v) uniform neuronal sampling. Finally, synaptosomal studies reveal pathways that are common to most nerve terminals, providing a level of generalization that is difficult to achieve by studying individual synapses.This simplified model, which lacks axons and cell bodies, is a...

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Section: Discussionmentioning

confidence: 99%

Properties of Synaptic Vesicle Pools in Mature Central Nerve Terminals

Ashton

Ushkaryov

2005

Journal of Biological Chemistry

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“…1Diii). This might reflect a form of kiss-and-run or 'kiss-and-hold' (Troyer and Wightman, 2002), in which exocytosis reversed or arrested after formation of a fusion pore. Alternatively the acrosomal matrix of these few cells might be effectively insoluble.…”

Section: Temporal and Spatial Characteristics Of The Acrosome Reactionmentioning

confidence: 99%

Dynamic resolution of acrosomal exocytosis in human sperm

Harper

Cummerson

White

et al. 2008

Journal of Cell Science

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An essential step in mammalian fertilisation is the sperm acrosome reaction (AR) – exocytosis of a single large vesicle (the acrosome) that surrounds the nucleus at the apical sperm head. The acrosomal and plasma membranes fuse, resulting in both the release of factors that might facilitate penetration of the zona pellucida (which invests the egg) and the externalisation of membrane components required for gamete fusion. Exocytosis in somatic cells is a rapid process – typically complete within milliseconds – yet acrosomal enzymes are required throughout zona penetration – a period of minutes. Here, we present the first studies of this crucial and complex event occurring in real-time in individual live sperm using time-lapse fluorescence microscopy. Simultaneous imaging of separate probes for acrosomal content and inner acrosomal membrane show that rapid membrane fusion, initiated at the cell apex, is followed by exceptionally slow dispersal of acrosomal content (up to 12 minutes). Cells that lose their acrosome prematurely (spontaneous AR), compromising their ability to penetrate the egg vestments, are those that are already subject to a loss of motility and viability. Cells undergoing stimulus-induced AR (progesterone or A23187) remain viable, with a proportion remaining motile (progesterone). These findings suggest that the AR is a highly adapted form of exocytosis.

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“…The formation of this pore is a complex event involving physical and chemical factors and is regulated by intracellular Ca 2ϩ and proteins (7). Studies on mast cells and chromaffin cells revealed that the opening of fusion pore and extrusion of granule content is influenced by an osmotic force depending upon the osmolarity of the surrounding solution (8).…”

mentioning

confidence: 99%

Fusion of Lamellar Body with Plasma Membrane Is Driven by the Dual Action of Annexin II Tetramer and Arachidonic Acid

Chattopadhyay

Sun

Wang

et al. 2003

Journal of Biological Chemistry

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Annexin II has been implicated in membrane fusion during the exocytosis of lamellar bodies from alveolar epithelial type II cells. Most previous studies were based on the fusion assays by using model membranes. In the present study, we investigated annexin II-mediated membrane fusion by using isolated lamellar bodies and plasma membrane as determined by the relief of octadecyl rhodamine B (R18) self-quenching. Immunodepletion of annexin II from type II cell cytosol reduced its fusion activity. Purified annexin II tetramer (AIIt) induced the fusion of lamellar bodies with the plasma membrane in a dose-dependent manner. This fusion is Ca 2؉ -dependent and is highly specific to AIIt because other annexins (I and II monomer, III, IV, V, and VI) were unable to induce the fusion. Modification of the different functional residues of AIIt by N-ethylmaleimide, nitric oxide, or peroxynitrite abolished AIIt-mediated fusion. Arachidonic acid enhanced AIIt-mediated fusion and reduced its Ca 2؉ requirement to an intracellularly achievable level. This effect is due to membranebound arachidonic acid, not free arachidonic acid. Other fatty acids including linolenic acid, palmitoleic acid, myristoleic acid, stearic acid, palmitic acid, and myristic acid had little effect. AIIt-mediated fusion was suppressed by the removal of arachidonic acid from lamellar body and plasma membrane using bovine serum albumin. The addition of arachidonic acid back to the arachidonic acid-depleted membranes restored its fusion activity. Our results suggest that the fusion between lamellar bodies with the plasma membrane is driven by the synergistic action of AIIt and arachidonic acid.Lung surfactant is a surface active material synthesized and secreted by cuboidal alveolar type II cells (1-3). It is composed of phospholipids, mainly dipalmitoylphosphatidylcholine, and surfactant proteins A-D. It minimizes the surface tension at the air-liquid interface by inserting phospholipids between water molecules. As a result, surfactant disrupts the cohesive hydrogen bonds and prevents them from exerting high molecular forces at the alveolar surface. Deficiency of dipalmitoylphosphatidylcholine in the alveolar surface has been associated with infant respiratory distress syndrome.The secretion of surfactant by type II cells involves the translocation, docking, and fusion of lamellar bodies to the plasma membrane. For the lamellar body content to reach its extracellular destination, a continuity of the vesicular lumen and extracellular fluid must be established. This continuity is maintained through the formation of a narrow pore similar to membrane channels (4 -6). The formation of this pore is a complex event involving physical and chemical factors and is regulated by intracellular Ca 2ϩ and proteins (7). Studies on mast cells and chromaffin cells revealed that the opening of fusion pore and extrusion of granule content is influenced by an osmotic force depending upon the osmolarity of the surrounding solution (8).Annexin II plays multidimensional roles in dif...

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Temporal Separation of Vesicle Release from Vesicle Fusion during Exocytosis

Cited by 52 publications

References 49 publications

Properties of Synaptic Vesicle Pools in Mature Central Nerve Terminals

Properties of Synaptic Vesicle Pools in Mature Central Nerve Terminals

Dynamic resolution of acrosomal exocytosis in human sperm

Fusion of Lamellar Body with Plasma Membrane Is Driven by the Dual Action of Annexin II Tetramer and Arachidonic Acid

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