a-Synuclein is a synaptic modulatory protein implicated in the pathogenesis of Parkinson disease. The precise functions of this small cytosolic protein are still under investigation. a-Synuclein has been proposed to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins involved in vesicle fusion. Interestingly, a-synuclein fails to interact with SNARE proteins in conventional protein-binding assays, thus suggesting an indirect mode of action. As the structural and functional properties of both a-synuclein and the SNARE proteins can be modified by arachidonic acid, a common lipid regulator, we analysed this possible tripartite link in detail. Here, we show that the ability of arachidonic acid to stimulate SNARE complex formation and exocytosis can be controlled by a-synuclein, both in vitro and in vivo. a-Synuclein sequesters arachidonic acid and thereby blocks the activation of SNAREs. Our data provide mechanistic insights into the action of a-synuclein in the modulation of neurotransmission.

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The expression of SNAP-25 fused to green fluorescent protein (GFP) has been instrumental in demonstrating SNARE role in exocytosis. The wild-type GFP-SNAP-25 and a D9 form, product of botulinum neurotoxin A activity, the main ingredient in the BOTOX preparation, were employed here to study SNARE implication in vesicle mobility and fusion in cultured bovine chromaffin cells, a neuroendocrine exocytotic model. Using total internal reflection fluorescent microscopy, we have identified membrane microdomains of 500-600 nm diameter that contain both SNAP-25 and syntaxin-1 and associate with synaptobrevin-2. Interestingly, while the SNAP-25 D9 formed similar clusters, they displayed increased mobility both laterally and in the axis perpendicular to the plasmalemma, and this correlates with the enhanced dynamics of associated chromaffin granules. SNARE cluster-enhanced motion is reversed by elevation of the intracellular calcium level. Furthermore, single vesicle fusion was unlikely in the highly mobile vesicles present in the cells expressing SNAP-25 D9, which, in addition, displayed in average slower fusion kinetics. Consequently, SNARE cluster dynamics is a new aspect to consider when determining the factors contributing to the mobility of the vesicles in close vicinity to the plasma membrane and also the probability of exocytosis of this granule population. Exocytosis is a key event in the regulated release of neurotransmitters in neurons and neuroendocrine cells. The characterization of various proteins (SNAREs) that form the core complex involved in the specific docking and fusion of neurotransmitter-containing vesicles has stimulated unprecedented progress toward elucidating the molecular bases of exocytosis (1-4). The assembly of plasma membrane proteins (t-SNAREs) such as syntaxin (2) and SNAP-25 (5), as well as vesicle-associated proteins (v-SNAREs) such as synaptobrevin-2 (6), provides the specificity required for vesicle docking and probably the basic machinery for membrane fusion (7). In addition to the use of classical tools such clostridial neurotoxins (8), the generation of engineered SNARE constructs fused to the green fluorescent protein (GFP) has proven instrumental in defining the involvement of these proteins in specific steps of exocytosis. For instance, the use of a functional GFP-SNAP-25 chimera demonstrated the importance of specific amino acids in the C-terminal domain of this protein for core complex assembly and normal vesicle fusion in chromaffin cells, an accepted model of neuroendocrine exocytosis (9).Subsequently, this and other constructs were employed to define the different stages of SNARE complex formation, its interaction with other proteins or factors and the recruitment of different vesicle reservoirs (10-13). Together, these studies demonstrated that ternary complex formation constitutes a late stage in membrane fusion. However, the chain of interactions between SNAREs and other proteins that controls this process (involving SNAPs, synaptotagmin, munc-18, etc.) essentially remain ...

SummaryWe have studied how the F-actin cytoskeleton is involved in establishing the heterogeneous intracellular Ca 2+ levels ([Ca 2+ ] i ) and in the organization of the exocytotic machinery in cultured bovine chromaffin cells. Simultaneous confocal visualization of [Ca 2+ ] i and transmitted light studies of the cytoskeleton showed that, following cell stimulation, the maximal signal from the Ca 2+ -sensitive fluorescent dye Fluo-3 was in the empty cytosolic spaces left by cytoskeletal cages. This was mostly due to the accumulation of the dye in spaces devoid of cytoskeletal components, as shown by the use of alternative Ca 2+ -insensitive fluorescent cytosolic markers. In addition to affecting the distribution of such compounds in the cytosol, the cytoskeleton influenced the location of L-and P-Q-type Ca 2+ channel clusters, which were associated with the borders of cytoskeletal cages in resting and stimulated cells. Indeed, syntaxin-1 and synaptotagmin-1, which are components of the secretory machinery, were present in the same location. Furthermore, granule exocytosis took place at these sites, indicating that the organization of the F-actin cytoskeletal cortex shapes the preferential sites for secretion by associating the secretory machinery with preferential sites for Ca 2+ entry. The influence of this cortical organization on the propagation of [Ca 2+ ] i can be modelled, illustrating how it serves to define rapid exocytosis.

Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca 2+ signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy. In contrast, ER was sparse in the cortex and more abundant in deep cytoplasmic regions. The mitochondrial distribution might be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to the position of SNARE microdomains and the granule fusion sites revealed that a third of the cortical mitochondria colocalized with exocytotic sites and another third located at a distance closer than two vesicle diameters. ER structures were also present in the vicinity of secretory sites but at a lower density. Therefore, mitochondria and ER have a spatial distribution that suggests a specialized role in modulation of exocytosis that fits with the role of cytosolic Ca 2+ microdomains described previously.

Neurotransmission and secretion of hormones involve a sequence of protein/lipid interactions with lipid turnover impacting on vesicle trafficking and ultimately fusion of secretory vesicles with the plasma membrane. We previously demonstrated that sphingosine, a sphingolipid metabolite, promotes formation of the SNARE complex required for membrane fusion and also increases the rate of exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and in hippocampal neurons. Recently a fungi-derived sphingosine homologue, FTY720, has been approved for treatment of multiple sclerosis. In its non-phosphorylated form FTY720 accumulates in the central nervous system, reaching high levels which could affect neuronal function. Considering close structural similarity of sphingosine and FTY720 we investigated whether FTY720 has an effect on regulated exocytosis. Our data demonstrate that FTY720 can activate vesicular synaptobrevin for SNARE complex formation and enhance exocytosis in neuroendocrine cells and neurons.

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Lipid molecules such as arachidonic acid (AA) and sphingolipid metabolites have been implicated in modulation of neuronal and endocrine secretion. Here we compare the effects of these lipids on secretion from cultured bovine chromaffin cells. First, we demonstrate that exogenous sphingosine and AA interact with the secretory apparatus as confirmed by FRET experiments. Examination of plasma membrane SNARE microdomains and chromaffin granule dynamics using total internal reflection fluorescent microscopy (TIRFM) suggests that sphingosine production promotes granule tethering while arachidonic acid promotes full docking. Our analysis of single granule release kinetics by amperometry demonstrated that both sphingomyelinase and AA treatments enhanced drastically the amount of catecholamines released per individual event by either altering the onset phase of or by prolonging the off phase of single granule catecholamine release kinetics. Together these results demonstrate that the kinetics and extent of the exocytotic fusion pore formation can be modulated by specific signalling lipids through related functional mechanisms.