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.
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.
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