α‐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. α‐Synuclein has been proposed to regulate soluble N ‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins involved in vesicle fusion. Interestingly, α‐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 α‐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 α‐synuclein, both in vitro and in vivo . α‐Synuclein sequesters arachidonic acid and thereby blocks the activation of SNAREs. Our data provide mechanistic insights into the action of α‐synuclein in the modulation of neurotransmission.

We have studied how the F-actin cytoskeleton is involved in establishing the heterogeneous intracellular Ca²⁺ levels ([Ca²⁺]_i) and in the organization of the exocytotic machinery in cultured bovine chromaffin cells. Simultaneous confocal visualization of [Ca²⁺]_i and transmitted light studies of the cytoskeleton showed that, following cell stimulation, the maximal signal from the Ca²⁺-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 Ca²⁺-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 Ca²⁺ 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 Ca²⁺ entry. The influence of this cortical organization on the propagation of [Ca²⁺]_i can be modelled, illustrating how it serves to define rapid exocytosis.

The distribution of mitochondria and ER in relation to exocytotic sites is relevant to understand the influence of these organelles in tuning calcium signals and secretion. Confocal images of probes tagged to mitochondria and F-actin cytoskeleton revealed the existence of two populations of mitochondria, one cortical and the other 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 may be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to SNARE microdomains or the granule fusion sites revealed that 1/3 of the cortical mitochondria co-localized with exocytotic sites whereas another 1/3 located at a distance smaller than 2 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 and fits with cytosolic Ca2+ microdomains described before.

Actin is one of the most ubiquitous protein playing fundamental roles in a variety of cellular processes. Since early in the 1980s, it was evident that filamentous actin (F-actin) formed a peripheral cortical barrier that prevented vesicles to access secretory sites in chromaffin cells in culture. Later, around 2000, it was described that the F-actin structure accomplishes a dual role serving both vesicle transport and retentive purposes and undergoing dynamic transient changes during cell stimulation. The complex role of the F-actin cytoskeleton in neuroendocrine secretion was further evidenced when it has been proved to participate in the scaffold structure holding together the secretory machinery at active sites and participate in the generation of mechanical forces that drive the opening of the fusion pore, during the first decade of the present century. The complex vision of the multiple roles of F-actin in secretion we have acquired to date comes largely from studies performed on traditional 2D cultures of primary cells; however, recent evidences suggest that these may not accurately mimic the 3D in vivo environment, and thus, more work is now needed on adrenomedullary cells kept in a more “native” configuration to fully understand the role of F-actin in regulating chromaffin granule transport and secretion under physiological conditions.