Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin’s ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent de novo cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.

In secretory cells, calcium-regulated exocytosis is rapidly followed by compensatory endocytosis. Neuroendocrine cells secrete hormones and neuropeptides through various modes of exo-endocytosis, including kiss-andrun, cavicapture and full-collapse fusion. During kissand-run and cavicapture modes, the granule membrane is maintained in an omega shape, whereas it completely merges with the plasma membrane during full-collapse mode. As the composition of the granule membrane is very different from that of the plasma membrane, a precise sorting process of granular proteins must occur. However, the fate of secretory granule membrane after full fusion exocytosis remains uncertain.Here, we investigated the mechanisms governing endocytosis of collapsed granule membranes by following internalization of antibodies labeling the granule membrane protein, dopamine-β-hydroxylase (DBH) in cultured chromaffin cells. Using immunofluorescence and electron microscopy, we observed that after full collapse, DBH remains clustered on the plasma membrane with other specific granule markers and is subsequently internalized through vesicular structures composed mainly of granule components. Moreover, the incorporation of this recaptured granule membrane into an early endosomal compartment is dependent on clathrin and actin. Altogether, these results suggest that after full collapse exocytosis, a selective sorting of granule membrane components is facilitated by the physical preservation of the granule membrane entity on the plasma membrane.

The molecular mechanisms for calcium-triggered membrane fusion have long been sought for, and detailed models now exist that account for at least some of the functions of the many proteins involved in the process. Key players in the fusion reaction are a group of proteins that, upon binding to calcium, trigger the merger of cargo-filled vesicles with the plasma membrane. Low-affinity, fast-kinetics calcium sensors of the synaptotagmin family -especially synaptotagmin-1 and synaptotagmin-2 -are the main calcium sensors for fast exocytosis triggering in many cell types. Their functions extend beyond fusion triggering itself, having been implicated in the calcium-dependent vesicle recruitment during activity, docking of vesicles to the plasma membrane and priming, and even in post-fusion steps, such as fusion pore expansion and endocytosis. Furthermore, synaptotagmin diversity imparts distinct properties to the release process itself. Other calcium-sensing proteins such as Munc13s and protein kinase C play important, but more indirect roles in calcium-triggered exocytosis. Because of their higher affinity, but intrinsic slower kinetics, they operate on longer temporal and spatial scales to organize assembly of the release machinery. Finally, the high-affinity synaptotagmin-7 and Doc2 (Double C2-domain) proteins are able to trigger membrane fusion in vitro, but cellular measurements in different systems show that they may participate in either fusion or vesicle priming. Here, we summarize the properties and possible interplay of (some of) the major C2-domain containing calcium sensors in calcium-triggered exocytosis. Keywords: calcium sensor, exocytosis, Munc13, neuroendocrine, synaptic transmission, synaptotagmin. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases". Cellular processes as distinct as neurotransmitter release, mast cell degranulation, and hormone release are brought about by calcium-dependent exocytosis (Lindau and Gomperts 1991;Bean et al. 1994;Sudhof 2013). Extensive research over several decades has elucidated the general mechanism behind those processes, and the functions of the key players involved. It is now clearly established that, both in synaptic and endocrine secretion, the vesicle fusion machinery is composed, at its core, by a set of proteins forming the soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) complex. The canonical (neuronal) SNARE complex consists of the vesicular SNARE protein synaptobrevin 2/VAMP-2 and the plasma membrane SNAREs synaptosomal-associated protein of 25 kDa (SNAP-25) and syntaxin-1 (Sollner et al. 1993). The~65-residue heptad-repeat SNARE motifs of these proteins form a parallel four-stranded helix bundle (Hanson et al. 1997;Sutton et al. 1998) that can zipper-up from its Nto C-terminus (Hua and Charlton 1999;Sorensen et al. 2006) in a stepwise manner (Gao et al. 2012;Min et al. 2013) to bring the two opposing membranes together. The formation of this trans alpha-helical ternary complex...