Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca-dependent release has been unclear. Here, we report that the Ca-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca-stimulated release. We propose a parsimonious model whereby Ca-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca Our data further suggest Ca-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery.

SummaryMutations in proline-rich transmembrane protein 2 (PRRT2) are associated with a range of paroxysmal neurological disorders. PRRT2 predominantly localizes to the pre-synaptic terminals and is believed to regulate neurotransmitter release. However, the mechanism of action is unclear. Here, we use reconstituted single vesicle and bulk fusion assays, combined with live cell imaging of single exocytotic events in PC12 cells and biophysical analysis, to delineate the physiological role of PRRT2. We report that PRRT2 selectively blocks the trans SNARE complex assembly and thus negatively regulates synaptic vesicle priming. This inhibition is actualized via weak interactions of the N-terminal proline-rich domain with the synaptic SNARE proteins. Furthermore, we demonstrate that paroxysmal dyskinesia-associated mutations in PRRT2 disrupt this SNARE-modulatory function and with efficiencies corresponding to the severity of the disease phenotype. Our findings provide insights into the molecular mechanisms through which loss-of-function mutations in PRRT2 result in paroxysmal neurological disorders.

On exocytosis, membrane fusion starts with the formation of a narrow fusion pore that must expand to allow the release of secretory compounds. The GTPase Cdc42 promotes fusion pore dilation in neuroendocrine cells by controlling membrane tension.

More transparency in bioanalysis: A microdevice based on transparent indium tin oxide (ITO) electrodes allows simultaneous total internal reflection fluorescence microscopy and amperometric measurements. Use of the device in the coupled optical and electrochemical detection of single exocytotic events is demonstrated with enterochromaffin BON cells (see picture).

Exocytosis of secretory granules (SGs) requires their delivery to the actin-rich cell cortex followed by their attachment to the plasma membrane (PM). How these reactions are executed and coordinated is still unclear. Myrip, which is also known as Slac-2c, binds to the SG-associated GTPase Rab27 and is thought to promote the delivery of SGs to the PM by recruiting the molecular motor myosin Va. Myrip also interacts with actin and the exocyst complex, suggesting that it may exert multiple roles in the secretory process. By combining total internal reflection fluorescence microscopy, single-particle tracking, a photoconversion-based assay, and mathematical modeling, we show that, in human enterochromaffin cells, Myrip (1) inhibits a class of SG motion characterized by fast and directed movement, suggesting that it facilitates the dissociation of SGs from microtubules; (2) enhances their motion toward the PM and the probability of SG attachment to the PM; and (3) increases the characteristic time of immobilization at the PM, indicating that it is a component of the molecular machinery that tether SGs to the PM. Remarkably, while the first two effects of Myrip depend on its ability to recruit myosin Va on SGs, the third is myosin Va independent but relies on the C-terminal domain of Myrip. We conclude that Myrip couples the retention of SGs in the cell cortex, their transport to the PM, and their attachment to the PM, and thus promotes secretion. These three steps of the secretory process are thus intimately coordinated.

Mehr Transparenz in der Bioanalytik: Ein Mikroinstrument bestehend aus transparenten Indiumzinnoxid(ITO)‐Elektroden ermöglicht simultane Messungen mittels Amperometrie und Fluoreszenzmikroskopie mit interner Totalreflexion. Die Anwendung dieses Instruments zum gekoppelten optischen und elektrochemischen Nachweis exozytotischer Einzelereignisse wird anhand von enterochromaffinen BON‐Zellen demonstriert (siehe Bild).

Exozytose ist ein Schlüsselmechanismus, durch den ein zellulärer Organismus molekulare Botenstoffe in das extrazelluläre Medium freisetzt. C. Amatore et al. berichten in ihrer Zuschrift auf über die Entwicklung eines ITO‐Mikroinstruments, das zwei leistungsfähige Techniken – Amperometrie an Mikroelektroden und Fluoreszenzmikroskopie mit interner Totalreflexion – kombiniert und die simultane Echtzeitüberwachung von einzelnen Exozytosereignissen an lebenden Zellen ermöglicht.

… is a key mechanism by which a cellular organism releases molecular messengers into the extracellular medium. C. Amatore et al. report in their Communication on page 5081 ff. the elaboration of an ITO microdevice that combines two powerful techniques--amperometry at microelectrodes and total internal reflection fluorescence microscopy--which allows the simultaneous real-time monitoring of complementary aspects of single exocytotic events at single living cells. Back CoverAnne Meunier, Ouardane Jouannot, RØmy Fulcrand, Isabelle Fanget, Marine Bretou, Erdem Karatekin, StØphane Arbault, Manon Guille, FranÅois Darchen, FrØdØric Lemaître, and Christian Amatore* Exocytosis is a key mechanism by which a cellular organism releases molecular messengers into the extracellular medium. C. Amatore et al. report in their Communication on page 5081 ff. the elaboration of an ITO microdevice that combines two powerful techniques--amperometry at microelectrodes and total internal reflection fluorescence microscopy--which allows the simultaneous real-time monitoring of complementary aspects of single exocytotic events at single living cells.