Push-and-pull regulation of the fusion pore by synaptotagmin-7

Segovia, Margarita; Alés, Eva; Montes, María Ángeles; Bonifas, Imelda; Jemal, Imane; Lindau, Manfred; Maximov, Anton; Südhof, Thomas C.; Toledo, G. Álvarez de

doi:10.1073/pnas.1014070107

Cited by 74 publications

(104 citation statements)

References 38 publications

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“…By amperometry, we observed serotonin release in spikelike form during small-capacitance flickers. These spikes are of lesser quantal charge than those from irreversible on-steps (full-fusion events), supporting the idea of a reduced outflow of transmitters through a narrow and transient fusion pore in these cells (7) as shown in chromaffin cells (41,42).…”

Section: Discussionsupporting

confidence: 49%

Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells

Deogracias

Acosta

Alés

2013

Journal of Biological Chemistry

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Section: Discussionsupporting

confidence: 49%

Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells

Deogracias

Acosta

Alés

2013

Journal of Biological Chemistry

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“…Synaptotagmin 7 was reported to be one of the Ca 2ϩ sensors for insulin exocytosis in ␤-cells (14), glucagon exocytosis in ␣-cells (19), and lysosomal exocytosis in fibroblasts (33,41). In chromaffin cells, synaptotagmin 7 was also demonstrated to control fusion pore opening during exocytosis (43).…”

Section: Discussionmentioning

confidence: 99%

Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

Falkowski¹,

Thomas²,

Messenger³

et al. 2011

American Journal of Physiology-Gastrointestinal and Liver Physi

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induced changes in intracellular Ca 2ϩ play a pivotal role in secretion in pancreatic acini yet the molecules that respond to Ca 2ϩ are uncertain. Zymogen granule (ZG) exocytosis is regulated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. In nerve and endocrine cells, Ca 2ϩ -stimulated exocytosis is regulated by the SNARE-associated family of proteins termed synaptotagmins. This study examined a potential role for synaptotagmins in acinar secretion. RT-PCR revealed that synaptotagmin isoforms 1, 3, 6, and 7 are present in isolated acini. Immunoblotting and immunofluorescence using three different antibodies demonstrated synaptotagmin 1 immunoreactivity in apical cytoplasm and ZG fractions of acini, where it colocalized with vesicle-associated membrane protein 2. Synaptotagmin 3 immunoreactivity was detected in membrane fractions and colocalized with an endolysosomal marker. A potential functional role for synaptotagmin 1 in secretion was indicated by results that introduction of synaptotagmin 1 C2AB domain into permeabilized acini inhibited Ca 2ϩ -dependent exocytosis by 35%. In contrast, constructs of synaptotagmin 3 had no effect. Confirmation of these findings was achieved by incubating intact acini with an antibody specific to the intraluminal domain of synaptotagmin 1, which is externalized following exocytosis. Externalized synaptotagmin 1 was detected exclusively along the apical membrane. Treatment with CCK-8 (100 pM, 5 min) enhanced immunoreactivity by fourfold, demonstrating that synaptotagmin is inserted into the apical membrane during ZG fusion. Collectively, these data indicate that acini express synaptotagmin 1 and support that it plays a functional role in secretion whereas synaptotagmin 3 has an alternative role in endolysosomal membrane trafficking. zymogen granules; exocytosis PANCREATIC ACINAR CELLS ARE a prototypical model of exocrine secretory cells that undergo stimulated exocytosis of digestive enzymes, which are necessary for the assimilation of the diet. Multiple intracellular signals including diacylglycerol, cAMP and inositol triphosphate (IP 3 )-mediated release of Ca 2ϩ have been shown to stimulate acinar exocytosis. However, the pivotal role of Ca 2ϩ in the overall secretory response is underscored by findings that double knockout of the type 2 and 3 IP 3 receptors fully abolished secretagogue-induced changes in cellular Ca 2ϩ and resulted in a complete lack of salivary and pancreatic acinar secretion (11). Although the critical importance of Ca 2ϩ signaling in acinar exocytosis is well established, the exocytic regulatory proteins that respond to elevated Ca 2ϩ in the cytoplasm are largely uncertain.Synaptotagmins are a family of membrane proteins composed of 15 known isoforms distributed widely in neuronal and nonneuronal tissues (for comprehensive reviews see Refs. 4,39,45). Family members have the same basic structure consisting of a short NH 2 -terminal intravesicular domain followed by a transmembrane domain and a large COOH-termina...

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“…However, a closer look reveals that the amount of material released from individual vesicles measured with patch amperometry is generally considerably larger than the amount measured using standard amperometry. A quick survey of the charges measured at chromaffin cells using amperometry gives values of ≈1 pC (Wightman et al 1991), 0·67 pC (Montesinos et al 2008) and 0·62 pC (Segovia et al 2010), and charges measured using patch amperometry are 2·2 pC (Albillos et al 1997), 1·8 pC (Montesinos et al 2008) and 3·2 pC (Segovia et al 2010). This suggests that each of these methods cater to a specific event, but it is difficult to make direct comparisons.…”

Section: Traditional Models Of Exocytosismentioning

confidence: 99%

“…This suggests that each of these methods cater to a specific event, but it is difficult to make direct comparisons. However, there are at least two experiments in the literature that carry both methods, one shows that the average charge measured using patch amperometry is nearly triple the values measured using amperometry (Montesinos et al 2008) and a second shows that the charge increases to more than five times when using patch amperometry (Segovia et al 2010). It appears that the patch influences the size of the quanta released.…”

Section: Traditional Models Of Exocytosismentioning

confidence: 99%

The evidence for open and closed exocytosis as the primary release mechanism

Ren

Mellander

Keighron

et al. 2016

Quart. Rev. Biophys.

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Abstract. Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss-and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.1. An overview of exocytosis and endocytosis 2 2. Techniques to monitor exocytosis 5 2.1. Patch clamp 6 2.2. Amperometry 6 2.3. Patch amperometry 7 2.4. Cyclic voltammetry 7 2.5. Fluorescence microscopy imaging 83. Traditional models of exocytosis 94. Evidence where partial release during exocytosis appears to be dominant 11 4.1. The total content of a secretory vesicle is greater than the amount released 11 4.2. The majority of exocytotic events are followed by immediate endocytosis 12 4.3. Full distention of the vesicle may not be necessary for quantal release 13 4.4. A flickering fusion pore might regulate quantal release and vesicle recycling 15 4.5. Nanometer-sized pores can be seen as 'feet' associated with amperometric spikes 17 3. Release appears to be only a fraction of vesicle content in mammalian brains 23 7.4. Impact cytometry of adrenal cell vesicles shows a significantly larger catecholamine content then that released 23 7.5. Impact cytometry has been used to measure vesicle content in live cells and this quantity is greater than the amount released 23 7.6. Full distention of the vesicle is not necessary for the measured amperometric current during exocytotic release 23 7.7. Fast changes in cell environment alter the amount of messenger released 23 7.8. Patch amperometry measurements reveal a greater amount of release 23 7.9. Postspike 'feet' are observed with an amperometric spike 23Acknowledgements 24

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Push-and-pull regulation of the fusion pore by synaptotagmin-7

Cited by 74 publications

References 38 publications

Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells

Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells

Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

The evidence for open and closed exocytosis as the primary release mechanism

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