Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells

Adrenal neuroendocrine chromaffin cells receive excitatory synaptic input from the sympathetic nervous system and secrete hormones into the peripheral circulation. Under basal sympathetic tone, modest amounts of freely soluble catecholamine are selectively released through a restricted fusion pore formed between the secretory granule and the plasma membrane. Upon activation of the sympathoadrenal stress reflex, elevated stimulation drives fusion pore expansion, resulting in increased catecholamine secretion and facilitating release of copackaged peptide hormones. Thus regulated expansion of the secretory fusion pore is a control point for differential hormone release of the sympathoadrenal stress response. Previous work has shown that syndapin 1 deletion alters transmitter release and that the dynamin 1-syndapin 1 interaction is necessary for coupled endocytosis in neurons. Dynamin has also been shown to be involved in regulation of fusion pore expansion in neuroendocrine chromaffin cells through an activity-dependent association with syndapin. However, it is not known which syndapin isoform(s) contributes to pore dynamics in neuroendocrine cells. Nor is it known at what stage of the secretion process dynamin and syndapin associate to modulate pore expansion. Here we investigate the expression and localization of syndapin isoforms and determine which are involved in mediating fusion pore expansion. We show that all syndapin isoforms are expressed in the adrenal medulla. Mutation of the SH3 dynamin-binding domain of all syndapin isoforms shows that fusion pore expansion and catecholamine release are limited specifically by mutation of syndapin 3. The mutation also disrupts targeting of syndapin 3 to the cell periphery. Syndapin 3 exists in a persistent colocalized state with dynamin 1.

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Activity-Dependent Fusion Pore Expansion Regulated by a Calcineurin-Dependent Dynamin-Syndapin Pathway in Mouse Adrenal Chromaffin Cells

Journal of Neuroscience ◽

10.1523/jneurosci.1299-12.2012 ◽

2012 ◽

Vol 32 (30) ◽

pp. 10438-10447 ◽

Cited By ~ 27

Author(s):

P. Samasilp ◽

S.-A. Chan ◽

C. Smith

Keyword(s):

Chromaffin Cells ◽

Fusion Pore ◽

Adrenal Chromaffin Cells ◽

Adrenal Chromaffin ◽

Activity Dependent ◽

Pore Expansion

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Chromogranin A, the major lumenal protein in chromaffin granules, controls fusion pore expansion

The Journal of General Physiology ◽

10.1085/jgp.201812182 ◽

2018 ◽

Vol 151 (2) ◽

pp. 118-130 ◽

Cited By ~ 3

Author(s):

Prabhodh S. Abbineni ◽

Mary A. Bittner ◽

Daniel Axelrod ◽

Ronald W. Holz

Keyword(s):

Plasma Membrane ◽

Small Molecules ◽

Chromogranin A ◽

Chromaffin Cells ◽

Fusion Pore ◽

Chromaffin Granules ◽

Chromaffin Granule ◽

Chromogranin B ◽

Tissue Plasminogen ◽

Pore Expansion

Upon fusion of the secretory granule with the plasma membrane, small molecules are discharged through the immediately formed narrow fusion pore, but protein discharge awaits pore expansion. Recently, fusion pore expansion was found to be regulated by tissue plasminogen activator (tPA), a protein present within the lumen of chromaffin granules in a subpopulation of chromaffin cells. Here, we further examined the influence of other lumenal proteins on fusion pore expansion, especially chromogranin A (CgA), the major and ubiquitous lumenal protein in chromaffin granules. Polarized TIRF microscopy demonstrated that the fusion pore curvature of granules containing CgA-EGFP was long lived, with curvature lifetimes comparable to those of tPA-EGFP–containing granules. This was surprising because fusion pore curvature durations of granules containing exogenous neuropeptide Y-EGFP (NPY-EGFP) are significantly shorter (80% lasting <1 s) than those containing CgA-EGFP, despite the anticipated expression of endogenous CgA. However, quantitative immunocytochemistry revealed that transiently expressed lumenal proteins, including NPY-EGFP, caused a down-regulation of endogenously expressed proteins, including CgA. Fusion pore curvature durations in nontransfected cells were significantly longer than those of granules containing overexpressed NPY but shorter than those associated with granules containing overexpressed tPA, CgA, or chromogranin B. Introduction of CgA to NPY-EGFP granules by coexpression converted the fusion pore from being transient to being longer lived, comparable to that found in nontransfected cells. These findings demonstrate that several endogenous chromaffin granule lumenal proteins are regulators of fusion pore expansion and that alteration of chromaffin granule contents affects fusion pore lifetimes. Importantly, the results indicate a new role for CgA. In addition to functioning as a prohormone, CgA plays an important role in controlling fusion pore expansion.

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Identification of β-synuclein on secretory granules in chromaffin cells and the effects of α- and β-synuclein on post-fusion BDNF discharge and fusion pore expansion

Neuroscience Letters ◽

10.1016/j.neulet.2019.01.056 ◽

2019 ◽

Vol 699 ◽

pp. 134-139 ◽

Cited By ~ 2

Author(s):

Prabhodh S. Abbineni ◽

Kevin P. Bohannon ◽

Mary A. Bittner ◽

Daniel Axelrod ◽

Ronald W. Holz

Keyword(s):

Chromaffin Cells ◽

Secretory Granules ◽

Fusion Pore ◽

Pore Expansion

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Fusion pore regulation by Epac2/cAMP controls cargo release during insulin exocytosis

10.1101/403253 ◽

2018 ◽

Cited By ~ 1

Author(s):

Alenka Gucek ◽

Nikhil R Gandasi ◽

Muhmmad Omar-Hmeadi ◽

Marit Bakke ◽

Stein O. Døskeland ◽

...

Keyword(s):

Type 2 Diabetes ◽

Transmitter Release ◽

Fusion Pore ◽

Hormone Release ◽

Peptide Release ◽

Insulin Granule ◽

Insulin Exocytosis ◽

Camp Elevation ◽

Pore Expansion

AbstractRegulated exocytosis establishes a narrow fusion pore as the initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of larger peptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in type-2 diabetes and neurodegenerative disease. Here we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation leads to pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- mice. Conversely, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

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Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis

eLife ◽

10.7554/elife.41711 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 7

Author(s):

Alenka Guček ◽

Nikhil R Gandasi ◽

Muhmmad Omar-Hmeadi ◽

Marit Bakke ◽

Stein O Døskeland ◽

...

Keyword(s):

Beta Cells ◽

Transmitter Release ◽

Extracellular Space ◽

Fusion Pore ◽

Hormone Release ◽

Peptide Release ◽

Insulin Granule ◽

Insulin Exocytosis ◽

Camp Elevation ◽

Pore Expansion

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- (Rapgef4-/-) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

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The synaptotagmin C2B domain calcium-binding loops modulate the rate of fusion pore expansion

Molecular Biology of the Cell ◽

10.1091/mbc.e17-11-0623 ◽

2018 ◽

Vol 29 (7) ◽

pp. 834-845 ◽

Cited By ~ 7

Author(s):

Mounir Bendahmane ◽

Kevin P. Bohannon ◽

Mazdak M. Bradberry ◽

Tejeshwar C. Rao ◽

Michael W. Schmidtke ◽

...

Keyword(s):

Plasma Membrane ◽

In Silico ◽

Calcium Binding ◽

Chromaffin Cells ◽

Fusion Pore ◽

Structural Specificity ◽

Fusion Pores ◽

Kinetics Of ◽

Pore Expansion

In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca2+-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca2+-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca2+-binding loops of Syt-7 (Syt-1:7C2B123) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2B123 and WT Syt-1 are likely to differ in their interactions with Ca2+ and membranes. Subsequent in vitro and in silico data demonstrated that the chimera exhibits a higher affinity for phospholipids than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma membrane, and the rate at which it releases the membrane, contribute in important ways to the rate of fusion pore expansion.

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