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

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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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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Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells

AJP Cell Physiology ◽

10.1152/ajpcell.00291.2013 ◽

2014 ◽

Vol 306 (9) ◽

pp. C831-C843 ◽

Cited By ~ 6

Author(s):

Prattana Samasilp ◽

Kyle Lopin ◽

Shyue-An Chan ◽

Rajesh Ramachandran ◽

Corey Smith

Keyword(s):

Chromaffin Cells ◽

Control Point ◽

Peripheral Circulation ◽

Neuroendocrine Cells ◽

Fusion Pore ◽

Cell Periphery ◽

Hormone Release ◽

Excitatory Synaptic Input ◽

Activity Dependent ◽

Pore Expansion

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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Fusion pores and their control of neurotransmitter and hormone release

The Journal of General Physiology ◽

10.1085/jgp.201611724 ◽

2017 ◽

Vol 149 (3) ◽

pp. 301-322 ◽

Cited By ~ 33

Author(s):

Che-Wei Chang ◽

Chung-Wei Chiang ◽

Meyer B. Jackson

Keyword(s):

Concentration Profile ◽

Extracellular Space ◽

Endocrine Cells ◽

Molecular Mechanisms ◽

Fusion Pore ◽

Hormone Release ◽

Cellular Regulation ◽

Underlying Mechanisms ◽

Multiple Signaling ◽

Fusion Pores

Ca2+-triggered exocytosis functions broadly in the secretion of chemical signals, enabling neurons to release neurotransmitters and endocrine cells to release hormones. The biological demands on this process can vary enormously. Although synapses often release neurotransmitter in a small fraction of a millisecond, hormone release can be orders of magnitude slower. Vesicles usually contain multiple signaling molecules that can be released selectively and conditionally. Cells are able to control the speed, concentration profile, and content selectivity of release by tuning and tailoring exocytosis to meet different biological demands. Much of this regulation depends on the fusion pore—the aqueous pathway by which molecules leave a vesicle and move out into the surrounding extracellular space. Studies of fusion pores have illuminated how cells regulate secretion. Furthermore, the formation and growth of fusion pores serve as a readout for the progress of exocytosis, thus revealing key kinetic stages that provide clues about the underlying mechanisms. Herein, we review the structure, composition, and dynamics of fusion pores and discuss the implications for molecular mechanisms as well as for the cellular regulation of neurotransmitter and hormone release.

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Repurposing molecular mechanisms of transmitter release: a new job for syndapin at the fusion pore. Focus on “Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells”

AJP Cell Physiology ◽

10.1152/ajpcell.00079.2014 ◽

2014 ◽

Vol 306 (9) ◽

pp. C792-C793 ◽

Cited By ~ 3

Author(s):

Annie Quan ◽

Phillip J. Robinson

Keyword(s):

Transmitter Release ◽

Chromaffin Cells ◽

Molecular Mechanisms ◽

Fusion Pore ◽

Pore Expansion

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Insulin granule mobility, cytosolic calcium concentration, and stimulated insulin secretion in MIN6 cells and beta cells: differences and similarities

Diabetologie und Stoffwechsel ◽

10.1055/s-0036-1580890 ◽

2016 ◽

Vol 11 (S 01) ◽

Author(s):

D Brüning ◽

K Reckers ◽

M Matz ◽

K Baumann ◽

I Rustenbeck

Keyword(s):

Insulin Secretion ◽

Beta Cells ◽

Calcium Concentration ◽

Cytosolic Calcium ◽

Cytosolic Calcium Concentration ◽

Min6 Cells ◽

Insulin Granule

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Lumenal Protein within Secretory Granules Affects Fusion Pore Expansion

Biophysical Journal ◽

10.1016/j.bpj.2014.04.064 ◽

2014 ◽

Vol 107 (1) ◽

pp. 26-33 ◽

Cited By ~ 19

Author(s):

Annita Ngatchou Weiss ◽

Arun Anantharam ◽

Mary A. Bittner ◽

Daniel Axelrod ◽

Ronald W. Holz

Keyword(s):

Secretory Granules ◽

Fusion Pore ◽

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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Insulin exocytosis in Goto-Kakizaki rat β-cells subjected to long-term glinide or sulfonylurea treatment

Biochemical Journal ◽

10.1042/bj20071282 ◽

2008 ◽

Vol 412 (1) ◽

pp. 93-101 ◽

Cited By ~ 13

Author(s):

Junko Kawai ◽

Mica Ohara-Imaizumi ◽

Yoko Nakamichi ◽

Tadashi Okamura ◽

Yoshihiro Akimoto ◽

...

Keyword(s):

Insulin Release ◽

Β Cells ◽

Gk Rats ◽

Insulin Granules ◽

Long Term Treatment ◽

Insulin Granule ◽

Granule Motion ◽

Insulin Exocytosis ◽

First Phase Insulin Release

Sulfonylurea and glinide drugs display different effects on insulin granule motion in single β-cells in vitro. We therefore investigated the different effects that these drugs manifest towards insulin release in an in vivo long-term treatment model. Diabetic GK (Goto-Kakizaki) rats were treated with nateglinide, glibenclamide or insulin for 6 weeks. Insulin granule motion in single β-cells and the expression of SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins were then analysed. Perifusion studies showed that decreased first-phase insulin release was partially recovered when GK rats were treated with nateglinide or insulin for 6 weeks, whereas no first-phase release occurred with glibenclamide treatment. In accord with the perifusion results, TIRF (total internal reflection fluorescence) imaging of insulin exocytosis showed restoration of the decreased number of docked insulin granules and the fusion events from them during first-phase release for nateglinide or insulin, but not glibenclamide, treatment; electron microscopy results confirmed the TIRF microscopy data. Relative to vehicle-treated GK β-cells, an increased number of SNARE clusters were evident in nateglinide- or insulin-treated cells; a lesser increase was observed in glibenclamide-treated cells. Immunostaining for insulin showed that nateglinide treatment better preserved pancreatic islet morphology than did glibenclamide treatment. However, direct exposure of GK β-cells to these drugs could not restore the decreased first-phase insulin release nor the reduced numbers of docked insulin granules. We conclude that treatment of GK rats with nateglinide and glibenclamide varies in long-term effects on β-cell functions; nateglinide treatment appears overall to be more beneficial.

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