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

2018 ◽  
Vol 151 (2) ◽  
pp. 118-130 ◽  
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.

EM localization of chromaffin cell ATPase

1992 ◽  
Vol 50 (1) ◽  
pp. 816-817
Author(s):  
V. Kriho ◽  
G. D. Pappas
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Immunogold Labelling ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Cells In Culture ◽  
Membrane Bound ◽  
Bovine Chromaffin Cells

During exocytosis of the chromaffin granules, ATP is released. ATP can then be hydrolyzed by the ecto-ATPases of the plasma membrane to provide adenosine for reuptake or for activation of P1 purinoceptors. Chromaffin granule membranes also possess ATPase activity. This activity is linked to the uptake of catecholamines from the cytoplasm into the membrane-bound granule compartment.In this report we combine EM cytochemistry and immunogold labelling to provide further evidence for the presence of ATPase on both the plasma membrane and granule membranes of bovine chromaffin cells in culture.

scholarly journals Chromogranin A, the Major Lumenal Protein in Chromaffin Granules, Controls Fusion Pore Expansion

2019 ◽  
Vol 116 (3) ◽  
pp. 524a ◽  
Author(s):  
Prabhodh S. Abbineni ◽  
Mary A. Bittner ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Chromogranin A ◽  
Fusion Pore ◽  
Chromaffin Granules ◽  
Pore Expansion

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

2018 ◽  
Vol 29 (7) ◽  
pp. 834-845 ◽  
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.

scholarly journals Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling.

1986 ◽  
Vol 102 (2) ◽  
pp. 510-515 ◽  
Author(s):  
A Patzak ◽  
H Winkler
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Direct Evidence ◽  
Ultrastructural Level ◽  
Multivesicular Bodies ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Golgi Region ◽  
Membrane Antigens ◽  
Dense Vesicles

The exocytotic exposure and retrieval of an antigen of chromaffin granule membranes were studied with chromaffin cells isolated from bovine adrenal medulla. Cells were incubated with an antiserum against glycoprotein III followed by fluorescein- or gold-labeled anti-IgG. Immunofluorescence on the cell surface was present in a patchy distribution irrespective of whether bivalent antibodies or Fab fragments were used. During subsequent incubation these fluorescent membrane patches were internalized within 45 min. At the ultrastructural level immunogold-labeled patches were present on the surface of stimulated cells. During incubation (5 min to 6 h) these immunolabeled membrane patches became coated, giving rise to coated vesicles and finally to smooth vesicles. These latter vesicles were found spread throughout the cytoplasm including the Golgi region, but Golgi stacks did not become labeled. Part of the immunolabel was transferred to multivesicular bodies, which probably represent a lysosomal pathway. 30 min after incubation immunolabel was also found in electron-dense vesicles apparently representing newly formed chromaffin granules. After 6 h of incubation immunolabel was found in vesicles indistinguishable from mature chromaffin granules. These results provide direct evidence that after exocytosis membranes of chromaffin granules are selectively retrieved from the plasma membrane and are partly recycled to newly formed chromaffin granules, providing a shuttle service from the Golgi region to the plasma membrane.

scholarly journals 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

2019 ◽  
Vol 699 ◽  
pp. 134-139 ◽  
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

scholarly journals Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells

2014 ◽  
Vol 306 (9) ◽  
pp. C831-C843 ◽  
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.

scholarly journals Targeted Ablation of the Chromogranin A (Chga) Gene: Normal Neuroendocrine Dense-Core Secretory Granules and Increased Expression of Other Granins

2006 ◽  
Vol 20 (8) ◽  
pp. 1935-1947 ◽  
Author(s):  
Geoffrey N. Hendy ◽  
Tong Li ◽  
Martine Girard ◽  
Richard C. Feldstein ◽  
Shree Mulay ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Chromogranin A ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Hormone Secretion ◽  
Proteolytic Processing ◽  
Dense Core ◽  
Secretory Process ◽  
Chromogranin B ◽  
Developmental Abnormalities

Abstract Chromogranin A (CgA), originally identified in adrenal chromaffin cells, is a member of the granin family of acidic secretory glycoproteins that are expressed in endocrine cells and neurons. CgA has been proposed to play multiple roles in the secretory process. Intracellularly, CgA may control secretory granule biogenesis and target neurotransmitters and peptide hormones to granules of the regulated pathway. Extracellularly, peptides formed as a result of proteolytic processing of CgA may regulate hormone secretion. To investigate the role of CgA in the whole animal, we created a mouse mutant null for the Chga gene. These mice are viable and fertile and have no obvious developmental abnormalities, and their neural and endocrine functions are not grossly impaired. Their adrenal glands were structurally unremarkable, and morphometric analyses of chromaffin cells showed vesicle size and number to be normal. However, the excretion of epinephrine, norepinephrine, and dopamine was significantly elevated in the Chga null mutants. Adrenal medullary mRNA and protein levels of other dense-core secretory granule proteins including chromogranin B, and secretogranins II to VI were up-regulated 2- to 3-fold in the Chga null mutant mice. Hence, the increased expression of the other granin family members is likely to compensate for the Chga deficiency.

scholarly journals Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2059 ◽  
Author(s):  
Marion Gabel ◽  
Cathy Royer ◽  
Tamou Thahouly ◽  
Valérie Calco ◽  
Stéphane Gasman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Chromaffin Cells ◽  
Spatial Organization ◽  
Lipid Binding ◽  
Annexin A2 ◽  
Neuroendocrine Cells ◽  
Outer Face ◽  
Tissue Plasminogen

Annexin A2 (AnxA2) is a calcium- and lipid-binding protein involved in neuroendocrine secretion where it participates in the formation and/or stabilization of lipid micro-domains required for structural and spatial organization of the exocytotic machinery. We have recently described that phosphorylation of AnxA2 on Tyr23 is critical for exocytosis. Considering that Tyr23 phosphorylation is known to promote AnxA2 externalization to the outer face of the plasma membrane in different cell types, we examined whether this phenomenon occurred in neurosecretory chromaffin cells. Using immunolabeling and biochemical approaches, we observed that nicotine stimulation triggered the egress of AnxA2 to the external leaflets of the plasma membrane in the vicinity of exocytotic sites. AnxA2 was found co-localized with tissue plasminogen activator, previously described on the surface of chromaffin cells following secretory granule release. We propose that AnxA2 might be a cell surface tissue plasminogen activator receptor for chromaffin cells, thus playing a role in autocrine or paracrine regulation of exocytosis.

scholarly journals Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes.

1992 ◽  
Vol 116 (2) ◽  
pp. 359-365 ◽  
Author(s):  
D Scheuner ◽  
C D Logsdon ◽  
R W Holz
Keyword(s):  
Chromaffin Cells ◽  
Secretory Vesicle ◽  
Xenopus Laevis Oocytes ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Vesicle Membrane ◽  
Sequence Of Events ◽  
Dopamine Beta Hydroxylase ◽  
Granule Membrane ◽  
Adrenal Chromaffin

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.

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