scholarly journals Two distinct secretory vesicle–priming steps in adrenal chromaffin cells

2010 ◽  
Vol 190 (6) ◽  
pp. 1067-1077 ◽  
Author(s):  
Yuanyuan Liu ◽  
Claudia Schirra ◽  
Ludwig Edelmann ◽  
Ulf Matti ◽  
JeongSeop Rhee ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Secretory Vesicle ◽  
Adrenal Chromaffin Cells ◽  
Attachment Protein ◽  
Calcium Dependent ◽  
Open Conformation ◽  
Large Dense Core Vesicles ◽  
Priming Process ◽  
Adrenal Chromaffin ◽  
Protein Attachment

Priming of large dense-core vesicles (LDCVs) is a Ca2+-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca2+ concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.

Red, yellow, green go! – a novel tool for microscopic segregation of secretory vesicle pools according to their age

2003 ◽  
Vol 31 (4) ◽  
pp. 851-856 ◽  
Author(s):  
U.K. Wiegand ◽  
R.R. Duncan ◽  
J. Greaves ◽  
R.H. Chow ◽  
M.J. Shipston ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Fluorescence Emission ◽  
Secretory Vesicle ◽  
Live Cells ◽  
Adrenal Chromaffin Cells ◽  
Vesicle Pools ◽  
Large Dense Core Vesicles ◽  
Confocal Fluorescence ◽  
Cargo Proteins ◽  
Adrenal Chromaffin

Large dense-core vesicles (LDCVs) were labelled in cultured bovine adrenal chromaffin cells expressing fluorescent chimaeric ‘cargo’ proteins that were targeted to these secretory vesicles. When the cells were stimulated with nicotine 48 h after transduction, the fractional loss of fluorescent LDCVs was much greater than the fractional catecholamine secretion, implying selective release of newly assembled vesicles. This was confirmed using a fluorescent ‘timer’ construct that changes its fluorescence emission from green to red over several hours, and by measurement of the location and mobility of LDCVs in live cells by confocal fluorescence microscopy. Newly assembled (green) LDCVs were located mostly in peripheral regions of the cells, were virtually immobile and could be released by nicotine, but not by Ba2+; in contrast, older (red) LDCVs were centrally located and relatively mobile, and their exocytotic release was triggered by Ba2+, but not by nicotine. We describe the image restoration procedure that is necessary in order to analyse the behaviour of LDCVs labelled with this construct.

A role for calpactin in calcium-dependent exocytosis in adrenal chromaffin cells

Nature ◽  
1989 ◽  
Vol 340 (6231) ◽  
pp. 313-315 ◽  
Author(s):  
Shahid M. Ali ◽  
Michael J. Geisow ◽  
Robert D. Burgoyne
Keyword(s):  
Chromaffin Cells ◽  
Adrenal Chromaffin Cells ◽  
Calcium Dependent ◽  
Adrenal Chromaffin

scholarly journals Impaired maturation of large dense-core vesicles in muted-deficient adrenal chromaffin cells

2015 ◽  
Vol 128 (7) ◽  
pp. 1365-1374 ◽  
Author(s):  
Z. Hao ◽  
L. Wei ◽  
Y. Feng ◽  
X. Chen ◽  
W. Du ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Dense Core ◽  
Adrenal Chromaffin Cells ◽  
Dense Core Vesicles ◽  
Large Dense Core Vesicles ◽  
Adrenal Chromaffin

scholarly journals Doc2B acts as a calcium sensor for vesicle priming requiring synaptotagmin-1, Munc13-2 and SNAREs

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sébastien Houy ◽  
Alexander J Groffen ◽  
Iwona Ziomkiewicz ◽  
Matthijs Verhage ◽  
Paulo S Pinheiro ◽  
...  
Keyword(s):  
Binding Sites ◽  
Chromaffin Cells ◽  
Protein Domain ◽  
Calcium Sensor ◽  
Adrenal Chromaffin Cells ◽  
Dynein Light Chain ◽  
C2 Domains ◽  
Calcium Dependent ◽  
Synaptotagmin 1 ◽  
Adrenal Chromaffin

Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca2+ and SNAREs. Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated. We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca2+-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca2+-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca2+-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B – inhibition of release during sustained calcium elevations – depends on an overlapping protein domain (the MID-domain), but is separate from its Ca2+-dependent priming function. We conclude that Doc2B acts as a vesicle priming protein.

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