Ultrastructural Organization of Bovine Chromaffin Cell Cortex—Analysis by Cryofixation and Morphometry of Aspects Pertinent to Exocytosis

Plattner, Helmut; Artalejo, Antonio R.; Neher, Erwin

doi:10.1083/jcb.139.7.1709

Cited by 192 publications

(151 citation statements)

References 45 publications

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“…This mechanism may alter quantal size. However, there is little evidence for homotypic fusion in chromaffin cells at this late stage of release (52). Whether chromaffin granules aggregate during recycling or during de novo vesicle synthesis was not addressed in this study, but we did not observe multiple granules in single membrane-bound vesicles in our electronmicroscopic studies.…”

Section: Vesicle Size Is Altered In a Manner That Mirrors Quantal Sizcontrasting

confidence: 34%

Regulation of large dense-core vesicle volume and neurotransmitter content mediated by adaptor protein 3

Grabner

Price

Lysakowski

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Adaptor protein 3 (AP-3) is a vesicle-coat protein that forms a heterotetrameric complex. Two types of AP-3 subunits are found in mammalian cells. Ubiquitous AP-3 subunits are expressed in all tissues of the body, including the brain. In addition, there are neuronal AP-3 subunits that are thought to serve neuron-specific functions such as neurotransmitter release. In this study, we show that overexpression of neuronal AP-3 in mouse chromaffin cells results in a striking decrease in the neurotransmitter content of individual vesicles (quantal size), whereas deletion of all AP-3 produces a dramatic increase in quantal size; these changes were correlated with alterations in dense-core vesicle size. AP-3 appears to localize in the trans-Golgi network and possibly immature secretory vesicles, where it may be involved in the formation of neurosecretory vesicles.amperometry ͉ electron microscopy ͉ quantal content ͉ vesicle size ͉ chromaffin cells

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Section: Vesicle Size Is Altered In a Manner That Mirrors Quantal Sizcontrasting

confidence: 34%

Regulation of large dense-core vesicle volume and neurotransmitter content mediated by adaptor protein 3

Grabner

Price

Lysakowski

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…3B), colocalization of the target SNAREs on the plasma membrane is likely irrespective of vesicular proteins. This was predictable, because previous electron microscopy studies demonstrated that the vast majority of secretory granules reside away from the plasma membrane in the resting cells (16).…”

Section: Resultsmentioning

confidence: 97%

High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E

Rickman

Meunier

Binz

et al. 2004

Journal of Biological Chemistry

105

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The release of hormones and neurotransmitters requires the fusion of cargo-containing vesicles with the plasma membrane. This process of exocytosis relies on three SNARE proteins, namely syntaxin and SNAP-25 on the target plasma membrane and synaptobrevin on the vesicular membrane. In this study we examined the molecular assembly pathway that leads to formation of the fusogenic SNARE complex. We now show that the plasma membrane syntaxin and SNAP-25 interact with high affinity and equimolar stoichiometry to form a stable dimer on the pathway to the ternary SNARE complex. In bovine chromaffin cells, syntaxin and SNAP-25 colocalize in defined clusters that average 700 nm in diameter and cover 10% of the plasma membrane. Removal of the C terminus of SNAP-25 by botulinum neurotoxin E, a known neuroparalytic agent, dissociates the target SNARE dimer in vitro and disrupts the SNARE clustering in vivo. Together, our data uncover formation of stable syntaxin/SNAP-25 dimers as a central principle of the SNARE assembly pathway underlying regulated exocytosis.Hormonal and neurotransmitter release occurs when vesicles fuse with the plasma membrane in a calcium-dependent manner. The three SNARE 1 proteins, namely synaptobrevin2 (also known as VAMP2) on the vesicular membrane and syntaxin1 and SNAP-25 on the target plasma membrane, are essential for the last step of vesicular exocytosis, the fusion of membranes (1, 2). These three proteins form a highly stable ternary complex that likely drives the fusion of the two apposing membranes (3). Botulinum toxins, which specifically cleave SNARE proteins prior to the formation of the ternary SNARE complex, block vesicle exocytosis in both neurons and endocrine cells (2, 4). The plasma membrane protein SNAP-25 is a molecular target for botulinum toxin E (5). This toxin removes the C-terminal 26 amino acids from the SNAP-25 molecule and has been used previously to dissect stages of calcium-triggered exocytosis (6, 7). Although these studies demonstrated that the ternary SNARE complex forms at a late stage of vesicle exocytosis, the molecular events preceding formation of this complex remain largely unknown.Several contradicting theories have been put forward with regard to the molecular pathway leading to SNARE complex assembly. One study suggested that SNAREs, although active, do not pre-assemble in resting cells into any intermediate complex but would form the ternary complex upon activation of exocytosis (8). It has also been proposed that SNAP-25 engages vesicular synaptobrevin in a priming step and that binding of syntaxin1 would be the pivotal event leading to membrane fusion (9). An alternative theory states that syntaxin1 and SNAP-25 pre-assemble into a stable heterodimer on the plasma membrane, and only then does vesicular synaptobrevin engage the two target SNAREs (10, 11). Importantly, it is not clear whether any SNARE intermediates actually exist in intact secretory cells as a prelude to SNARE-mediated fusion.To address these outstanding issues, we analyzed SNARE binary re...

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“…However, rise and decay times are not correlated with quantal size. Furthermore, the size of dense core granules rather closely follows a Gaussian distribution (Plattner et al, 1997). Therefore, these two populations of amperometric spikes probably correspond to two modes of fusion with distinct rates of fusion pore dilation and not to two groups of vesicles.…”

Section: Two Modes Of Membrane Fusionmentioning

confidence: 96%

A Fast Mode of Membrane Fusion Dependent on Tight SNARE Zippering

Bretou¹,

Anne²,

Darchen³

2008

J. Neurosci.

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SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have a key role in membrane fusion. It is commonly assumed that pairing of SNARE proteins anchored in opposing membranes overcomes the repulsion energy between membranes, thereby catalyzing fusion. In this study, we have increased the distance between the coiled-coil SNARE motif and the transmembrane domain of the vesicular SNARE synaptobrevin-2 by insertion of a flexible linker to analyze how an increased intermembrane distance affects exocytosis. Synaptobrevin-2 lengthening did not change the frequency of exocytotic events measured at 1 M free calcium but prevented the increase in the secretory activity triggered by higher calcium concentration. Exocytotic events monitored in adrenal chromaffin cells by means of carbon fiber amperometry were classified in two groups according to the rate and extent of fusion pore expansion. Lengthening the juxtamembrane region of synaptobrevin-2 severely reduced the occurrence of rapid single events, leaving slow ones unchanged. It also impaired the increase in the fast-fusion mode that normally follows elevation of intracellular Ca 2ϩ levels. We conclude that mild stimuli trigger slow fusion events that do not rely on a short intermembrane distance. In contrast, a short intermembrane distance mediated by tight zippering of SNAREs is essential to a component of the secretory response elicited by robust stimuli and characterized by rapid dilation of the fusion pore.

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Ultrastructural Organization of Bovine Chromaffin Cell Cortex—Analysis by Cryofixation and Morphometry of Aspects Pertinent to Exocytosis

Cited by 192 publications

References 45 publications

Regulation of large dense-core vesicle volume and neurotransmitter content mediated by adaptor protein 3

Regulation of large dense-core vesicle volume and neurotransmitter content mediated by adaptor protein 3

High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E

A Fast Mode of Membrane Fusion Dependent on Tight SNARE Zippering

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