Dense-core secretory vesicle docking and exocytotic membrane fusion in Paramecium cells

: The fusion of secretory vesicles with the plasma membrane depends on the assembly of v-SNAREs (VAMP2/synaptobrevin2) and t-SNAREs (SNAP25/syntaxin1) into the SNARE complex. Vesicles go through several upstream steps, referred to as docking and priming, to gain fusion competence. The vesicular protein synaptotagmin-1 (Syt-1) is the principal Ca2+ sensor for fusion in several central nervous system neurons and neuroendocrine cells and part of the docking complex for secretory granules. Syt-1 binds to the acceptor complex such as synaxin1, SNAP-25 on the plasma membrane to facilitate secretory vesicle docking, and upon Ca2+-influx promotes vesicle fusion. This review assesses the role of the Syt-1 protein involved in the secretory vesicle docking, priming, and fusion.

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Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells.

The Journal of Cell Biology ◽

10.1083/jcb.127.5.1419 ◽

1994 ◽

Vol 127 (5) ◽

pp. 1419-1433 ◽

Cited By ~ 97

Author(s):

Y Liu ◽

E S Schweitzer ◽

M J Nirenberg ◽

V M Pickel ◽

C J Evans ◽

...

Keyword(s):

Pc12 Cells ◽

Gradient Centrifugation ◽

Secretory Vesicle ◽

Dense Core ◽

Gamma Aminobutyric Acid ◽

Dense Core Vesicles ◽

Protein Marker ◽

Preferential Localization ◽

Large Dense Core Vesicles ◽

Pheochromocytoma Pc12

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH-terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.

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Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion

The Journal of Cell Biology ◽

10.1083/jcb.201104079 ◽

2011 ◽

Vol 195 (7) ◽

pp. 1159-1170 ◽

Cited By ~ 72

Author(s):

Zhao Wang ◽

Huisheng Liu ◽

Yiwen Gu ◽

Edwin R. Chapman

Keyword(s):

Synaptic Transmission ◽

Membrane Fusion ◽

Synaptic Vesicle ◽

Cytoplasmic Domain ◽

Snare Proteins ◽

Antagonist Activity ◽

Vesicle Docking ◽

Synaptotagmin I ◽

Target Membrane

The synaptic vesicle protein synaptotagmin I (syt) promotes exocytosis via its ability to penetrate membranes in response to binding Ca2+ and through direct interactions with SNARE proteins. However, studies using full-length (FL) membrane-embedded syt in reconstituted fusion assays have yielded conflicting results, including a lack of effect, or even inhibition of fusion, by Ca2+. In this paper, we show that reconstituted FL syt promoted rapid docking of vesicles (<1 min) followed by a priming step (3–9 min) that was required for subsequent Ca2+-triggered fusion between v- and t-SNARE liposomes. Moreover, fusion occurred only when phosphatidylinositol 4,5-bisphosphate was included in the target membrane. This system also recapitulates some of the effects of syt mutations that alter synaptic transmission in neurons. Finally, we demonstrate that the cytoplasmic domain of syt exhibited mixed agonist/antagonist activity during regulated membrane fusion in vitro and in cells. Together, these findings reveal further convergence of reconstituted and cell-based systems.

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Molecular mechanism of secretory vesicle docking

Biochemical Society Transactions ◽

10.1042/bst0380192 ◽

2010 ◽

Vol 38 (1) ◽

pp. 192-198 ◽

Cited By ~ 20

Author(s):

Heidi de Wit

Keyword(s):

Plasma Membrane ◽

Cell Model ◽

Secretory Vesicle ◽

Model Systems ◽

Secretory Vesicles ◽

Filamentous Actin ◽

Embryonic Mouse ◽

Vesicle Docking ◽

Bovine Chromaffin Cell ◽

Stable Association

Docking, the stable association of secretory vesicles with the plasma membrane, is considered to be the necessary first step before vesicles gain fusion-competence, but it is unclear how vesicles dock. In adrenal medullary chromaffin cells, access of secretory vesicles to docking sites is controlled by dense F-actin (filamentous actin) beneath the plasma membrane. Recently, we found that, in the absence of Munc18-1, the number of docked vesicles and the thickness of cortical F-actin are affected. In the present paper, I discuss the possible mechanism by which Munc18-1 modulates cortical F-actin and how it orchestrates the docking machinery via an interaction with syntaxin-1. Finally, a comparison of Munc18's role in embryonic mouse and adult bovine chromaffin cell model systems will be made to clarify observed differences in cortical F-actin as well as docking phenotypes.

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The Role of Rab3a in Secretory Vesicle Docking Requires Association/Dissociation of Guanidine Phosphates and Munc18-1

PLoS ONE ◽

10.1371/journal.pone.0000616 ◽

2007 ◽

Vol 2 (7) ◽

pp. e616 ◽

Cited By ~ 21

Author(s):

Jan R.T. van Weering ◽

Ruud F. Toonen ◽

Matthijs Verhage

Keyword(s):

Secretory Vesicle ◽

Vesicle Docking

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Munc18-1 domain-1 controls vesicle docking and secretion by interacting with syntaxin-1 and chaperoning it to the plasma membrane

Molecular Biology of the Cell ◽

10.1091/mbc.e11-02-0135 ◽

2011 ◽

Vol 22 (21) ◽

pp. 4134-4149 ◽

Cited By ~ 31

Author(s):

Gayoung A. Han ◽

Nancy T. Malintan ◽

Ner Mu Nar Saw ◽

Lijun Li ◽

Liping Han ◽

...

Keyword(s):

Molecular Chaperone ◽

Dense Core ◽

Dense Core Vesicle ◽

Attachment Protein ◽

Vesicle Docking ◽

Protein Receptor ◽

Sensitive Factor ◽

Intracellular Expression ◽

Factor Attachment Protein Receptor

Munc18-1 plays pleiotropic roles in neurosecretion by acting as 1) a molecular chaperone of syntaxin-1, 2) a mediator of dense-core vesicle docking, and 3) a priming factor for soluble N-ethylmaleimide–sensitive factor attachment protein receptor–mediated membrane fusion. However, how these functions are executed and whether they are correlated remains unclear. Here we analyzed the role of the domain-1 cleft of Munc18-1 by measuring the abilities of various mutants (D34N, D34N/M38V, K46E, E59K, K46E/E59K, K63E, and E66A) to bind and chaperone syntaxin-1 and to restore the docking and secretion of dense-core vesicles in Munc18-1/-2 double-knockdown cells. We identified striking correlations between the abilities of these mutants to bind and chaperone syntaxin-1 with their ability to restore vesicle docking and secretion. These results suggest that the domain-1 cleft of Munc18-1 is essential for binding to syntaxin-1 and thereby critical for its chaperoning, docking, and secretory functions. Our results demonstrate that the effect of the alleged priming mutants (E59K, D34N/M38V) on exocytosis can largely be explained by their reduced syntaxin-1–chaperoning functions. Finally, our data suggest that the intracellular expression and distribution of syntaxin-1 determines the level of dense-core vesicle docking.

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Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e16-01-0057 ◽

2016 ◽

Vol 27 (15) ◽

pp. 2423-2434 ◽

Cited By ~ 27

Author(s):

Adam J. Trexler ◽

Kem A. Sochacki ◽

Justin W. Taraska

Keyword(s):

Plasma Membrane ◽

Fluorescence Microscopy ◽

Membrane Fusion ◽

Beta Cells ◽

Total Internal Reflection ◽

Living Cells ◽

Dense Core ◽

Fusion Pore ◽

Dense Core Vesicles ◽

Fusion Site

How and when the dozens of molecules that control exocytosis assemble in living cells to regulate the fusion of a vesicle with the plasma membrane is unknown. Here we image with two-color total internal reflection fluorescence microscopy the local changes of 27 proteins at single dense-core vesicles undergoing calcium-triggered fusion. We identify two broad dynamic behaviors of exocytic molecules. First, proteins enriched at exocytic sites are associated with DCVs long before exocytosis, and near the time of membrane fusion, they diffuse away. These proteins include Rab3 and Rab27, rabphilin3a, munc18a, tomosyn, and CAPS. Second, we observe a group of classical endocytic proteins and lipids, including dynamins, amphiphysin, syndapin, endophilin, and PIP2, which are rapidly and transiently recruited to the exocytic site near the time of membrane fusion. Dynamin mutants unable to bind amphiphysin were not recruited, indicating that amphiphysin is involved in localizing dynamin to the fusion site. Expression of mutant dynamins and knockdown of endogenous dynamin altered the rate of cargo release from single vesicles. Our data reveal the dynamics of many key proteins involved in exocytosis and identify a rapidly recruited dynamin/PIP2/BAR assembly that regulates the exocytic fusion pore of dense-core vesicles in cultured endocrine beta cells.

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