Faculty Opinions recommendation of CAPS and syntaxin dock dense core vesicles to the plasma membrane in neurons.

2008 ◽  
Author(s):  
Jens Rettig
Keyword(s):  
Plasma Membrane ◽  
Dense Core ◽  
Dense Core Vesicles

scholarly journals Synaptotagmin VII Is Targeted to Dense-core Vesicles and Regulates Their Ca2+-dependent Exocytosis in PC12 Cells

2004 ◽  
Vol 279 (50) ◽  
pp. 52677-52684 ◽  
Author(s):  
Mitsunori Fukuda ◽  
Eiko Kanno ◽  
Megumi Satoh ◽  
Chika Saegusa ◽  
Akitsugu Yamamoto
Keyword(s):  
Plasma Membrane ◽  
Neuropeptide Y ◽  
Cell Line ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Small Interfering Rna ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Pc12 Cell Line ◽  
Interfering Rna

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membraneversussecretory granules). In this study we established a PC12 cell line “stably expressing” the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as thetrans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+-dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (∼60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (∼85–90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expressionversusstable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+sensor for exocytosis in endocrine cells.

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

2016 ◽  
Vol 27 (15) ◽  
pp. 2423-2434 ◽  
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.

scholarly journals The Slp4-a Linker Domain Controls Exocytosis through Interaction with Munc18-1·Syntaxin-1a Complex

2006 ◽  
Vol 17 (5) ◽  
pp. 2101-2112 ◽  
Author(s):  
Takashi Tsuboi ◽  
Mitsunori Fukuda
Keyword(s):  
Plasma Membrane ◽  
Specific Interaction ◽  
Inhibitory Effect ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Dense Core Vesicle ◽  
Dense Core Vesicles ◽  
Syntaxin 1A ◽  
Vesicle Exocytosis ◽  
Linker Domain

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1·syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly inhibited high-KCl–induced dense-core vesicle exocytosis. The inhibitory effect by Slp4-a is absolutely dependent on the linker domain of Slp4-a, because substitution of the linker domain of Slp4-a by that of Slp5 (the closest isoform of Slp4-a that cannot bind the Munc18-1·syntaxin-1a complex) completely abrogated the inhibitory effect. Our findings reveal a novel docking machinery for dense-core vesicle exocytosis: Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane.

scholarly journals Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

2016 ◽  
Vol 27 (4) ◽  
pp. 654-668 ◽  
Author(s):  
Greg Kabachinski ◽  
D. Michelle Kielar-Grevstad ◽  
Xingmin Zhang ◽  
Declan J. James ◽  
Thomas F. J. Martin
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Protein Complexes ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Snare Complex ◽  
Snare Protein ◽  
Dense Core Vesicles ◽  
Vesicle Docking ◽  
Single Vesicle

The Ca2+-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.

scholarly journals CAPS and syntaxin dock dense core vesicles to the plasma membrane in neurons

2008 ◽  
Vol 180 (3) ◽  
pp. 483-491 ◽  
Author(s):  
Marc Hammarlund ◽  
Shigeki Watanabe ◽  
Kim Schuske ◽  
Erik M. Jorgensen
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Motor Neurons ◽  
Dense Core ◽  
Dense Core Vesicle ◽  
Dense Core Vesicles ◽  
Vesicle Docking ◽  
Rapid Release ◽  
Related Proteins ◽  
Protein Attachment

Docking to the plasma membrane prepares vesicles for rapid release. Here, we describe a mechanism for dense core vesicle docking in neurons. In Caenorhabditis elegans motor neurons, dense core vesicles dock at the plasma membrane but are excluded from active zones at synapses. We have found that the calcium-activated protein for secretion (CAPS) protein is required for dense core vesicle docking but not synaptic vesicle docking. In contrast, we see that UNC-13, a docking factor for synaptic vesicles, is not essential for dense core vesicle docking. Both the CAPS and UNC-13 docking pathways converge on syntaxin, a component of the SNARE (soluble N-ethyl-maleimide–sensitive fusion protein attachment receptor) complex. Overexpression of open syntaxin can bypass the requirement for CAPS in dense core vesicle docking. Thus, CAPS likely promotes the open state of syntaxin, which then docks dense core vesicles. CAPS function in dense core vesicle docking parallels UNC-13 in synaptic vesicle docking, which suggests that these related proteins act similarly to promote docking of independent vesicle populations.

scholarly journals The nanoscale anatomy of exocytic dense-core vesicles in neuroendocrine cells

2020 ◽  
Author(s):  
Bijeta Prasai ◽  
Gideon J. Haber ◽  
Marie-Paule Strub ◽  
John A. Ciemniecki ◽  
Kem A. Sochacki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Metal Binding ◽  
Electron Tomography ◽  
Molecular Model ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Rab Gtpases ◽  
Dense Core Vesicles

AbstractRab-GTPases and their interacting partners are key regulators of secretory vesicle trafficking, docking, and fusion to the plasma membrane in neurons and neuroendocrine cells. Where and how these proteins are positioned and organized with respect to the vesicle and plasma membrane are unknown. Here, we use correlative super-resolution light and platinum replica electron microscopy to map Rab-GTPases (Rab27a and Rab3a) and their effectors (Granuphilin-a, Rabphilin3a, and Rim2) at the nanoscale in 2D. Next, we develop a targetable genetically-encoded electron microscopy labeling method that uses histidine based affinity-tags and metal-binding gold-nanoparticles to determine the axial location of exocytic proteins using electron tomography. Our data show that Rab-GTPases and their effectors are distributed across the entire surface of individual docked vesicles. This circumferential distribution likely aids in the efficient transport, capture, docking, and rapid fusion of vesicles in excitable cells. The nanoscale molecular model of dense core vesicles generated from our methods reveals how key proteins assemble at the plasma membrane to regulate membrane trafficking and exocytosis.

scholarly journals Munc18-1 Is Critical for Plasma Membrane Localization of Syntaxin1 but Not of SNAP-25 in PC12 Cells

2008 ◽  
Vol 19 (2) ◽  
pp. 722-734 ◽  
Author(s):  
Lakshmanan Arunachalam ◽  
Liping Han ◽  
Nardos G. Tassew ◽  
Yu He ◽  
Li Wang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Expression Level ◽  
Membrane Localization ◽  
Dense Core Vesicles ◽  
Interacting Protein ◽  
Plasma Membrane Localization ◽  
Mutant Phenotypes

Although Munc18-1 was originally identified as a syntaxin1–interacting protein, the physiological significance of this interaction remains unclear. In fact, recent studies of Munc18-1 mutants have suggested that Munc18-1 plays a critical role for docking of secretory vesicles, independent of syntaxin1 regulation. Here we investigated the role of Munc18-1 in syntaxin1 localization by generating stable neuroendocrine cell lines in which Munc18-1 was strongly down-regulated. In these cells, the secretion capability, as well as the docking of dense-core vesicles, was significantly reduced. More importantly, not only was the expression level of syntaxin1 reduced, but the localization of syntaxin1 at the plasma membrane was also severely perturbed. The mislocalized syntaxin1 resided primarily in the perinuclear region of the cells, in which it was highly colocalized with Secretogranin II, a marker protein for dense-core vesicles. In contrast, the expression level and the plasma membrane localization of SNAP-25 were not affected. Furthermore, the syntaxin1 localization and the secretion capability were restored upon transfection-mediated reintroduction of Munc18-1. Our results indicate that endogenous Munc18-1 plays a critical role for the plasma membrane localization of syntaxin1 in neuroendocrine cells and therefore necessitates the interpretation of Munc18-1 mutant phenotypes to be in terms of mislocalized syntaxin1.

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