scholarly journals Dynamin controls neuropeptide secretion by organizing dense-core vesicle fusion sites

2021 ◽  
Vol 7 (21) ◽  
pp. eabf0659
Author(s):  
Alessandro Moro ◽  
Anne van Nifterick ◽  
Ruud F. Toonen ◽  
Matthijs Verhage
Keyword(s):  
Secretory Pathway ◽  
Dense Core ◽  
Dense Core Vesicle ◽  
Attachment Protein ◽  
Neuropeptide Secretion ◽  
Sensitive Factor ◽  
Fusion Site ◽  
Repeated Use ◽  
Guanosine Triphosphatase ◽  
Organizing Principles

Synaptic vesicles (SVs) release neurotransmitters at specialized active zones, but release sites and organizing principles for the other major secretory pathway, neuropeptide/neuromodulator release from dense-core vesicles (DCVs), remain elusive. We identify dynamins, yeast Vps1 orthologs, as DCV fusion site organizers in mammalian neurons. Genetic or pharmacological inactivation of all three dynamins strongly impaired DCV exocytosis, while SV exocytosis remained unaffected. Wild-type dynamin restored normal exocytosis but not guanosine triphosphatase–deficient or membrane-binding mutants that cause neurodevelopmental syndromes. During prolonged stimulation, repeated use of the same DCV fusion location was impaired in dynamin 1-3 triple knockout neurons. The syntaxin-1 staining efficiency, but not its expression level, was reduced. αSNAP (α–soluble N-ethylmaleimide–sensitive factor attachment protein) expression restored this. We conclude that mammalian dynamins organize DCV fusion sites, downstream of αSNAP, by regulating the equilibrium between fusogenic and non-fusogenic syntaxin-1 promoting its availability for SNARE (SNAP receptor) complex formation and DCV exocytosis.

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

2011 ◽  
Vol 22 (21) ◽  
pp. 4134-4149 ◽  
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.

scholarly journals The activity of Golgi transport vesicles depends on the presence of the N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF attachment protein (alpha SNAP) during vesicle formation.

1992 ◽  
Vol 118 (6) ◽  
pp. 1321-1332 ◽  
Author(s):  
B W Wattenberg ◽  
T J Raub ◽  
R R Hiebsch ◽  
P J Weidman
Keyword(s):  
Protein Transport ◽  
Vesicle Formation ◽  
Attachment Protein ◽  
Direct Role ◽  
Transport Vesicles ◽  
Sensitive Factor ◽  
Fusion Site ◽  
Transport Vesicle ◽  
Target Membrane ◽  
A Cell

An assay designed to measure the formation of functional transport vesicles was constructed by modifying a cell-free assay for protein transport between compartments of the Golgi (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:405-416). A 35-kD cytosolic protein that is immunologically and functionally indistinguishable from alpha SNAP (soluble NSF attachment protein) was found to be required during vesicle formation. SNAP, together with the N-ethylmaleimide-sensitive factor (NSF) have previously been implicated in the attachment and/or fusion of vesicles with their target membrane. We show that NSF is also required during the formation of functional vesicles. Strikingly, we found that after vesicle formation, the NEM-sensitive function of NSF was no longer required for transport to proceed through the ensuing steps of vesicle attachment and fusion. In contrast to these functional tests of vesicle formation, SNAP was not required for the morphological appearance of vesicular structures on the Golgi membranes. If SNAP and NSF have a direct role in transport vesicle attachment and/or fusion, as previously suggested, these results indicate that these proteins become incorporated into the vesicle membranes during vesicle formation and are brought to the fusion site on the transport vesicles.

scholarly journals Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1

2012 ◽  
Vol 23 (2) ◽  
pp. 337-346 ◽  
Author(s):  
Francesca Morgera ◽  
Margaret R. Sallah ◽  
Michelle L. Dubuke ◽  
Pallavi Gandhi ◽  
Daniel N. Brewer ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Secretory Pathway ◽  
Secretory Vesicle ◽  
Snare Complex ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Membrane Bound ◽  
Sm Protein ◽  
Snare Complex Formation

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function—it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6–Sec1 interaction is exclusive of Sec6–Sec9 but compatible with Sec6–exocyst assembly. In contrast, the Sec6–exocyst interaction is incompatible with Sec6–Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6–exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.

scholarly journals Atg16L1, an essential factor for canonical autophagy, participates in hormone secretion from PC12 cells independently of autophagic activity

2012 ◽  
Vol 23 (16) ◽  
pp. 3193-3202 ◽  
Author(s):  
Koutaro Ishibashi ◽  
Takefumi Uemura ◽  
Satoshi Waguri ◽  
Mitsunori Fukuda
Keyword(s):  
Pc12 Cells ◽  
Secretory Pathway ◽  
Biological Activities ◽  
Hormone Secretion ◽  
Cell Types ◽  
Small Gtpase ◽  
Dense Core ◽  
Dense Core Vesicle ◽  
Dense Core Vesicles ◽  
Autophagic Activity

Autophagy is a bulk degradation system in all eukaryotic cells and regulates a variety of biological activities in higher eukaryotes. Recently involvement of autophagy in the regulation of the secretory pathway has also been reported, but the molecular mechanism linking autophagy with the secretory pathway remains largely unknown. Here we show that Atg16L1, an essential protein for canonical autophagy, is localized on hormone-containing dense-core vesicles in neuroendocrine PC12 cells and that knockdown of Atg16L1 causes a dramatic reduction in the level of hormone secretion independently of autophagic activity. We also find that Atg16L1 interacts with the small GTPase Rab33A and that this interaction is required for the dense-core vesicle localization of Atg16L1 in PC12 cells. Our findings indicate that Atg16L1 regulates not only autophagy in all cell types, but also secretion from dense-core vesicles, presumably by acting as a Rab33A effector, in particular cell types.

Copper modulates the large dense core vesicle secretory pathway in PC12 cells

Metallomics ◽  
2013 ◽  
Vol 5 (6) ◽  
pp. 700 ◽  
Author(s):  
Clare Duncan ◽  
Laura Bica ◽  
Peter J. Crouch ◽  
Aphrodite Caragounis ◽  
Grace E. Lidgerwood ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Secretory Pathway ◽  
Dense Core ◽  
Dense Core Vesicle ◽  
Large Dense Core Vesicle

scholarly journals AtVPS45 Complex Formation at thetrans-Golgi Network

2000 ◽  
Vol 11 (7) ◽  
pp. 2251-2265 ◽  
Author(s):  
Diane C. Bassham ◽  
Anton A. Sanderfoot ◽  
Valentina Kovaleva ◽  
Haiyan Zheng ◽  
Natasha V. Raikhel
Keyword(s):  
Secretory Pathway ◽  
Gene Families ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Attachment Protein ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Target Membrane ◽  
Functional Complexity ◽  
Golgi Network

The Sec1p family of proteins are thought to be involved in the regulation of vesicle fusion reactions through interaction with t-SNAREs (target soluble N-ethylmaleimide–sensitive factor attachment protein receptors) at the target membrane. AtVPS45 is a member of this family from Arabidopsis thaliana that we now demonstrate to be present on the trans-Golgi network (TGN), where it colocalizes with the vacuolar cargo receptor AtELP. Unlike yeast Vps45p, AtVPS45 does not interact with, or colocalize with, the prevacuolar t-SNARE AtPEP12. Instead, AtVPS45 interacts with two t-SNAREs, AtTLG2a and AtTLG2b, that show similarity to the yeast t-SNARE Tlg2p. AtTLG2a and -b each colocalize with AtVPS45 at the TGN; however, AtTLG2a is in a different region of the TGN than AtTLG2b by immunogold electron microscopy. Therefore, we propose that complexes containing AtVPS45 and either AtTLG2a or -b define functional subdomains of the TGN and may be required for different trafficking events. Among other Arabidopsis SNAREs, AtVPS45 antibodies preferentially coprecipitate AtVTI1b over the closely related isoform AtVTI1a, implying that AtVTI1a and AtVTI1b also have distinct functions within the cell. These data point to a functional complexity within the plant secretory pathway, where proteins encoded by gene families have specialized functions, rather than functional redundancy.

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