Tomosyn negatively regulates arginine vasopressin secretion in embryonic stem cell-derived neurons

Seiji Takeuchi, Shintaro Iwama, Hiroshi Takagi, Atsushi Kiyota, Kohtaro Nakashima, Hisakazu Izumida, Haruki Fujisawa, Naoko Iwata, Hidetaka Suga, Takashi Watanabe, Kozo Kaibuchi, Yutaka Oiso, Hiroshi Arima, Yoshihisa Sugimura

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP remains to be elucidated. To better understand the mechanisms of AVP secretion, in our study we have identified proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25). SNAP25 plays a crucial role in exocytosis, in the posterior pituitary. Embryonic stem (ES) cell-derived AVP neurons were established to investigate the functions of the identified proteins. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, we identified tomosyn-1 (syntaxinbinding protein 5) as a SNAP25-binding protein in the posterior pituitary. Coimmunoprecipitation assays indicated that tomosyn formed N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with SNAP25 and syntaxin1. Immunohistochemistry showed that tomosyn localized to the posterior pituitary. Mouse ES cells self-differentiated into AVP neurons (mES-AVP) that expressed tomosyn and two transmembrane SNARE proteins, including SNAP25 and syntaxin1. KCl increased AVP secretion in mES-AVP, and overexpression of tomosyn-1 reduced KCl-stimulated AVP secretion. Downregulation of tomosyn-1 with siRNA increased KCl-stimulated AVP secretion. These results suggested that tomosyn-1 negatively regulated AVP secretion in mES-AVP and further suggest the possibility of using mES-AVP culture systems to evaluate the role of synaptic proteins from AVP neurons.

Original languageEnglish
Article numbere0164544
JournalPloS one
Volume11
Issue number10
DOIs
Publication statusPublished - 01-10-2016

Fingerprint

arginine vasopressin
Arginine Vasopressin
embryonic stem cells
Embryonic Stem Cells
Stem cells
Neurons
neurons
secretion
posterior pituitary
exocytosis
Exocytosis
Synaptosomal-Associated Protein 25
SNARE Proteins
proteins
Proteins
Assays
N-Ethylmaleimide-Sensitive Proteins
transmembrane proteins
assays
small interfering RNA

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Takeuchi, Seiji ; Iwama, Shintaro ; Takagi, Hiroshi ; Kiyota, Atsushi ; Nakashima, Kohtaro ; Izumida, Hisakazu ; Fujisawa, Haruki ; Iwata, Naoko ; Suga, Hidetaka ; Watanabe, Takashi ; Kaibuchi, Kozo ; Oiso, Yutaka ; Arima, Hiroshi ; Sugimura, Yoshihisa. / Tomosyn negatively regulates arginine vasopressin secretion in embryonic stem cell-derived neurons. In: PloS one. 2016 ; Vol. 11, No. 10.
@article{e6c6f56c6c534be5b5ad3fa2bcc9abc7,
title = "Tomosyn negatively regulates arginine vasopressin secretion in embryonic stem cell-derived neurons",
abstract = "Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP remains to be elucidated. To better understand the mechanisms of AVP secretion, in our study we have identified proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25). SNAP25 plays a crucial role in exocytosis, in the posterior pituitary. Embryonic stem (ES) cell-derived AVP neurons were established to investigate the functions of the identified proteins. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, we identified tomosyn-1 (syntaxinbinding protein 5) as a SNAP25-binding protein in the posterior pituitary. Coimmunoprecipitation assays indicated that tomosyn formed N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with SNAP25 and syntaxin1. Immunohistochemistry showed that tomosyn localized to the posterior pituitary. Mouse ES cells self-differentiated into AVP neurons (mES-AVP) that expressed tomosyn and two transmembrane SNARE proteins, including SNAP25 and syntaxin1. KCl increased AVP secretion in mES-AVP, and overexpression of tomosyn-1 reduced KCl-stimulated AVP secretion. Downregulation of tomosyn-1 with siRNA increased KCl-stimulated AVP secretion. These results suggested that tomosyn-1 negatively regulated AVP secretion in mES-AVP and further suggest the possibility of using mES-AVP culture systems to evaluate the role of synaptic proteins from AVP neurons.",
author = "Seiji Takeuchi and Shintaro Iwama and Hiroshi Takagi and Atsushi Kiyota and Kohtaro Nakashima and Hisakazu Izumida and Haruki Fujisawa and Naoko Iwata and Hidetaka Suga and Takashi Watanabe and Kozo Kaibuchi and Yutaka Oiso and Hiroshi Arima and Yoshihisa Sugimura",
year = "2016",
month = "10",
day = "1",
doi = "10.1371/journal.pone.0164544",
language = "English",
volume = "11",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "10",

}

Takeuchi, S, Iwama, S, Takagi, H, Kiyota, A, Nakashima, K, Izumida, H, Fujisawa, H, Iwata, N, Suga, H, Watanabe, T, Kaibuchi, K, Oiso, Y, Arima, H & Sugimura, Y 2016, 'Tomosyn negatively regulates arginine vasopressin secretion in embryonic stem cell-derived neurons', PloS one, vol. 11, no. 10, e0164544. https://doi.org/10.1371/journal.pone.0164544

Tomosyn negatively regulates arginine vasopressin secretion in embryonic stem cell-derived neurons. / Takeuchi, Seiji; Iwama, Shintaro; Takagi, Hiroshi; Kiyota, Atsushi; Nakashima, Kohtaro; Izumida, Hisakazu; Fujisawa, Haruki; Iwata, Naoko; Suga, Hidetaka; Watanabe, Takashi; Kaibuchi, Kozo; Oiso, Yutaka; Arima, Hiroshi; Sugimura, Yoshihisa.

In: PloS one, Vol. 11, No. 10, e0164544, 01.10.2016.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Tomosyn negatively regulates arginine vasopressin secretion in embryonic stem cell-derived neurons

AU - Takeuchi, Seiji

AU - Iwama, Shintaro

AU - Takagi, Hiroshi

AU - Kiyota, Atsushi

AU - Nakashima, Kohtaro

AU - Izumida, Hisakazu

AU - Fujisawa, Haruki

AU - Iwata, Naoko

AU - Suga, Hidetaka

AU - Watanabe, Takashi

AU - Kaibuchi, Kozo

AU - Oiso, Yutaka

AU - Arima, Hiroshi

AU - Sugimura, Yoshihisa

PY - 2016/10/1

Y1 - 2016/10/1

N2 - Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP remains to be elucidated. To better understand the mechanisms of AVP secretion, in our study we have identified proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25). SNAP25 plays a crucial role in exocytosis, in the posterior pituitary. Embryonic stem (ES) cell-derived AVP neurons were established to investigate the functions of the identified proteins. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, we identified tomosyn-1 (syntaxinbinding protein 5) as a SNAP25-binding protein in the posterior pituitary. Coimmunoprecipitation assays indicated that tomosyn formed N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with SNAP25 and syntaxin1. Immunohistochemistry showed that tomosyn localized to the posterior pituitary. Mouse ES cells self-differentiated into AVP neurons (mES-AVP) that expressed tomosyn and two transmembrane SNARE proteins, including SNAP25 and syntaxin1. KCl increased AVP secretion in mES-AVP, and overexpression of tomosyn-1 reduced KCl-stimulated AVP secretion. Downregulation of tomosyn-1 with siRNA increased KCl-stimulated AVP secretion. These results suggested that tomosyn-1 negatively regulated AVP secretion in mES-AVP and further suggest the possibility of using mES-AVP culture systems to evaluate the role of synaptic proteins from AVP neurons.

AB - Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP remains to be elucidated. To better understand the mechanisms of AVP secretion, in our study we have identified proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25). SNAP25 plays a crucial role in exocytosis, in the posterior pituitary. Embryonic stem (ES) cell-derived AVP neurons were established to investigate the functions of the identified proteins. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, we identified tomosyn-1 (syntaxinbinding protein 5) as a SNAP25-binding protein in the posterior pituitary. Coimmunoprecipitation assays indicated that tomosyn formed N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with SNAP25 and syntaxin1. Immunohistochemistry showed that tomosyn localized to the posterior pituitary. Mouse ES cells self-differentiated into AVP neurons (mES-AVP) that expressed tomosyn and two transmembrane SNARE proteins, including SNAP25 and syntaxin1. KCl increased AVP secretion in mES-AVP, and overexpression of tomosyn-1 reduced KCl-stimulated AVP secretion. Downregulation of tomosyn-1 with siRNA increased KCl-stimulated AVP secretion. These results suggested that tomosyn-1 negatively regulated AVP secretion in mES-AVP and further suggest the possibility of using mES-AVP culture systems to evaluate the role of synaptic proteins from AVP neurons.

UR - http://www.scopus.com/inward/record.url?scp=84991454563&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84991454563&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0164544

DO - 10.1371/journal.pone.0164544

M3 - Article

C2 - 27732637

AN - SCOPUS:84991454563

VL - 11

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 10

M1 - e0164544

ER -

Takeuchi S, Iwama S, Takagi H, Kiyota A, Nakashima K, Izumida H et al. Tomosyn negatively regulates arginine vasopressin secretion in embryonic stem cell-derived neurons. PloS one. 2016 Oct 1;11(10). e0164544. https://doi.org/10.1371/journal.pone.0164544