Tomosyn associates with secretory vesicles in neurons through its N- and C-terminal domains

PLoS One. 2017 Jul 26;12(7):e0180912. doi: 10.1371/journal.pone.0180912. eCollection 2017.

Abstract

The secretory pathway in neurons requires efficient targeting of cargos and regulatory proteins to their release sites. Tomosyn contributes to synapse function by regulating synaptic vesicle (SV) and dense-core vesicle (DCV) secretion. While there is large support for the presynaptic accumulation of tomosyn in fixed preparations, alternative subcellular locations have been suggested. Here we studied the dynamic distribution of tomosyn-1 (Stxbp5) and tomosyn-2 (Stxbp5l) in mouse hippocampal neurons and observed a mixed diffuse and punctate localization pattern of both isoforms. Tomosyn-1 accumulations were present in axons and dendrites. As expected, tomosyn-1 was expressed in about 75% of the presynaptic terminals. Interestingly, also bidirectional moving tomosyn-1 and -2 puncta were observed. Despite the lack of a membrane anchor these puncta co-migrated with synapsin and neuropeptide Y, markers for respectively SVs and DCVs. Genetic blockade of two known tomosyn interactions with synaptotagmin-1 and its cognate SNAREs did not abolish its vesicular co-migration, suggesting an interplay of protein interactions mediated by the WD40 and SNARE domains. We hypothesize that the vesicle-binding properties of tomosyns may control the delivery, pan-synaptic sharing and secretion of neuronal signaling molecules, exceeding its canonical role at the plasma membrane.

MeSH terms

  • Adaptor Proteins, Vesicular Transport
  • Animals
  • Axons / metabolism
  • Binding Sites
  • Blotting, Western
  • Cells, Cultured
  • Dendrites / metabolism
  • Hippocampus / cytology
  • Hippocampus / metabolism*
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Microscopy, Confocal
  • Microscopy, Immunoelectron
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Neurons / metabolism*
  • Neurons / ultrastructure
  • Neuropeptide Y / metabolism
  • Presynaptic Terminals / metabolism
  • Protein Binding
  • Protein Transport
  • R-SNARE Proteins / chemistry
  • R-SNARE Proteins / genetics
  • R-SNARE Proteins / metabolism*
  • Secretory Vesicles / metabolism
  • Synapsins / genetics
  • Synapsins / metabolism
  • Synaptic Vesicles / metabolism
  • Synaptotagmin I / genetics
  • Synaptotagmin I / metabolism

Substances

  • Adaptor Proteins, Vesicular Transport
  • Nerve Tissue Proteins
  • Neuropeptide Y
  • R-SNARE Proteins
  • STXBP5L protein, mouse
  • Synapsins
  • Synaptotagmin I
  • tomosyn protein, mouse

Grant support

The research was supported by the Centre for Medical Systems Biology (CMSB2 project 3.3.5) to MV, the EU (FP7 SYNSYS HEALTH-F2-2009-242167 to AS, FP7-PEOPLE-2013-607508 and the Erasmus Mundus ENC network to AG) and the Netherlands Organization for Health Research and Development (ZonMW project 91111009 to the VU/VUmc EM facility and 91113022 to AG).