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

Date Published: July 26, 2017

Publisher: Public Library of Science

Author(s): Cornelia J. Geerts, Roberta Mancini, Ning Chen, Frank T. W. Koopmans, Ka Wan Li, August B. Smit, Jan R. T. van Weering, Matthijs Verhage, Alexander J. A. Groffen, Jiajie Diao.


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.

Partial Text

Neural communication is established by the controlled release of signaling molecules from synaptic vesicles (SVs) and large dense-core vesicles (DCVs). Coordinated transport is essential to deliver secretory vesicles and their cargos to sites of release. For synapse formation in young neurons, multiple active zone proteins are packaged and co-transported in piccolo-bassoon transport vesicles (PTVs) [1,2], while synaptic vesicle components are transported by synaptic vesicle precursor (SVP) organelles [3,4]. Lateral axonal transport in mature neurons is central to dynamic sharing of vesicles across adjacent presynaptic boutons, implicated in synaptic plasticity [5–7]. Interestingly, vesicular organelles with different destinations co-migrate in neurites [8,9], while the final subcellular targeting steps are likely encoded by molecules on the vesicle surface [10–12].

Tomosyn is a cytosolic inhibitor of secretion that localizes both pre- and postsynaptically in diverse model systems [17,20,28]. Despite the absence of a membrane anchor, some evidence points to an association of tomosyn with secretory vesicles [16,21,32]. Here we studied the localization of tomosyn in cultured hippocampal neurons. Besides a diffuse distribution in neurites and accumulation at synapses, tomosyn co-localized with moving SVs and DCVs in living neurons. The presence of at least a third type of tomosyn-containing transport organelles was suggested by fast-moving tomosyn puncta that did not co-migrate with synapsin- or NPY-mCherry (Fig 5E and 5F). In line with the broad distribution of neuronal secretory vesicles [42,50], tomosyn puncta were observed in both axons and dendrites. The association of tomosyn with secretory vesicles is apparently driven by multiple redundant interactions in the N- and C-terminal domains. The observation that the SNARE domain alone is sufficient for co-migration with secretory vesicles suggests a contribution of the reported SNARE interaction [25,44,45,51]. In addition however, the isolated N-terminal domain is also able to bind to vesicles. This activity is not attributable to Syt-1 binding [41], leaving Rab3 as the most likely candidate [29]. Interestingly, the yeast tomosyn ortholog Sro7p also associates with secretory vesicles through an interaction with the Rab GTPase Sec4p [46,52]. This interaction is GTP-dependent and was mapped to the boundary between the two WD40 propellers of Sro7p, suggesting a highly conserved role in vesicular trafficking. Compared to full length tomosyn, reduced co-migration of tail-CC and CC tomosyn-m1 fragments with synapsin puncta, but not NPY puncta, was observed, suggesting that the tomosyn binding modes may differ between SVs and DCVs.




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