A kymograph representation shows bidirectional movement of these puncta (Fig 3C). Dual-colour images were acquired for 60 frames at 1 Hz.(MP4) pone.0180912.s005.mp4 (1.1M) GUID:?DC068626-0CFD-4E39-BCE6-E996C59DDDA7 S4 File: Live imaging movie of construct Tail-CC. EYFP-tomosyn-1 and synapsin-mCherry are depicted in green and red, respectively. Dual-colour images were acquired for 60 frames at 1 Hz.(MP4) pone.0180912.s006.mp4 (945K) GUID:?36878062-89DE-465D-BBF3-31D7CCE3374D S5 File: Live imaging CCK2R Ligand-Linker Conjugates 1 movie of construct CC. EYFP-tomosyn-1 and synapsin-mCherry are depicted in green and red, respectively. Dual-colour images were acquired for 60 frames at 1 Hz.(MP4) pone.0180912.s007.mp4 (982K) GUID:?182E940B-E48E-4C04-8EC3-6FAEB08477BB Data Availability StatementAll data are deposited in the VU Institutional Research Data Management system at https://research.vu.nl with accession number 32936588. Abstract The secretory pathway in neurons MDNCF 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 CCK2R Ligand-Linker Conjugates 1 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 CCK2R Ligand-Linker Conjugates 1 and secretion of neuronal signaling molecules, exceeding its canonical role at the plasma membrane. Introduction 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 [5C7]. 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 [10C12]. Neurotransmitter release is mediated by a complex of VAMP2 on the vesicular membrane and syntaxin-1/SNAP25 on the plasma membrane, although the latter molecules were also observed on the vesicle surface [13C16]. Tomosyn is an inhibitor of such SNARE (Soluble NSF Attachment Protein Receptor)-mediated secretion from SVs [16C20] and DCVs [21,22] that fuse with the plasma membrane in axons and dendrites [23,24]. It competes with the vesicular SNARE for t-SNARE-binding, does not contain a vesicle-binding motif itself and was suggested to thereby prevent priming of vesicles [20,25,26]. By splice variation, two paralogous genes (tomosyn-1/STXBP5 and tomosyn-2/STXBP5L) give rise to at least seven tomosyn isoforms in the mammalian brain [27]. In line with a presynaptic function, tomosyn localizes with synaptic markers in mouse hippocampal tissue [28], hippocampal neurons in primary culture [29], superior cervical ganglion cells [17] and motor neurons [19]. Dendritic localization has been observed in mouse hippocampal tissue slices [28]. In both HEK293 and PC12 cells, fluorescent-tagged tomosyn exhibits a diffuse cytoplasmic distribution, whereas co-expression of syntaxin-1A induces plasma membrane binding [30,31]. In insulin-secreting INS-1E cells [32] and MIN6 cells [29], tomosyn expression partly co-localizes with secretory granules. Amisyn, a tomosyn homologous protein, is mainly cytosolic, but a fraction associates with membranes in rat brain extract, partly independent of syntaxin [33]. Both tomosyn and amisyn are present on SVs according to proteomic analysis [16]. Tomosyn also CCK2R Ligand-Linker Conjugates 1 associates with DCVs in immuno-electron microscopy [21]. Thus, while there is CCK2R Ligand-Linker Conjugates 1 large support for the synaptic targeting of tomosyn in fixed preparations, a number of other localizations have also been described, prompting a need for a more detailed localization of tomosyn in living neurons. In this study we show that tomosyn is targeted to migrating SVs and DCVs by multiple redundant interactions located in different domains of the protein. These data suggest an intricate role of tomosyn beyond the conventional model in which it inhibits neurotransmitter release by competing with VAMP2 for.