Formation of Golgi-Derived Active Zone Precursor Vesicles
ABSTRACT Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellular mechanism for the delivery of scaffold proteins, ion channels, and receptors to nascent and mature synapses. However, the molecular mechanisms leading to the selection of cargos and their differential transport to subneuronal compartments are not well understood, in part because of the mixing of cargos at the plasma membrane and/or within endosomal compartments. In the present study, we have explored the cellular mechanisms of active zone precursor vesicle assembly at the Golgi in dissociated hippocampal neurons of Rattus norvegicus. Our studies show that Piccolo, Bassoon, and ELKS2/CAST exit the trans-Golgi network on a common vesicle that requires Piccolo and Bassoon for its proper assembly. In contrast, Munc13 and synaptic vesicle proteins use distinct sets of Golgi-derived transport vesicles, while RIM1α associates with vesicular membranes in a post-Golgi compartment. Furthermore, Piccolo and Bassoon are necessary for ELKS2/CAST to leave the Golgi in association with vesicles, and a core domain of Bassoon is sufficient to facilitate formation of these vesicles. While these findings support emerging principles regarding active zone differentiation, the cellular and molecular analyses reported here also indicate that the Piccolo-Bassoon transport vesicles leaving the Golgi may undergo further changes in protein composition before arriving at synaptic sites.
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- "Bassoon and its paralogue Piccolo are transported into the axon on membranous organelles , , , . To check whether the defect in supply of mobile GFP-BsnS2845A contributes to observed decreased recovery we analyzed the mobility properties of fluorescently-labeled vesicles in neurons transfected with wt and mutant constructs. "
ABSTRACT: The proper organization of the presynaptic cytomatrix at the active zone is essential for reliable neurotransmitter release from neurons. Despite of the virtual stability of this tightly interconnected proteinaceous network it becomes increasingly clear that regulated dynamic changes of its composition play an important role in the processes of synaptic plasticity. Bassoon, a core component of the presynaptic cytomatrix, is a key player in structural organization and functional regulation of presynaptic release sites. It is one of the most highly phosphorylated synaptic proteins. Nevertheless, to date our knowledge about functions mediated by any one of the identified phosphorylation sites of Bassoon is sparse. In this study, we have identified an interaction of Bassoon with the small adaptor protein 14-3-3, which depends on phosphorylation of the 14-3-3 binding motif of Bassoon. In vitro phosphorylation assays indicate that phosphorylation of the critical Ser-2845 residue of Bassoon can be mediated by a member of the 90-kDa ribosomal S6 protein kinase family. Elimination of Ser-2845 from the 14-3-3 binding motif results in a significant decrease of Bassoon's molecular exchange rates at synapses of living rat neurons. We propose that the phosphorylation-induced 14-3-3 binding to Bassoon modulates its anchoring to the presynaptic cytomatrix. This regulation mechanism might participate in molecular and structural presynaptic remodeling during synaptic plasticity.PLoS ONE 03/2013; 8(3):e58814. DOI:10.1371/journal.pone.0058814 · 3.23 Impact Factor
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ABSTRACT: The location, size, and number of synapses critically influence the specificity and strength of neural connections. In axons, synaptic vesicle (SV) and active zone (AZ) proteins are transported by molecular motors and accumulate at discrete presynaptic loci. Little is known about the mechanisms coordinating presynaptic protein transport and deposition to achieve proper distribution of synaptic material. Here we show that SV and AZ proteins exhibit extensive cotransport and undergo frequent pauses. At the axonal and synaptic pause sites, the balance between the capture and dissociation of mobile transport packets determines the extent of presynaptic assembly. The small G protein ARL-8 inhibits assembly by promoting dissociation, while a JNK kinase pathway and AZ assembly proteins inhibit dissociation. Furthermore, ARL-8 directly binds to the UNC-104/KIF1A motor to limit the capture efficiency. Together, molecular regulation of the dichotomy between axonal trafficking and local assembly controls vital aspects of synapse formation and maintenance.Neuron 05/2013; 78(6). DOI:10.1016/j.neuron.2013.04.035 · 15.98 Impact Factor
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ABSTRACT: Synapse formation is a highly regulated process that requires the coordination of many cell biological events. Decades of research have identified a long list of molecular components involved in assembling a functioning synapse. Yet how the various steps, from transporting synaptic components to adhering synaptic partners and assembling the synaptic structure, are regulated and precisely executed during development and maintenance is still unclear. With the improvement of imaging and molecular tools, recent work in vertebrate and invertebrate systems has provided important insight into various aspects of presynaptic development, maintenance, and trans-synaptic signals, thereby increasing our understanding of how extrinsic organizers and intracellular mechanisms contribute to presynapse formation.The Journal of Cell Biology 10/2013; 203(1):11-22. DOI:10.1083/jcb.201307020 · 9.69 Impact Factor