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High- and low-mobility stages in the synaptic vesicle cycle.

ABSTRACT: Synaptic vesicles need to be mobile to reach their release sites during synaptic activity. We investigated vesicle mobility throughout the synaptic vesicle cycle using both conventional and subdiffraction-resolution stimulated emission depletion fluorescence microscopy. Vesicle tracking revealed that recently endocytosed synaptic vesicles are highly mobile for a substantial time period after endocytosis. They later undergo a maturation process and integrate into vesicle clusters where they exhibit little mobility. Despite the differences in mobility, both recently endocytosed and mature vesicles are exchanged between synapses. Electrical stimulation does not seem to affect the mobility of the two types of vesicles. After exocytosis, the vesicle material is mobile in the plasma membrane, although the movement appears to be somewhat limited. Increasing the proportion of fused vesicles (by stimulating exocytosis while simultaneously blocking endocytosis) leads to substantially higher mobility. We conclude that both high- and low-mobility states are characteristic of synaptic vesicle movement.

SUBMITTER: Kamin D

PROVIDER: S-EPMC2905121 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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High- and low-mobility stages in the synaptic vesicle cycle.

Kamin Dirk D Lauterbach Marcel A MA Westphal Volker V Keller Jan J Schönle Andreas A Hell Stefan W SW Rizzoli Silvio O SO

Biophysical journal 20100701 2

Synaptic vesicles need to be mobile to reach their release sites during synaptic activity. We investigated vesicle mobility throughout the synaptic vesicle cycle using both conventional and subdiffraction-resolution stimulated emission depletion fluorescence microscopy. Vesicle tracking revealed that recently endocytosed synaptic vesicles are highly mobile for a substantial time period after endocytosis. They later undergo a maturation process and integrate into vesicle clusters where they exhib ...[more]

PMID: 20643088

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Project description:Synaptic vesicles (SVs) undergo a cycle of biogenesis and membrane fusion to release transmitter, followed by recycling. How exocytosis and endocytosis are coupled is intensively investigated. We describe an all-optical method for identification of neurotransmission genes that can directly distinguish SV recycling factors in C. elegans, by motoneuron photostimulation and muscular RCaMP Ca2+ imaging. We verified our approach on mutants affecting synaptic transmission. Mutation of genes affecting SV recycling (unc-26 synaptojanin, unc-41 stonin, unc-57 endophilin, itsn-1 intersectin, snt-1 synaptotagmin) showed a distinct 'signature' of muscle Ca2+ dynamics, induced by cholinergic motoneuron photostimulation, i.e. faster rise, and earlier decrease of the signal, reflecting increased synaptic fatigue during ongoing photostimulation. To facilitate high throughput, we measured (3-5 times) ~1000 nematodes for each gene. We explored if this method enables RNAi screening for SV recycling genes. Previous screens for synaptic function genes, based on behavioral or pharmacological assays, allowed no distinction of the stage of the SV cycle in which a protein might act. We generated a strain enabling RNAi specifically only in cholinergic neurons, thus resulting in healthier animals and avoiding lethal phenotypes resulting from knockdown elsewhere. RNAi of control genes resulted in Ca2+ measurements that were consistent with results obtained in the respective genomic mutants, albeit to a weaker extent in most cases, and could further be confirmed by opto-electrophysiological measurements for mutants of some of the genes, including synaptojanin. We screened 95 genes that were previously implicated in cholinergic transmission, and several controls. We identified genes that clustered together with known SV recycling genes, exhibiting a similar signature of their Ca2+ dynamics. Five of these genes (C27B7.7, erp-1, inx-8, inx-10, spp-10) were further assessed in respective genomic mutants; however, while all showed electrophysiological phenotypes indicative of reduced cholinergic transmission, no obvious SV recycling phenotypes could be uncovered for these genes.

Dataset Information

High- and low-mobility stages in the synaptic vesicle cycle.

Publications

High- and low-mobility stages in the synaptic vesicle cycle.

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