Morphologically Docked Synaptic Vesicles Are Reduced insynaptotagminMutants ofDrosophila (original) (raw)
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Morphologically docked synaptic vesicles are reduced in synaptotagmin mutants of Drosophila
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1998
Nerve terminal specializations include mechanisms for maintaining a subpopulation of vesicles in a docked, fusion-ready state. We have investigated the relationship between synaptotagmin and the number of morphologically docked vesicles by an electron microscopic analysis of Drosophila synaptotagmin (syt) mutants. The overall number of synaptic vesicles in a terminal was reduced, although each active zone continued to have a cluster of vesicles in its vicinity. In addition, there was an increase in the number of large vesicles near synapses. Examining the clusters, we found that the pool of synaptic vesicles immediately adjacent to the presynaptic membrane, the pool that includes the docked population, was reduced to 24 +/- 5% (means +/- SEM) of control in the sytnull mutation. To separate contributions of overall vesicle depletion and increased spontaneous release from direct effects of synaptotagmin on morphological docking, we examined syt mutants in an altered genetic background...
Neuron, 2000
Valdivia terminal during sustained neuronal activity, we asked the following questions: what is the size of vesicle pools? Chile † Escuela de Postgrado What is the rate of vesicle mobilization from these pools? And to what extent vesicle recycling is contributing in Fac. de Ciencias Universidad de Chile maintaining synaptic transmission during prolonged tetanic stimulation? Combining measurements of nerve- ‡ Gunma University School of Medicine 3-39-22 Showa-machi evoked synaptic currents and imaging of FM1-43 in synaptic boutons, these questions can be answered by Maebashi 371-8511 Japan taking advantage of a temperature-sensitive paralytic mutant, shibire ts (shi ts ). In shi ts endocytosis ceases and synaptic vesicles are completely depleted after synaptic activation at nonpermissive temperatures, while synap-Summary tic transmission at room temperature is normal (Koenig and Ikeda, 1989, 1996; Koenig et al., 1989; Estes et al., Two vesicle pools, readily releasable (RRP) and re -1996). This mutant provides a means to estimate the serve (RP) pools, are present at Drosophila neuromussize of vesicle pools and their rates of mobilization at cular junctions. Using a temperature-sensitive mutant, the larval neuromuscular junction. The contribution of shibire ts , we studied pool sizes and vesicle mobilizarecycling for synaptic transmission can be examined in tion rates. In shibire ts , due to lack of endocytosis at wild-type larvae at steady state during repetitive nerve nonpermissive temperatures, synaptic currents constimulation.
Synaptic transmission persists in synaptotagmin mutants of Drosophila
Cell, 1993
Synaptotagmin is one of the major integral membrane proteins of synaptic vesicles. It has been postulated to dock vesicles to their release sites, to act as the Ca*+ sensor for the release process, and to be a fusion protein during exocytosis. To clarify the function of this protein, we have undertaken a genetic analysis of the synaptotagmin gene in Drosophila. We have identified five lethal alleles of synapfotagmin, at least one of which lacksdetectable protein. Surprisingly, however, many embryos homozygous for this null allele hatch and, as larvae, crawl, feed, and respond to stimuli. Electrophyslological recordings in embryonic cultures confirmed that synaptic transmission persists in the null allele. Therefore, synapfofagmin is not absolutely required for the regulated exocytosis of synaptic vesicles. The lethality of synapfofagmin in late first instar larvae is probably due to a perturbation of transmission that leaves the main apparatus for vesicle docking and fusion intact.
Bio-protocol, 2019
Presynaptic boutons at nerve terminals are densely packed with synaptic vesicles, specialized organelles for rapid and regulated neurotransmitter secretion. Upon depolarization of the nerve terminal, synaptic vesicles fuse at specializations called active zones that are localized at discrete compartments in the plasma membrane to initiate synaptic transmission. A small proportion of synaptic vesicles are docked and primed for immediate fusion upon synaptic stimulation, which together comprise the readily releasable pool. The size of the readily releasable pool is an important property of synapses, which influences release probability and can dynamically change during various forms of plasticity. Here we describe a detailed protocol for estimating the readily releasable pool at a model glutamatergic synapse, the Drosophila neuromuscular junction. This synapse is experimentally robust and amenable to sophisticated genetic, imaging, electrophysiological, and pharmacological approaches. We detail the experimental design, electrophysiological recording procedure, and quantitative analysis necessary to determine the readily releasable pool size. This technique requires the use of a two-electrode voltageclamp recording configuration in elevated external Ca 2+ with high frequency stimulation. We have used this assay to measure the readily releasable pool size and reveal that a form of homeostatic plasticity modulates this pool with synapse-specific and compartmentalized precision. This powerful approach can be utilized to illuminate the dynamics of synaptic vesicle trafficking and plasticity and determine how synaptic function adapts and deteriorates during states of altered development, stress and neuromuscular disease.
Proceedings of the National Academy of Sciences, 1994
Since the demonstration that Ca2+ influx into the presynaptic terminal is essential for neurotransmitter release, there has been much speculation about the Ca2+ receptor responsible for initiating exocytosis. Numerous experiments have shown that the protein, or protein complex, binds multiple Ca2+ ions, resides near the site of Ca2+ influx, and has a relatively low affinity for Ca2+. Synaptotagmin is an integral membrane protein of synaptic vesicles that contains two copies of a domain known to be involved in Ca(2+)-dependent membrane interactions. Synaptotagmin has been shown to bind Ca2+ in vitro with a relatively low affinity. In addition, synaptotagmin has been shown to bind indirectly to Ca2+ channels, positioning the protein close to the site of Ca2+ influx. Recently, a negative regulatory role for synaptotagmin has been proposed, in which it functions as a clamp to prevent fusion of synaptic vesicles with the presynaptic membrane. Release of the clamp would allow exocytosis. ...
Development, 1993
Synaptotagmin is a synaptic vesicle-specific integral membrane protein that has been suggested to play a key role in synaptic vesicle docking and fusion. By monitoring Synaptotagmin’s cellular and subcellular distribution during development, it is possible to study synaptic vesicle localization and transport, and synapse formation. We have initiated the study of Synaptotag-min’s expression during Drosophila neurogenesis in order to follow synaptic vesicle movement prior to and during synapse formation, as well as to localize synaptic sites in Drosophila. In situ hybridizations to whole-mount embryos show that synaptotagmin (syt) message is present in the cell bodies of all peripheral nervous system neurons and many, if not all, central nervous system neurons during neurite outgrowth and synapse formation, and in mature neurons. Immunocytochemical staining with antisera specific to Synaptotagmin indicates that the protein is present at all stages of the Drosophila life cycle followin...
Syntaxin and synaptobrevin function downstream of vesicle docking in drosophila
Neuron, 1995
In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.
The Journal of Comparative Neurology, 2006
Synaptotagmin I is a synaptic vesicle protein postulated to mediate vesicle docking, vesicle recycling, and the Ca 2ϩ sensing required to trigger vesicle fusion. Analysis of synaptotagmin I knockouts (sytI NULL mutants) in both Drosophila and mice led to these hypotheses. Although much research on the mechanisms of synaptic transmission in Drosophila is performed at the third instar neuromuscular junction, the ultrastructure of this synapse has never been analyzed in sytI NULL mutants. Here we report severe synaptic vesicle depletion, an accumulation of large vesicles, and decreased vesicle docking at sytI NULL third instar neuromuscular junctions. Mutations in synaptotagmin I's C 2 B Ca 2ϩ -binding motif nearly abolish synaptic transmission and decrease the apparent Ca 2ϩ affinity of neurotransmitter release. Although this result is consistent with disruption of the Ca 2ϩ sensor, synaptic vesicle depletion and/or redistribution away from the site of Ca 2ϩ influx could produce a similar phenotype. To address this question, we examined vesicle distributions at neuromuscular junctions from third instar C 2 B Ca 2ϩ -binding motif mutants and transgenic wild-type controls. The number of docked vesicles and the overall number of synaptic vesicles in the vicinity of active zones was unchanged in the mutants. We conclude that the near elimination of synaptic transmission and the decrease in the Ca 2ϩ affinity of release observed in C 2 B Ca 2ϩ -binding motif mutants is not due to altered synaptic vesicle distribution but rather is a direct result of disrupting synaptotagmin I's ability to bind Ca 2ϩ . Thus, Ca 2ϩ binding by the C 2 B domain mediates a postdocking step in fusion.
FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction
2008
To maintain transmitter release during intense stimulation, neurons need to efficiently recycle vesicles at the synapse. Following membrane fusion, vesicles are reshaped and formed from the plasma membrane by bulk or clathrin-mediated endocytosis. Most synapses, including the Drosophila neuromuscular junction (NMJ), can also recycle synaptic vesicles directly by closing the fusion pore, a process referred to as "kiss and run." While the process of clathrin-mediated vesicle retrieval is under intense investigation, the kiss-and-run phenomenon remains much less accepted. To gain better insight into the mechanisms of synaptic vesicle recycling, it is therefore critical not only to identify and characterize novel players involved in the process, but also to develop novel methods to study vesicle recycling. Although in recent years numerous techniques to study vesicle traffic have been developed (see also this volume), in this chapter we outline established procedures that use the fluorescent dye FM 1-43 or related compounds to study vesicle cycling. We describe how FM 1-43 can be used to study and visualize clathrin-mediated or bulk endocytosis from the presynaptic membrane as well as exocytosis of labeled vesicles at the Drosophila NMJ, one of the best-characterized model synapses to study synaptic function in a genetic model system.
—The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals, at crayfish and larval Drosophila neuromuscular junctions (NMJs), were examined for physiological differentiation into distinctly separate functional groups. These NMJs are glutamatergic and produce graded excitatory postsynap-tic potentials (EPSPs). The packaging of glutamate was perturbed by blocking the vesicular glutamate transporter (VGlut) with bafilomycin A1. Various frequencies of motor nerve stimulation, exposure time, and concentration of bafilomycin A1 were examined. The low-output tonic opener NMJs in crayfish exposed to 4 lM bafilomycin A1 and 20-Hz continuous stimulation decreased the EPSP amplitude to 50% in 30minwithcontrolslasting3h.AfteractivityandbafilomycinA1−inducedsynapticdepression,theEPSPswererapidlyrevitalizedbyserotonin(5−HT,1lM)inthecrayfishpreparations.The5−HTactioncanbeblockedwithaPLCinhibitor.Wepostulate5−HTrecruitsunusedvesiclesfromtheRP.TheperceptionistheRRPisselectivelyactivatedduringrapidelectricalstimulation(20Hz)sparingtheRP.Whenstimulationfrequencyishigh(40Hz)theRPisrecruitedtotheRRPanddampenssubsequentrecruitmentwith5−HT.ThehigheroutputsynapsesofthelarvalDrosophilaNMJwhenstimulatedat1Hzor5Hzandexposedto4lMofbafilo−mycinA1showedadepressionrateof5030 min with controls lasting 3 h. After activity and bafilomycin A1-induced synaptic depression, the EPSPs were rapidly revitalized by serotonin (5-HT, 1 lM) in the crayfish preparations. The 5-HT action can be blocked with a PLC inhibitor. We postulate 5-HT recruits unused vesicles from the RP. The perception is the RRP is selectively activated during rapid electrical stimulation (20 Hz) sparing the RP. When stimulation frequency is high (40 Hz) the RP is recruited to the RRP and dampens subsequent recruitment with 5-HT. The higher output synapses of the larval Drosophila NMJ when stimulated at 1 Hz or 5 Hz and exposed to 4 lM of bafilo-mycin A1 showed a depression rate of 50% within 30minwithcontrolslasting3h.AfteractivityandbafilomycinA1−inducedsynapticdepression,theEPSPswererapidlyrevitalizedbyserotonin(5−HT,1lM)inthecrayfishpreparations.The5−HTactioncanbeblockedwithaPLCinhibitor.Wepostulate5−HTrecruitsunusedvesiclesfromtheRP.TheperceptionistheRRPisselectivelyactivatedduringrapidelectricalstimulation(20Hz)sparingtheRP.Whenstimulationfrequencyishigh(40Hz)theRPisrecruitedtotheRRPanddampenssubsequentrecruitmentwith5−HT.ThehigheroutputsynapsesofthelarvalDrosophilaNMJwhenstimulatedat1Hzor5Hzandexposedto4lMofbafilo−mycinA1showedadepressionrateof5010 min with controls lasting $40 min. After low frequency depression and/or exposure to bafilomycin A1 a burst of higher frequency (10 Hz) can recruit vesicles from the RP to the RRP.