Abstract
The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.
Highlights
Active zones (AZs) allow exquisite spatial and temporal control of vesicle fusion
excitatory postsynaptic current (EPSC) evoked by 0.2 Hz nerve stimulation were recorded at brp69 and wt larval neuromuscular junctions (NMJs) on muscles 6/7 to measure half-duration of the positive action potentials (AP) deflection and synaptic delay (Figure 1A)
To determine the impact of the putative calcium sensor Syt on synchronous transmitter release in the context of different AZ states we combined these genotypes with sytKD
Summary
Large multidomain proteins rich in coiled-coil sequences such as Bassoon, Piccolo and the CAST/ERC family member Brp are major structural and functional organizers of AZs (Südhof, 2012). Their abundance appears to correlate positively with neurotransmitter release (Graf et al, 2009; Matz et al, 2010; Weyhersmüller et al, 2011; Ehmann et al, 2014; Peled et al, 2014). Linking the amount of Brp or Bassoon per AZ to the number and spatial arrangement of calcium channels may account for the correlation with release probability, e.g., in the context of synaptic homeostasis (Matz et al, 2010; Weyhersmüller et al, 2011; Ehmann et al, 2014). Differences in coupling distance are ideal for scaling the amount of release, whereas controlling its time course appears to require additional molecular mechanisms
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
