Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics
- University of California, San Diego, United States
- Howard Hughes Medical Institute, University of California, San Diego, United States
Figures
Figure 1 with 4 supplements
The C2A domain of UNC-13L regulates the release probability of evoked synaptic vesicle release.
(A) Illustration of UNC-13 long and short isoforms, and location of unc-13 mutations. * marks possible initiation methionines downstream of n2609 mutation. The purple domain is the calmodulin …
Figure 1—figure supplement 1
Alignment of C2A domains among UNC-13/Munc13 isoforms.
Alignment of C2A domains among worm UNC-13L, Rat Munc13-1 and Rat ubMunc13-2. Residues that are identical are shown on a black background, and residues that are similar are shaded. The C2A domain of …
Figure 1—figure supplement 2
Transcripts of unc-13(n2609).
Transcripts of unc-13 long and short isoforms were detected by RT-PCR. The long isoform contains exon 1–13 and exon 15–31. The short isoform contains exon 14–31. The primers of RT-PCR1 are designed …
Figure 1—figure supplement 3
The effects of loss of the C2A domain on locomotion speeds.
(A) Schematics of single copy insertion (Si) for full length UNC-13L and UNC-13LC2A- lacking the C2A domain, driven by pan-neuronal promoter Prgef-1. (B) The locomotion speeds in wild type and unc-13…
Figure 1—figure supplement 4
Ratios of mean charge transfers during eEPSC and during sucrose application and the rescue effects of overexpression of UNC-13L and UNC-13LC2A− in unc-13(s69).
(A). Summary of ratios of mean charge transfers during eEPSCs within 50 ms and during sucrose applications within 5 s after triggers for each given genotype. (B). Overexpression of UNC-13LC2A− did …
Figure 2
The C2A domain of UNC-13L promotes the docking of synaptic vesicles at the active zone.
(A) Ultrastructural organization of cholinergic presynaptic terminals in wild type and unc-13(n2609). The dense projections were outlined by light green. The 165 nm, 231 nm and 330 nm regions along …
Figure 3 with 2 supplements
The C2A domain of UNC-13L is required for the precise localization of UNC-13L at active zones.
(A1) Representative confocal Z-stack images of co-immunostaining for ELKS-1 and UNC-10/RIM from wild type and unc-13(n2609). (A2) Average fluorescence intensities in six-pixel wide regions along a …
Figure 3—figure supplement 1
Loss of C2A domain does not change the co-localization between Ca2+ channel and UNC-10/RIM.
(A1–4) Representative confocal Z-stack images (A1), average pixel-by-pixel fluorescence intensity correlation coefficients (A3), peak distance calculation from images shown in A1 (A2) and summary (A4…
Figure 3—figure supplement 2
Presynaptic localization of UNC-13 is not solely dependent on UNC-10/RIM.
Representative confocal Z-stack images of co-immunostaining for UNC-13L and UNC-10/RIM from unc-10(md1117) null mutants. Scale bar: 5 µm.
Figure 4 with 3 supplements
The N-terminal region of UNC-13L determines the presynaptic active zone localization of UNC-13L and is necessary for fast kinetics of evoked release.
(A) Schematics and images in dorsal nerve cords of GFP tagged full length UNC-13L, UNC-13LN− lacking the entire N-terminal region (amino acids 632–1816), N-terminal amino acids 1–157 fragment and …
Figure 4—figure supplement 1
Locomotion speeds of unc-13(s69) rescue strains.
The locomotion speeds in animals of genotypes indicated without OP50 bacteria. Number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Statistics, one way ANOVA. ***p<0.00…
Figure 4—figure supplement 2
Ratios of mean charge transfers during eEPSC and during sucrose application.
Summary of ratios of mean charge transfers during eEPSCs within 50 ms and during sucrose applications within 5 s after triggers in animals of genotype indicated.
Figure 4—figure supplement 3
Higher [Ca2+]ex partially rescue eEPSC of unc-13(n2609).
(A) Shown are average recording traces of eEPSCs from wild type and unc-13(n2609) in 2 mM and 5 mM extracellular Ca2+ concentrations, as well as peak amplitudes of eEPSCs and fractions of increased …
Figure 5 with 1 supplement
The C2A domain of UNC-13L is required for tonic synaptic vesicle release.
(A and B) Representative recording traces (left) and summary (right) of tEPSC frequency in animals of genotype indicated. (C) Average recording traces and mean peak amplitudes of eEPSCs in animals …
Figure 5—figure supplement 1
Tonic EPSC amplitudes and decay times of unc-13(s69) rescue strains and cpx-1 mutants, and the rescue effects of overexpression of UNC-13L and UNC-13LC2A− on tEPSC in unc-13(s69).
(A and B) Summary of the amplitudes and decay times of tEPSCs in animals of genotypes indicated. (C) Overexpression UNC-13LC2A− did not fully rescue tEPSC frequency, but had no effects on tEPSC …
Figure 6 with 1 supplement
MiniSOG-mediated acute abalation supports a specific role of UNC-13L in fast phase of evoked release and in tonic release.
(A) Average recording traces of eEPSCs in animals of genotype indicated without or with blue light treatment. (B and C) Summaries of the peak amplitude, transferred charge of fast component and slow …
Figure 6—figure supplement 1
Effects of acute miniSOG-mediated CALI of UNC-13L and UNC-13LN− on locomotion speeds and on SV release in unc-13(s69).
(A) Summary of the effects of blue light treatment on normalized locomotion speeds in L4 stage animals of genotype indicated. (B and C) Rescue and inactivation effect of miniSOG tagged UNC-13L and …
Figure 7 with 2 supplements
The C2A domain-containing N-terminal region of UNC-13L is required for acr-2(gf)-induced epileptic-like convulsions.
(A) Summary of the suppression of unc-13(n2609), unc-13(n2813) on acr-2(gf)-induced convulsions, and the effects of unc-13 genomic DNA cosmid C44E1, UNC-13L and UNC-13N− transgene on convulsions in a…
Figure 7—figure supplement 1
Tonic release in acr-2(gf) mutants is reduced by unc-13(n2609).
Representative recording traces and mean frequencies of tEPSCs in animals of genotype indicated. The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Statistics, …
Figure 7—figure supplement 2
Recovery of convulsions in acr-2(gf); UNC-13L-miniSOG after blue light treatment.
Recovery of convulsions in acr-2(gf); UNC-13L-miniSOG animals after blue light treatment.
Figure 8
Model for the C2A domain-containing N-terminal region of UNC-13L support spontaneous release and fast kinetics of evoked release.
N-terminal sequences subsequent to the C2A domain interact with unknown targets (represented by a question mark) to facilitate the presynaptic localization of UNC-13L. The C2A domain binding to the …
Additional files
-
Supplementary file 1
(A) Genetic mutations. (B) Strains with genetic mutations. (C) Transgenes, plasmids, and strains.
- https://doi.org/10.7554/eLife.01180.024
