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. 2023 May 23;120(21):e2220856120.
doi: 10.1073/pnas.2220856120. Epub 2023 May 15.

C. elegans Clarinet/CLA-1 recruits RIMB-1/RIM-binding protein and UNC-13 to orchestrate presynaptic neurotransmitter release

Mia Krout  1 , Kelly H Oh  2 , Ame Xiong  2 , Elisa B Frankel  3 , Peri T Kurshan  3 , Hongkyun Kim  3 , Janet E Richmond  1
Affiliations

C. elegans Clarinet/CLA-1 recruits RIMB-1/RIM-binding protein and UNC-13 to orchestrate presynaptic neurotransmitter release

Mia Krout et al. Proc Natl Acad Sci U S A. .

Abstract

Synaptic transmission requires the coordinated activity of multiple synaptic proteins that are localized at the active zone (AZ). We previously identified a Caenorhabditis elegans protein named Clarinet (CLA-1) based on homology to the AZ proteins Piccolo, Rab3-interactingmolecule (RIM)/UNC-10 and Fife. At the neuromuscular junction (NMJ), cla-1 null mutants exhibit release defects that are greatly exacerbated in cla-1;unc-10 double mutants. To gain insights into the coordinated roles of CLA-1 and UNC-10, we examined the relative contributions of each to the function and organization of the AZ. Using a combination of electrophysiology, electron microscopy, and quantitative fluorescence imaging we explored the functional relationship of CLA-1 to other key AZ proteins including: RIM1, Cav2.1 channels, RIM1-binding protein, and Munc13 (C. elegans UNC-10, UNC-2, RIMB-1 and UNC-13, respectively). Our analyses show that CLA-1 acts in concert with UNC-10 to regulate UNC-2 calcium channel levels at the synapse via recruitment of RIMB-1. In addition, CLA-1 exerts a RIMB-1-independent role in the localization of the priming factor UNC-13. Thus C. elegans CLA-1/UNC-10 exhibit combinatorial effects that have overlapping design principles with other model organisms: RIM/RBP and RIM/ELKS in mouse and Fife/RIM and BRP/RBP in Drosophila. These data support a semiconserved arrangement of AZ scaffolding proteins that are necessary for the localization and activation of the fusion machinery within nanodomains for precise coupling to Ca2+ channels.

Keywords: cytomatrix of the active zone; exocytosis; neuromuscular junction; synaptic proteins; synaptic transmission.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Behavioral and electrophysiology data show further reduction in cla-1(ΔS/M/L) and cla-1(ΔL) in an unc-10 mutant background. (A) Full-length CLA-1 with allele designations and areas of the deletions for mutants used in this study. (B) Though the effect is mild, thrashing rates are significantly reduced in cla-1(ΔS/M/L) (P < 0.0001) and cla-1(ΔS) (P < 0.01). In an unc-10 background both cla-1(ΔS/M/L) (P < 0.0001) and cla-1(ΔL) (P < 0.01) are additively reduced beyond unc-10 mutants alone, whereas cla-1(ΔS) mutants were not. (C–H) In 5 Ca2+ ringer none of the cla-1 single mutants exhibit a change in evoked amplitude; however, both cla-1(ΔS/M/L) (P < 0.01) and cla-1(ΔL) (P < 0.05) exhibit a reduction in evoked charged integral. The mini frequency is only reduced in cla-1(ΔS/M/L) (P < 0.001). However, in an unc-10 mutant background we see a dramatic defect in evoked amplitude and integral in both the cla-1(ΔS/M/L) (amp P < 0.0001; int P < 0.001) and cla-1(ΔL) (amp P < 0.0001; int P < 0.001). The cla-1(ΔS/M/L);unc-10 (P < 0.001) and cla-1(ΔL);unc-10 (P < 0.001) double mutants have a significant reduction in mini frequency as compared to the unc-10 mutant, and surprisingly the cla-1(ΔS);unc-10 double mutants exhibit a slight but significant increase (P < 0.05).
Fig. 2.
Fig. 2.
Analysis of cla-1 isoform single and double unc-10 mutants using HPF/FS electron microscopy demonstrates that loss of all CLA-1 isoforms is necessary to disrupt synaptic morphology. (A) A single 40-nm cross-section of a synapse from a young adult worm prepared by HPF/FS. Examples of a dense projection and docked SVs (SVs 0 nm distance to the plasma membrane) measured and quantified in this study. (B–D) These data show that loss of CLA-1(L) or (S) alone does not result in the same morphological defects as observed in the cla-1(ΔS/M/L) mutant, indicating that loss of all CLA-1 protein is necessary to disrupt the (B) dense projection and reduce the number of (C) cytosolic and (D) docked SVs. *P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001 (C) SV number in cla-1(ΔS/M/L);unc-10 are significantly reduced when compared to unc-10 (####P < 0.0001) and not different from cla-1(ΔS/M/L). One way ANOVA with Tukey’s post hoc analysis.
Fig. 3.
Fig. 3.
Endogenously tagged GFP::UNC-2 levels are additively reduced in a cla-1(ΔS/M/L);unc-10 double mutant. (A and B) GFP::UNC-2 average peak intensities (A) and peak numbers (B) were determined based on analysis of a 30-μm section of the dorsal nerve cord (DNC) of wild-type, rimb-1(ce828)cla-1(ΔS), cla-1(ΔL), and cla-1(ΔS/M/L) single-mutant animals. The solid and dotted lines in the violin plot indicate the median and quantile values, respectively. Individual dots represent biological replicates from single animals. ns, not significant, **P < 0.01, One-way ANOVA, Tukey’s post hoc analysis. (C) Representative DNC images from wild-type and single-mutant animals. (Scale bar, 5 μm.) (D and E) GFP::UNC-2 average peak intensities (D) and peak numbers (E) were determined based on analysis of a 30-μm section of the dorsal nerve cord (DNC) of wild-type, unc-10(md1117)rimb-1;unc-10, cla-1(ΔS);unc-10, cla-1(ΔL);unc-10, and cla-1(ΔS/M/L); unc-10 double-mutant animals. *P < 0.05, ****P < 0.0001, One-way ANOVA, Tukey’s post hoc analysis. (F) Representative DNC images from the indicated mutants. (Scale bar, 5 μm.)
Fig. 4.
Fig. 4.
Endogenous RIMB-1 levels are drastically reduced in cla-1(ΔS/M/L) mutants. (A and B) GFP::RIMB-1 average peak intensities (A) and peak numbers (B) were determined based on analysis of a 30-μm section of the DNC of wild-type, unc-10(md1117)cla-1(ΔS), cla-1(ΔL), and cla-1(ΔS/M/L) single-mutant animals. RIMB-1 peak number and intensity are dramatically reduced in cla-1(ΔS/M/L) mutants (****P < 0.0001), more so than unc-10 mutants. (C) Representative GFP::RIMB-1 images from wild-type, unc-10, and cla-1 mutant animals. (Scale bar, 5 μm.) (D and E) GFP::RIMB-1 average peak intensities (D) and peak numbers (E) in unc-10 and cla-1 double mutants. *P < 0.05, **P < 0.01, ****P < 0.0001, One-way ANOVA, Tukey’s post-hoc analysis. (F) Representative GFP::RIMB-1 images from unc-10 and cla-1 double mutants. (Scale bar, 5 μm.)
Fig. 5.
Fig. 5.
Thrashing rates, electrophysiological recordings, and ultrastructural analysis in cla-1;unc-10 and rimb-1;unc-10 doubles demonstrate that cla-1 mutants have a more severe synaptic defect than rimb-1 mutants. (A–E) These data show that while rimb-1;unc-10 exhibits more severe behavioral and release defects than unc-10 mutants alone, there is still a significant difference in the severity of release in a cla-1;unc-10 double. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) Ultrastructural analysis shows no significant difference in the area of DP between the two double mutants. However, we observed that rimb-1;unc-10 double-mutant synapses have more cytosolic and docked SVs. Additionally, while docking distributions are similar in both double mutants, the docked SVs in rimb-1;unc-10 mutants appear to accumulate proximal to the active zone. One way ANOVA, Tukey’s post hoc analysis.
Fig. 6.
Fig. 6.
UNC-13 is reduced in cla-1 and unc-10 single mutants and shows a further reduction in cla-1;unc-10 double mutants. (A and B) UNC-13::GFP average puncta max intensities (A) and puncta numbers (B) were determined based on analysis of a 30 μm section of the DNC of the indicated wild-type and single-mutant animals. While UNC-13 puncta intensity and number are reduced in unc-10 animals, the puncta numbers are significantly reduced in cla-1(ΔL) and cla-1(ΔS/M/L) mutants. *P < 0.05, ***P < 0.001, ****P < 0.0001, One-way ANOVA, Tukey’s post hoc analysis. (C) Representative UNC-13::GFP images from the indicated wild-type and single-mutant animals. (Scale bar, 5 μm.) (D and E) UNC-13::GFP average puncta max intensities (D) and puncta numbers (E) were determined based on analysis of a 30-μm section of the DNC of the indicated unc-10 and double-mutant animals. **P < 0.01, One-way ANOVA, Tukey’s post hoc analysis. (F) Representative GFP::RIMB-1 images from unc-10 and cla-1 double mutants. (Scale bar, 5 μm.) (G) Examples of straightened DNC images (60 μm) in cla-1(ΔS/M/L);unc-10 double-mutant animals in comparison to wild-type animals.
Fig. 7.
Fig. 7.
UNC-13L and S isoforms are reduced in cla-1 mutants. (A–C) The fluorescence and peak intensities of the UNC-13L isoform are significantly reduced in cla-1(ΔL)cla-1(ΔS/M/L)unc-10 and both double mutants. *P < 0.05, ****P < 0.0001, One-way ANOVA, Dunnett’s post hoc analysis. (D–F) The fluorescence and peak intensities of the UNC-13S isoform are only reduced in the cla-1(ΔL) and cla-1(ΔS/M/L). *P < 0.05, **P < 0.01, ****P < 0.0001, One-way ANOVA, Dunnett’s post hoc analysis. (Scale bar, 10 µm.) Extraneous fluorescent puncta outside the nerve cord represent gut granule autofluorescence and were not included in the analysis. (G) A genetic model for CLA-1- and UNC-10-dependent regulation of downstream AZ components.
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