Mechanism that regulates the mutual inhibition controls functional switch of UNC-13

Joint Public Review:

Summary:

In this manuscript, the authors investigate how different domains of the presynaptic protein UNC-13 regulate synaptic vesicle release in the nematode C. elegans. By generating numerous point mutations and domain deletions, they propose that two membrane-binding domains (C1 and C2B) can exhibit "mutual inhibition," enabling either domain to enhance or restrain transmission depending on its conformation. The authors also explore additional N-terminal regions, suggesting that these domains may modulate both miniature and evoked synaptic responses. From their electrophysiological data, they present a "functional switch" model in which UNC-13 potentially toggles between a basal state and a gain-of-function state, though the physiological basis for this switch remains partly speculative.

Strengths:

(1) The authors conduct a thorough exploration of how mutations in the C1, C2B, and other regulatory domains affect synaptic transmission. This includes single, double, and triple mutations, as well as domain truncations, yielding a large, informative dataset.

(2) The study includes systematically measuring both spontaneous and evoked synaptic currents at neuromuscular junctions, under various experimental conditions (e.g., different Ca²⁺ levels), which strengthens the reliability of their functional conclusions.

(3) Findings that different domain disruptions produce distinct effects on mEPSCs, mIPSCs, and evoked EPSCs suggest UNC-13 may adopt an elevated functional state to regulate synaptic transmission.

Weaknesses:

It remains unclear whether the various domain alterations truly converge on a single "gain-of-function" state or instead represent multiple pathways for enhancing UNC-13 activity. Different mutations selectively affect spontaneous or evoked release, suggesting that each variant may not share the same underlying mechanism. Moreover, many conclusions rely on combining domain deletions or point mutations, yet the electrophysiological data show distinct outcomes across EPSCs, IPSCs, mini, and evoked responses. This raises questions about whether these manipulations all act on the same pathway and whether their observed additivity or suppression genuinely reflects a single mechanistic process. A unifying model-or at least a clearer explanation of why the authors infer one mechanistic state across different domain manipulations would strengthen the paper's conclusions.

The manuscript proposes that UNC-13 toggles from a basal to a "gain-of-function" state under normal synaptic activity. However, it does not address when or how this switch might occur in vivo, since it is demonstrated principally via artificial mutations. Providing direct evidence or additional discussion of such switching under physiological conditions would be particularly informative.

What is the physiological significance of the proposed gain-of-function state? The data suggest that certain mutants (e.g., HK+D1-5N) lacking the gain-of-function state can still support synaptic transmission at wild-type levels. How do the authors reconcile this with the idea that the gain-of-function state plays a critical role at the synapse?

The authors determined the fluorescence intensity of mApple-tagged UNC-13 variants (Figure 1J-K and Figure 7J-K), finding no significant changes compared to the wild-type. However, a more detailed analysis of the density or distribution of fluorescent puncta in axons could clarify whether certain mutations alter the localization of UNC-13 at synapses. Demonstrating colocalization with wild-type UNC-13 (or another presynaptic marker) would help rule out mislocalization effects.

The study mainly relies on extrachromosomal transgenes, which can show variable copy numbers and expression levels among individual worm strains. This variability might complicate interpretation, as differences in expression could mask or exaggerate certain phenotypes.

Finally, the discussion is somewhat diffused. Streamlining the text to focus on the most direct connections would help readers pinpoint the key conclusions and open questions.