Heterodimerization of UNC-13/RIM regulates synaptic vesicle release probability but not priming in C. elegans

Essential revisions:

1) Earlier studies showed that a truncation of mammalian Munc13-1 that abolishes RIM1 binding leads to a priming defect that can be (partially) overcome by prolonged strong overexpression of the truncated Munc13-1 variant (Betz et al., 2001).[…] Furthermore, the present study would have benefited from using CRISPR or some other form of homologous recombination throughout, as was done for their UNC-10 (K77/79E) construct so that expression level effects do not confound the interpretation of the results. Of particular concern is the possibility that overexpression of UNC-13 K49E in the rescue of release in the UNC-10(K77/79E) background may not be physiologically relevant.

For these reasons, the present study and its interpretation would profit tremendously from a comparative analysis of the expression levels and sub-cellular localization of the different UNC-13 variants at synapses (as well as of UNC-13 levels and positioning in the presence of the different UNC-10 variants). If analyzing sub-synaptic UNC-13 localization cannot be performed within a reasonable timeframe for this communication, the authors should at least quantify the synaptic UNC-13 levels. The authors should also discuss these various issues and tone down their conclusions accordingly (including the definitive nature of the title).

We agree that the synaptic levels and localization of Munc13/UNC-13 proteins at the nerve terminals are essential for SV exocytosis. The use of extrachromosomal arrays in this study raised the question of whether the UNC-13 protein levels from distinct transgenic lines (UNC-13L, UNC-13LK49E, and UNC-13LDC2A rescues) are similar or not.

To determine this, we made all the rescue constructs with a fluorescence protein tag (mApple). Re-introducing these constructs into the unc-13 null mutants rescued the locomotion defects (data not shown). Indeed, the UNC-13 with a C-terminal tag is fully functional in mediating SV exocytosis (Hu et al., 2013). By analyzing the fluorescence intensity of mApple, we showed that the average proteins levels of UNC-13L, UNC13LK49E, and UNC-13LDC2A are not significantly different (Figure 4A, B). These results exclude the possibility that the decreased SV release in the UNC-13LDC2A rescue is due to a change in the protein level, and that disrupting the C2A/C2A homodimerization has no effect on protein level. It should be noted that we didn’t compare release in these new mApple transgenic strains, as prior studies have shown that a fluorescence tag does not affect UNC-13 protein’s function (Madison et al., 2005; Hu et al., 2013).

To examine whether the sub-localization of UNC-13L is altered when heterodimerization with RIM is disrupted, we analyzed UNC-13L co-localization with ELKS1, another active zone protein. In wild-type background, UNC-13L (tagged with mCherry) and ELKS^-1 (tagged with Cerulean) exhibit an obvious colocalization (Figure 4C, D). This colocalization was still observed in unc-10(K77/79E) mutants (Figure 4E, F). These results suggest that disrupting the UNC-13L/RIM interaction does not dramatically alter UNC-13L synaptic localization and that UNC-13L likely has other binding partners that promote its active zone localization.

All the above results have been incorporated into the figures and text.

“The decreased release probability in the UNC-13LDC2A rescue and the unc-10(K77/79E) mutant could arise from a change of the expression level of UNC-13, or a change of the sublocalization of the UNC-13 proteins at the nerve terminals. […] These results suggest that disrupting UNC-13L binding to RIM does not prevent synaptic localization of UNC-13L.”

The new generated imaging lines have been added into the “Strain names” in the Materials and methods section.

2) Crucial differences observed between the results described by Liu et al. and by Camacho et al., 2017 correspond to the measurements of the readily-releasable pool (RRP) of vesicles by treatment with hypertonic sucrose. It is unclear whether these are indeed real differences between the mammalian and worm systems, or the different results arise from differences in experimental protocols. For instance, the authors use 1 M sucrose instead of 500 mM sucrose, which is more common and was used by Camacho et al. It is also plausible that differences might arise from the method of application of sucrose. The authors should perform experiments with 500 mM sucrose to examine whether their results stand at this lower concentration, provide more experimental details on these experiments and discuss how experimental details might influence the results obtained. Raw traces for both concentrations should be provided and, when collecting the 500 mOsm data, the leak current for each trace used in the analysis should be noted.

Since Rosemund and colleagues first established the assay of using hypertonic sucrose-evoked synaptic current to estimate the size of the readily releasable vesicular pool (RRP), this assay has been widely used in both vertebrates and invertebrates. The sucrose concentrations and puffing durations, however, vary in different studies (Aravamudan et al., 1999; Richmond et al., 1999; Yoshihara and Littleton, 2002; Pang et al., 2006; Zhou et al., 2013). It is unlikely that the different results in SV priming in the unc-10(K77/79E) arise from the experimental protocols we used, as the large priming defect in the unc-13 mutants has been consistently observed in our lab. Nevertheless, to address the reviewer’s question, we compared the synaptic currents elicited by 0.5M sucrose between wild type and the unc10(K77/79E) mutants. To record a relatively stable sucrose response under this condition, a 5s puffing duration was used (Author response image 1A, B). As shown in Author response image 1, the charge transfer of the sucrose currents in the unc-10(K77/79E) mutants were comparable to those in wild-type worms (Author response image 1C), consistent with the observation when using 1M sucrose. We have added these results in the main text. All the raw traces recorded in both 0.5M and 1M sucrose were shown in Author response image 1A, B, D, E). These results confirmed the notion that disrupting the UNC-13L/RIM heterodimerization does not affect SV priming.

We have described more details how to perform the sucrose puff experiments at the worm NMJ in the “Materials and methods”.

“A pulsed application of sucrose (1M, 2s) was applied onto the ventral nerve cord using a Picospritzer III (Parker). The glass pipette containing sucrose was placed at the end of the patched muscle (around half muscle distance from the recording pipette).”

“Nevertheless, we assessed the RRP size in the UNC-13L(K49E) rescue animals and the unc10(K77/79E) mutant by puffing hypertonic sucrose (1M, 2s). […] The unchanged SV priming in the unc-10(K77/79E) mutant was also confirmed by puffing a lower concentration of sucrose (0.5M, 5s; data not shown).”

Author response image 1

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[Editors' note: further revisions were requested prior to acceptance, as described below.]

The reviewers have discussed the reviews with one another and with the Reviewing Editor. They found most revisions satisfactory, but there are still some concerns that are summarized below to help you prepare a revised submission.

1) The authors produced new extrachromasomal lines for the UNC-13 constructs tagged with C-terminal Apple, which allowed them to measure comparable expression levels, but these were not the arrays used to do the functional electrophysiological analysis in the paper. It is the case that arrays can vary in expression levels from one injection to the next. The authors only provide assurances that the behavior was rescued but did not show the data or the extent of the rescue. More concerning, they did not repeat the electrophysiological analyses on these strains. Without this, how can we be certain that these are similar to the original array-expressing strains and that the same results would be found.

Both mEPSCs and evoked EPSCs were analyzed in the unc-13 mutants rescued by mApple-tagged UNC-13 proteins (UNC-13L, DC2A, and K49E). Basically, we found very similar results in these rescue strains, compared to those observed untagged UNC-13 rescue strains, indicating that the UNC-13 with C-terminal tag is fully functional. These results have been incorporated into the revised manuscript as a supplementary figure (Figure 4—figure supplement 1).

“All mApple-tagged UNC-13L constructs rescued the SV release in the unc-13 mutants, and the phenotypes were similar to those rescued by corresponding untagged UNC13L constructs (Figure 4—figure supplement 1)”

2) As for the hyperosmotic traces, only those for the unc-10(k77/79E) were reexamined in 0.5M sucrose and the requested leak currents for the two data sets (i.e. 0.5 and 1M) were not provided. The concern is that at 1M sucrose, if there were substantial leak, the large inward currents observed would arise not from sucrose-induced release but poor seals. This problem has arisen in the past in analyses of fly embryos in which large inward currents were observed in mutants that were subsequently shown to have no primed vesicles; the reason was leaky recordings. If the authors could have produced these data for the unc-13 null and the UNC-13 rescuing arrays tagged with Apple, this would have greatly improved confidence in the conclusions of the paper. The leak currents should be provided and the data should be interpreted considering these issues.

We now added the quantification for two more mutants, unc-13(s69) and UNC-13L rescue, under the lower sucrose condition (0.5M, 5s). The results are similar with what we obtained when puffing 1M sucrose (see Author response image 2). Moreover, Zhou et al. have shown that removing the C2A domain from UNC-13L does not cause a change in the RRP size measured by puffing 0.5M sucrose (Zhou et al., 2013, eLife), consistent with our findings in this study. We hope that the reviews can agree that our current protocol for sucrose puffing (1M, 2s) is reasonable.

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We remade the following figures with all sucrose traces. The leak current can be seen on each individual traces. We also quantified the leak current for all the sucrose recordings (1M, 2s). The results have been incorporated into the revised manuscript as a supplementary figure (Figure 3—figure supplement 1). As you can see, the leak current is controlled very well in our recordings. The averaged leak current for each genotype is between -20pA and -30pA.

“It should be noted that recording of the sucrose-evoked current requires a tight gigaohm seal between the recording pipette and the cell, as a loose seal often leads to an unreal current. We therefore analyzed the leak currents for each sucrose recordings. Our data showed that all recordings were controlled well with acceptable leak currents (Figure 3—figure supplement 1).”

3) In the decision letter a critical discussion of the use of extrachromosomal-array-based expression was provided. Clearly, overexpression via this approach could mask effects that would become detectable with WT-like expression levels. This issue should be discussed briefly as a possible caveat.

We agree with this point and have discussed the issue of rescue using extrachromosomal arrays.

“It should be noted that all the UNC-13 transgenes in this study are extrachromosomal arrays, and that the UNC-13 proteins are overexpressed in these transgenic animals. The overexpression of UNC-13 may cause differential effects with wild-type expression of this protein. Despite this, our findings (e.g., DC2A) are similar with Zhou et al. results which were obtained from unc-13 mutants rescued by single copy of UNC-13.”

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