Synaptotagmin 1 directs repetitive release by coupling vesicle exocytosis to the Rab3 cycle

First, the notion that SNT inhibits Rab3-GAP needs more support. One approach would be to examine RBD RAB-3 pulldowns in snt-1; rbg-1 double mutants. This experiment is a better test for the hypothesis that SNT-1's effects on RAB-3/GTP levels are caused by a change in RBG-1 activity. Even better (if feasible) would be to carry out GAP assays in vitro.

We have performed the RBD RAB-3 pulldown in snt-1;rbg1 double mutants and the corresponding data has been included in Figure 4C. Basically, we found that the GTP-bound RAB-3 is increased in the double mutant compared with snt-1 single mutant animals, further supporting the idea that SNT-1 can protect GTP-RAB-3 by inhibiting RAB-3 GAP activity. We have tried the in vitro GAP assay in our laboratory for a year. However, the extremely low expression level of Rab3GAP1/RBG-1 protein in both bacteria and insect cell lines prevents us from carrying out any further analysis. Hence, the GAP assays are not currently feasible.

Second, the reviewers questioned whether RAB-3 was cytosolic or plasma membrane associated in snt-1 mutants. If the diffuse RAB-3 is on the plasma membrane, that would not support the model for increased GAP activity. If RAB-3 is cytosolic, that would provide independent support for the altered GTP state (i.e. confirming the RBD pull down). For these reasons the reviewers request direct fractionation experiments (analyzing Rab3 levels in the membrane and soluble fractions) to solidify this key conclusion.

We have performed the cell fractionation experiments and the results are shown in Figure 3C. Basically, the level of Rab-3 in the cytosolic fraction is greatly increased in snt-1 mutants compared to wild type, which is consistent with our conclusion that SNT-1 regulates RAB-3 localization by altering its GTP status.

Third, the sensitivity of the snt-1 and rab3 single mutants and the snt-1/rab3 double mutant to the inhibitor aldicarb should be tested. If the authors are correct, there should be no increased sensitivity of the double mutant.

The snt-1 (II:0.12 +/− 0.000 cM) and rab-3 (II:-0.96 +/− 0.001 cM) genes are in a close proximity on the same chromosome. Therefore, we used two approaches to obtain the snt-1 rab-3 double mutant. Firstly, we tried to use CRISPR-Cas9 to mutate the snt-1 gene in the rab-3(js49) background. After guidance RNA sequence design, cloning and injection, we obtained 4 heritable transgenic lines. However, PCR and sequence analysis showed that none of the transgenic lines was carrying the corresponding snt-1 mutation. Second, we tried to get the snt-1 rab-3 recombinant directly. After crossing rab-3(js49) males to snt-1(md290), from the heterozygote progenies, we obtained a total of 300 snt-1(md290) homozygotes. Among the 300 animals, three were rab-3(js49) heterozygotes. We singled out 72 animals from these three plates. However, PCR and sequencing analysis could not identify any rab-3(js49) homozygote animals, suggesting that the snt-1 rab-3 double animals are probably lethal. Indeed, after monitoring the progeny from a single snt-1 rab-3/snt-1 + animal, we found that a quarter of the progeny were arrested at the later larval stage. Thus, the lethality of snt-1 rab-3 doubles prevents us from doing the aldicarb assay.

Fourth, add an rbg-1 rescue experiment.

We have now included the rbg-1 rescue data in Figure 4–figure supplement 1C and D.

Finally, some of the referees were concerned about the nonphysiologically high concentrations of calcium that are required to see the effect. This should be at least commented on by the authors.

We have now commented on this point in the Discussion.

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

The manuscript has been improved but there was one minor issue raised by one of the referees that needs to be addressed before acceptance, as outlined below: “The methods state that worm extracts were prepared by dounce homogenization for the biochemistry experiments. I thought that worms are not well disrupted by dounce, and that one needs to use sonication, french press, or a microfluidizer. Could you check with the authors that the methods are correctly reported?”

We indeed used the Dounce homogenizer to break worms. This method was used by other researchers in worm community as well. Worms can be easily and well disrupted by this method. For instance, after homogenization 10 min on ice, protein concentration 10 µg/µl was obtained from worm sample composed of ∼100 µl worms and 300 µl buffer, which is comparable to other methods including sonication, French press, or microfluidizer. We now included the name of the company, which sells the homogenizers and the citation in the current manuscript in the subsection headed “Cell fractionation”.