Synaptotagmin 7 transiently docks synaptic vesicles to support facilitation and Doc2α-triggered asynchronous release

Reviewer #1 (Public Review):

Summary:

In the current manuscript, the authors find distinct roles for the calcium sensors Syt7 and Doc2alpha in the regulation of asynchronous release and calcium-dependent synaptic vesicle docking in hippocampal neurons. The authors' data indicate that Doc2 functions in activating a component of asynchronous release beginning with the initial stimulus, while Syt7 does not appear to have a role at this early stage. A role for Syt7 in supporting both synchronous and asynchronous release appears during stimulation trains, where Syt7 is proposed to promote synaptic vesicle docking or capture during stimulation. Doc2 mutants show facilitation initially during a train and display higher levels of synchronous release initially, before reaching a similar plateau to controls later in the train. The authors contribute the increased synchronous release in Doc2 mutants to Syt1 having access to more SVs that can fuse synchronously. In contrast, Syt7 mutants show depression during a train, and continue to decline during stimulation. The authors contribute this to a role for Syt7 in promoting calcium-dependent SV docking and capture that feeds SVs to both synchronous and asynchronous fusion pathways. Importantly, phenotypes of a double Doc2/Syt7 mutant collapse onto the Doc2 phenotype, suggesting the two proteins are not additive in their role in supporting distinct aspects of SV release. Rapid freeze EM after stimulation provides support for a role for Syt7 in SV docking/capture at release sites, as they display less docked SVs after stimulation. In the case of Doc2, EM reveals fewer SVs fusion pits later during a stimulation, consistent with fewer asynchronous fusion events. The authors also provide modeling that supports aspects of their conclusions from the experimental data. I cannot evaluate the modeling data or the specific experimental subtleties of the GluSnFR quantification approach, as these are outside of my reviewer expertise.

Strengths:

The use of multiple approaches (optical imaging, physiology, rapid freeze EM, modeling, double mutant analysis) provides compelling support for the distinct roles of the two proteins in regulating SV release.

Weaknesses:

Some of the phenotypes for both Doc2 and Syt7 mutants have been reported in the authors' prior publications. It is not clear how well the GluSnFR approach is for accurately separating synchronous versus asynchronous release kinetics. The authors also tend to overstate the significance of the two proteins for asynchronous release in general, as a significant fraction of this release component is still intact in the double mutant, indicating these two proteins are only part of the asynchronous release mechanism.

Reviewer #2 (Public Review):

Summary:

The goal of this study is to provide a deeper understanding of the roles of syt7 and Doc2 in synaptic vesicle fusion. Depending on the system studied, and the nature of the preparation, it appears that syt7 functions as a sensor for asynchronous release, synaptic facilitation, both processes, or neither. The perspective offered by Chapman, Watanabe, and colleagues varies from those previously published and is therefore novel and interesting. However, the study is also burdened by some weaknesses which should be acknowledged and addressed.

Strengths:

The strengths of the study include the complementary imaging and electrophysiology approaches for assessing the function of syt7, and the use of appropriate knockout lines.

Weaknesses:

First, the manuscript strongly overstates the significance of the EM data which is interesting but not as definitive as the authors would suggest. As a consequence, the conclusion offered by the authors of syt7 "feeding" vesicles to Doc2 for asynchronous release is weakened. Second, it is not clear to this reviewer that the mathematical model is necessary or justified.