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  1. Author Response:

    Reviewer #1 (Public Review):

    Munc13 is a key regulator of synaptic vesicle (SV) fusion that is thought to mediate SV tethering and regulate SNARE assembly. Based primarily on Munc13 crystal structures, the authors design a set of four charge reversal mutations in the C1C2B region that are predicted to affect the interaction of Munc13 with the plasma membrane (PM). Various in vivo and in vitro consequences of these mutations are studied, leading to two main conclusions: (1) an interaction between the PM and a polybasic surface of Munc13 is likely important for SV tethering, and (2) two residues in the Ca2+-binding loops of the C2B domain are important for SV fusion.

    So far, so good - I think the data strongly support the two main conclusions noted above. It is less clear that these studies support (or could falsify) the main hypothesis, stated in the title, that re-orientation of membrane-bound Munc13 controls neurotransmitter release. Primed vesicles appear to exist in dynamic equilibrium between two states, one of them "loosely" primed (LS) and the other "tightly" primed (TS). Inasmuch as this simple model is correct, one could characterize the various players - SNAREs, synaptotagmin, complexin and of course Munc13 - in terms of their ability to influence the LS/TS equilibrium, perhaps in response to Ca2+ or other small molecules. This manuscript postulates that the orientation of Munc13 relative to the membrane has a major impact on the LS/TS equilibrium, with a perpendicular orientation favoring LS and a slanted orientation favoring TS.

    The authors' previous structure (Xu et al., 2017) suggested that two partially-discrete faces of C1C2B, one polybasic and the other centered around the Ca2+-binding loops of C2B, are likely involved in PM binding. In that paper they hypothesized that the polybasic face would dominate in the absence of Ca2+ whereas the 'Ca2+-binding face' [not a very good name, but the authors haven't suggested a better one] would dominate in the presence of Ca2+. Binding to the PM via the polybasic face would yield a more erect or 'perpendicular' binding orientation, whereas binding to the PM by the Ca2+-binding face would yield a more tilted or 'slanted' binding orientation.

    In revised manuscript we use the term DAG/Ca2+/PIP2-binding face, or Ca2+-dependent face when we discuss the effects of Ca2+ in particular.

    Here the authors performed two molecular dynamics simulations, one without and one with bound Ca2+. In the Results section, they correctly point out that their findings cannot be used to support their hypothesis because, in each case, Munc13 was placed in the hypothesized orientation - perpendicular for minus Ca2+, slanted for plus Ca2+ - at the beginning of the simulation. In the Discussion however the authors argue that the MD simulations support their model. I disagree because the simulations needed to falsify the model have not yet been conducted. In addition, an opportunity was seemingly missed by not doing MD simulations on the mutants.

    We have removed the sentence stating that the MD simulations support the model in the corresponding paragraph of the discussion (page 22). A meaningful analysis of the effects of the mutations would have required much longer simulations of this large system, which would take several months for each mutant in the UT Southwestern BioHPC facility or acquisition of a dedicated allocation at the Texas Advanced Computing Center.

    Of the four mutations studied, two (K603E and K720E) should specifically destabilize PM binding by the polybasic face, one (K706E) should destabilize binding by the Ca2+-binding face, and one (R769E) is expected to destabilize both. Two of the mutants (K603E and R769E) in fact abrogate priming. This result, along with biochemical experiments, implicates the polybasic face in SV tethering and thus represents the main evidence supporting the first of the main conclusions (see Evaluation Summary above). However, since an unprimed vesicle does not participate in the LS/TS equilibrium, these mutants are in this respect uninformative. Only the remaining mutants, K720E and K706E, would therefore appear to have the potential of yielding information about the LS/TS equilibrium and its relationship to Munc13 orientation.

    Although we understand the concern expressed by the reviewer, we do not fully agree with the last sentence. If we accept that the K603E and R769E mutations impair priming, this result implies that binding through the polybasic face occurs for WT Munc13-1. This conclusion does not demonstrate the LS/TS equilibrium, but it does support the notion that one of the proposed states exists.

    Both K720E and K706E support normal priming but have opposite effects on vesicular release probability and evoked release. These results can be rationalized in terms of an LS/TS equilibrium. The K720E mutation, which selectively destabilizes binding by the polybasic face, would shift the equilibrium toward TS and thereby increase the release probability. Conversely the K706E mutation, which destabilizes binding by the Ca2+-binding face, would shift the equilibrium toward LS and thereby reduce the release probability.

    However, the authors themselves cast serious doubt on this straightforward interpretation. In the case of K720E, they point out that the other 'polybasic mutant', K603E, has no effect of release probability. (I argued above that, perhaps, K603E is best viewed as uninformative about the LS/TS equilibrium owing to its strong upstream priming defect.) In the case of K706E, the authors point out that phorbol ester potentiation was similar for K706E and wild-type, suggesting to them "that the effects of the K706E mutation might not be related to the transition to slanted orientations but rather to another mechanism that directly influences fusion. For instance, the Munc13-1 C2B domain might cause membrane perturbations analogous to those that are believed to underlie the function of the Syt1 C2 domains in triggering release (Fernandez-Chacon et al., 2001; Rhee et al., 2005). It is also possible that the phenotypes caused by the K706E mutation and other mutations studied here reflect effects of Munc13-1 in more than one step leading to release, which complicates the interpretation of the data." If this is indeed the case, we are down to one mutant - K720E - that can be informative about the LS/TS equilibrium. (For the most part, I did not find the double and quadruple mutants informative, especially because each of them contains at least one mutation that strongly abrogates priming.)

    We again understand the concerns expressed by the reviewer but do not agree that the K706E mutant does not provide any information on the LS/TS equilibrium. If we accept that the K706E mutation does have an effect on evoked release and that K603E has an effect on priming, these results support the notion that both proposed binding modes occur and are functionally relevant. We do agree however that this conclusion does not prove that there is an equilibrium between two primed states.

    It looks like K720E is right in the center of the polybasic surface (although it's hard to tell from a single 'projection' image) so it would have been expected to impair Ca2+-independent liposome binding, and it does. However the liposome clustering effects are very weird, displaying a much broader distribution than any other experiment, an observation which the authors disregard. However, overall, I would say that the authors' K720E findings offer modest support for their overall main hypothesis. But for me it's not enough to justify making that hypothesis the title of the paper.

    We agree with the reviewer that it is a stretch to include the hypothesis in the title of the paper. We have changed the title to: ‘Control of neurotransmitter release by two distinct membrane-binding faces of the Munc13-1 C1C2B region’, which emphasizes the notion that there are two functional membrane-binding faces of the Munc13-1 C1C2B region without making a specific claim on a role of two faces on presynaptic plasticity. We note that the notion that the Ca2+- and DAG-dependent face of the C1C2B region is functionally relevant was already supported by previous studies (Rhee et al. 2002; Shin et al. 2010), which we now cite in additional sentences to emphasize this point (e.g. pages 22, 23). Hence, we believe that, together with the previous data, our results strongly support the conclusion that two faces of the C1C2B region are functionally important. We still present the LS/TS model and use it often to interpret our results, but have tried to be careful throughout the manuscript to not overstate our conclusions and point out when our results are consistent with the model without concluding that they prove it.

    For the most part I could not follow the discussion of figures 4 and 5. But I am struck by strong similarity between the data for K603E and K706E (comparing Fig. 4B/C to Fig. 4H/I). How can these results be reconciled with the opposite roles predicted for K603 and K706?

    The normalized data obtained for K603E and K706E mutants do look similar (new Fig. 8C,I), but the absolute amplitudes are lower for the former (new Fig. 8B,H). Nevertheless, we agree that it not straightforward to interpret some of the data obtained in the repetitive stimulation experiments. To acknowledge this difficulty, we have included the following sentence at the end of the first paragraph of the corresponding section (line 416): ‘Nevertheless, interpretation of some of the data was not straightforward, and there may be alternative explanations to those offered below, which are based in part on the proposed LS-TS equilibrium.’

    I'm not sure how the results of the PDBu experiments contribute to the conclusion that "two faces of the C1-C2B region are critical for Munc13-1-dependent short-term plasticity" (p. 15), since the only mutant that selectively affects one of the faces, K706E, has no impact (Fig. 6).

    We have toned down the sentence at the end of the section describing the PDB data, which now reads (line 475): ‘Overall, these results show that basic residues in the Munc13-1 C1-C2B region influence the potentiation of synaptic responses by PDBu and, together with the data obtained with repetitive stimulation, they support the notion that two faces of the C1-C2B region are involved in Munc13-1-dependent short-term plasticity.’

    Why are the liposome-binding assays in Fig. 7 done with C2C present - isn't that just a confounding factor? And if Ca2+-independent binding by C2C is as weak as suggested by the results in Fig. 7, how do any of the Munc13 constructs cluster liposomes in Fig. 8? (Note that, according to my reading of the methods, V-type liposomes are simply T-type liposomes without the DAG and PIP2.)

    Binding of the C2C domain to liposomes is indeed weak but still can contribute to liposome clustering because multiple C1C2BMUNC2C molecules can cooperate in this activity (see Quade et al. 2019). We used C1C2BMUNC2C mutants in the binding assays because they were also employed for the liposome clustering and fusion assays, in which C1C2BMUN is much less active (see Quade et al., 2019). We agree that having the C2C domain present could be a confounding factor, but we included the binding results because the effects of the mutations did correlate, albeit qualitatively, with those of the clustering and fusion assays.

    What is the basis for the claim (p. 22) that "the perpendicular orientation of Munc13-1 is expected to facilitate initiation of SNARE core complex assembly"?

    The perpendicular orientation may hinder the initiation of SNARE complex assembly if Munc13-1 is located between the vesicle and the plasma membrane, but can facilitate initiation of assembly if the bridging Munc13-1 molecules are located further from the center of the vesicle-plasma membrane interface (e.g. as in Fig. 1D; see also cryo electron tomography images of Quade et al. 2019). We agree that the term ‘expected’ is too strong and now state that the perpendicular orientation ‘may facilitate initiation of SNARE complex assembly’ (line 523).

    Reviewer #2 (Public Review):

    In this manuscript, Rosenmund and colleagues describe new results regarding the mode of action of Munc13 in neurotransmitter release. Based on molecular dynamics simulations of Munc13 (C1C2BMun) with phospholipid membranes, the authors selected promising point mutations and comparatively investigated their functional impact with electrophysiological experiments in hippocampal neurons and with a variety of in vitro experiments (lipid binding assay, liposome clustering and fusion). The results show that specific mutations in the C1C2B-domain (also referred to as polybasic face) of Munc13 (K603E, R769E) strongly inhibit vesicle priming, a property that correlates well with their re duced ability to bind to phospholipid membranes in a calcium-independent manner.

    The manuscript describes comprehensive electrophysiological and biochemical experiments that are complemented and extended by thoughtful analyses. The direct combination of electrophysiological and biochemical expertise from the Rosenmund and Rizo laboratories, respectively, represents a particular strength of this study, allowing the authors to develop new insights into the function of the Munc13 protein. A welcome (but not necessary) extension of the data presented would be the demonstration that the mutants in question (K603E, R769E) also show altered phospholipid binding in the MD simulations. In any case, the presentation of the data is clear and the authors' conclusions are convincing.

    Taken together, the manuscript and the results represent a significant advance in the understanding of the molecular mechanisms underlying synaptic vesicle priming.

    We thank the reviewer for the very positive evaluation of our study. As mentioned above, a meaningful analysis of the effects of the mutations would have required much longer MD simulations of this large system.

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  2. Evaluation Summary:

    This study will be of high interest to readers in the field of the molecular mechanisms of synaptic operation. The functional demonstration of two different topological states of Munc13 involved in, Ca2+-independent and Ca2+-dependent, synaptic vesicle priming is a remarkable contribution to further understand key mechanisms of neurotransmitter release and its modulation. A multidisciplinary, solid and careful study supported by simulations of molecular dynamics, in vitro assays of membrane fusion and synaptic electrophysiology of mouse hippocampal neurons.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  3. Reviewer #1 (Public Review):

    Munc13 is a key regulator of synaptic vesicle (SV) fusion that is thought to mediate SV tethering and regulate SNARE assembly. Based primarily on Munc13 crystal structures, the authors design a set of four charge reversal mutations in the C1C2B region that are predicted to affect the interaction of Munc13 with the plasma membrane (PM). Various in vivo and in vitro consequences of these mutations are studied, leading to two main conclusions: (1) an interaction between the PM and a polybasic surface of Munc13 is likely important for SV tethering, and (2) two residues in the Ca2+-binding loops of the C2B domain are important for SV fusion.

    So far, so good - I think the data strongly support the two main conclusions noted above. It is less clear that these studies support (or could falsify) the main hypothesis, stated in the title, that re-orientation of membrane-bound Munc13 controls neurotransmitter release. Primed vesicles appear to exist in dynamic equilibrium between two states, one of them "loosely" primed (LS) and the other "tightly" primed (TS). Inasmuch as this simple model is correct, one could characterize the various players - SNAREs, synaptotagmin, complexin and of course Munc13 - in terms of their ability to influence the LS/TS equilibrium, perhaps in response to Ca2+ or other small molecules. This manuscript postulates that the orientation of Munc13 relative to the membrane has a major impact on the LS/TS equilibrium, with a perpendicular orientation favoring LS and a slanted orientation favoring TS.

    The authors' previous structure (Xu et al., 2017) suggested that two partially-discrete faces of C1C2B, one polybasic and the other centered around the Ca2+-binding loops of C2B, are likely involved in PM binding. In that paper they hypothesized that the polybasic face would dominate in the absence of Ca2+ whereas the 'Ca2+-binding face' [not a very good name, but the authors haven't suggested a better one] would dominate in the presence of Ca2+. Binding to the PM via the polybasic face would yield a more erect or 'perpendicular' binding orientation, whereas binding to the PM by the Ca2+-binding face would yield a more tilted or 'slanted' binding orientation.

    Here the authors performed two molecular dynamics simulations, one without and one with bound Ca2+. In the Results section, they correctly point out that their findings cannot be used to support their hypothesis because, in each case, Munc13 was placed in the hypothesized orientation - perpendicular for minus Ca2+, slanted for plus Ca2+ - at the beginning of the simulation. In the Discussion however the authors argue that the MD simulations support their model. I disagree because the simulations needed to falsify the model have not yet been conducted. In addition, an opportunity was seemingly missed by not doing MD simulations on the mutants.

    Of the four mutations studied, two (K603E and K720E) should specifically destabilize PM binding by the polybasic face, one (K706E) should destabilize binding by the Ca2+-binding face, and one (R769E) is expected to destabilize both. Two of the mutants (K603E and R769E) in fact abrogate priming. This result, along with biochemical experiments, implicates the polybasic face in SV tethering and thus represents the main evidence supporting the first of the main conclusions (see Evaluation Summary above). However, since an unprimed vesicle does not participate in the LS/TS equilibrium, these mutants are in this respect uninformative. Only the remaining mutants, K720E and K706E, would therefore appear to have the potential of yielding information about the LS/TS equilibrium and its relationship to Munc13 orientation.

    Both K720E and K706E support normal priming but have opposite effects on vesicular release probability and evoked release. These results can be rationalized in terms of an LS/TS equilibrium. The K720E mutation, which selectively destabilizes binding by the polybasic face, would shift the equilibrium toward TS and thereby increase the release probability. Conversely the K706E mutation, which destabilizes binding by the Ca2+-binding face, would shift the equilibrium toward LS and thereby reduce the release probability.

    However, the authors themselves cast serious doubt on this straightforward interpretation. In the case of K720E, they point out that the other 'polybasic mutant', K603E, has no effect of release probability. (I argued above that, perhaps, K603E is best viewed as uninformative about the LS/TS equilibrium owing to its strong upstream priming defect.) In the case of K706E, the authors point out that phorbol ester potentiation was similar for K706E and wild-type, suggesting to them "that the effects of the K706E mutation might not be related to the transition to slanted orientations but rather to another mechanism that directly influences fusion. For instance, the Munc13-1 C2B domain might cause membrane perturbations analogous to those that are believed to underlie the function of the Syt1 C2 domains in triggering release (Fernandez-Chacon et al., 2001; Rhee et al., 2005). It is also possible that the phenotypes caused by the K706E mutation and other mutations studied here reflect effects of Munc13-1 in more than one step leading to release, which complicates the interpretation of the data." If this is indeed the case, we are down to one mutant - K720E - that can be informative about the LS/TS equilibrium. (For the most part, I did not find the double and quadruple mutants informative, especially because each of them contains at least one mutation that strongly abrogates priming.)

    It looks like K720E is right in the center of the polybasic surface (although it's hard to tell from a single 'projection' image) so it would have been expected to impair Ca2+-independent liposome binding, and it does. However the liposome clustering effects are very weird, displaying a much broader distribution than any other experiment, an observation which the authors disregard. However, overall, I would say that the authors' K720E findings offer modest support for their overall main hypothesis. But for me it's not enough to justify making that hypothesis the title of the paper.
    For the most part I could not follow the discussion of figures 4 and 5. But I am struck by strong similarity between the data for K603E and K706E (comparing Fig. 4B/C to Fig. 4H/I). How can these results be reconciled with the opposite roles predicted for K603 and K706?

    I'm not sure how the results of the PDBu experiments contribute to the conclusion that "two faces of the C1-C2B region are critical for Munc13-1-dependent short-term plasticity" (p. 15), since the only mutant that selectively affects one of the faces, K706E, has no impact (Fig. 6).

    Why are the liposome-binding assays in Fig. 7 done with C2C present - isn't that just a confounding factor? And if Ca2+-independent binding by C2C is as weak as suggested by the results in Fig. 7, how do any of the Munc13 constructs cluster liposomes in Fig. 8? (Note that, according to my reading of the methods, V-type liposomes are simply T-type liposomes without the DAG and PIP2.)

    What is the basis for the claim (p. 22) that "the perpendicular orientation of Munc13-1 is expected to facilitate initiation of SNARE core complex assembly"?

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  4. Reviewer #2 (Public Review):

    In this manuscript, Rosenmund and colleagues describe new results regarding the mode of action of Munc13 in neurotransmitter release.
    Based on molecular dynamics simulations of Munc13 (C1C2BMun) with phospholipid membranes, the authors selected promising point mutations and comparatively investigated their functional impact with electrophysiological experiments in hippocampal neurons and with a variety of in vitro experiments (lipid binding assay, liposome clustering and fusion). The results show that specific mutations in the C1C2B-domain (also referred to as polybasic face) of Munc13 (K603E, R769E) strongly inhibit vesicle priming, a property that correlates well with their re duced ability to bind to phospholipid membranes in a calcium-independent manner.

    The manuscript describes comprehensive electrophysiological and biochemical experiments that are complemented and extended by thoughtful analyses. The direct combination of electrophysiological and biochemical expertise from the Rosenmund and Rizo laboratories, respectively, represents a particular strength of this study, allowing the authors to develop new insights into the function of the Munc13 protein. A welcome (but not necessary) extension of the data presented would be the demonstration that the mutants in question (K603E, R769E) also show altered phospholipid binding in the MD simulations. In any case, the presentation of the data is clear and the authors' conclusions are convincing.

    Taken together, the manuscript and the results represent a significant advance in the understanding of the molecular mechanisms underlying synaptic vesicle priming.

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  5. Reviewer #3 (Public Review):

    Camacho et al. have tested a model to understand the mechanism by which the active zone protein Munc-13 operates in synaptic vesicle priming and Ca2+-dependent neurotransmitter release. Their model is based on the crystal structure -previously solved by authors- of a major fragment of Munc-13-1, including the C1, C2B and MUN domains and the C-terminal C2C domains (C1C2BMUNC2C fragment) but missing the N-terminal part of the protein. An insightful molecular dynamic analysis developed to investigate the interaction of the C1C2B segment with the plasma membrane has led to refine the model and to design mutations of specific residues to test and to validate the model experimentally. A tenet of the proposed mechanism is that Munc-13-1 bridges the synaptic vesicle (SV) to the plasma membrane. The major conclusion is that Munc-13-1 adopts two conformations: (a) a conformation perpendicular to the synaptic vesicle, which is Ca2+-independent, it involves the interaction of negatively charged plasma membrane phospholipids with basic residues (K603, at C1 domain; R769, at C2B domain Ca2+-binding loop) and it is essential for SV priming and (b) a slanted conformation that occurs when Ca2+ binds to the C2B domain and it is key to progress toward the full assembly of the SNARE complex previous to neurotransmitter release. This model is well supported by studies based on the behavior of different versions of Munc-13-1 in which basic residues have been mutated to a negatively charged residue (specially K603E and R769E) to impair membrane binding. These studies include liposome interaction and fusion assays and electrophysiological analysis of synapses (measurements of the readily releasable pool (RRP), vesicular release probability and short-term synaptic plasticity) in Munc-13-1/2 DKO mouse hippocampal neurons in which either Munc-13 WT or mutant versions have been re-introduced. The results obtained based on the analysis of the K603 and R769E are clear, supports the conclusions and clearly advances the knowledge regarding the details of how Munc-13-1 mediates SV priming and Ca2+-dependent full assembly of SNARE complex.

    On the other hand, the results pertaining some aspects of the contribution of other residues investigated in the study (K720 and K706, at the C2 domain Ca2+-binding loops) are less obvious to fit within a rather simple model but they open interesting perspectives for future investigations. The residue K720 seems not to be a critic residue for Ca2+-independent interaction with the plasma membrane, the mutation K720E does not interfere with priming and, surprisingly, it increases Ca2+-dependent release. Consistently with the model, the K706E mutation did not affect SV priming but it decreased the probability of release. In any case, the authors provided careful, however, open interpretations that are supported by the results obtained.

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