Molecular determinants of complexin clamping and activation function

  1. Manindra Bera
  2. Sathish Ramakrishnan
  3. Jeff Coleman
  4. Shyam S Krishnakumar
  5. James E Rothman

  • Curated by eLife

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    Evaluation Summary:

    Berra and colleagues revisit several mechanistic questions mainly centered on the accessory helix of mouse complexin (mCpx) and its contribution to the 'fusion clamp' property of mCpx whereby mCpx-SNARE interactions prevent full assembly and subsequent membrane fusion. This clamping function is believed to help generate a metastable pool of release-ready vesicles at the synapse, and it has been studied in a wide variety of systems including mouse, fly, worm, squid, fish, and diverse in vitro biochemical preps over the past ~ 20 years. The authors derive several conclusions from their efforts, but most relevant is a reiteration of a previous proposal that the accessory helix region of mCpx stabilizes a pre-fusion clamped state via interactions with SNAREs.

    (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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Abstract

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca 2+ influx (Ramakrishnan et al., 2020). Here, using the same in vitro single-vesicle fusion assay, we determine the molecular details of the Complexin-mediated fusion clamp and its role in Ca 2+ -activation. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins enhances this functionality. The C-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Independent of their clamping functions, the accessory-central helical domains of Complexin also contribute to rapid Ca 2+ -synchronized vesicle release by increasing the probability of fusion from the clamped state.

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  1. Version published to 10.7554/elife.71938 on eLife
  2. eLife

    Evaluation Summary:

    Berra and colleagues revisit several mechanistic questions mainly centered on the accessory helix of mouse complexin (mCpx) and its contribution to the 'fusion clamp' property of mCpx whereby mCpx-SNARE interactions prevent full assembly and subsequent membrane fusion. This clamping function is believed to help generate a metastable pool of release-ready vesicles at the synapse, and it has been studied in a wide variety of systems including mouse, fly, worm, squid, fish, and diverse in vitro biochemical preps over the past ~ 20 years. The authors derive several conclusions from their efforts, but most relevant is a reiteration of a previous proposal that the accessory helix region of mCpx stabilizes a pre-fusion clamped state via interactions with SNAREs.

    (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.)

  3. eLife

    Reviewer #1 (Public Review):

    Berra and colleagues revisit several mechanistic questions mainly centered on the accessory helix of mouse complexin (mCpx) and its contribution to the 'fusion clamp' property of mCpx whereby mCpx-SNARE interactions prevent full assembly and subsequent membrane fusion. This clamping function is believed to help generate a metastable pool of release-ready vesicles at the synapse, and it has been studied in a wide variety of systems including mouse, fly, worm, squid, fish, and diverse in vitro biochemical preps over the past ~ 20 years. The authors derive several conclusions from their efforts, but most relevant is a reiteration of a previous proposal that the accessory helix region of mCpx stabilizes a pre-fusion clamped state via interactions with SNAREs.

    Strengths:

    There are several attractive features of this study. The single-vesicle fusion assay (which this lab has now published several papers on) provides an impressive minimal model fusion system with nearly complete control over all the relevant components including their absolute and relative concentrations, as well as a quantitative assessment of both docking and vesicle persistence time prior to fusion using mouse, worm and fly Cpx orthologs. The authors substantiate a claim made in a previous publication that efficient clamping by Cpx requires some sort of kinetic delay in vesicle fusion by employing DNA assemblies to slow fusion via a means entirely independent of SNARE assembly. By changing the Cpx concentration, the authors reveal a range of dose-dependent clamping that may be important for interpreting results from previous Cpx studies and further support the notion that the C-terminal domain of Cpx plays a role in its local enrichment.

    Weaknesses:

    A major selling point of this study is its reassessment of a long-standing conundrum in the complexin field: what is the mechanism of Cpx acting as a fusion clamp and why does its function appear to be so different between species? The results described here more or less echo previous claims using this new in vitro assay without providing much resolution. One the one hand, this in vitro assay suggests that mCpx1 is a potent fusion clamp while fly and worm orthologs are less efficient. Whereas using the currently best available synaptic data, one would conclude that the reverse is true: fly/worm Cpx is a potent fusion clamp while mCpx doesn't strongly clamp. Given that the apparent discrepancy between vertebrate and invertebrate Cpx mechanism remains an outstanding question in the field, it was a bit of a missed opportunity to compare them. Did the worm and fly Cpx proteins perform poorly because they were not paired up with their conspecific SNAREs and Syt1s? Would mCpx1 fail to clamp efficiently using invertebrate SNAREs/Syt1? We know that mCpx doesn't suppress spontaneous neurotransmitter release efficiently in the fly (Xue 2009, Cho 2014) or worm (Wragg 2017). And fly Cpx potently suppresses spontaneous fusion in mouse cultured neurons much more than mCpx (Xue 2009), suggesting that, in the context of synapses, fly Cpx can efficiently clamp. None of these past examples specifically and precisely controlled for expression levels, so a rigorous comparison has yet to be done. However, this current study does not clarify these long-standing questions that have confounded the field for years. Moreover, while this in vitro assay has many advantages, it is not entirely clear whether it is a sufficiently good model of clamping at the synapse to resolve some major questions. Perhaps using pre-assembled tSNAREs rather than relying on accessory fusion proteins such as Munc18 and Munc13 and proteins that remodel/diassemble the SNARE complex such as NSF/alpha-SNAP lead to significantly different energy barriers for SNARE assembly and fusion. In this scenario, the relative impact of Cpx may differ from its impact at the synapse. Regardless of these details, the study is technically nice and provides some interesting assessments of various Cpx accessory helix mutations and invertebrate Cpx for researchers of synaptic molecular mechanisms to explore further.

  4. eLife

    Reviewer #2 (Public Review):

    Fast SNARE-mediated membrane fusion triggered by calcium is tightly regulated by accessory proteins including synaptotagmin and complexin (Cpx). In this paper, the authors used vesicles containing ssDNAs to demonstrate the clamping effect of Cpx. Moreover, besides previous known clamping effect of Cpx variants, the author also showed that Cpx doesn't promote triggered fusion with 1 mM Ca++. Several major concerns have to be addressed; particularly, the high Ca++ level and low effective Cpx concentration are two critical technical issues.

  5. eLife

    Reviewer #3 (Public Review):

    This work builds, albeit incrementally, on three recent papers - Ramakrishnan 2018, 2019, and 2020 - the most recent of which was published in eLife. The eLife paper led to an interesting model in which complexin was found to operate synergistically with synaptotagmin, with synaptotagmin delaying fusion long enough for complexin to clamp most of the SNARE complexes and synaptotagmin clamping the rest in a Ca2+-releasable form. The goal of the current manuscript was to gain a better understanding of this interesting system.

    What then are the main contributions of this manuscript? First, for the purpose of delaying fusion long enough to allow the complexin clamp to form, the authors are able to replace synaptotagmin with duplex DNA. Unlike the complexin/synaptotagmin machine, however, this system once clamped cannot proceed to fusion, so there's no way to know whether, or in what way(s), the clamped state resembles the physiologically clamped state.

    Second, the authors compare two Ca2+-triggered fusion reactions, with and without complexin. (In the absence of complexin, synaptotagmin will clamp provided it is superstoichiometric to the SNAREs.) They conclude that complexin does not contribute to the probability or speed of Ca2+-triggered fusion. However, they only test a single, high concentration of Ca2+ that leads to almost complete fusion in both cases, leaving open the possibility that at physiological Ca2+ they would find a difference in release probability after all. As for the speed of fusion being the same, the authors acknowledge that they don't have the temporal resolution to measure that speed - it's basically over in a single time step, so again it's not possible to know whether the speeds with and without complexin might differ.

    The other main goal of the paper is to gain more insight into the molecular nature of the complexin clamp by studying complexin mutants. The authors favor the "zigzag array" model they proposed in 2011, but I the new data really move the needle very far in resolving that debate. All but one of the mutations studied compromise complexin's clamping ability. As the authors know, it is relatively easy to break a machine, and that would seem especially true when all of the mutants they studied involve quadruple substitutions (or a quadruple insertion) within a single ~25-amino acid stretch. Thus, although the results seem to be consistent with a zigzag array model, they cannot in my view be used to rule out alternative models (such as the model proposed by Malsam, 2020).

    I am puzzled by the authors' claim (in the Abstract) that their in vitro work "establishes" the "physiological relevance" of the complexin clamp. Since no physiological experiments are performed, it would seem that such a claim must be based on new and improved agreement between experiments performed and literature data. Yet I was unable to find evidence of this.

    Finally, how is the reader to evaluate the claim (lines 288ff) that "the only plausible way for both CPX(cen) and CPX(acc) to interact with the SNAREpins is if CPX interacts with different pre-fusion SNAREpins as observed in the pre-fusion CPX-SNARE X-ray crystal structure (Kummel et al. 2011)"? Doesn't Malsam et al. (2020), Fig. 5 (which seems to be undercited) present an alternative model?

  6. Version published to 10.1101/2021.07.05.451112v1 on bioRxiv