Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion

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Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion

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DOI: December 13, 2012 Cite as eLife 2012;1:e00109


The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle–vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca2+-injection at 250–500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca2+-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca2+-triggered immediate fusion started from a membrane–membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca2+-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca2+-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.


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  1. Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms

    1. Ying Lai
    2. Jiajie Diao
    3. Daniel J Cipriano
    4. Yunxiang Zhang
    5. Richard A Pfuetzner
    6. Mark S Padolina
    7. Axel T Brunger
    Building on previous work (Diao et al., 2012), we show that the mechanism by which complexin suppresses spontaneous fusion is distinct from the mechanism by which it synchronizes Ca2+-triggered fusion.
    eLife 2014;3:e03756

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We thank Drs. James Rothman, Reinhard Jahn, and Alex Hoepker for stimulating discussions, Joseph Rizo for providing the coordinates of the NMR-derived model of the SNARE/synaptotagmin complex, Dr. Taekjip Ha for single molecule software, and Drs. Chirlmin Joo and Minglei Zhao for figure preparation.

Decision letter

Richard Aldrich, Reviewing editor, The University of Texas at Austin, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for choosing to send your work entitled “Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion” for consideration at eLife. Your article has been evaluated by a Senior Editor and 3 reviewers, one of whom is a member of eLife's Board of Reviewing Editors. The following individuals responsible for the peer review of your submission want to reveal their identity: Jose Rizo-Ray and James Rothman.

The Reviewing Editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing Editor has assembled the following comments based on the reviewers' reports. Our goal is to provide the essential revision requirements as a single set of instructions, so that you have a clear view of the revisions that are necessary for us to publish your work.

General assessment:

The reviewers find that the paper describes a very interesting study of the mechanism of membrane fusion induced by the neuronal SNAREs syntaxin-1, synaptobrevin and SNAP-25, together with the Ca2+ sensor synaptotagmin-1 and the SNARE complex-binding protein complexin.

Central conclusions:

1. Calcium-triggered immediate fusion starts from a membrane-membrane point-contact and proceeds to complete fusion within “milliseconds”, without discernible intermediates.

2. Pathways that involve a stable hemifusion diaphragm only result in fusion after seconds.

3. Complexin shifts the fusion pathways towards the fast pathway.

4. Assays that only measure membrane component mixing, without content mixing measurements, may give misleading results.

The idea of multiple fusion pathways, fast fusion events with no discernible intermediates, and the slow hemi-fusion driven events is a novel concept. The strength of this work is the single vesicle-vesicle system, which is now functional under more physiological levels of Ca2+ and provides a level of resolution that is unparalleled. The combination of the fusion assay with the cryoEM studies is a noted strength of the study.

The effect of CPX in increasing the probability of the immediate fusion events is in line with recent findings on the role of the CPX in synchronizing the Ca2+-triggered fusion, and quite nicely explains how the zig zag array in addition to clamping fusion might aid in the enhancing the rate of fusion itself and is quite appealing, but this could be explained more clearly in the manuscript.

Required revisions:

The reviewers raise a number of concerns that must be adequately addressed before the paper can be accepted. Some of the required revisions will likely require further experimentation within the framework of the presented studies and techniques.

1. An essential assumption for all of the work is that the two types of reconstituted vesicles (donor and acceptor) are homogeneous populations, all with the same make up of lipids and proteins. Significant heterogeneity in terms of protein constituents would completely invalidate the conclusions. Convincing evidence of high homogeneity, or a reference to a convincing study showing such, must be included.

2. The results are for the most part presented in a “semiquantitative” manner, without proper attention to definition of quantitative criteria for classification into the various classes of contacts in the EM and release patterns in the optical measurements. Additionally some of the conclusions are based only on very simple measurements or visual inspection of data, particularly the histograms in Figures 6 and 8. Presenting only the parameters of exponential fits is not adequate. Further treatment of the range of acceptable parameters is necessary. There is also inadequate statistical treatment of the data, even though the term "significant: is often used to describe differences between data from different treatments.

3. The histograms in figure 6, and to a lesser extent figure 8, are very noisy and there are several time bins with no occurrences. The differences are not convincing with only this much data, especially given that the differences between both sets of histograms seem to depend only on the values in the first time bin. These histograms must include a greater number of entries to be convincing.

4. “We performed a quantitative analysis of all observed cryo-EM images” onwards: histograms should be shown for the diameter distributions mentioned, along with a description of classification criteria and statistical analysis.

5. It should be at least mentioned that changes in steady state calcium concentration for many minutes is quite different than the transient increases involved in calcium-dependent transmitter release.

6. The discussion section requires some revision – it is not very accessible and hard to understand at times.

7. Although not necessary, the paper would be greatly strengthened by CryoEM data with CPX.

8. The claimed millisecond time resolution seems inappropriate as the fastest sample interval seems to be 200 ms and 600 ms binning is often used.

9. Figure 4 presents a nice experiment, but numbers of occurrences, classification criteria, and statistics need to be included.


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