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Cited by 107Cellular transport and secretionNeuroscience and Neuropharmacology ResearchLipid Membrane Structure and Behavior

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

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Lai, Ying, Diao, Jiajie, Cipriano, Daniel J, Zhang, Yunxiang, Pfuetzner, Richard A, Padolina, Mark S, Brunger, Axel T·2014·eLife
DOI 10.7554/elife.03756PMC PMC4130161
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Abstract

Previously we showed that fast Ca2+-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle–vesicle lipid/content mixing assay (Diao et al., 2012). When complexin-1 was included, a more pronounced Ca2+-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several complexin-1 truncation mutants on spontaneous and Ca2+-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca2+-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca2+-triggered fusion. Taken together, complexin-1 utilizes distinct mechanisms for synchronization of Ca2+-triggered fusion and inhibition of spontaneous fusion.

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Figure 1. Reconstituted single vesicle-vescile fusion assay. (A) Protein composition and labeling of proteoliposomes. (B) SDS-PAGE analysis of reconstituted vesicles mimicking the plasma membrane (PM) and synaptic vesicles (SV). (C) Schema of the extended single vesicle–vesicle content mixing assay. The shaded backgrounds indicate the three subsequent 1 min time periods of the procedure and the short intervals (5 s) for buffer exchanges. Representative content fluorescence intensity time traces are shown for an associated pair of SV and PM vesicles without any fusion (top trace), for an associated pair that undergoes spontaneous fusion, and for an associated pair that undergoes fusion after Ca2+-injection. The association of an SV vesicle to a surface-immobilized PM vesicle is characterized by fluorescence intensity increases during the 1 min period after initial SV vesicle loading. Spontaneous and Ca2+-triggered fusion events are characterized by a subsequent stepwise increase of the content dye fluorescence intensity during the respective 1 min observation periods. The time point of the arrival of Ca2+ is determined by the appearance of fluorescence intensity from soluble Cy5 dyes that are part of the injected solution (bottom trace). DOI: http://dx.doi.org/10.7554/eLife.03756.004
Figure 1. Reconstituted single vesicle-vescile fusion assay. (A) Protein composition and labeling of proteoliposomes. (B) SDS-PAGE analysis of reconstituted vesicles mimicking the plasma membrane (PM) and synaptic vesicles (SV). (C) Schema of the extended single vesicle–vesicle content mixing assay. The shaded backgrounds indicate the three subsequent 1 min time periods of the procedure and the short intervals (5 s) for buffer exchanges. Representative content fluorescence intensity time traces are shown for an associated pair of SV and PM vesicles without any fusion (top trace), for an associated pair that undergoes spontaneous fusion, and for an associated pair that undergoes fusion after Ca2+-injection. The association of an SV vesicle to a surface-immobilized PM vesicle is characterized by fluorescence intensity increases during the 1 min period after initial SV vesicle loading. Spontaneous and Ca2+-triggered fusion events are characterized by a subsequent stepwise increase of the content dye fluorescence intensity during the respective 1 min observation periods. The time point of the arrival of Ca2+ is determined by the appearance of fluorescence intensity from soluble Cy5 dyes that are part of the injected solution (bottom trace). DOI: http://dx.doi.org/10.7554/eLife.03756.004CC BY · PubMed Central
Figure 2. Complexin-1 synchronizes Ca2+-triggered fusion and inhibits spontaneous fusion in concert with synaptotagmin-1 and neuronal SNAREs. (A and B) Probability of fusion vs time upon 500 μM Ca2+-injection and spontaneous fusion, both in the presence of full-length synaptotagmin-1 and neuronal SNAREs, but with and without 2 μM complexin-1 (Cpx) as indicated. (A) The histograms of Ca2+-triggered fusion events (1 s time bin) were normalized by the number of associated SV vesicles and fit to an exponential decay function. The decay rate with and without complexin-1 is 0.43 s−1 and 0.11 s−1 respectively. (B) The histograms of spontaneous fusion events were normalized by the number of associated SV vesicles. (C) Domain structure of complexin-1. To probe the roles of the four domains of complexin-1 we used the truncation mutant Cpx26–134 for the N-terminal domain, the truncation mutant Cpx41–134 for the accessory α-helical domain, the Cpx4M mutant for the SNARE binding domain, and the truncation mutant Cpx1–88 for the C-terminal domain. We chose these particular truncations/mutations based on studies of cortical neuronal cultures (Maximov et al., 2009; Kaeser-Woo et al., 2012). (D–G) The bar graphs show the effects of complexin-1 and its mutants on SV-PM vesicle association (D), the number of spontaneous fusion events over the 1-min observation period divided by the number of associated SV vesicles (E), the decay rate of the histogram upon Ca2+-injection (F), and the amplitude of the first 1-sec time bin upon Ca2+-injection (G). Each value in panel G was normalized by the respective number of fusion events after Ca2+-injection. Moreover, only those single vesicle–vesicle fluorescence intensity traces that exhibited a distinct event during the association period were analyzed for fusion events during the subsequent observation periods. Error bars in D, E, G are standard deviations for 6–13 independent repeat experiments. Error bars in F are error estimates computed from the covariance matrix upon fitting the corresponding histograms with a single exponential decay function using a Levenberg-Marquardt technique. DOI: http://dx.doi.org/10.7554/eLife.03756.002
Figure 2. Complexin-1 synchronizes Ca2+-triggered fusion and inhibits spontaneous fusion in concert with synaptotagmin-1 and neuronal SNAREs. (A and B) Probability of fusion vs time upon 500 μM Ca2+-injection and spontaneous fusion, both in the presence of full-length synaptotagmin-1 and neuronal SNAREs, but with and without 2 μM complexin-1 (Cpx) as indicated. (A) The histograms of Ca2+-triggered fusion events (1 s time bin) were normalized by the number of associated SV vesicles and fit to an exponential decay function. The decay rate with and without complexin-1 is 0.43 s−1 and 0.11 s−1 respectively. (B) The histograms of spontaneous fusion events were normalized by the number of associated SV vesicles. (C) Domain structure of complexin-1. To probe the roles of the four domains of complexin-1 we used the truncation mutant Cpx26–134 for the N-terminal domain, the truncation mutant Cpx41–134 for the accessory α-helical domain, the Cpx4M mutant for the SNARE binding domain, and the truncation mutant Cpx1–88 for the C-terminal domain. We chose these particular truncations/mutations based on studies of cortical neuronal cultures (Maximov et al., 2009; Kaeser-Woo et al., 2012). (D–G) The bar graphs show the effects of complexin-1 and its mutants on SV-PM vesicle association (D), the number of spontaneous fusion events over the 1-min observation period divided by the number of associated SV vesicles (E), the decay rate of the histogram upon Ca2+-injection (F), and the amplitude of the first 1-sec time bin upon Ca2+-injection (G). Each value in panel G was normalized by the respective number of fusion events after Ca2+-injection. Moreover, only those single vesicle–vesicle fluorescence intensity traces that exhibited a distinct event during the association period were analyzed for fusion events during the subsequent observation periods. Error bars in D, E, G are standard deviations for 6–13 independent repeat experiments. Error bars in F are error estimates computed from the covariance matrix upon fitting the corresponding histograms with a single exponential decay function using a Levenberg-Marquardt technique. DOI: http://dx.doi.org/10.7554/eLife.03756.002CC BY · PubMed Central
Figure 3. Suppression of spontaneous fusion and synchronization of Ca2+-triggered fusion vs complexin-1 concentration (in concert with synaptotagmin-1 and neuronal SNAREs). The bar graphs show the effect of wildtype complexin-1 at specified concentrations on SV-PM vesicle association (A), the number of spontaneous fusion events divided by the number of associated SV vesicles (B), the decay rate of the histogram of fusion events upon Ca2+-injection (C), and the amplitude of the first 1-sec time bin upon Ca2+-injection (D). Each value in panel D was normalized by the respective number of fusion events after Ca2+-injection. Moreover, only those single vesicle–vesicle fluorescence intensity traces that exhibited a distinct event during the association period were analyzed for fusion events during the subsequent observation periods. Error bars in A, B, D show the standard deviation for 6–10 independent repeat experiments. Error bars in C are error estimates computed from the covariance matrix upon fitting the corresponding histograms with a single exponential decay function using a Levenberg-Marquardt technique. ** indicates p<0.01 using the Student's t test. DOI: http://dx.doi.org/10.7554/eLife.03756.005
Figure 3. Suppression of spontaneous fusion and synchronization of Ca2+-triggered fusion vs complexin-1 concentration (in concert with synaptotagmin-1 and neuronal SNAREs). The bar graphs show the effect of wildtype complexin-1 at specified concentrations on SV-PM vesicle association (A), the number of spontaneous fusion events divided by the number of associated SV vesicles (B), the decay rate of the histogram of fusion events upon Ca2+-injection (C), and the amplitude of the first 1-sec time bin upon Ca2+-injection (D). Each value in panel D was normalized by the respective number of fusion events after Ca2+-injection. Moreover, only those single vesicle–vesicle fluorescence intensity traces that exhibited a distinct event during the association period were analyzed for fusion events during the subsequent observation periods. Error bars in A, B, D show the standard deviation for 6–10 independent repeat experiments. Error bars in C are error estimates computed from the covariance matrix upon fitting the corresponding histograms with a single exponential decay function using a Levenberg-Marquardt technique. ** indicates p<0.01 using the Student's t test. DOI: http://dx.doi.org/10.7554/eLife.03756.005CC BY · PubMed Central
Figure 4. Suppression of spontaneous fusion by complexin-1 with or without synaptotagmin-1. The bar graphs summarize the effect of 2 μM complexin-1 on SV-PM vesicle association (A) and the number of spontaneous fusion events divided by the number of associated SV vesicles (B); the conditions that did not include synaptotagmin-1 used SV vesicles that were reconstituted with synaptobrevin-2 only. Moreover, only those single vesicle–vesicle fluorescence intensity traces that exhibited a distinct event during the association period were analyzed for spontaneous fusion events. DOI: http://dx.doi.org/10.7554/eLife.03756.007
Figure 4. Suppression of spontaneous fusion by complexin-1 with or without synaptotagmin-1. The bar graphs summarize the effect of 2 μM complexin-1 on SV-PM vesicle association (A) and the number of spontaneous fusion events divided by the number of associated SV vesicles (B); the conditions that did not include synaptotagmin-1 used SV vesicles that were reconstituted with synaptobrevin-2 only. Moreover, only those single vesicle–vesicle fluorescence intensity traces that exhibited a distinct event during the association period were analyzed for spontaneous fusion events. DOI: http://dx.doi.org/10.7554/eLife.03756.007CC BY · PubMed Central

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  1. Reim, 2001, Complexins regulate a late step in Ca2+-dependent neurotransmitter release, Cell, 104, 71, 10.1016/S0092-8674(01)00192-1 · DOI

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Metadata

Authors
Lai, Y., Diao, J., Cipriano, D. J., Zhang, Y., Pfuetzner, R. A., Padolina, M. S., & Brunger, A. T.
Affiliations
  • Stanford University (US)
  • Howard Hughes Medical Institute (US)
  • Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2014-08-13
Volume
3
Pages
e03756-e03756
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.03756
PMC
PMC4130161
OpenAlex
W2128246609
Cited by
107
References
40
Citations by year
2026: 1 · 2025: 7 · 2024: 13 · 2023: 8 · 2022: 9 · 2021: 2 · 2020: 3 · 2019: 4 · 2018: 16 · 2017: 17 · 2016: 12 · 2015: 11 · 2014: 4
FWCI
7.31
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Howard Hughes Medical Institute
  • National Institutes of Health (R37-MH63105)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
Keywords
complexin-1, synaptic vesicle fusion, spontaneous release, calcium-triggered release, SNARE proteins, synaptotagmin-1, hemifusion, truncation mutants, N-terminal domain, C-terminal domain, neurotransmitter release
MeSH terms
Animals, Biological Assay, Biological Transport, Calcium, Escherichia coli, Exocytosis, Membrane Fusion, Models, Biological, Mutation, Nerve Tissue Proteins, Neurons, Protein Binding, Recombinant Proteins, Synaptic Vesicles, Gene Expression, Protein Structure, Tertiary, Adaptor Proteins, Vesicular Transport, Synaptosomal-Associated Protein 25, Syntaxin 1, Synaptotagmin I, Vesicle-Associated Membrane Protein 2, Rats, Protein Refolding

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