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Concurrent binding of complexin and synaptotagmin to liposome-embedded SNARE complexes.

Chicka MC, Chapman ER - Biochemistry (2009)

Bottom Line: Synaptotagmin and complexin regulate SNARE-mediated synaptic vesicle exocytosis.It has been proposed that complexin clamps membrane fusion and that Ca(2+)-synaptotagmin displaces complexin from SNARE complexes to relieve this clamping activity.Moreover, the clamping ability of apo-synaptotagmin occluded the clamping activity of complexin until the arrival of a Ca(2+) trigger, at which point synaptotagmin accelerated fusion while high concentrations of complexin inhibited fusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Programs in Cellular and Molecular Biology, University of Wisconsin, 1300 University Avenue, SMI 129, Madison, Wisconsin 53706, USA.

ABSTRACT
Synaptotagmin and complexin regulate SNARE-mediated synaptic vesicle exocytosis. It has been proposed that complexin clamps membrane fusion and that Ca(2+)-synaptotagmin displaces complexin from SNARE complexes to relieve this clamping activity. Using a reconstituted system, we demonstrate that complexin and synaptotagmin simultaneously bind to neuronal SNARE complexes and that both apo-synaptotagmin and complexin inhibit SNARE-mediated membrane fusion. Moreover, the clamping ability of apo-synaptotagmin occluded the clamping activity of complexin until the arrival of a Ca(2+) trigger, at which point synaptotagmin accelerated fusion while high concentrations of complexin inhibited fusion. Thus, the inhibitory patterns of synaptotagmin and complexin are different, suggesting that SNAREs assemble into distinct states along the fusion pathway. These data also suggest that during synaptotagmin-regulated vesicle-vesicle fusion, complexin does not function as a fusion clamp that is relieved by Ca(2+)-synaptotagmin.

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In vitro membrane fusion regulated by cpx-I and syt. (a) Increasing concentrations of cpx-I were added to fusion reaction mixtures containing 30 μM syt. t+v denotes fusion reaction mixtures lacking cpx-I and syt. As a control, the cytoplasmic domain of synaptobrevin 2 (cd-syb, 10 μM) was added to t+v to inhibit SNARE-mediated fusion. Fusion was monitored for 120 min at 37 °C, normalized to the maximum donor fluorescence signal (% Max. fluorescence), and plotted as a function of time. Reactions were carried out in 0.2 mM EGTA. (b) The final extent of fusion at each cpx-I concentration tested in panel a was normalized to the final extent of fusion obtained by t+v (% t+v). (c) Experiments were conducted as described for panel a except Ca2+ (1 mM) was added to reaction mixtures at 20 min. (d) Data from panel c were normalized as in panel b. The inset shows the final extent of fusion at each cpx-I concentration tested in panels a and c normalized to the extent of fusion obtained in reaction mixtures containing syt, but lacking cpx-I (% t+v+syt). All fusion traces are representative from n ≥ 3. Data in panels b and d represent the mean ± the standard error of the mean from n ≥ 3.
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fig2: In vitro membrane fusion regulated by cpx-I and syt. (a) Increasing concentrations of cpx-I were added to fusion reaction mixtures containing 30 μM syt. t+v denotes fusion reaction mixtures lacking cpx-I and syt. As a control, the cytoplasmic domain of synaptobrevin 2 (cd-syb, 10 μM) was added to t+v to inhibit SNARE-mediated fusion. Fusion was monitored for 120 min at 37 °C, normalized to the maximum donor fluorescence signal (% Max. fluorescence), and plotted as a function of time. Reactions were carried out in 0.2 mM EGTA. (b) The final extent of fusion at each cpx-I concentration tested in panel a was normalized to the final extent of fusion obtained by t+v (% t+v). (c) Experiments were conducted as described for panel a except Ca2+ (1 mM) was added to reaction mixtures at 20 min. (d) Data from panel c were normalized as in panel b. The inset shows the final extent of fusion at each cpx-I concentration tested in panels a and c normalized to the extent of fusion obtained in reaction mixtures containing syt, but lacking cpx-I (% t+v+syt). All fusion traces are representative from n ≥ 3. Data in panels b and d represent the mean ± the standard error of the mean from n ≥ 3.

Mentions: In the final series of experiments, cpx-I was titrated into an in vitro fusion assay in the presence of a saturating concentration of syt (30 μM syt; see ref (17)). Fusion was monitored in the absence of Ca2+ (Figure 2a,b), and after addition of 1 mM Ca2+ to reaction mixtures at 20 min (Figure 2c,d) (similar to a method used in ref (6)). Under these conditions, if cpx-I acts as a clamp as reported in refs (4)−(6), (8), and (13), it would be expected to inhibit fusion only in the absence of Ca2+ and not after a Ca2+ trigger since Ca2+-syt would overcome the cpx-I clamp to drive fusion. As shown in Figure 2a,b, in the absence of Ca2+, syt alone inhibited fusion by ∼25%. Addition of cpx-I to reaction mixtures containing apo-syt (Ca2+ free) resulted in a small but reproducible amount of stimulation at low cpx-I concentrations analogous to the findings obtained using SNAREs alone in the fusion assay (Figure 2a,b and Figure S2b,c of the Supporting Information). However, in contrast to what was observed when cpx-I was added to reaction mixtures containing SNAREs alone, addition of higher concentrations of cpx-I to reaction mixtures containing apo-syt only modestly inhibited fusion beyond what was already achieved by apo-syt alone (Figure 2a,b). Thus, cpx-I does not appear to play a major role in clamping SNARE-mediated vesicle−vesicle fusion prior to a Ca2+ signal when syt is also present (see the Supporting Information for more details). In duplicate samples, we triggered syt-stimulated fusion by adding Ca2+ at 20 min (Figure 2c). From these traces, it is evident that Ca2+-syt stimulates fusion but also that high concentrations of cpx-I inhibit Ca2+-syt-triggered fusion (Figure 2c,d).


Concurrent binding of complexin and synaptotagmin to liposome-embedded SNARE complexes.

Chicka MC, Chapman ER - Biochemistry (2009)

In vitro membrane fusion regulated by cpx-I and syt. (a) Increasing concentrations of cpx-I were added to fusion reaction mixtures containing 30 μM syt. t+v denotes fusion reaction mixtures lacking cpx-I and syt. As a control, the cytoplasmic domain of synaptobrevin 2 (cd-syb, 10 μM) was added to t+v to inhibit SNARE-mediated fusion. Fusion was monitored for 120 min at 37 °C, normalized to the maximum donor fluorescence signal (% Max. fluorescence), and plotted as a function of time. Reactions were carried out in 0.2 mM EGTA. (b) The final extent of fusion at each cpx-I concentration tested in panel a was normalized to the final extent of fusion obtained by t+v (% t+v). (c) Experiments were conducted as described for panel a except Ca2+ (1 mM) was added to reaction mixtures at 20 min. (d) Data from panel c were normalized as in panel b. The inset shows the final extent of fusion at each cpx-I concentration tested in panels a and c normalized to the extent of fusion obtained in reaction mixtures containing syt, but lacking cpx-I (% t+v+syt). All fusion traces are representative from n ≥ 3. Data in panels b and d represent the mean ± the standard error of the mean from n ≥ 3.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651691&req=5

fig2: In vitro membrane fusion regulated by cpx-I and syt. (a) Increasing concentrations of cpx-I were added to fusion reaction mixtures containing 30 μM syt. t+v denotes fusion reaction mixtures lacking cpx-I and syt. As a control, the cytoplasmic domain of synaptobrevin 2 (cd-syb, 10 μM) was added to t+v to inhibit SNARE-mediated fusion. Fusion was monitored for 120 min at 37 °C, normalized to the maximum donor fluorescence signal (% Max. fluorescence), and plotted as a function of time. Reactions were carried out in 0.2 mM EGTA. (b) The final extent of fusion at each cpx-I concentration tested in panel a was normalized to the final extent of fusion obtained by t+v (% t+v). (c) Experiments were conducted as described for panel a except Ca2+ (1 mM) was added to reaction mixtures at 20 min. (d) Data from panel c were normalized as in panel b. The inset shows the final extent of fusion at each cpx-I concentration tested in panels a and c normalized to the extent of fusion obtained in reaction mixtures containing syt, but lacking cpx-I (% t+v+syt). All fusion traces are representative from n ≥ 3. Data in panels b and d represent the mean ± the standard error of the mean from n ≥ 3.
Mentions: In the final series of experiments, cpx-I was titrated into an in vitro fusion assay in the presence of a saturating concentration of syt (30 μM syt; see ref (17)). Fusion was monitored in the absence of Ca2+ (Figure 2a,b), and after addition of 1 mM Ca2+ to reaction mixtures at 20 min (Figure 2c,d) (similar to a method used in ref (6)). Under these conditions, if cpx-I acts as a clamp as reported in refs (4)−(6), (8), and (13), it would be expected to inhibit fusion only in the absence of Ca2+ and not after a Ca2+ trigger since Ca2+-syt would overcome the cpx-I clamp to drive fusion. As shown in Figure 2a,b, in the absence of Ca2+, syt alone inhibited fusion by ∼25%. Addition of cpx-I to reaction mixtures containing apo-syt (Ca2+ free) resulted in a small but reproducible amount of stimulation at low cpx-I concentrations analogous to the findings obtained using SNAREs alone in the fusion assay (Figure 2a,b and Figure S2b,c of the Supporting Information). However, in contrast to what was observed when cpx-I was added to reaction mixtures containing SNAREs alone, addition of higher concentrations of cpx-I to reaction mixtures containing apo-syt only modestly inhibited fusion beyond what was already achieved by apo-syt alone (Figure 2a,b). Thus, cpx-I does not appear to play a major role in clamping SNARE-mediated vesicle−vesicle fusion prior to a Ca2+ signal when syt is also present (see the Supporting Information for more details). In duplicate samples, we triggered syt-stimulated fusion by adding Ca2+ at 20 min (Figure 2c). From these traces, it is evident that Ca2+-syt stimulates fusion but also that high concentrations of cpx-I inhibit Ca2+-syt-triggered fusion (Figure 2c,d).

Bottom Line: Synaptotagmin and complexin regulate SNARE-mediated synaptic vesicle exocytosis.It has been proposed that complexin clamps membrane fusion and that Ca(2+)-synaptotagmin displaces complexin from SNARE complexes to relieve this clamping activity.Moreover, the clamping ability of apo-synaptotagmin occluded the clamping activity of complexin until the arrival of a Ca(2+) trigger, at which point synaptotagmin accelerated fusion while high concentrations of complexin inhibited fusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Programs in Cellular and Molecular Biology, University of Wisconsin, 1300 University Avenue, SMI 129, Madison, Wisconsin 53706, USA.

ABSTRACT
Synaptotagmin and complexin regulate SNARE-mediated synaptic vesicle exocytosis. It has been proposed that complexin clamps membrane fusion and that Ca(2+)-synaptotagmin displaces complexin from SNARE complexes to relieve this clamping activity. Using a reconstituted system, we demonstrate that complexin and synaptotagmin simultaneously bind to neuronal SNARE complexes and that both apo-synaptotagmin and complexin inhibit SNARE-mediated membrane fusion. Moreover, the clamping ability of apo-synaptotagmin occluded the clamping activity of complexin until the arrival of a Ca(2+) trigger, at which point synaptotagmin accelerated fusion while high concentrations of complexin inhibited fusion. Thus, the inhibitory patterns of synaptotagmin and complexin are different, suggesting that SNAREs assemble into distinct states along the fusion pathway. These data also suggest that during synaptotagmin-regulated vesicle-vesicle fusion, complexin does not function as a fusion clamp that is relieved by Ca(2+)-synaptotagmin.

Show MeSH
Related in: MedlinePlus