Structured Review

Ipsen Group synaptotagmin 1
Representative fluorescence emission spectra used in the experiments of Figure 5B to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1, <t>synaptotagmin-1</t> and Ca 2+ in different combinations protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP. V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), synaptotagmin-1 C 2 AB (C 2 AB) and Ca 2+ . Black curves show spectra acquired before addition of NSF-αSNAP, and red curves the spectra obtained after addition of NSF-αSNAP.
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Images

1) Product Images from "Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP"

Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

Journal: eLife

doi: 10.7554/eLife.38880

Representative fluorescence emission spectra used in the experiments of Figure 5B to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1, synaptotagmin-1 and Ca 2+ in different combinations protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP. V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), synaptotagmin-1 C 2 AB (C 2 AB) and Ca 2+ . Black curves show spectra acquired before addition of NSF-αSNAP, and red curves the spectra obtained after addition of NSF-αSNAP.
Figure Legend Snippet: Representative fluorescence emission spectra used in the experiments of Figure 5B to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1, synaptotagmin-1 and Ca 2+ in different combinations protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP. V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), synaptotagmin-1 C 2 AB (C 2 AB) and Ca 2+ . Black curves show spectra acquired before addition of NSF-αSNAP, and red curves the spectra obtained after addition of NSF-αSNAP.

Techniques Used: Fluorescence, Incubation

Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1 and synaptotagmin-1 C 2 AB do not protect cis-SNARE complexes against disassembly by NSF-αSNAP. ( A ) Kinetic assays where cis-SNARE complex formation was catalyzed by Syb49-93, as in Figure 6D , and different concentrations of complexin-1 (Cpx) were added five minutes before disassembly with NSF-αSNAP. ( B ) Kinetic assays analogous to those of Figure 6D , but using WT SNAP-25 instead of SNAP-25m to ensure that the mutation in SNAP-25m did not affect the disassembly of cis-SNARE complexes by NSF-αSNAP in the presence of Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1, synaptotagmin-1 C 2 AB and Ca 2+ . ( C ) Kinetic assays analogous to those of panels ( A ), but adding 1 μM complexin-1 five minutes before disassembly with NSF-αSNAP (red and orange traces). In these experiments, the concentrations of NSF and αSNAP were 0.1 μM and 0.5 μM, respectively, which were lower than those of our standard conditions (0.5 μM and 2 μM, respectively) to test whether complexin-1 might hinder disassembly at a higher molar ratio with respect to αSNAP. The experiments were performed with SNAP-25m (black and red traces) or WT SNAP-25 (gray and orange traces). The black and gray traces are controls where complexin-1 was not added. In the experiments shown in ( A–C ), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces of ( A–C ), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents to make the data comparable.
Figure Legend Snippet: Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1 and synaptotagmin-1 C 2 AB do not protect cis-SNARE complexes against disassembly by NSF-αSNAP. ( A ) Kinetic assays where cis-SNARE complex formation was catalyzed by Syb49-93, as in Figure 6D , and different concentrations of complexin-1 (Cpx) were added five minutes before disassembly with NSF-αSNAP. ( B ) Kinetic assays analogous to those of Figure 6D , but using WT SNAP-25 instead of SNAP-25m to ensure that the mutation in SNAP-25m did not affect the disassembly of cis-SNARE complexes by NSF-αSNAP in the presence of Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1, synaptotagmin-1 C 2 AB and Ca 2+ . ( C ) Kinetic assays analogous to those of panels ( A ), but adding 1 μM complexin-1 five minutes before disassembly with NSF-αSNAP (red and orange traces). In these experiments, the concentrations of NSF and αSNAP were 0.1 μM and 0.5 μM, respectively, which were lower than those of our standard conditions (0.5 μM and 2 μM, respectively) to test whether complexin-1 might hinder disassembly at a higher molar ratio with respect to αSNAP. The experiments were performed with SNAP-25m (black and red traces) or WT SNAP-25 (gray and orange traces). The black and gray traces are controls where complexin-1 was not added. In the experiments shown in ( A–C ), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces of ( A–C ), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents to make the data comparable.

Techniques Used: Mutagenesis, Fluorescence

Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) Models showing ribbon diagrams of the cryo-electron microscopy structure of the 20S complex (PDB accession code 3J96) ( Zhao et al., 2015 ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.
Figure Legend Snippet: Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) Models showing ribbon diagrams of the cryo-electron microscopy structure of the 20S complex (PDB accession code 3J96) ( Zhao et al., 2015 ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.

Techniques Used: Electron Microscopy, Binding Assay

Control experiments acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes containing Alexa488-synaptobrevin. Spectra were acquired before (V) (black traces) or after (red traces) addition of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), Ca 2+ -bound synaptotagmin-1 C 2 AB, NSF, αSNAP or NSF+αSNAP.
Figure Legend Snippet: Control experiments acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes containing Alexa488-synaptobrevin. Spectra were acquired before (V) (black traces) or after (red traces) addition of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), Ca 2+ -bound synaptotagmin-1 C 2 AB, NSF, αSNAP or NSF+αSNAP.

Techniques Used: Fluorescence

Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of T-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing synaptobrevin (V) were incubated for five hours with Syb49-93 and T-liposomes containing TMR-syntaxin-1-SNAP-25m (T*) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for five minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.
Figure Legend Snippet: Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of T-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing synaptobrevin (V) were incubated for five hours with Syb49-93 and T-liposomes containing TMR-syntaxin-1-SNAP-25m (T*) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for five minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.

Techniques Used: Fluorescence, Incubation

Ca 2+ -dependent fusion between VSyt1- and T-liposomes. ( A,B ) Lipid mixing ( A ) between VSyt1-liposomes (synaptobrevin-to-lipid ratio 1:10,000; synaptotagmin-1-to-lipid ratio 1:1,000) and T-liposomes was monitored from the fluorescence de-quenching of Marina Blue lipids and content mixing ( B ) was monitored from the increase in the fluorescence signal of Cy5-streptavidin trapped in the V-liposomes caused by FRET with PhycoE-biotin trapped in the T-liposomes upon liposome fusion. The assays were performed in the presence of NSF-αSNAP, and Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13) or both. Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin, and Ca 2+ (600 μM) was added at 300 s.
Figure Legend Snippet: Ca 2+ -dependent fusion between VSyt1- and T-liposomes. ( A,B ) Lipid mixing ( A ) between VSyt1-liposomes (synaptobrevin-to-lipid ratio 1:10,000; synaptotagmin-1-to-lipid ratio 1:1,000) and T-liposomes was monitored from the fluorescence de-quenching of Marina Blue lipids and content mixing ( B ) was monitored from the increase in the fluorescence signal of Cy5-streptavidin trapped in the V-liposomes caused by FRET with PhycoE-biotin trapped in the T-liposomes upon liposome fusion. The assays were performed in the presence of NSF-αSNAP, and Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13) or both. Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin, and Ca 2+ (600 μM) was added at 300 s.

Techniques Used: Fluorescence

Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing Alexa488-synaptobrevin (V*) were incubated for five hours with Syb49-93 and T-liposomes containing syntaxin-1-SNAP-25m (T) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for 5 minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.
Figure Legend Snippet: Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing Alexa488-synaptobrevin (V*) were incubated for five hours with Syb49-93 and T-liposomes containing syntaxin-1-SNAP-25m (T) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for 5 minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.

Techniques Used: Fluorescence, Incubation

2) Product Images from "Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP"

Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

Journal: eLife

doi: 10.7554/eLife.38880

Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.
Figure Legend Snippet: Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.

Techniques Used: Binding Assay

3) Product Images from "Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP"

Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

Journal: eLife

doi: 10.7554/eLife.38880

Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.
Figure Legend Snippet: Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.

Techniques Used: Binding Assay

4) Product Images from "Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP"

Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

Journal: eLife

doi: 10.7554/eLife.38880

Representative fluorescence emission spectra used in the experiments of Figure 5B to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1, synaptotagmin-1 and Ca 2+ in different combinations protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP. V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), synaptotagmin-1 C 2 AB (C 2 AB) and Ca 2+ . Black curves show spectra acquired before addition of NSF-αSNAP, and red curves the spectra obtained after addition of NSF-αSNAP.
Figure Legend Snippet: Representative fluorescence emission spectra used in the experiments of Figure 5B to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1, synaptotagmin-1 and Ca 2+ in different combinations protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP. V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), synaptotagmin-1 C 2 AB (C 2 AB) and Ca 2+ . Black curves show spectra acquired before addition of NSF-αSNAP, and red curves the spectra obtained after addition of NSF-αSNAP.

Techniques Used: Fluorescence, Incubation

Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1 and synaptotagmin-1 C 2 AB do not protect cis-SNARE complexes against disassembly by NSF-αSNAP. ( A ) Kinetic assays where cis-SNARE complex formation was catalyzed by Syb49-93, as in Figure 6D , and different concentrations of complexin-1 (Cpx) were added five minutes before disassembly with NSF-αSNAP. ( B ) Kinetic assays analogous to those of Figure 6D , but using WT SNAP-25 instead of SNAP-25m to ensure that the mutation in SNAP-25m did not affect the disassembly of cis-SNARE complexes by NSF-αSNAP in the presence of Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1, synaptotagmin-1 C 2 AB and Ca 2+ . ( C ) Kinetic assays analogous to those of panels ( A ), but adding 1 μM complexin-1 five minutes before disassembly with NSF-αSNAP (red and orange traces). In these experiments, the concentrations of NSF and αSNAP were 0.1 μM and 0.5 μM, respectively, which were lower than those of our standard conditions (0.5 μM and 2 μM, respectively) to test whether complexin-1 might hinder disassembly at a higher molar ratio with respect to αSNAP. The experiments were performed with SNAP-25m (black and red traces) or WT SNAP-25 (gray and orange traces). The black and gray traces are controls where complexin-1 was not added. In the experiments shown in ( A–C ), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces of ( A–C ), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents to make the data comparable.
Figure Legend Snippet: Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1 and synaptotagmin-1 C 2 AB do not protect cis-SNARE complexes against disassembly by NSF-αSNAP. ( A ) Kinetic assays where cis-SNARE complex formation was catalyzed by Syb49-93, as in Figure 6D , and different concentrations of complexin-1 (Cpx) were added five minutes before disassembly with NSF-αSNAP. ( B ) Kinetic assays analogous to those of Figure 6D , but using WT SNAP-25 instead of SNAP-25m to ensure that the mutation in SNAP-25m did not affect the disassembly of cis-SNARE complexes by NSF-αSNAP in the presence of Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1, synaptotagmin-1 C 2 AB and Ca 2+ . ( C ) Kinetic assays analogous to those of panels ( A ), but adding 1 μM complexin-1 five minutes before disassembly with NSF-αSNAP (red and orange traces). In these experiments, the concentrations of NSF and αSNAP were 0.1 μM and 0.5 μM, respectively, which were lower than those of our standard conditions (0.5 μM and 2 μM, respectively) to test whether complexin-1 might hinder disassembly at a higher molar ratio with respect to αSNAP. The experiments were performed with SNAP-25m (black and red traces) or WT SNAP-25 (gray and orange traces). The black and gray traces are controls where complexin-1 was not added. In the experiments shown in ( A–C ), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces of ( A–C ), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents to make the data comparable.

Techniques Used: Mutagenesis, Fluorescence

Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) Models showing ribbon diagrams of the cryo-electron microscopy structure of the 20S complex (PDB accession code 3J96) ( Zhao et al., 2015 ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.
Figure Legend Snippet: Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) Models showing ribbon diagrams of the cryo-electron microscopy structure of the 20S complex (PDB accession code 3J96) ( Zhao et al., 2015 ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.

Techniques Used: Electron Microscopy, Binding Assay

Control experiments acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes containing Alexa488-synaptobrevin. Spectra were acquired before (V) (black traces) or after (red traces) addition of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), Ca 2+ -bound synaptotagmin-1 C 2 AB, NSF, αSNAP or NSF+αSNAP.
Figure Legend Snippet: Control experiments acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes containing Alexa488-synaptobrevin. Spectra were acquired before (V) (black traces) or after (red traces) addition of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), Ca 2+ -bound synaptotagmin-1 C 2 AB, NSF, αSNAP or NSF+αSNAP.

Techniques Used: Fluorescence

Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of T-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing synaptobrevin (V) were incubated for five hours with Syb49-93 and T-liposomes containing TMR-syntaxin-1-SNAP-25m (T*) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for five minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.
Figure Legend Snippet: Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of T-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing synaptobrevin (V) were incubated for five hours with Syb49-93 and T-liposomes containing TMR-syntaxin-1-SNAP-25m (T*) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for five minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.

Techniques Used: Fluorescence, Incubation

Ca 2+ -dependent fusion between VSyt1- and T-liposomes. ( A,B ) Lipid mixing ( A ) between VSyt1-liposomes (synaptobrevin-to-lipid ratio 1:10,000; synaptotagmin-1-to-lipid ratio 1:1,000) and T-liposomes was monitored from the fluorescence de-quenching of Marina Blue lipids and content mixing ( B ) was monitored from the increase in the fluorescence signal of Cy5-streptavidin trapped in the V-liposomes caused by FRET with PhycoE-biotin trapped in the T-liposomes upon liposome fusion. The assays were performed in the presence of NSF-αSNAP, and Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13) or both. Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin, and Ca 2+ (600 μM) was added at 300 s.
Figure Legend Snippet: Ca 2+ -dependent fusion between VSyt1- and T-liposomes. ( A,B ) Lipid mixing ( A ) between VSyt1-liposomes (synaptobrevin-to-lipid ratio 1:10,000; synaptotagmin-1-to-lipid ratio 1:1,000) and T-liposomes was monitored from the fluorescence de-quenching of Marina Blue lipids and content mixing ( B ) was monitored from the increase in the fluorescence signal of Cy5-streptavidin trapped in the V-liposomes caused by FRET with PhycoE-biotin trapped in the T-liposomes upon liposome fusion. The assays were performed in the presence of NSF-αSNAP, and Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13) or both. Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin, and Ca 2+ (600 μM) was added at 300 s.

Techniques Used: Fluorescence

Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing Alexa488-synaptobrevin (V*) were incubated for five hours with Syb49-93 and T-liposomes containing syntaxin-1-SNAP-25m (T) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for 5 minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.
Figure Legend Snippet: Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing Alexa488-synaptobrevin (V*) were incubated for five hours with Syb49-93 and T-liposomes containing syntaxin-1-SNAP-25m (T) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for 5 minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.

Techniques Used: Fluorescence, Incubation

5) Product Images from "Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP"

Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

Journal: eLife

doi: 10.7554/eLife.38880

Representative fluorescence emission spectra used in the experiments of Figure 5B to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1, synaptotagmin-1 and Ca 2+ in different combinations protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP. V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), synaptotagmin-1 C 2 AB (C 2 AB) and Ca 2+ . Black curves show spectra acquired before addition of NSF-αSNAP, and red curves the spectra obtained after addition of NSF-αSNAP.
Figure Legend Snippet: Representative fluorescence emission spectra used in the experiments of Figure 5B to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1, synaptotagmin-1 and Ca 2+ in different combinations protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP. V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), synaptotagmin-1 C 2 AB (C 2 AB) and Ca 2+ . Black curves show spectra acquired before addition of NSF-αSNAP, and red curves the spectra obtained after addition of NSF-αSNAP.

Techniques Used: Fluorescence, Incubation

Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1 and synaptotagmin-1 C 2 AB do not protect cis-SNARE complexes against disassembly by NSF-αSNAP. ( A ) Kinetic assays where cis-SNARE complex formation was catalyzed by Syb49-93, as in Figure 6D , and different concentrations of complexin-1 (Cpx) were added five minutes before disassembly with NSF-αSNAP. ( B ) Kinetic assays analogous to those of Figure 6D , but using WT SNAP-25 instead of SNAP-25m to ensure that the mutation in SNAP-25m did not affect the disassembly of cis-SNARE complexes by NSF-αSNAP in the presence of Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1, synaptotagmin-1 C 2 AB and Ca 2+ . ( C ) Kinetic assays analogous to those of panels ( A ), but adding 1 μM complexin-1 five minutes before disassembly with NSF-αSNAP (red and orange traces). In these experiments, the concentrations of NSF and αSNAP were 0.1 μM and 0.5 μM, respectively, which were lower than those of our standard conditions (0.5 μM and 2 μM, respectively) to test whether complexin-1 might hinder disassembly at a higher molar ratio with respect to αSNAP. The experiments were performed with SNAP-25m (black and red traces) or WT SNAP-25 (gray and orange traces). The black and gray traces are controls where complexin-1 was not added. In the experiments shown in ( A–C ), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces of ( A–C ), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents to make the data comparable.
Figure Legend Snippet: Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1 and synaptotagmin-1 C 2 AB do not protect cis-SNARE complexes against disassembly by NSF-αSNAP. ( A ) Kinetic assays where cis-SNARE complex formation was catalyzed by Syb49-93, as in Figure 6D , and different concentrations of complexin-1 (Cpx) were added five minutes before disassembly with NSF-αSNAP. ( B ) Kinetic assays analogous to those of Figure 6D , but using WT SNAP-25 instead of SNAP-25m to ensure that the mutation in SNAP-25m did not affect the disassembly of cis-SNARE complexes by NSF-αSNAP in the presence of Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1, synaptotagmin-1 C 2 AB and Ca 2+ . ( C ) Kinetic assays analogous to those of panels ( A ), but adding 1 μM complexin-1 five minutes before disassembly with NSF-αSNAP (red and orange traces). In these experiments, the concentrations of NSF and αSNAP were 0.1 μM and 0.5 μM, respectively, which were lower than those of our standard conditions (0.5 μM and 2 μM, respectively) to test whether complexin-1 might hinder disassembly at a higher molar ratio with respect to αSNAP. The experiments were performed with SNAP-25m (black and red traces) or WT SNAP-25 (gray and orange traces). The black and gray traces are controls where complexin-1 was not added. In the experiments shown in ( A–C ), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces of ( A–C ), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents to make the data comparable.

Techniques Used: Mutagenesis, Fluorescence

Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) Models showing ribbon diagrams of the cryo-electron microscopy structure of the 20S complex (PDB accession code 3J96) ( Zhao et al., 2015 ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.
Figure Legend Snippet: Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) Models showing ribbon diagrams of the cryo-electron microscopy structure of the 20S complex (PDB accession code 3J96) ( Zhao et al., 2015 ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.

Techniques Used: Electron Microscopy, Binding Assay

Control experiments acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes containing Alexa488-synaptobrevin. Spectra were acquired before (V) (black traces) or after (red traces) addition of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), Ca 2+ -bound synaptotagmin-1 C 2 AB, NSF, αSNAP or NSF+αSNAP.
Figure Legend Snippet: Control experiments acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes containing Alexa488-synaptobrevin. Spectra were acquired before (V) (black traces) or after (red traces) addition of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), Ca 2+ -bound synaptotagmin-1 C 2 AB, NSF, αSNAP or NSF+αSNAP.

Techniques Used: Fluorescence

Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of T-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing synaptobrevin (V) were incubated for five hours with Syb49-93 and T-liposomes containing TMR-syntaxin-1-SNAP-25m (T*) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for five minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.
Figure Legend Snippet: Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of T-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing synaptobrevin (V) were incubated for five hours with Syb49-93 and T-liposomes containing TMR-syntaxin-1-SNAP-25m (T*) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for five minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.

Techniques Used: Fluorescence, Incubation

Ca 2+ -dependent fusion between VSyt1- and T-liposomes. ( A,B ) Lipid mixing ( A ) between VSyt1-liposomes (synaptobrevin-to-lipid ratio 1:10,000; synaptotagmin-1-to-lipid ratio 1:1,000) and T-liposomes was monitored from the fluorescence de-quenching of Marina Blue lipids and content mixing ( B ) was monitored from the increase in the fluorescence signal of Cy5-streptavidin trapped in the V-liposomes caused by FRET with PhycoE-biotin trapped in the T-liposomes upon liposome fusion. The assays were performed in the presence of NSF-αSNAP, and Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13) or both. Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin, and Ca 2+ (600 μM) was added at 300 s.
Figure Legend Snippet: Ca 2+ -dependent fusion between VSyt1- and T-liposomes. ( A,B ) Lipid mixing ( A ) between VSyt1-liposomes (synaptobrevin-to-lipid ratio 1:10,000; synaptotagmin-1-to-lipid ratio 1:1,000) and T-liposomes was monitored from the fluorescence de-quenching of Marina Blue lipids and content mixing ( B ) was monitored from the increase in the fluorescence signal of Cy5-streptavidin trapped in the V-liposomes caused by FRET with PhycoE-biotin trapped in the T-liposomes upon liposome fusion. The assays were performed in the presence of NSF-αSNAP, and Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13) or both. Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin, and Ca 2+ (600 μM) was added at 300 s.

Techniques Used: Fluorescence

Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing Alexa488-synaptobrevin (V*) were incubated for five hours with Syb49-93 and T-liposomes containing syntaxin-1-SNAP-25m (T) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for 5 minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.
Figure Legend Snippet: Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing Alexa488-synaptobrevin (V*) were incubated for five hours with Syb49-93 and T-liposomes containing syntaxin-1-SNAP-25m (T) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for 5 minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.

Techniques Used: Fluorescence, Incubation

6) Product Images from "Vti1a/b regulate synaptic vesicle and dense core vesicle secretion via protein sorting at the Golgi"

Article Title: Vti1a/b regulate synaptic vesicle and dense core vesicle secretion via protein sorting at the Golgi

Journal: Nature Communications

doi: 10.1038/s41467-018-05699-z

Reduced synaptic levels of proteins that mediate secretion in Vti1a/b-deficient neurons. a – d Representative examples of single DIV-14 neurons immunostained for Synaptophysin-1 as synaptic marker and Syntaxin-1 and SNAP25 ( a ), Munc18-1 and Munc13-1 ( b ), RIM1/2 and Bassoon ( c ), and Synaptobrevin-2 and Synaptophysin-1 ( d ). e – l Intensity distribution of the levels of Syntaxin-1 ( e ), SNAP25 ( f ), Munc13-1 ( g ), Munc18-1 ( h ), Bassoon ( i ), RIM1/2 ( j ), Synaptotagmin-1 ( k ), Synaptobrevin-2 ( l ) in all synapses ( n = Over 15,000 synapses per protein and group). Statistical significance tested in Supplementary Figure 5 . Scale bar = 2 µm
Figure Legend Snippet: Reduced synaptic levels of proteins that mediate secretion in Vti1a/b-deficient neurons. a – d Representative examples of single DIV-14 neurons immunostained for Synaptophysin-1 as synaptic marker and Syntaxin-1 and SNAP25 ( a ), Munc18-1 and Munc13-1 ( b ), RIM1/2 and Bassoon ( c ), and Synaptobrevin-2 and Synaptophysin-1 ( d ). e – l Intensity distribution of the levels of Syntaxin-1 ( e ), SNAP25 ( f ), Munc13-1 ( g ), Munc18-1 ( h ), Bassoon ( i ), RIM1/2 ( j ), Synaptotagmin-1 ( k ), Synaptobrevin-2 ( l ) in all synapses ( n = Over 15,000 synapses per protein and group). Statistical significance tested in Supplementary Figure 5 . Scale bar = 2 µm

Techniques Used: Marker

7) Product Images from "Vti1a/b regulate synaptic vesicle and dense core vesicle secretion via protein sorting at the Golgi"

Article Title: Vti1a/b regulate synaptic vesicle and dense core vesicle secretion via protein sorting at the Golgi

Journal: Nature Communications

doi: 10.1038/s41467-018-05699-z

Reduced synaptic levels of proteins that mediate secretion in Vti1a/b-deficient neurons. a – d Representative examples of single DIV-14 neurons immunostained for Synaptophysin-1 as synaptic marker and Syntaxin-1 and SNAP25 ( a ), Munc18-1 and Munc13-1 ( b ), RIM1/2 and Bassoon ( c ), and Synaptobrevin-2 and Synaptophysin-1 ( d ). e – l Intensity distribution of the levels of Syntaxin-1 ( e ), SNAP25 ( f ), Munc13-1 ( g ), Munc18-1 ( h ), Bassoon ( i ), RIM1/2 ( j ), Synaptotagmin-1 ( k ), Synaptobrevin-2 ( l ) in all synapses ( n . Scale bar = 2 µm
Figure Legend Snippet: Reduced synaptic levels of proteins that mediate secretion in Vti1a/b-deficient neurons. a – d Representative examples of single DIV-14 neurons immunostained for Synaptophysin-1 as synaptic marker and Syntaxin-1 and SNAP25 ( a ), Munc18-1 and Munc13-1 ( b ), RIM1/2 and Bassoon ( c ), and Synaptobrevin-2 and Synaptophysin-1 ( d ). e – l Intensity distribution of the levels of Syntaxin-1 ( e ), SNAP25 ( f ), Munc13-1 ( g ), Munc18-1 ( h ), Bassoon ( i ), RIM1/2 ( j ), Synaptotagmin-1 ( k ), Synaptobrevin-2 ( l ) in all synapses ( n . Scale bar = 2 µm

Techniques Used: Marker

8) Product Images from "Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission"

Article Title: Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission

Journal: The EMBO Journal

doi: 10.15252/embj.201798044

Increased synaptic activity accelerates the loss of Synaptotagmin 1 molecules from the actively recycling pool, and their entry into the inactive pool We investigated the effect of increasing neuronal activity on the aging and inactivation of vesicles as follows. We tagged Synaptotagmin 1 epitopes with non‐conjugated antibodies, and then, after a 12‐h interval, we applied fluorophore‐conjugated secondary antibodies (Cy5) to the living cultures for 1 h. These antibodies revealed all of the Synaptotagmin 1 antibodies that were exposed during vesicle recycling. We then fixed and permeabilized the neurons and applied secondary antibodies conjugated to a different fluorophore (Cy3), to thereby reveal all of the remaining, no longer releasable, Synaptotagmin 1 antibodies. This procedure thus indicates the proportion of the tagged Synaptotagmin 1 epitopes that are still recycling in the synapses under normal activity, as well as the proportion that are present in the synapses. Scale bar: 50 μm. Imaging was performed with a Leica SP5 confocal microscope. Data from images such as those shown in (A) were quantified, with the fluorescence intensity from releasable (Cy5) and inactive (Cy3) epitopes plotted in (B). The experiment was performed in untreated control cultures, or in cultures incubated for the 12 h with bicuculline or 8 mM Ca 2+ to increase synaptic activity ( n = 30 neurons for control, 29 neurons for bicuculline, and 30 neurons for 8 mM Ca 2+ , from three independent experiments per condition). The intensity of the signal ascribed to releasable or inactive vesicles is shown. All data represent the mean ± SEM. Ratio of releasable vs. inactive vesicles, in arbitrary units, from (A); * P
Figure Legend Snippet: Increased synaptic activity accelerates the loss of Synaptotagmin 1 molecules from the actively recycling pool, and their entry into the inactive pool We investigated the effect of increasing neuronal activity on the aging and inactivation of vesicles as follows. We tagged Synaptotagmin 1 epitopes with non‐conjugated antibodies, and then, after a 12‐h interval, we applied fluorophore‐conjugated secondary antibodies (Cy5) to the living cultures for 1 h. These antibodies revealed all of the Synaptotagmin 1 antibodies that were exposed during vesicle recycling. We then fixed and permeabilized the neurons and applied secondary antibodies conjugated to a different fluorophore (Cy3), to thereby reveal all of the remaining, no longer releasable, Synaptotagmin 1 antibodies. This procedure thus indicates the proportion of the tagged Synaptotagmin 1 epitopes that are still recycling in the synapses under normal activity, as well as the proportion that are present in the synapses. Scale bar: 50 μm. Imaging was performed with a Leica SP5 confocal microscope. Data from images such as those shown in (A) were quantified, with the fluorescence intensity from releasable (Cy5) and inactive (Cy3) epitopes plotted in (B). The experiment was performed in untreated control cultures, or in cultures incubated for the 12 h with bicuculline or 8 mM Ca 2+ to increase synaptic activity ( n = 30 neurons for control, 29 neurons for bicuculline, and 30 neurons for 8 mM Ca 2+ , from three independent experiments per condition). The intensity of the signal ascribed to releasable or inactive vesicles is shown. All data represent the mean ± SEM. Ratio of releasable vs. inactive vesicles, in arbitrary units, from (A); * P

Techniques Used: Activity Assay, Imaging, Microscopy, Fluorescence, Incubation

The Synaptotagmin 1 molecules that left the recycling population do not return to it spontaneously To determine the rate at which new Synaptotagmin 1 epitopes come into the recycling pool, we saturated the lumenal Synaptotagmin 1 epitopes on active synaptic vesicles by incubating with an unconjugated monoclonal antibody for 2 h at 37°C and then followed the appearance of new epitopes by applying a fluorophore‐conjugated (Atto647N) version of the same monoclonal antibody, at different time points after saturating the initial epitopes. Individual coverslips were only used to investigate single time points and were fixed before imaging. Exemplary images taken after applying the fluorophore‐conjugated antibodies at 0, 12, or 24 h after saturating the initial Synaptotagmin 1 epitopes. Scale bar: 20 μm. Imaging was performed with a Leica SP5 confocal microscope. The same experiment was performed, but the cultures were incubated with drugs that disrupt protein synthesis (anisomycin) or microtubule‐based transport (colchicine). The fluorescence was measured at different time points and was expressed as percentage of the fluorescence at the initial time point. n = 4 independent experiments per data point for untreated 0 and 24 h, and n = 3 for all else, at least 10 neurons sampled per experiment; * P = 0.0070; ** P = 0.0022. Statistical significance was evaluated using one‐way ANOVA ( P = 0.014, F (2, 9) = 19.26), followed by the Bonferroni procedure. To determine whether the drugs impair synaptic activity, neurons were treated with the drugs for 24 h and were then stimulated in the presence of fluorescently conjugated Synaptotagmin 1 antibodies (20 Hz, 30 s), to label the entire recycling pool. Only a limited decrease in recycling was observed in drug‐treated preparations ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.2205, F (2, 8) = 1.97). To assess the spontaneous network activity of our cultures after 24 h of drug treatment, we measured vesicle recycling during the last hour of the treatment. The network activity did not change significantly ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.8835, F (2, 8) = 0.13). The total synaptic vesicle pool size after 24 h of drug treatment. This value was determined by immunostaining for a major synaptic vesicle marker, Synaptophysin. There were no significant changes in synaptic vesicle pool size ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.5519, F (2, 8) = 0.66). Data information: All data represent the mean ± SEM. Imaging was performed with a Leica SP5 confocal microscope. Source data are available online for this figure.
Figure Legend Snippet: The Synaptotagmin 1 molecules that left the recycling population do not return to it spontaneously To determine the rate at which new Synaptotagmin 1 epitopes come into the recycling pool, we saturated the lumenal Synaptotagmin 1 epitopes on active synaptic vesicles by incubating with an unconjugated monoclonal antibody for 2 h at 37°C and then followed the appearance of new epitopes by applying a fluorophore‐conjugated (Atto647N) version of the same monoclonal antibody, at different time points after saturating the initial epitopes. Individual coverslips were only used to investigate single time points and were fixed before imaging. Exemplary images taken after applying the fluorophore‐conjugated antibodies at 0, 12, or 24 h after saturating the initial Synaptotagmin 1 epitopes. Scale bar: 20 μm. Imaging was performed with a Leica SP5 confocal microscope. The same experiment was performed, but the cultures were incubated with drugs that disrupt protein synthesis (anisomycin) or microtubule‐based transport (colchicine). The fluorescence was measured at different time points and was expressed as percentage of the fluorescence at the initial time point. n = 4 independent experiments per data point for untreated 0 and 24 h, and n = 3 for all else, at least 10 neurons sampled per experiment; * P = 0.0070; ** P = 0.0022. Statistical significance was evaluated using one‐way ANOVA ( P = 0.014, F (2, 9) = 19.26), followed by the Bonferroni procedure. To determine whether the drugs impair synaptic activity, neurons were treated with the drugs for 24 h and were then stimulated in the presence of fluorescently conjugated Synaptotagmin 1 antibodies (20 Hz, 30 s), to label the entire recycling pool. Only a limited decrease in recycling was observed in drug‐treated preparations ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.2205, F (2, 8) = 1.97). To assess the spontaneous network activity of our cultures after 24 h of drug treatment, we measured vesicle recycling during the last hour of the treatment. The network activity did not change significantly ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.8835, F (2, 8) = 0.13). The total synaptic vesicle pool size after 24 h of drug treatment. This value was determined by immunostaining for a major synaptic vesicle marker, Synaptophysin. There were no significant changes in synaptic vesicle pool size ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.5519, F (2, 8) = 0.66). Data information: All data represent the mean ± SEM. Imaging was performed with a Leica SP5 confocal microscope. Source data are available online for this figure.

Techniques Used: Imaging, Microscopy, Incubation, Fluorescence, Activity Assay, Immunostaining, Marker

9) Product Images from "Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission"

Article Title: Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission

Journal: The EMBO Journal

doi: 10.15252/embj.201798044

Increased synaptic activity accelerates the loss of Synaptotagmin 1 molecules from the actively recycling pool, and their entry into the inactive pool We investigated the effect of increasing neuronal activity on the aging and inactivation of vesicles as follows. We tagged Synaptotagmin 1 epitopes with non‐conjugated antibodies, and then, after a 12‐h interval, we applied fluorophore‐conjugated secondary antibodies (Cy5) to the living cultures for 1 h. These antibodies revealed all of the Synaptotagmin 1 antibodies that were exposed during vesicle recycling. We then fixed and permeabilized the neurons and applied secondary antibodies conjugated to a different fluorophore (Cy3), to thereby reveal all of the remaining, no longer releasable, Synaptotagmin 1 antibodies. This procedure thus indicates the proportion of the tagged Synaptotagmin 1 epitopes that are still recycling in the synapses under normal activity, as well as the proportion that are present in the synapses. Scale bar: 50 μm. Imaging was performed with a Leica SP5 confocal microscope. Data from images such as those shown in (A) were quantified, with the fluorescence intensity from releasable (Cy5) and inactive (Cy3) epitopes plotted in (B). The experiment was performed in untreated control cultures, or in cultures incubated for the 12 h with bicuculline or 8 mM Ca 2+ to increase synaptic activity ( n = 30 neurons for control, 29 neurons for bicuculline, and 30 neurons for 8 mM Ca 2+ , from three independent experiments per condition). The intensity of the signal ascribed to releasable or inactive vesicles is shown. All data represent the mean ± SEM. Ratio of releasable vs. inactive vesicles, in arbitrary units, from (A); * P
Figure Legend Snippet: Increased synaptic activity accelerates the loss of Synaptotagmin 1 molecules from the actively recycling pool, and their entry into the inactive pool We investigated the effect of increasing neuronal activity on the aging and inactivation of vesicles as follows. We tagged Synaptotagmin 1 epitopes with non‐conjugated antibodies, and then, after a 12‐h interval, we applied fluorophore‐conjugated secondary antibodies (Cy5) to the living cultures for 1 h. These antibodies revealed all of the Synaptotagmin 1 antibodies that were exposed during vesicle recycling. We then fixed and permeabilized the neurons and applied secondary antibodies conjugated to a different fluorophore (Cy3), to thereby reveal all of the remaining, no longer releasable, Synaptotagmin 1 antibodies. This procedure thus indicates the proportion of the tagged Synaptotagmin 1 epitopes that are still recycling in the synapses under normal activity, as well as the proportion that are present in the synapses. Scale bar: 50 μm. Imaging was performed with a Leica SP5 confocal microscope. Data from images such as those shown in (A) were quantified, with the fluorescence intensity from releasable (Cy5) and inactive (Cy3) epitopes plotted in (B). The experiment was performed in untreated control cultures, or in cultures incubated for the 12 h with bicuculline or 8 mM Ca 2+ to increase synaptic activity ( n = 30 neurons for control, 29 neurons for bicuculline, and 30 neurons for 8 mM Ca 2+ , from three independent experiments per condition). The intensity of the signal ascribed to releasable or inactive vesicles is shown. All data represent the mean ± SEM. Ratio of releasable vs. inactive vesicles, in arbitrary units, from (A); * P

Techniques Used: Activity Assay, Imaging, Microscopy, Fluorescence, Incubation

Antibody‐tagged Synaptotagmin 1 and VGAT molecules are lost from synapses over a few days To determine the time interval that synaptic vesicle proteins spend in synapses, we incubated living hippocampal cultured neurons with fluorophore‐conjugated (green stars) antibodies against the lumenal domain of synaptic vesicle proteins. Fluorophore‐conjugated Synaptotagmin 1 (Atto647N‐conjugated) or VGAT (CypHer5E‐conjugated) antibodies were applied (diluted 1:120 from 1 mg/ml stock) to the neurons in their own culture medium, for 1 h at 37°C in a cell culture incubator. The unbound antibodies were then washed off, and the neurons were placed again in the incubator. Individual coverslips were then chased and retrieved at different time intervals after labeling, fixed, and imaged, to determine the amount of Synaptotagmin 1 and VGAT labeling remaining in synapses. Exemplary images of neurons labeled with Synaptotagmin 1 antibodies, imaged at different time points after labeling. Scale bar: 20 μm. All images were taken using a Leica SP5 confocal microscope. Exemplary images of neurons labeled with VGAT antibodies, imaged at different time points after labeling. Scale bar: 20 μm. The images are taken after fixation and permeabilization, in buffers with pH 5.5, to enable the entire CypHer5E fluorescence to be detected. All images were taken using a Nikon Ti‐E epifluorescence microscope. The loss of synaptic vesicle proteins from the synapse was monitored by imaging the Synaptotagmin 1 antibody fluorescence at serial time points after tagging Synaptotagmin 1 ( n = 3, 3, 2, 3, 3, and 2 independent experiments per respective time point, at least 10 neurons sampled per experiment). To determine fluorescence intensities specifically in synapses, the neurons were immunostained for the synaptic vesicle marker Synaptophysin, and only the fluorescence found within Synaptophysin spots (synapses) was analyzed. We did not observe any significant changes in the co‐localization of fluorophore‐conjugated antibodies in respect to Synaptophysin over time ( Appendix Fig S8 ). Similar intensity analysis for VGAT ( n = 3, 4, 2, 4, 4, and 3 independent experiments per respective time point, at least 10 neurons sampled per experiment). Data information: All data represent the mean ± SEM. Source data are available online for this figure.
Figure Legend Snippet: Antibody‐tagged Synaptotagmin 1 and VGAT molecules are lost from synapses over a few days To determine the time interval that synaptic vesicle proteins spend in synapses, we incubated living hippocampal cultured neurons with fluorophore‐conjugated (green stars) antibodies against the lumenal domain of synaptic vesicle proteins. Fluorophore‐conjugated Synaptotagmin 1 (Atto647N‐conjugated) or VGAT (CypHer5E‐conjugated) antibodies were applied (diluted 1:120 from 1 mg/ml stock) to the neurons in their own culture medium, for 1 h at 37°C in a cell culture incubator. The unbound antibodies were then washed off, and the neurons were placed again in the incubator. Individual coverslips were then chased and retrieved at different time intervals after labeling, fixed, and imaged, to determine the amount of Synaptotagmin 1 and VGAT labeling remaining in synapses. Exemplary images of neurons labeled with Synaptotagmin 1 antibodies, imaged at different time points after labeling. Scale bar: 20 μm. All images were taken using a Leica SP5 confocal microscope. Exemplary images of neurons labeled with VGAT antibodies, imaged at different time points after labeling. Scale bar: 20 μm. The images are taken after fixation and permeabilization, in buffers with pH 5.5, to enable the entire CypHer5E fluorescence to be detected. All images were taken using a Nikon Ti‐E epifluorescence microscope. The loss of synaptic vesicle proteins from the synapse was monitored by imaging the Synaptotagmin 1 antibody fluorescence at serial time points after tagging Synaptotagmin 1 ( n = 3, 3, 2, 3, 3, and 2 independent experiments per respective time point, at least 10 neurons sampled per experiment). To determine fluorescence intensities specifically in synapses, the neurons were immunostained for the synaptic vesicle marker Synaptophysin, and only the fluorescence found within Synaptophysin spots (synapses) was analyzed. We did not observe any significant changes in the co‐localization of fluorophore‐conjugated antibodies in respect to Synaptophysin over time ( Appendix Fig S8 ). Similar intensity analysis for VGAT ( n = 3, 4, 2, 4, 4, and 3 independent experiments per respective time point, at least 10 neurons sampled per experiment). Data information: All data represent the mean ± SEM. Source data are available online for this figure.

Techniques Used: Incubation, Cell Culture, Labeling, Microscopy, Fluorescence, Imaging, Marker

Aging antibody‐labeled synaptic vesicle proteins cease to participate in exocytosis To determine whether the antibody‐tagged synaptic vesicle proteins that are present in synapses at different time intervals after labeling are exocytosis‐competent, we labeled neurons with CypHer5E‐conjugated antibodies for Synaptotagmin 1 or VGAT for 1 h exactly as described in Fig 1 A. At different time intervals after labeling, individual cultures were removed from the incubator, mounted in a live‐stimulation chamber, and subjected to a stimulation paradigm designed to release the entire recycling pool (20 Hz, 30 s; Wilhelm et al , 2010 ). Exocytosis is determined by monitoring the fluorescence of the pH‐sensitive fluorophore CypHer5E, which is fluorescent at low pH within vesicles, but is quenched by exposure to the neutral extracellular pH. To determine the proportion of the labeled vesicles that is still able to exocytose, we measured the fluorescence signal corresponding to all labeled vesicles by fixing and permeabilizing the neurons and then exposing them to a pH of 5.5, obtained with TES‐buffered solutions. Typical images of fluorescence either before or after stimulation, for Synaptotagmin 1 (B) or VGAT (C). Numerous individual images were taken of different fields for every condition and were averaged for the statistics, as imaging the same field under all conditions proved unfeasible, due to the high bleaching tendency of CypHer5E. Scale bar: 50 μm. All images in (B) were taken using an Olympus epifluorescence microscope, and all images in (C) were taken using a Nikon Ti‐E epifluorescence microscope. The following numbers of experiments were quantified: for Synaptotagmin 1, n = 4, 3, 4, 4, 2, and 2 independent experiments per respective time point, with at least 12 neurons sampled per experiment; and for VGAT, n = 4, 4, 2, 4, 4, and 3 independent experiments per respective time point, with at least eight neurons sampled per experiment. The VGAT signal follows the same approximate dynamic as Synaptotagmin 1, but is faster. VGAT is present in only ˜ 5–10% of the neurons in our cultures, and therefore, this difference may reflect a cell type‐specific effect. All data represent the mean ± SEM. Source data are available online for this figure.
Figure Legend Snippet: Aging antibody‐labeled synaptic vesicle proteins cease to participate in exocytosis To determine whether the antibody‐tagged synaptic vesicle proteins that are present in synapses at different time intervals after labeling are exocytosis‐competent, we labeled neurons with CypHer5E‐conjugated antibodies for Synaptotagmin 1 or VGAT for 1 h exactly as described in Fig 1 A. At different time intervals after labeling, individual cultures were removed from the incubator, mounted in a live‐stimulation chamber, and subjected to a stimulation paradigm designed to release the entire recycling pool (20 Hz, 30 s; Wilhelm et al , 2010 ). Exocytosis is determined by monitoring the fluorescence of the pH‐sensitive fluorophore CypHer5E, which is fluorescent at low pH within vesicles, but is quenched by exposure to the neutral extracellular pH. To determine the proportion of the labeled vesicles that is still able to exocytose, we measured the fluorescence signal corresponding to all labeled vesicles by fixing and permeabilizing the neurons and then exposing them to a pH of 5.5, obtained with TES‐buffered solutions. Typical images of fluorescence either before or after stimulation, for Synaptotagmin 1 (B) or VGAT (C). Numerous individual images were taken of different fields for every condition and were averaged for the statistics, as imaging the same field under all conditions proved unfeasible, due to the high bleaching tendency of CypHer5E. Scale bar: 50 μm. All images in (B) were taken using an Olympus epifluorescence microscope, and all images in (C) were taken using a Nikon Ti‐E epifluorescence microscope. The following numbers of experiments were quantified: for Synaptotagmin 1, n = 4, 3, 4, 4, 2, and 2 independent experiments per respective time point, with at least 12 neurons sampled per experiment; and for VGAT, n = 4, 4, 2, 4, 4, and 3 independent experiments per respective time point, with at least eight neurons sampled per experiment. The VGAT signal follows the same approximate dynamic as Synaptotagmin 1, but is faster. VGAT is present in only ˜ 5–10% of the neurons in our cultures, and therefore, this difference may reflect a cell type‐specific effect. All data represent the mean ± SEM. Source data are available online for this figure.

Techniques Used: Labeling, Fluorescence, Imaging, Microscopy

SNAP 25 overexpression reduces exocytosis, while overexpression of CSP α enhances it Neurons were transfected with wild‐type SNAP25 (left panel) or co‐transfected with SNAP25 and with wild‐type CSPα (CSPα WT , right panel). The transfected cells are shown in magenta, which corresponds to a YFP moiety that is coupled to SNAP25, for detection purposes. At 3–4 days after transfection, the neurons were incubated with Atto647N‐conjugated Synaptotagmin 1 antibodies, for 1 h, to label the actively recycling vesicles, as in Fig 1 A. The neurons were then fixed, and the Synaptotagmin 1 antibody intensity from control neurites and from neurites containing the expressed proteins was analyzed. Exemplary images show reduced vesicle recycling (reduced Synaptotagmin 1 antibody levels) in neurons overexpressing SNAP25 (left panel; very limited green intensity within the magenta areas), compared to neurons overexpressing both SNAP25 and CSPα WT (right panel); the arrowheads point to areas of high overlap of Synaptotagmin 1 and SNAP25 signals. Scale bar: 2 μm. We quantified the size of the actively recycling pool, determined by incubation with Atto647N‐conjugated Synaptotagmin 1 antibodies, as in Fig 1 A, in neurons expressing different constructs, or combinations of constructs. We compared the size of the recycling pool in the neurons expressing the constructs to the size of this pool in the non‐transfected neurons from the same coverslips. The following conditions were used: (B) SNAP25 ( n = 10 transfected neurons from four independent experiments); (C) Syntaxin 1, as a control ( n = 13 transfected neurons from seven independent experiments); (D) neurons overexpressing CSPα WT ( n = 14 transfected neurons from four independent experiments); (E) CSPα mut , a mutated version of CSPα unable to target to vesicles and thus incapable of interacting with SNAP25 (Sharma et al , 2012 ; n = 11 transfected neurons from four independent experiments); (F) SNAP 25 + CSPα WT ( n = 20 transfected neurons from eight independent experiments); and (G) SNAP25 + CSPα mut ( n = 20 transfected neurons from six independent experiments). The significance levels determined are as follows: (B) * P = 0.0017, t (3) = 10.8862; (C) * P = 0.0003, t (6) = 7.5661; (D) * P = 0.0034, t (3) = 8.5006; (E) n.s P = 0.6004, t (3) = 0.5837; (F) * P = 0.0173, t (7) = 3.0992; and (G) * P = 0.0165, t (5) = 3.5410. Statistical significance was evaluated using paired t ‐tests. All data represent the mean ± SEM. A hypothetical model of SNAP25 and CSPα activity. They interact with SGTα and Hsc70 in trans , promoting synaptic vesicle fusion (Sharma et al , 2012 ). A hypothetical model of how SNAP25 on the aged vesicles may interact in cis with CSPα, thus sequestering it from its trans interaction with SNAP25 molecules from the plasma membrane. Data information: All imaging was performed with a Leica SP5 confocal microscope (A, B, D–F) or a Nikon Ti‐E epifluorescence microscope (C). Source data are available online for this figure.
Figure Legend Snippet: SNAP 25 overexpression reduces exocytosis, while overexpression of CSP α enhances it Neurons were transfected with wild‐type SNAP25 (left panel) or co‐transfected with SNAP25 and with wild‐type CSPα (CSPα WT , right panel). The transfected cells are shown in magenta, which corresponds to a YFP moiety that is coupled to SNAP25, for detection purposes. At 3–4 days after transfection, the neurons were incubated with Atto647N‐conjugated Synaptotagmin 1 antibodies, for 1 h, to label the actively recycling vesicles, as in Fig 1 A. The neurons were then fixed, and the Synaptotagmin 1 antibody intensity from control neurites and from neurites containing the expressed proteins was analyzed. Exemplary images show reduced vesicle recycling (reduced Synaptotagmin 1 antibody levels) in neurons overexpressing SNAP25 (left panel; very limited green intensity within the magenta areas), compared to neurons overexpressing both SNAP25 and CSPα WT (right panel); the arrowheads point to areas of high overlap of Synaptotagmin 1 and SNAP25 signals. Scale bar: 2 μm. We quantified the size of the actively recycling pool, determined by incubation with Atto647N‐conjugated Synaptotagmin 1 antibodies, as in Fig 1 A, in neurons expressing different constructs, or combinations of constructs. We compared the size of the recycling pool in the neurons expressing the constructs to the size of this pool in the non‐transfected neurons from the same coverslips. The following conditions were used: (B) SNAP25 ( n = 10 transfected neurons from four independent experiments); (C) Syntaxin 1, as a control ( n = 13 transfected neurons from seven independent experiments); (D) neurons overexpressing CSPα WT ( n = 14 transfected neurons from four independent experiments); (E) CSPα mut , a mutated version of CSPα unable to target to vesicles and thus incapable of interacting with SNAP25 (Sharma et al , 2012 ; n = 11 transfected neurons from four independent experiments); (F) SNAP 25 + CSPα WT ( n = 20 transfected neurons from eight independent experiments); and (G) SNAP25 + CSPα mut ( n = 20 transfected neurons from six independent experiments). The significance levels determined are as follows: (B) * P = 0.0017, t (3) = 10.8862; (C) * P = 0.0003, t (6) = 7.5661; (D) * P = 0.0034, t (3) = 8.5006; (E) n.s P = 0.6004, t (3) = 0.5837; (F) * P = 0.0173, t (7) = 3.0992; and (G) * P = 0.0165, t (5) = 3.5410. Statistical significance was evaluated using paired t ‐tests. All data represent the mean ± SEM. A hypothetical model of SNAP25 and CSPα activity. They interact with SGTα and Hsc70 in trans , promoting synaptic vesicle fusion (Sharma et al , 2012 ). A hypothetical model of how SNAP25 on the aged vesicles may interact in cis with CSPα, thus sequestering it from its trans interaction with SNAP25 molecules from the plasma membrane. Data information: All imaging was performed with a Leica SP5 confocal microscope (A, B, D–F) or a Nikon Ti‐E epifluorescence microscope (C). Source data are available online for this figure.

Techniques Used: Over Expression, Transfection, Incubation, Expressing, Construct, Activity Assay, Imaging, Microscopy

Metabolic imaging based on the unnatural amino acid AHA reveals that the actively recycling Synaptotagmin 1 molecules are found within younger protein environments than the inactive ones To label newly produced proteins, we used the unnatural amino acid AHA, which was detected in STED imaging. AHA was fed to the neurons in culture medium free of methionine, which is the amino acid AHA replaces during protein biogenesis. We used two experimental paradigms. First (A), to correlate the presence of newly synthesized proteins with recycling vesicles, we fed AHA to the neurons for 9 h (AHA), before labeling Synaptotagmin 1 from the recycling pool by applying Atto647N‐conjugated antibodies for 1 h at 37°C, as in Fig 1 A. Second (B), to compare the co‐localization of newly synthesized proteins with inactive synaptic vesicles, we performed the same experiment, but with 3–4 days between antibody tagging and metabolic labeling: We first labeled the recycling vesicles by Synaptotagmin 1 antibody incubation, as in Fig 1 A, and then returned the cell cultures to the incubator. We then waited for 4 days for the inactivation of the vesicles to take place, and then fed the neurons with AHA, to label newly synthesized proteins. Using this approach, we analyzed the co‐localization of AHA with releasable or inactive synaptic vesicles in STED microscopy. The neurons were fixed and subjected to a click chemistry procedure that reveals all AHA moieties, coupling them to the fluorophore Chromeo494 (a procedure known as FUNCAT; Dieterich et al , 2011 ). The neurons were then embedded in melamine resin and were sectioned into 20‐nm sections on an ultramicrotome. The sections were then imaged by two‐color STED microscopy. The images are shown after processing by deconvolution, to improve signal‐to‐noise ratios. The line scans indicated in light blue in the rightmost panels are plotted in (D). Scale bar: 2 μm. Line scans through pairs of vesicles found close to each other are shown, as examples. The line scans were performed on the original raw data files, to eliminate any artifacts due to deconvolution. The amount of AHA fluorescence co‐localizing with the releasable or inactive vesicles was determined and was expressed as fold over baseline signals ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment, * P = 0.0037, t (4) = 6.09). The baseline signal refers here to the average AHA signal within the respective synaptic boutons. All data represent the mean ± SEM. Statistical significance was evaluated using unpaired t ‐tests. Data information: All images were taken using a Leica SP5 STED microscope. Source data are available online for this figure.
Figure Legend Snippet: Metabolic imaging based on the unnatural amino acid AHA reveals that the actively recycling Synaptotagmin 1 molecules are found within younger protein environments than the inactive ones To label newly produced proteins, we used the unnatural amino acid AHA, which was detected in STED imaging. AHA was fed to the neurons in culture medium free of methionine, which is the amino acid AHA replaces during protein biogenesis. We used two experimental paradigms. First (A), to correlate the presence of newly synthesized proteins with recycling vesicles, we fed AHA to the neurons for 9 h (AHA), before labeling Synaptotagmin 1 from the recycling pool by applying Atto647N‐conjugated antibodies for 1 h at 37°C, as in Fig 1 A. Second (B), to compare the co‐localization of newly synthesized proteins with inactive synaptic vesicles, we performed the same experiment, but with 3–4 days between antibody tagging and metabolic labeling: We first labeled the recycling vesicles by Synaptotagmin 1 antibody incubation, as in Fig 1 A, and then returned the cell cultures to the incubator. We then waited for 4 days for the inactivation of the vesicles to take place, and then fed the neurons with AHA, to label newly synthesized proteins. Using this approach, we analyzed the co‐localization of AHA with releasable or inactive synaptic vesicles in STED microscopy. The neurons were fixed and subjected to a click chemistry procedure that reveals all AHA moieties, coupling them to the fluorophore Chromeo494 (a procedure known as FUNCAT; Dieterich et al , 2011 ). The neurons were then embedded in melamine resin and were sectioned into 20‐nm sections on an ultramicrotome. The sections were then imaged by two‐color STED microscopy. The images are shown after processing by deconvolution, to improve signal‐to‐noise ratios. The line scans indicated in light blue in the rightmost panels are plotted in (D). Scale bar: 2 μm. Line scans through pairs of vesicles found close to each other are shown, as examples. The line scans were performed on the original raw data files, to eliminate any artifacts due to deconvolution. The amount of AHA fluorescence co‐localizing with the releasable or inactive vesicles was determined and was expressed as fold over baseline signals ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment, * P = 0.0037, t (4) = 6.09). The baseline signal refers here to the average AHA signal within the respective synaptic boutons. All data represent the mean ± SEM. Statistical significance was evaluated using unpaired t ‐tests. Data information: All images were taken using a Leica SP5 STED microscope. Source data are available online for this figure.

Techniques Used: Imaging, Produced, Synthesized, Labeling, Incubation, Microscopy, Fluorescence

SNAP 25 co‐localizes better with 4‐day‐old antibody‐labeled Synaptotagmin 1 molecules Two‐color STED analysis of changes in synaptic vesicle protein levels during the transition from the releasable state to the inactive state. Living neurons were incubated with Atto647N‐conjugated Synaptotagmin 1 lumenal domain antibodies for 1 h at 37°C, to label the actively recycling vesicles, as in Fig 1 A, and were then fixed and co‐immunostained for different proteins of interest directly, or were placed in a cell culture incubator for 3–4 days, to enable the antibody‐labeled molecules to enter the inactive pool, before fixation and co‐immunostaining. The samples were embedded in melamine and cut in ultrathin (50 nm) sections, before two‐color STED imaging, as in Fig 2 B. Exemplary images are shown in (A) for SNAP25 and in (B) for Syntaxin 1, with the protein of interest signal next to the signal from the live tagging of Synaptotagmin 1, and a merged image of both, for day 0 (releasable vesicles) and day 4 (inactive vesicles). Scale bar: 1 μm. We analyzed the amount of fluorescence corresponding to the protein of interest that overlapped with the Synaptotagmin 1 signal (i.e., the two signals were presented within the same voxels, which are substantially below the synaptic vesicle volume in this experiment). The only protein whose levels changed significantly is SNAP25 (SNAP25, n (day 0) = 4, n (day 4) = 3, * P = 0.0124, t (5) = 3.8200; Syntaxin 1, n (day 0) = 3, n (day 4) = 3, P = 0.8850, t (4) = 0.1541; VGlut 1/2, n (day 0) = 2, n (day 4) = 3, P = 0.1986, t (3) = 1.6447; vATPase, n (day 0) = 3, n (day 4) = 4, P = 0.7340, t (5) = 0.3594; VAMP2, n (day 0) = 4, n (day 4) = 4, P = 0.8837, t (6) = 0.1527; Synaptotagmin 1, n (day 0) = 3, n (day 4) = 3, P = 0.1604, t (4) = 1.7208; Syntaxin 16, n (day 0) = 4, n (day 4) = 4, P = 0.7406, t (6) = 0.3468; VAMP4, n (day 0) = 3, n (day 4) = 3, P = 0.9863, t (4) = 0.0183; Synapsin I/II, n (day 0) = 3, n (day 4) = 3, P = 0.6638, t (4) = 0.4685; at least 10 neurons sampled per experiment). Statistical significance was evaluated using unpaired t ‐tests. All data represent the mean ± SEM. Data information: Imaging was performed with a Leica SP5 STED microscope. Source data are available online for this figure.
Figure Legend Snippet: SNAP 25 co‐localizes better with 4‐day‐old antibody‐labeled Synaptotagmin 1 molecules Two‐color STED analysis of changes in synaptic vesicle protein levels during the transition from the releasable state to the inactive state. Living neurons were incubated with Atto647N‐conjugated Synaptotagmin 1 lumenal domain antibodies for 1 h at 37°C, to label the actively recycling vesicles, as in Fig 1 A, and were then fixed and co‐immunostained for different proteins of interest directly, or were placed in a cell culture incubator for 3–4 days, to enable the antibody‐labeled molecules to enter the inactive pool, before fixation and co‐immunostaining. The samples were embedded in melamine and cut in ultrathin (50 nm) sections, before two‐color STED imaging, as in Fig 2 B. Exemplary images are shown in (A) for SNAP25 and in (B) for Syntaxin 1, with the protein of interest signal next to the signal from the live tagging of Synaptotagmin 1, and a merged image of both, for day 0 (releasable vesicles) and day 4 (inactive vesicles). Scale bar: 1 μm. We analyzed the amount of fluorescence corresponding to the protein of interest that overlapped with the Synaptotagmin 1 signal (i.e., the two signals were presented within the same voxels, which are substantially below the synaptic vesicle volume in this experiment). The only protein whose levels changed significantly is SNAP25 (SNAP25, n (day 0) = 4, n (day 4) = 3, * P = 0.0124, t (5) = 3.8200; Syntaxin 1, n (day 0) = 3, n (day 4) = 3, P = 0.8850, t (4) = 0.1541; VGlut 1/2, n (day 0) = 2, n (day 4) = 3, P = 0.1986, t (3) = 1.6447; vATPase, n (day 0) = 3, n (day 4) = 4, P = 0.7340, t (5) = 0.3594; VAMP2, n (day 0) = 4, n (day 4) = 4, P = 0.8837, t (6) = 0.1527; Synaptotagmin 1, n (day 0) = 3, n (day 4) = 3, P = 0.1604, t (4) = 1.7208; Syntaxin 16, n (day 0) = 4, n (day 4) = 4, P = 0.7406, t (6) = 0.3468; VAMP4, n (day 0) = 3, n (day 4) = 3, P = 0.9863, t (4) = 0.0183; Synapsin I/II, n (day 0) = 3, n (day 4) = 3, P = 0.6638, t (4) = 0.4685; at least 10 neurons sampled per experiment). Statistical significance was evaluated using unpaired t ‐tests. All data represent the mean ± SEM. Data information: Imaging was performed with a Leica SP5 STED microscope. Source data are available online for this figure.

Techniques Used: Labeling, Incubation, Cell Culture, Immunostaining, Imaging, Fluorescence, Microscopy

Metabolic imaging based on 15 N‐leucine confirms that actively recycling Synaptotagmin 1 molecules are found within younger protein environments than the inactive ones To label newly produced proteins, we used here 15 N‐leucine, which was detected in nanoSIMS imaging. 15 N leucine was fed to the cultured neurons in threefold molar excess over 14 N leucine, in their normal culture medium, for 24–72 h prior to processing the samples. The extended feeding time compared to AHA (Fig 3 ) was necessary to obtain a reliable signal in nanoSIMS imaging. We used the same experimental paradigms as in Fig 3 . First, to correlate the presence of newly synthesized proteins with recycling vesicles, we fed 15 N‐leucine to the neurons for 24–72 h, before labeling before labeling Synaptotagmin 1 from the recycling pool, as in Figs 1 A and 3 . Second, to compare the co‐localization of newly synthesized proteins with inactive synaptic vesicles, we performed the same experiment, but with 3–4 days between antibody tagging and metabolic labeling: We labeled the recycling vesicles by Synaptotagmin 1 antibody incubation and then returned the cell cultures to the incubator. We then waited for 4 days for the inactivation of the vesicles to take place and then fed the neurons with 15 N‐leucine, to label newly synthesized proteins. We analyzed the co‐localization of 15 N‐leucine with releasable and inactive synaptic vesicles in correlated fluorescence and isotopic microscopy (COIN), as follows. The neurons were fixed and were embedded in LR White resin, a vinyl resin that is usable in both fluorescence and isotopic secondary ion mass spectrometry imaging. The samples were then sectioned into 200‐nm sections on an ultramicrotome, as this thickness is ideal for secondary ion mass spectrometry. The sections were mounted on silicon wafers and were imaged on a Nikon Ti‐E microscope, using 150× magnification, to detect the synaptic vesicles (black‐on‐white images). The same areas were then imaged in a nanoSIMS instrument, recording both the 15 N and 14 N signals (color images). The lowest panels show an overlay between the 15 N/ 14 N ratios and the Synaptotagmin 1 labeling. In this overlay, only the pixels with the highest value of the two overlaid images are indicated (showing color where the 15 N/ 14 N ratio images have the higher value; showing black‐on‐white where the fluorescence images of synaptic vesicles have the higher value). This means that wherever color is visible, synaptic vesicles co‐localize well with newly produced proteins. Wherever black or gray is visible, synaptic vesicles co‐localize poorly with newly produced proteins. The white arrowheads point to several vesicles co‐localizing well with newly produced proteins. The black arrowheads point to several vesicles co‐localizing poorly with newly produced proteins. Scale bar: 2 μm. The 15 N/ 14 N ratio was then determined both within the vesicle areas and elsewhere and was then presented as fold over the baseline ratio in the axons. n = 57 synapses from three independent experiments for releasable synaptic vesicles, and n = 47 synapses from two independent experiments for inactive vesicles, * P = 0.0001, t (102) = 5.5378. The ratio is a direct indication of the amount of 15 N‐leucine in the vesicles. The inactive vesicles contain substantially fewer newly synthesized proteins than the rest of the axon (the 15 N/ 14 N ratio of which served as baseline; P = 0.0001, t (96) = 4.0691), while the releasable ones contain substantially more newly synthesized proteins ( P = 0.0004, t (116) = 3.6156). Statistical significance was evaluated using unpaired t ‐tests. All data are represented as box plots with median and upper and lower quartile boundaries, plus 1.5 times inter‐quartile range (whiskers) and outliers (dots). Source data are available online for this figure.
Figure Legend Snippet: Metabolic imaging based on 15 N‐leucine confirms that actively recycling Synaptotagmin 1 molecules are found within younger protein environments than the inactive ones To label newly produced proteins, we used here 15 N‐leucine, which was detected in nanoSIMS imaging. 15 N leucine was fed to the cultured neurons in threefold molar excess over 14 N leucine, in their normal culture medium, for 24–72 h prior to processing the samples. The extended feeding time compared to AHA (Fig 3 ) was necessary to obtain a reliable signal in nanoSIMS imaging. We used the same experimental paradigms as in Fig 3 . First, to correlate the presence of newly synthesized proteins with recycling vesicles, we fed 15 N‐leucine to the neurons for 24–72 h, before labeling before labeling Synaptotagmin 1 from the recycling pool, as in Figs 1 A and 3 . Second, to compare the co‐localization of newly synthesized proteins with inactive synaptic vesicles, we performed the same experiment, but with 3–4 days between antibody tagging and metabolic labeling: We labeled the recycling vesicles by Synaptotagmin 1 antibody incubation and then returned the cell cultures to the incubator. We then waited for 4 days for the inactivation of the vesicles to take place and then fed the neurons with 15 N‐leucine, to label newly synthesized proteins. We analyzed the co‐localization of 15 N‐leucine with releasable and inactive synaptic vesicles in correlated fluorescence and isotopic microscopy (COIN), as follows. The neurons were fixed and were embedded in LR White resin, a vinyl resin that is usable in both fluorescence and isotopic secondary ion mass spectrometry imaging. The samples were then sectioned into 200‐nm sections on an ultramicrotome, as this thickness is ideal for secondary ion mass spectrometry. The sections were mounted on silicon wafers and were imaged on a Nikon Ti‐E microscope, using 150× magnification, to detect the synaptic vesicles (black‐on‐white images). The same areas were then imaged in a nanoSIMS instrument, recording both the 15 N and 14 N signals (color images). The lowest panels show an overlay between the 15 N/ 14 N ratios and the Synaptotagmin 1 labeling. In this overlay, only the pixels with the highest value of the two overlaid images are indicated (showing color where the 15 N/ 14 N ratio images have the higher value; showing black‐on‐white where the fluorescence images of synaptic vesicles have the higher value). This means that wherever color is visible, synaptic vesicles co‐localize well with newly produced proteins. Wherever black or gray is visible, synaptic vesicles co‐localize poorly with newly produced proteins. The white arrowheads point to several vesicles co‐localizing well with newly produced proteins. The black arrowheads point to several vesicles co‐localizing poorly with newly produced proteins. Scale bar: 2 μm. The 15 N/ 14 N ratio was then determined both within the vesicle areas and elsewhere and was then presented as fold over the baseline ratio in the axons. n = 57 synapses from three independent experiments for releasable synaptic vesicles, and n = 47 synapses from two independent experiments for inactive vesicles, * P = 0.0001, t (102) = 5.5378. The ratio is a direct indication of the amount of 15 N‐leucine in the vesicles. The inactive vesicles contain substantially fewer newly synthesized proteins than the rest of the axon (the 15 N/ 14 N ratio of which served as baseline; P = 0.0001, t (96) = 4.0691), while the releasable ones contain substantially more newly synthesized proteins ( P = 0.0004, t (116) = 3.6156). Statistical significance was evaluated using unpaired t ‐tests. All data are represented as box plots with median and upper and lower quartile boundaries, plus 1.5 times inter‐quartile range (whiskers) and outliers (dots). Source data are available online for this figure.

Techniques Used: Imaging, Produced, Cell Culture, Synthesized, Labeling, Incubation, Fluorescence, Microscopy, Mass Spectrometry

The Synaptotagmin 1 molecules that left the recycling population do not return to it spontaneously To determine the rate at which new Synaptotagmin 1 epitopes come into the recycling pool, we saturated the lumenal Synaptotagmin 1 epitopes on active synaptic vesicles by incubating with an unconjugated monoclonal antibody for 2 h at 37°C and then followed the appearance of new epitopes by applying a fluorophore‐conjugated (Atto647N) version of the same monoclonal antibody, at different time points after saturating the initial epitopes. Individual coverslips were only used to investigate single time points and were fixed before imaging. Exemplary images taken after applying the fluorophore‐conjugated antibodies at 0, 12, or 24 h after saturating the initial Synaptotagmin 1 epitopes. Scale bar: 20 μm. Imaging was performed with a Leica SP5 confocal microscope. The same experiment was performed, but the cultures were incubated with drugs that disrupt protein synthesis (anisomycin) or microtubule‐based transport (colchicine). The fluorescence was measured at different time points and was expressed as percentage of the fluorescence at the initial time point. n = 4 independent experiments per data point for untreated 0 and 24 h, and n = 3 for all else, at least 10 neurons sampled per experiment; * P = 0.0070; ** P = 0.0022. Statistical significance was evaluated using one‐way ANOVA ( P = 0.014, F (2, 9) = 19.26), followed by the Bonferroni procedure. To determine whether the drugs impair synaptic activity, neurons were treated with the drugs for 24 h and were then stimulated in the presence of fluorescently conjugated Synaptotagmin 1 antibodies (20 Hz, 30 s), to label the entire recycling pool. Only a limited decrease in recycling was observed in drug‐treated preparations ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.2205, F (2, 8) = 1.97). To assess the spontaneous network activity of our cultures after 24 h of drug treatment, we measured vesicle recycling during the last hour of the treatment. The network activity did not change significantly ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.8835, F (2, 8) = 0.13). The total synaptic vesicle pool size after 24 h of drug treatment. This value was determined by immunostaining for a major synaptic vesicle marker, Synaptophysin. There were no significant changes in synaptic vesicle pool size ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.5519, F (2, 8) = 0.66). Data information: All data represent the mean ± SEM. Imaging was performed with a Leica SP5 confocal microscope. Source data are available online for this figure.
Figure Legend Snippet: The Synaptotagmin 1 molecules that left the recycling population do not return to it spontaneously To determine the rate at which new Synaptotagmin 1 epitopes come into the recycling pool, we saturated the lumenal Synaptotagmin 1 epitopes on active synaptic vesicles by incubating with an unconjugated monoclonal antibody for 2 h at 37°C and then followed the appearance of new epitopes by applying a fluorophore‐conjugated (Atto647N) version of the same monoclonal antibody, at different time points after saturating the initial epitopes. Individual coverslips were only used to investigate single time points and were fixed before imaging. Exemplary images taken after applying the fluorophore‐conjugated antibodies at 0, 12, or 24 h after saturating the initial Synaptotagmin 1 epitopes. Scale bar: 20 μm. Imaging was performed with a Leica SP5 confocal microscope. The same experiment was performed, but the cultures were incubated with drugs that disrupt protein synthesis (anisomycin) or microtubule‐based transport (colchicine). The fluorescence was measured at different time points and was expressed as percentage of the fluorescence at the initial time point. n = 4 independent experiments per data point for untreated 0 and 24 h, and n = 3 for all else, at least 10 neurons sampled per experiment; * P = 0.0070; ** P = 0.0022. Statistical significance was evaluated using one‐way ANOVA ( P = 0.014, F (2, 9) = 19.26), followed by the Bonferroni procedure. To determine whether the drugs impair synaptic activity, neurons were treated with the drugs for 24 h and were then stimulated in the presence of fluorescently conjugated Synaptotagmin 1 antibodies (20 Hz, 30 s), to label the entire recycling pool. Only a limited decrease in recycling was observed in drug‐treated preparations ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.2205, F (2, 8) = 1.97). To assess the spontaneous network activity of our cultures after 24 h of drug treatment, we measured vesicle recycling during the last hour of the treatment. The network activity did not change significantly ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.8835, F (2, 8) = 0.13). The total synaptic vesicle pool size after 24 h of drug treatment. This value was determined by immunostaining for a major synaptic vesicle marker, Synaptophysin. There were no significant changes in synaptic vesicle pool size ( n = 3 independent experiments per data point, at least 10 neurons sampled per experiment; no statistically significant differences as determined via one‐way ANOVA, P = 0.5519, F (2, 8) = 0.66). Data information: All data represent the mean ± SEM. Imaging was performed with a Leica SP5 confocal microscope. Source data are available online for this figure.

Techniques Used: Imaging, Microscopy, Incubation, Fluorescence, Activity Assay, Immunostaining, Marker

10) Product Images from "Molecular mechanisms of synaptic vesicle priming by Munc13 and Munc18"

Article Title: Molecular mechanisms of synaptic vesicle priming by Munc13 and Munc18

Journal: Neuron

doi: 10.1016/j.neuron.2017.07.004

The MUN domain improves the efficiency of Ca 2+ -triggered fusion (A) Single vesicle content mixing assay. PM: plasma membrane mimic vesicles with reconstituted syntaxin-1A and SNAP-25A; SV: synaptic vesicle mimic with reconstituted synaptobrevin-2 and synaptotagmin-1. After SV-PM vesicle association, vesicle pairs either undergo Ca 2+ -independent fusion or remain associated until fusion is triggered by Ca 2+ addition. The MUN domain is only present during the specified stages. “0 μM MUN” refers to the absence of the MUN domain in all stages. In protocol “Pre” the MUN domain is only present before and during SV-PM vesicle association. In protocol “Post” the MUN domain is present only after SV-PM vesicle association. (B–E) The bar graphs show the effects of MUN domain on the average probability of Ca 2+ -independent fusion events per second (B), the amplitude of the first 1-sec time bin upon 500 μM Ca 2+ -injection (C), the ratio of the Ca 2+ -triggered amplitude to the average probably of Ca 2+ -independent fusion per second (D), and the decay rate (1/τ) of the Ca 2+ ). (E) Decay constants and error estimates computed from the covariance matrix upon fitting the corresponding histograms with a single exponential decay function using the Levenberg-Marquardt algorithm. * p
Figure Legend Snippet: The MUN domain improves the efficiency of Ca 2+ -triggered fusion (A) Single vesicle content mixing assay. PM: plasma membrane mimic vesicles with reconstituted syntaxin-1A and SNAP-25A; SV: synaptic vesicle mimic with reconstituted synaptobrevin-2 and synaptotagmin-1. After SV-PM vesicle association, vesicle pairs either undergo Ca 2+ -independent fusion or remain associated until fusion is triggered by Ca 2+ addition. The MUN domain is only present during the specified stages. “0 μM MUN” refers to the absence of the MUN domain in all stages. In protocol “Pre” the MUN domain is only present before and during SV-PM vesicle association. In protocol “Post” the MUN domain is present only after SV-PM vesicle association. (B–E) The bar graphs show the effects of MUN domain on the average probability of Ca 2+ -independent fusion events per second (B), the amplitude of the first 1-sec time bin upon 500 μM Ca 2+ -injection (C), the ratio of the Ca 2+ -triggered amplitude to the average probably of Ca 2+ -independent fusion per second (D), and the decay rate (1/τ) of the Ca 2+ ). (E) Decay constants and error estimates computed from the covariance matrix upon fitting the corresponding histograms with a single exponential decay function using the Levenberg-Marquardt algorithm. * p

Techniques Used: Size-exclusion Chromatography, Injection

11) Product Images from "Tomosyn associates with secretory vesicles in neurons through its N- and C-terminal domains"

Article Title: Tomosyn associates with secretory vesicles in neurons through its N- and C-terminal domains

Journal: PLoS ONE

doi: 10.1371/journal.pone.0180912

EYFP-tomosyn puncta co-localized with various synaptic and secretory vesicle markers. (A-J) Images show expression of EYFP-tomosyn-m1 (‘Tom-1’; green) and endogenous synaptic and secretory vesicle markers (red). Fluorescence intensity profiles along depicted neurites are displayed below each image. Co-localization of EYFP-tomosyn-m1 puncta with antibodies recognizing endogenous (A) SNARE protein syntaxin, (B) the presynaptic SNARE-associated protein munc18 as well as the active zone protein bassoon was observed, confirming presynaptic localization. Moreover, tom-1 puncta co-localized with synaptic vesicle (SV) markers (D) synaptotagmin-1 (Syt-1), (E) VAMP2 and (F) synapsin, (G) vesicular glutamate transporter-1 (VGLUT1) and (H) the DCV marker chromogranin B. (I) CAPS, implicated in release from both SVs and DCVs, additionally co-localized with tomosyn-1 puncta. (J) Synapsin / VAMP2 co-localization was used as a positive control. (K-M) Co-localization was quantified using (K) Pearson’s correlation and Manders’ overlap (L) M1 and (M) M2. As a negative control, tomosyn images were rotated relative to syntaxin. (N-Q) Ultrastructural localization of endogenous tomosyn-1 in (N) presynaptic boutons (O) vesicles in neurites and (P) dense core vesicles. (Q) Overexpressed tomosyn-1 was predominantly localized to presynaptic boutons. Arrowheads indicate post-synaptic densities.
Figure Legend Snippet: EYFP-tomosyn puncta co-localized with various synaptic and secretory vesicle markers. (A-J) Images show expression of EYFP-tomosyn-m1 (‘Tom-1’; green) and endogenous synaptic and secretory vesicle markers (red). Fluorescence intensity profiles along depicted neurites are displayed below each image. Co-localization of EYFP-tomosyn-m1 puncta with antibodies recognizing endogenous (A) SNARE protein syntaxin, (B) the presynaptic SNARE-associated protein munc18 as well as the active zone protein bassoon was observed, confirming presynaptic localization. Moreover, tom-1 puncta co-localized with synaptic vesicle (SV) markers (D) synaptotagmin-1 (Syt-1), (E) VAMP2 and (F) synapsin, (G) vesicular glutamate transporter-1 (VGLUT1) and (H) the DCV marker chromogranin B. (I) CAPS, implicated in release from both SVs and DCVs, additionally co-localized with tomosyn-1 puncta. (J) Synapsin / VAMP2 co-localization was used as a positive control. (K-M) Co-localization was quantified using (K) Pearson’s correlation and Manders’ overlap (L) M1 and (M) M2. As a negative control, tomosyn images were rotated relative to syntaxin. (N-Q) Ultrastructural localization of endogenous tomosyn-1 in (N) presynaptic boutons (O) vesicles in neurites and (P) dense core vesicles. (Q) Overexpressed tomosyn-1 was predominantly localized to presynaptic boutons. Arrowheads indicate post-synaptic densities.

Techniques Used: Expressing, Fluorescence, Marker, Positive Control, Negative Control

12) Product Images from "N-terminal domain of complexin independently activates calcium-triggered fusion"

Article Title: N-terminal domain of complexin independently activates calcium-triggered fusion

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1604348113

Model of the prefusion state of the SNARE/synaptotagmin/Cpx supercomplex. We propose that the Ca 2+ sensitivity, cooperativity, and high speed of SV fusion are achieved by the synergistic actions of synaptotagmin-1–SNARE membrane interactions (
Figure Legend Snippet: Model of the prefusion state of the SNARE/synaptotagmin/Cpx supercomplex. We propose that the Ca 2+ sensitivity, cooperativity, and high speed of SV fusion are achieved by the synergistic actions of synaptotagmin-1–SNARE membrane interactions (

Techniques Used:

Normalized histograms of the probability of spontaneous fusion. Experiments were performed in the presence of both full-length synaptotagmin-1 and neuronal SNAREs, and in the absence or presence of 2 μM full-length Cpx, Cpx fragments, or Cpx chimeras
Figure Legend Snippet: Normalized histograms of the probability of spontaneous fusion. Experiments were performed in the presence of both full-length synaptotagmin-1 and neuronal SNAREs, and in the absence or presence of 2 μM full-length Cpx, Cpx fragments, or Cpx chimeras

Techniques Used:

Normalized histograms of the probability of Ca 2+ -triggered fusion. Experiments were performed in the presence of both full-length synaptotagmin-1 and neuronal SNAREs, and in the absence or presence of 2 μM full-length Cpx, Cpx fragments, or Cpx
Figure Legend Snippet: Normalized histograms of the probability of Ca 2+ -triggered fusion. Experiments were performed in the presence of both full-length synaptotagmin-1 and neuronal SNAREs, and in the absence or presence of 2 μM full-length Cpx, Cpx fragments, or Cpx

Techniques Used:

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    Ipsen Group synaptotagmin 1
    Representative fluorescence emission spectra used in the experiments of Figure 5B to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1, <t>synaptotagmin-1</t> and Ca 2+ in different combinations protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP. V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), synaptotagmin-1 C 2 AB (C 2 AB) and Ca 2+ . Black curves show spectra acquired before addition of NSF-αSNAP, and red curves the spectra obtained after addition of NSF-αSNAP.
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    Representative fluorescence emission spectra used in the experiments of Figure 5B to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1, synaptotagmin-1 and Ca 2+ in different combinations protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP. V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), synaptotagmin-1 C 2 AB (C 2 AB) and Ca 2+ . Black curves show spectra acquired before addition of NSF-αSNAP, and red curves the spectra obtained after addition of NSF-αSNAP.

    Journal: eLife

    Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

    doi: 10.7554/eLife.38880

    Figure Lengend Snippet: Representative fluorescence emission spectra used in the experiments of Figure 5B to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1, synaptotagmin-1 and Ca 2+ in different combinations protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP. V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), synaptotagmin-1 C 2 AB (C 2 AB) and Ca 2+ . Black curves show spectra acquired before addition of NSF-αSNAP, and red curves the spectra obtained after addition of NSF-αSNAP.

    Article Snippet: Disassembly of cis-SNARE complexes To further investigate the functional interplay between NSF, αSNAP, Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 in the SNARE complex assembly-disassembly cycle, we performed kinetic assays where we analyzed the assembly and disassembly of cis-SNARE complexes mixing V-liposomes containing Alexa488-synaptobrevin with SNAP-25m and a soluble fragment spanning the cytoplasmic region of syntaxin-1 (residues 2–253) labeled with TMR at residue 186.

    Techniques: Fluorescence, Incubation

    Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1 and synaptotagmin-1 C 2 AB do not protect cis-SNARE complexes against disassembly by NSF-αSNAP. ( A ) Kinetic assays where cis-SNARE complex formation was catalyzed by Syb49-93, as in Figure 6D , and different concentrations of complexin-1 (Cpx) were added five minutes before disassembly with NSF-αSNAP. ( B ) Kinetic assays analogous to those of Figure 6D , but using WT SNAP-25 instead of SNAP-25m to ensure that the mutation in SNAP-25m did not affect the disassembly of cis-SNARE complexes by NSF-αSNAP in the presence of Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1, synaptotagmin-1 C 2 AB and Ca 2+ . ( C ) Kinetic assays analogous to those of panels ( A ), but adding 1 μM complexin-1 five minutes before disassembly with NSF-αSNAP (red and orange traces). In these experiments, the concentrations of NSF and αSNAP were 0.1 μM and 0.5 μM, respectively, which were lower than those of our standard conditions (0.5 μM and 2 μM, respectively) to test whether complexin-1 might hinder disassembly at a higher molar ratio with respect to αSNAP. The experiments were performed with SNAP-25m (black and red traces) or WT SNAP-25 (gray and orange traces). The black and gray traces are controls where complexin-1 was not added. In the experiments shown in ( A–C ), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces of ( A–C ), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents to make the data comparable.

    Journal: eLife

    Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

    doi: 10.7554/eLife.38880

    Figure Lengend Snippet: Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1 and synaptotagmin-1 C 2 AB do not protect cis-SNARE complexes against disassembly by NSF-αSNAP. ( A ) Kinetic assays where cis-SNARE complex formation was catalyzed by Syb49-93, as in Figure 6D , and different concentrations of complexin-1 (Cpx) were added five minutes before disassembly with NSF-αSNAP. ( B ) Kinetic assays analogous to those of Figure 6D , but using WT SNAP-25 instead of SNAP-25m to ensure that the mutation in SNAP-25m did not affect the disassembly of cis-SNARE complexes by NSF-αSNAP in the presence of Munc18-1, Munc13-1 C 1 C 2 BMUNC 2 C, complexin-1, synaptotagmin-1 C 2 AB and Ca 2+ . ( C ) Kinetic assays analogous to those of panels ( A ), but adding 1 μM complexin-1 five minutes before disassembly with NSF-αSNAP (red and orange traces). In these experiments, the concentrations of NSF and αSNAP were 0.1 μM and 0.5 μM, respectively, which were lower than those of our standard conditions (0.5 μM and 2 μM, respectively) to test whether complexin-1 might hinder disassembly at a higher molar ratio with respect to αSNAP. The experiments were performed with SNAP-25m (black and red traces) or WT SNAP-25 (gray and orange traces). The black and gray traces are controls where complexin-1 was not added. In the experiments shown in ( A–C ), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces of ( A–C ), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents to make the data comparable.

    Article Snippet: Disassembly of cis-SNARE complexes To further investigate the functional interplay between NSF, αSNAP, Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 in the SNARE complex assembly-disassembly cycle, we performed kinetic assays where we analyzed the assembly and disassembly of cis-SNARE complexes mixing V-liposomes containing Alexa488-synaptobrevin with SNAP-25m and a soluble fragment spanning the cytoplasmic region of syntaxin-1 (residues 2–253) labeled with TMR at residue 186.

    Techniques: Mutagenesis, Fluorescence

    Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) Models showing ribbon diagrams of the cryo-electron microscopy structure of the 20S complex (PDB accession code 3J96) ( Zhao et al., 2015 ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.

    Journal: eLife

    Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

    doi: 10.7554/eLife.38880

    Figure Lengend Snippet: Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly. ( A,B ) Models showing ribbon diagrams of the cryo-electron microscopy structure of the 20S complex (PDB accession code 3J96) ( Zhao et al., 2015 ) assembled on a cis-SNARE complex on one membrane ( A ) or on a trans-SNARE complex between two membranes ( B ). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in ( A ) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In ( B ), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.

    Article Snippet: Disassembly of cis-SNARE complexes To further investigate the functional interplay between NSF, αSNAP, Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 in the SNARE complex assembly-disassembly cycle, we performed kinetic assays where we analyzed the assembly and disassembly of cis-SNARE complexes mixing V-liposomes containing Alexa488-synaptobrevin with SNAP-25m and a soluble fragment spanning the cytoplasmic region of syntaxin-1 (residues 2–253) labeled with TMR at residue 186.

    Techniques: Electron Microscopy, Binding Assay

    Control experiments acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes containing Alexa488-synaptobrevin. Spectra were acquired before (V) (black traces) or after (red traces) addition of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), Ca 2+ -bound synaptotagmin-1 C 2 AB, NSF, αSNAP or NSF+αSNAP.

    Journal: eLife

    Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

    doi: 10.7554/eLife.38880

    Figure Lengend Snippet: Control experiments acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes containing Alexa488-synaptobrevin. Spectra were acquired before (V) (black traces) or after (red traces) addition of Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx), Ca 2+ -bound synaptotagmin-1 C 2 AB, NSF, αSNAP or NSF+αSNAP.

    Article Snippet: Disassembly of cis-SNARE complexes To further investigate the functional interplay between NSF, αSNAP, Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 in the SNARE complex assembly-disassembly cycle, we performed kinetic assays where we analyzed the assembly and disassembly of cis-SNARE complexes mixing V-liposomes containing Alexa488-synaptobrevin with SNAP-25m and a soluble fragment spanning the cytoplasmic region of syntaxin-1 (residues 2–253) labeled with TMR at residue 186.

    Techniques: Fluorescence

    Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of T-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing synaptobrevin (V) were incubated for five hours with Syb49-93 and T-liposomes containing TMR-syntaxin-1-SNAP-25m (T*) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for five minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.

    Journal: eLife

    Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

    doi: 10.7554/eLife.38880

    Figure Lengend Snippet: Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of T-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing synaptobrevin (V) were incubated for five hours with Syb49-93 and T-liposomes containing TMR-syntaxin-1-SNAP-25m (T*) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for five minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.

    Article Snippet: Disassembly of cis-SNARE complexes To further investigate the functional interplay between NSF, αSNAP, Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 in the SNARE complex assembly-disassembly cycle, we performed kinetic assays where we analyzed the assembly and disassembly of cis-SNARE complexes mixing V-liposomes containing Alexa488-synaptobrevin with SNAP-25m and a soluble fragment spanning the cytoplasmic region of syntaxin-1 (residues 2–253) labeled with TMR at residue 186.

    Techniques: Fluorescence, Incubation

    Ca 2+ -dependent fusion between VSyt1- and T-liposomes. ( A,B ) Lipid mixing ( A ) between VSyt1-liposomes (synaptobrevin-to-lipid ratio 1:10,000; synaptotagmin-1-to-lipid ratio 1:1,000) and T-liposomes was monitored from the fluorescence de-quenching of Marina Blue lipids and content mixing ( B ) was monitored from the increase in the fluorescence signal of Cy5-streptavidin trapped in the V-liposomes caused by FRET with PhycoE-biotin trapped in the T-liposomes upon liposome fusion. The assays were performed in the presence of NSF-αSNAP, and Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13) or both. Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin, and Ca 2+ (600 μM) was added at 300 s.

    Journal: eLife

    Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

    doi: 10.7554/eLife.38880

    Figure Lengend Snippet: Ca 2+ -dependent fusion between VSyt1- and T-liposomes. ( A,B ) Lipid mixing ( A ) between VSyt1-liposomes (synaptobrevin-to-lipid ratio 1:10,000; synaptotagmin-1-to-lipid ratio 1:1,000) and T-liposomes was monitored from the fluorescence de-quenching of Marina Blue lipids and content mixing ( B ) was monitored from the increase in the fluorescence signal of Cy5-streptavidin trapped in the V-liposomes caused by FRET with PhycoE-biotin trapped in the T-liposomes upon liposome fusion. The assays were performed in the presence of NSF-αSNAP, and Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13) or both. Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin, and Ca 2+ (600 μM) was added at 300 s.

    Article Snippet: Disassembly of cis-SNARE complexes To further investigate the functional interplay between NSF, αSNAP, Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 in the SNARE complex assembly-disassembly cycle, we performed kinetic assays where we analyzed the assembly and disassembly of cis-SNARE complexes mixing V-liposomes containing Alexa488-synaptobrevin with SNAP-25m and a soluble fragment spanning the cytoplasmic region of syntaxin-1 (residues 2–253) labeled with TMR at residue 186.

    Techniques: Fluorescence

    Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing Alexa488-synaptobrevin (V*) were incubated for five hours with Syb49-93 and T-liposomes containing syntaxin-1-SNAP-25m (T) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for 5 minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.

    Journal: eLife

    Article Title: Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

    doi: 10.7554/eLife.38880

    Figure Lengend Snippet: Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes incorporated into trans-SNARE complexes in the absence of FRET. V-liposomes containing Alexa488-synaptobrevin (V*) were incubated for five hours with Syb49-93 and T-liposomes containing syntaxin-1-SNAP-25m (T) (1:4 V- to T-liposome ratio). The mixture was then incubated with Munc18-1 (M18), Munc13-1 C 1 C 2 BMUNC 2 C (M13), complexin-1 (Cpx) or synaptotagmin-1 C 2 AB/Ca 2+ for 5 minutes, and spectra were acquired before (black traces) or after (red traces) addition of NSF-αSNAP.

    Article Snippet: Disassembly of cis-SNARE complexes To further investigate the functional interplay between NSF, αSNAP, Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 in the SNARE complex assembly-disassembly cycle, we performed kinetic assays where we analyzed the assembly and disassembly of cis-SNARE complexes mixing V-liposomes containing Alexa488-synaptobrevin with SNAP-25m and a soluble fragment spanning the cytoplasmic region of syntaxin-1 (residues 2–253) labeled with TMR at residue 186.

    Techniques: Fluorescence, Incubation