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Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin.

Zhang Y, Diao J, Colbert KN, Lai Y, Pfuetzner RA, Padolina MS, Vivona S, Ressl S, Cipriano DJ, Choi UB, Shah N, Weis WI, Brunger AT - J. Biol. Chem. (2015)

Bottom Line: Moreover, a phosphorylation mimic mutant of Munc18a with reduced affinity to syntaxin-1A results in less reduction of vesicle association.In summary, Munc18a does not directly affect fusion, although it has an effect on the t-SNARE complex, depending on the presence of other factors and experimental conditions.Our results suggest that Munc18a primarily acts at the prefusion stage.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.

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Related in: MedlinePlus

Vesicle association efficiency between SV vesicles and immobilized PM vesicles.A–F, SV vesicle association efficiency was measured by counting associated SV vesicles per field of view (45 μm × 90 μm) in the presence of 2 μm complexin. Each panel corresponds to measurements that were performed on the same surface and with exactly the same vesicle concentration and incubation time (i.e. experiments within a particular panel are comparable, whereas caution should be used when comparing different panels because they were from different protein and surface preparations). Consequently, each bar chart was normalized by the mean value of the respective condition without Munc18. For details of the incubation periods, see “Experimental Procedures.” A, SV vesicles associated with PM vesicles with no Munc18, with Munc18, and with Munc18a(S306E/S313E) or supplemented with 10 μm soluble synaptobrevin(1–96) in addition to the reconstituted full-length synaptobrevin-2. B, SV vesicles associated with PM vesicles with reconstituted Stx1a·SNAP-25 but not containing PIP2. C, SV vesicles preassociated with PM vesicles and then incubated in the flow chamber with or without Munc18a for (see “Experimental Procedures” for details). D, same as A but with PM vesicles with reconstituted Stx1a(10–288)·SNAP-25. E, SV vesicles with reconstituted synaptobrevin-2, but without synaptotagmin-1, associated with PM vesicles with reconstituted Stx1a·SNAP-25. F, same as E but with PM vesicles with reconstituted Stx1a(10–288)·SNAP-25. Error bars, S.D. values over 10–15 experiments in random locations of the same flow channel. **, p < 0.01 using Student's t test.
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Figure 7: Vesicle association efficiency between SV vesicles and immobilized PM vesicles.A–F, SV vesicle association efficiency was measured by counting associated SV vesicles per field of view (45 μm × 90 μm) in the presence of 2 μm complexin. Each panel corresponds to measurements that were performed on the same surface and with exactly the same vesicle concentration and incubation time (i.e. experiments within a particular panel are comparable, whereas caution should be used when comparing different panels because they were from different protein and surface preparations). Consequently, each bar chart was normalized by the mean value of the respective condition without Munc18. For details of the incubation periods, see “Experimental Procedures.” A, SV vesicles associated with PM vesicles with no Munc18, with Munc18, and with Munc18a(S306E/S313E) or supplemented with 10 μm soluble synaptobrevin(1–96) in addition to the reconstituted full-length synaptobrevin-2. B, SV vesicles associated with PM vesicles with reconstituted Stx1a·SNAP-25 but not containing PIP2. C, SV vesicles preassociated with PM vesicles and then incubated in the flow chamber with or without Munc18a for (see “Experimental Procedures” for details). D, same as A but with PM vesicles with reconstituted Stx1a(10–288)·SNAP-25. E, SV vesicles with reconstituted synaptobrevin-2, but without synaptotagmin-1, associated with PM vesicles with reconstituted Stx1a·SNAP-25. F, same as E but with PM vesicles with reconstituted Stx1a(10–288)·SNAP-25. Error bars, S.D. values over 10–15 experiments in random locations of the same flow channel. **, p < 0.01 using Student's t test.

Mentions: Although we did not observe an effect of Munc18a on the Ca2+-triggered fusion histograms and spontaneous fusion probabilities (Fig. 1), we found that the number of associated SV vesicles was reduced when Munc18a had been preincubated with the immobilized PM vesicles; in fact, we compensated for this reduction in associated SV vesicles by increasing the SV vesicle concentration accordingly compared with control without Munc18a (see “Experimental Procedures”). We studied this effect quantitatively by using a single vesicle-vesicle association assay that enabled higher throughput (53, 54). Similar to the fusion experiments discussed above, immobilized PM vesicles were preincubated with Munc18a before SV vesicles along with complexin-1 (see “Experimental Procedures” for details). The number of associated SV vesicles per field of view was counted and used as a measure of association efficiency between pairs of free SV and immobilized PM vesicles. For a series of conditions, the same surface preparation and immobilized PM vesicles were used, and the free SV vesicle concentrations and incubation times were kept the same as well. Because experiments with different protein and surface preparations cannot be compared directly, we plotted the results as fractional probabilities relative to the respective controls without Munc18a for each series of experiments that used the same surface and PM vesicle preparation (Fig. 7).


Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin.

Zhang Y, Diao J, Colbert KN, Lai Y, Pfuetzner RA, Padolina MS, Vivona S, Ressl S, Cipriano DJ, Choi UB, Shah N, Weis WI, Brunger AT - J. Biol. Chem. (2015)

Vesicle association efficiency between SV vesicles and immobilized PM vesicles.A–F, SV vesicle association efficiency was measured by counting associated SV vesicles per field of view (45 μm × 90 μm) in the presence of 2 μm complexin. Each panel corresponds to measurements that were performed on the same surface and with exactly the same vesicle concentration and incubation time (i.e. experiments within a particular panel are comparable, whereas caution should be used when comparing different panels because they were from different protein and surface preparations). Consequently, each bar chart was normalized by the mean value of the respective condition without Munc18. For details of the incubation periods, see “Experimental Procedures.” A, SV vesicles associated with PM vesicles with no Munc18, with Munc18, and with Munc18a(S306E/S313E) or supplemented with 10 μm soluble synaptobrevin(1–96) in addition to the reconstituted full-length synaptobrevin-2. B, SV vesicles associated with PM vesicles with reconstituted Stx1a·SNAP-25 but not containing PIP2. C, SV vesicles preassociated with PM vesicles and then incubated in the flow chamber with or without Munc18a for (see “Experimental Procedures” for details). D, same as A but with PM vesicles with reconstituted Stx1a(10–288)·SNAP-25. E, SV vesicles with reconstituted synaptobrevin-2, but without synaptotagmin-1, associated with PM vesicles with reconstituted Stx1a·SNAP-25. F, same as E but with PM vesicles with reconstituted Stx1a(10–288)·SNAP-25. Error bars, S.D. values over 10–15 experiments in random locations of the same flow channel. **, p < 0.01 using Student's t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Vesicle association efficiency between SV vesicles and immobilized PM vesicles.A–F, SV vesicle association efficiency was measured by counting associated SV vesicles per field of view (45 μm × 90 μm) in the presence of 2 μm complexin. Each panel corresponds to measurements that were performed on the same surface and with exactly the same vesicle concentration and incubation time (i.e. experiments within a particular panel are comparable, whereas caution should be used when comparing different panels because they were from different protein and surface preparations). Consequently, each bar chart was normalized by the mean value of the respective condition without Munc18. For details of the incubation periods, see “Experimental Procedures.” A, SV vesicles associated with PM vesicles with no Munc18, with Munc18, and with Munc18a(S306E/S313E) or supplemented with 10 μm soluble synaptobrevin(1–96) in addition to the reconstituted full-length synaptobrevin-2. B, SV vesicles associated with PM vesicles with reconstituted Stx1a·SNAP-25 but not containing PIP2. C, SV vesicles preassociated with PM vesicles and then incubated in the flow chamber with or without Munc18a for (see “Experimental Procedures” for details). D, same as A but with PM vesicles with reconstituted Stx1a(10–288)·SNAP-25. E, SV vesicles with reconstituted synaptobrevin-2, but without synaptotagmin-1, associated with PM vesicles with reconstituted Stx1a·SNAP-25. F, same as E but with PM vesicles with reconstituted Stx1a(10–288)·SNAP-25. Error bars, S.D. values over 10–15 experiments in random locations of the same flow channel. **, p < 0.01 using Student's t test.
Mentions: Although we did not observe an effect of Munc18a on the Ca2+-triggered fusion histograms and spontaneous fusion probabilities (Fig. 1), we found that the number of associated SV vesicles was reduced when Munc18a had been preincubated with the immobilized PM vesicles; in fact, we compensated for this reduction in associated SV vesicles by increasing the SV vesicle concentration accordingly compared with control without Munc18a (see “Experimental Procedures”). We studied this effect quantitatively by using a single vesicle-vesicle association assay that enabled higher throughput (53, 54). Similar to the fusion experiments discussed above, immobilized PM vesicles were preincubated with Munc18a before SV vesicles along with complexin-1 (see “Experimental Procedures” for details). The number of associated SV vesicles per field of view was counted and used as a measure of association efficiency between pairs of free SV and immobilized PM vesicles. For a series of conditions, the same surface preparation and immobilized PM vesicles were used, and the free SV vesicle concentrations and incubation times were kept the same as well. Because experiments with different protein and surface preparations cannot be compared directly, we plotted the results as fractional probabilities relative to the respective controls without Munc18a for each series of experiments that used the same surface and PM vesicle preparation (Fig. 7).

Bottom Line: Moreover, a phosphorylation mimic mutant of Munc18a with reduced affinity to syntaxin-1A results in less reduction of vesicle association.In summary, Munc18a does not directly affect fusion, although it has an effect on the t-SNARE complex, depending on the presence of other factors and experimental conditions.Our results suggest that Munc18a primarily acts at the prefusion stage.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.

Show MeSH
Related in: MedlinePlus