Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin.
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.
Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.Show MeSH
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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).
Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.