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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

Munc18a sequesters syntaxin-1A and releases SNAP-25 from t-SNARE complex that is reconstituted in PM vesicles.Top panels, shown are the average number of labeled SNAP-25 molecules from 10 random imaging locations in the same sample channel before and after (120 min) 1 μm Munc18 addition. Bottom panels, representative fields of view. Error bars, S.D. Note that a purple spot indicates higher brightness. **, p < 0.01 using Student's t test.
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Figure 8: Munc18a sequesters syntaxin-1A and releases SNAP-25 from t-SNARE complex that is reconstituted in PM vesicles.Top panels, shown are the average number of labeled SNAP-25 molecules from 10 random imaging locations in the same sample channel before and after (120 min) 1 μm Munc18 addition. Bottom panels, representative fields of view. Error bars, S.D. Note that a purple spot indicates higher brightness. **, p < 0.01 using Student's t test.

Mentions: To test this hypothesis, we labeled SNAP-25 at residue 76 and reconstituted a mixture of labeled and unlabeled SNAP-25 molecules into PM vesicles together with unlabeled syntaxin-1A. The same reconstitution and immobilization protocols were performed as for all other single vesicle experiments in this work. We adjusted the ratio of labeled to unlabeled SNAP-25 molecules in order to obtain at most one labeled SNAP-25 molecule per PM vesicle. Note that we used a soluble SNAP-25 construct (i.e. without palmitoylation), so once a SNAP-25 molecule was dislodged from a t-SNARE complex it was expected to diffuse away from the surface. We monitored fluorescence from single SNAP-25 molecules before and 120 min after the Munc18a addition (Fig. 8). Consistent with our hypothesis of syntaxin-1A sequestration, we observed that incubation of Munc18a results in reduction of about 50% of surface-localized SNAP-25 molecules.


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)

Munc18a sequesters syntaxin-1A and releases SNAP-25 from t-SNARE complex that is reconstituted in PM vesicles.Top panels, shown are the average number of labeled SNAP-25 molecules from 10 random imaging locations in the same sample channel before and after (120 min) 1 μm Munc18 addition. Bottom panels, representative fields of view. Error bars, S.D. Note that a purple spot indicates higher brightness. **, p < 0.01 using Student's t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400359&req=5

Figure 8: Munc18a sequesters syntaxin-1A and releases SNAP-25 from t-SNARE complex that is reconstituted in PM vesicles.Top panels, shown are the average number of labeled SNAP-25 molecules from 10 random imaging locations in the same sample channel before and after (120 min) 1 μm Munc18 addition. Bottom panels, representative fields of view. Error bars, S.D. Note that a purple spot indicates higher brightness. **, p < 0.01 using Student's t test.
Mentions: To test this hypothesis, we labeled SNAP-25 at residue 76 and reconstituted a mixture of labeled and unlabeled SNAP-25 molecules into PM vesicles together with unlabeled syntaxin-1A. The same reconstitution and immobilization protocols were performed as for all other single vesicle experiments in this work. We adjusted the ratio of labeled to unlabeled SNAP-25 molecules in order to obtain at most one labeled SNAP-25 molecule per PM vesicle. Note that we used a soluble SNAP-25 construct (i.e. without palmitoylation), so once a SNAP-25 molecule was dislodged from a t-SNARE complex it was expected to diffuse away from the surface. We monitored fluorescence from single SNAP-25 molecules before and 120 min after the Munc18a addition (Fig. 8). Consistent with our hypothesis of syntaxin-1A sequestration, we observed that incubation of Munc18a results in reduction of about 50% of surface-localized SNAP-25 molecules.

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