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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

Effect of Munc18a on Ca2+-triggered fusion and spontaneous fusion with concurrent addition of Munc18a and SV vesicles to immobilized PM vesicles. Shown are histograms of Ca2+-triggered fusion events without Munc18a (A) and with 1 μm Munc18a (B) using PM vesicles containing full-length Stx1a. Munc18a and SV vesicles were introduced concurrently. All experiments were performed in the presence of 2 μm complexin. The histograms were calculated with a bin size of 1 s, normalized by the total of fusion events observed upon Ca2+ injection, and fitted to two-exponential decay functions (solid lines). The synchronization time constants (faster time constant of the fitted two-exponential fit) are shown in C. Spontaneous fusion events were observed for 1 min before Ca2+ injection and shown in D. Error bars in C, are 1σ errors computed from the covariance matrix during least square fitting using a Levenberg-Marquardt technique; error bars in D, S.E. value over 4–6 experiments.
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Figure 2: Effect of Munc18a on Ca2+-triggered fusion and spontaneous fusion with concurrent addition of Munc18a and SV vesicles to immobilized PM vesicles. Shown are histograms of Ca2+-triggered fusion events without Munc18a (A) and with 1 μm Munc18a (B) using PM vesicles containing full-length Stx1a. Munc18a and SV vesicles were introduced concurrently. All experiments were performed in the presence of 2 μm complexin. The histograms were calculated with a bin size of 1 s, normalized by the total of fusion events observed upon Ca2+ injection, and fitted to two-exponential decay functions (solid lines). The synchronization time constants (faster time constant of the fitted two-exponential fit) are shown in C. Spontaneous fusion events were observed for 1 min before Ca2+ injection and shown in D. Error bars in C, are 1σ errors computed from the covariance matrix during least square fitting using a Levenberg-Marquardt technique; error bars in D, S.E. value over 4–6 experiments.

Mentions: Munc18a had no effect on the normalized Ca2+-triggered fusion histograms (Fig. 1, A–F), the synchronization time constant (Fig. 1G), or the probability of spontaneous fusion (Fig. 1H), regardless of whether full-length Stx1a or the N-terminally truncated Stx1a(10–288) fragment were used. Moreover, there was no effect if Munc18a was incubated concurrently when adding free SV vesicles to the immobilized PM vesicles (Fig. 2). We note that the histograms were normalized with respect to the total number of fusion events, so they measure the effect of Munc18a on the intrinsic spontaneous and Ca2+-triggered fusion kinetics of associated vesicle-vesicle pairs. The ability to measure intrinsic fusion kinetics is an advantage compared with ensemble fusion experiments, where effects on docking and fusion cannot be separated (21).


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)

Effect of Munc18a on Ca2+-triggered fusion and spontaneous fusion with concurrent addition of Munc18a and SV vesicles to immobilized PM vesicles. Shown are histograms of Ca2+-triggered fusion events without Munc18a (A) and with 1 μm Munc18a (B) using PM vesicles containing full-length Stx1a. Munc18a and SV vesicles were introduced concurrently. All experiments were performed in the presence of 2 μm complexin. The histograms were calculated with a bin size of 1 s, normalized by the total of fusion events observed upon Ca2+ injection, and fitted to two-exponential decay functions (solid lines). The synchronization time constants (faster time constant of the fitted two-exponential fit) are shown in C. Spontaneous fusion events were observed for 1 min before Ca2+ injection and shown in D. Error bars in C, are 1σ errors computed from the covariance matrix during least square fitting using a Levenberg-Marquardt technique; error bars in D, S.E. value over 4–6 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of Munc18a on Ca2+-triggered fusion and spontaneous fusion with concurrent addition of Munc18a and SV vesicles to immobilized PM vesicles. Shown are histograms of Ca2+-triggered fusion events without Munc18a (A) and with 1 μm Munc18a (B) using PM vesicles containing full-length Stx1a. Munc18a and SV vesicles were introduced concurrently. All experiments were performed in the presence of 2 μm complexin. The histograms were calculated with a bin size of 1 s, normalized by the total of fusion events observed upon Ca2+ injection, and fitted to two-exponential decay functions (solid lines). The synchronization time constants (faster time constant of the fitted two-exponential fit) are shown in C. Spontaneous fusion events were observed for 1 min before Ca2+ injection and shown in D. Error bars in C, are 1σ errors computed from the covariance matrix during least square fitting using a Levenberg-Marquardt technique; error bars in D, S.E. value over 4–6 experiments.
Mentions: Munc18a had no effect on the normalized Ca2+-triggered fusion histograms (Fig. 1, A–F), the synchronization time constant (Fig. 1G), or the probability of spontaneous fusion (Fig. 1H), regardless of whether full-length Stx1a or the N-terminally truncated Stx1a(10–288) fragment were used. Moreover, there was no effect if Munc18a was incubated concurrently when adding free SV vesicles to the immobilized PM vesicles (Fig. 2). We note that the histograms were normalized with respect to the total number of fusion events, so they measure the effect of Munc18a on the intrinsic spontaneous and Ca2+-triggered fusion kinetics of associated vesicle-vesicle pairs. The ability to measure intrinsic fusion kinetics is an advantage compared with ensemble fusion experiments, where effects on docking and fusion cannot be separated (21).

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