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The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming

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ABSTRACT

Kasula et al. use single-molecule imaging to reveal the diffusional signature for the SNARE proteins Munc18-1 and syntaxin-1A during secretory vesicle priming. The authors show that a conformational change in the Munc18-1 domain 3a hinge-loop regulates engagement of syntaxin-1A in the SNARE complex.

No MeSH data available.


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BoNT/E-LC expression prevents the activity-dependent decrease in syntaxin-1A mobility. (A–L) DKD-PC12 cells transfected with the indicated plasmids were imaged at 50 Hz (16,000 frames) in either unstimulated or stimulated (2 mM Ba2+) conditions using the uPAINT technique. Mean MSD as a function of time, mean distribution of the diffusion coefficients, and ratio of mobile versus immobile fraction in indicated cells and conditions. (n = 10–19 cells for each condition). Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (*, P < 0.05). Mean ± SEM.
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fig6: BoNT/E-LC expression prevents the activity-dependent decrease in syntaxin-1A mobility. (A–L) DKD-PC12 cells transfected with the indicated plasmids were imaged at 50 Hz (16,000 frames) in either unstimulated or stimulated (2 mM Ba2+) conditions using the uPAINT technique. Mean MSD as a function of time, mean distribution of the diffusion coefficients, and ratio of mobile versus immobile fraction in indicated cells and conditions. (n = 10–19 cells for each condition). Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (*, P < 0.05). Mean ± SEM.

Mentions: We predicted that the reduced mobility of syntaxin-1A elicited by activity might stem from its incorporation into the trans-SNARE complex with SNAP-25. To test this hypothesis, we used BoNT/E-LC. BoNT/E-LC enzymatic activity targets SNAP-25 and cleaves 26 residues from the C-terminus of the molecule, thereby blocking neuroexocytosis (Schiavo et al., 1993). The light chains prevent SNARE complex formation (Hayashi et al., 1994), and BoNT/E has an additional effect on the syntaxin-1A/SNAP-25 heterodimer (Rickman et al., 2004). BoNT/E-LC was cotransfected with syntaxin-1A-GFP in DKD-PC12 cells. Our results revealed that the activity-dependent decrease in syntaxin-1A-GFP mobility (Fig. 6, A–C) was blocked by coexpression of Munc18-1WT-mCherry and BoNT/E-LC (Fig. 3 C and Fig. 6, D–F). This strongly suggests that the reduction in syntaxin-1A mobility results from syntaxin-1A engagement in the SNARE complex. A similar result was obtained in PC12 cells containing endogenous Munc18-1 (Fig. S5), further highlighting the importance of Munc18-1 in mediating this effect. To assess whether the domain 3a hinge-loop was responsible for the activity-dependent reduction in syntaxin-1A-GFP mobility, we expressed Munc18-1Δ317-333-mCherry either alone (pCMV) or in conjunction with BoNT/E-LC in DKD-PC12 cells and found that both conditions equally inhibited this confining effect and that no additive change was detected upon coexpression of Munc18-1Δ317-333-mCherry and BoNT/E-LC (Fig. 6, G–L).


The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming
BoNT/E-LC expression prevents the activity-dependent decrease in syntaxin-1A mobility. (A–L) DKD-PC12 cells transfected with the indicated plasmids were imaged at 50 Hz (16,000 frames) in either unstimulated or stimulated (2 mM Ba2+) conditions using the uPAINT technique. Mean MSD as a function of time, mean distribution of the diffusion coefficients, and ratio of mobile versus immobile fraction in indicated cells and conditions. (n = 10–19 cells for each condition). Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (*, P < 0.05). Mean ± SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037406&req=5

fig6: BoNT/E-LC expression prevents the activity-dependent decrease in syntaxin-1A mobility. (A–L) DKD-PC12 cells transfected with the indicated plasmids were imaged at 50 Hz (16,000 frames) in either unstimulated or stimulated (2 mM Ba2+) conditions using the uPAINT technique. Mean MSD as a function of time, mean distribution of the diffusion coefficients, and ratio of mobile versus immobile fraction in indicated cells and conditions. (n = 10–19 cells for each condition). Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (*, P < 0.05). Mean ± SEM.
Mentions: We predicted that the reduced mobility of syntaxin-1A elicited by activity might stem from its incorporation into the trans-SNARE complex with SNAP-25. To test this hypothesis, we used BoNT/E-LC. BoNT/E-LC enzymatic activity targets SNAP-25 and cleaves 26 residues from the C-terminus of the molecule, thereby blocking neuroexocytosis (Schiavo et al., 1993). The light chains prevent SNARE complex formation (Hayashi et al., 1994), and BoNT/E has an additional effect on the syntaxin-1A/SNAP-25 heterodimer (Rickman et al., 2004). BoNT/E-LC was cotransfected with syntaxin-1A-GFP in DKD-PC12 cells. Our results revealed that the activity-dependent decrease in syntaxin-1A-GFP mobility (Fig. 6, A–C) was blocked by coexpression of Munc18-1WT-mCherry and BoNT/E-LC (Fig. 3 C and Fig. 6, D–F). This strongly suggests that the reduction in syntaxin-1A mobility results from syntaxin-1A engagement in the SNARE complex. A similar result was obtained in PC12 cells containing endogenous Munc18-1 (Fig. S5), further highlighting the importance of Munc18-1 in mediating this effect. To assess whether the domain 3a hinge-loop was responsible for the activity-dependent reduction in syntaxin-1A-GFP mobility, we expressed Munc18-1Δ317-333-mCherry either alone (pCMV) or in conjunction with BoNT/E-LC in DKD-PC12 cells and found that both conditions equally inhibited this confining effect and that no additive change was detected upon coexpression of Munc18-1Δ317-333-mCherry and BoNT/E-LC (Fig. 6, G–L).

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Kasula et al. use single-molecule imaging to reveal the diffusional signature for the SNARE proteins Munc18-1 and syntaxin-1A during secretory vesicle priming. The authors show that a conformational change in the Munc18-1 domain 3a hinge-loop regulates engagement of syntaxin-1A in the SNARE complex.

No MeSH data available.


Related in: MedlinePlus