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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.


Related in: MedlinePlus

Area under the MSD curves. (A–C) 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. Area under the MSD curves for each cell in each condition. Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (*, P < 0.05; **, P < 0.01). Mean ± SEM.
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fig3: Area under the MSD curves. (A–C) 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. Area under the MSD curves for each cell in each condition. Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (*, P < 0.05; **, P < 0.01). Mean ± SEM.

Mentions: We next analyzed the MSD of the sptPALM trajectories of individual Munc18-1WT-mEos2 molecules to assess changes in mobility in response to Ba2+ secretagogue stimulation (Fig. 2, B–G). We compared the MSD of trajectories of Munc18-1WT-mEos2 molecules from unstimulated and stimulated DKD-PC12 cells (Fig. 2 B) and found that the areas under these curves were significantly different (Fig. 3 A). Analysis of the distribution of the diffusion coefficients of Munc18-1WT-mEos2 single-molecule trajectories in unstimulated cells revealed the presence of two distinct populations: an immobile fraction and a mobile fraction (Fig. 2 C; Constals et al., 2015). Importantly, Munc18-1WT-mEos2 mobility was significantly altered in stimulated DKD-PC12 cells, as indicated by changes in the MSD and diffusion coefficient (Fig. 2, B and C). Stimulation resulted in a significant increase in the mobile fraction (Fig. 2 D). This suggests that a population of Munc18-1WT-mEos2 molecules is released from the confinement of nanodomains (Fig. 2, B–D). In comparison to Munc18-1WT-mEos2, the MSD of Munc18-1Δ317-333-mEos2 was largely unchanged in the resting cells (Fig. 2 E). Strikingly, we did not detect the same shift toward a more mobile fraction for the hinge-loop deletion mutant after stimulation (Fig. 2, F and G), suggesting that the mutation prevented the activity-dependent release of Munc18-1 from the confinement of nanodomains.


The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming
Area under the MSD curves. (A–C) 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. Area under the MSD curves for each cell in each condition. Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (*, P < 0.05; **, P < 0.01). Mean ± SEM.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037406&req=5

fig3: Area under the MSD curves. (A–C) 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. Area under the MSD curves for each cell in each condition. Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (*, P < 0.05; **, P < 0.01). Mean ± SEM.
Mentions: We next analyzed the MSD of the sptPALM trajectories of individual Munc18-1WT-mEos2 molecules to assess changes in mobility in response to Ba2+ secretagogue stimulation (Fig. 2, B–G). We compared the MSD of trajectories of Munc18-1WT-mEos2 molecules from unstimulated and stimulated DKD-PC12 cells (Fig. 2 B) and found that the areas under these curves were significantly different (Fig. 3 A). Analysis of the distribution of the diffusion coefficients of Munc18-1WT-mEos2 single-molecule trajectories in unstimulated cells revealed the presence of two distinct populations: an immobile fraction and a mobile fraction (Fig. 2 C; Constals et al., 2015). Importantly, Munc18-1WT-mEos2 mobility was significantly altered in stimulated DKD-PC12 cells, as indicated by changes in the MSD and diffusion coefficient (Fig. 2, B and C). Stimulation resulted in a significant increase in the mobile fraction (Fig. 2 D). This suggests that a population of Munc18-1WT-mEos2 molecules is released from the confinement of nanodomains (Fig. 2, B–D). In comparison to Munc18-1WT-mEos2, the MSD of Munc18-1Δ317-333-mEos2 was largely unchanged in the resting cells (Fig. 2 E). Strikingly, we did not detect the same shift toward a more mobile fraction for the hinge-loop deletion mutant after stimulation (Fig. 2, F and G), suggesting that the mutation prevented the activity-dependent release of Munc18-1 from the confinement of nanodomains.

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