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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.

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The Munc18-1 domain 3a hinge-loop controls an activity-dependent decrease in syntaxin-1A mobility. DKD-PC12 cells expressing syntaxin-1A-GFP alone or with either Munc18-1WT-mCherry or Munc18-1Δ317-333-mCherry were imaged at 50 Hz (16,000 frames) in either unstimulated or stimulated (2 mM Ba2+) conditions using the uPAINT technique. (A) TIRF image (A1), spt-PALM mean intensity map (A2), and diffusion coefficient map (A3) of a DKD-PC12 cell (detections range from 10−5 to 101 µm2/s) and trajectory maps (A4) calculated from 16,000 images (trajectory color-coding in arbitrary units). (A5 and A6) Nanodomains of syntaxin-1A-GFP from the mean intensity map (outlined) were overlaid with low-mobility and freely diffusing trajectories. Nanodomains preferentially contain low-mobility molecules. Bars: 1 µm; (insets) 22 nm. (B–J) Mean MSD as a function of time, mean distribution of the diffusion coefficients, and the mobile fraction in indicated cells and conditions (n = 12–14 cells for each condition). Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (**, P < 0.01). Mean ± SEM.
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fig5: The Munc18-1 domain 3a hinge-loop controls an activity-dependent decrease in syntaxin-1A mobility. DKD-PC12 cells expressing syntaxin-1A-GFP alone or with either Munc18-1WT-mCherry or Munc18-1Δ317-333-mCherry were imaged at 50 Hz (16,000 frames) in either unstimulated or stimulated (2 mM Ba2+) conditions using the uPAINT technique. (A) TIRF image (A1), spt-PALM mean intensity map (A2), and diffusion coefficient map (A3) of a DKD-PC12 cell (detections range from 10−5 to 101 µm2/s) and trajectory maps (A4) calculated from 16,000 images (trajectory color-coding in arbitrary units). (A5 and A6) Nanodomains of syntaxin-1A-GFP from the mean intensity map (outlined) were overlaid with low-mobility and freely diffusing trajectories. Nanodomains preferentially contain low-mobility molecules. Bars: 1 µm; (insets) 22 nm. (B–J) Mean MSD as a function of time, mean distribution of the diffusion coefficients, and the mobile fraction in indicated cells and conditions (n = 12–14 cells for each condition). Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (**, P < 0.01). Mean ± SEM.

Mentions: We next examined the effect of Munc18-1 expression on syntaxin-1A mobility in DKD-PC12 cells. We first expressed syntaxin-1A-GFP in these cells. TIRF imaging of syntaxin-1A-GFP revealed a relatively homogeneous staining of the plasma membrane with some apparent small puncta (Fig. 5 A1), similar to the pattern observed for Munc18-1 (Fig. 2 A1). To investigate syntaxin-1A-GFP mobility, we used universal point accumulation imaging in nanoscale topography (uPAINT; Giannone et al., 2010), as the C-terminal GFP tag is exposed to the extracellular space. When a low concentration of ATTO 647N–coupled anti-GFP nanobodies (Kubala et al., 2010) were added to the buffer, stochastic binding of the nanobodies to syntaxin-1A-GFP could be detected during live imaging (Giannone et al., 2010). The uPAINT superresolved intensity images revealed syntaxin-1A nanodomains on the plasma membrane (Fig. 5 A2), as previously described (Lang et al., 2001). Tracking of individual molecules and mapping of their trajectories and instantaneous diffusion coefficients also revealed a high level of heterogeneity (Fig. 5, A3 and A4). Most punctate structures were formed by trajectories exhibiting low diffusion coefficients (Fig. 5 A5). Freely moving molecules were less associated with these nanodomains (Fig. 5 A6). The MSD was calculated for the trajectories of individual syntaxin-1A-GFP molecules to assess potential changes in caging in response to Ba2+ stimulation. In DKD-PC12 cells, no significant change was detected in either the MSD or the distribution of diffusion coefficients in response to stimulation (Fig. 5, B and C), as also quantified using the mobile fraction (Fig. 5 D) and area under the MSD curves (Fig. 3 B). Expression of Munc18-1WT-mCherry resulted in a reduced MSD of syntaxin-1A-GFP diffusion in unstimulated cells (Fig. 5, E–G). Moreover, the MSD of syntaxin-1A-GFP was significantly reduced in response to Ba2+ stimulation, demonstrating that the cage size of syntaxin-1A was also reduced during activity (Figs. 3 B and 5 E). Importantly, the distribution of the diffusion coefficients was also altered by stimulation, leading to a concomitant increase in the immobile fraction and a decrease in the mobile fraction (Fig. 5, F and G). We previously demonstrated that the expression of Munc18-1Δ317-333 is able to fully rescue syntaxin-1A transport to the plasma membrane in DKD-PC12 cells (Martin et al., 2013). We therefore asked whether this mutant could also control the activity-dependent change in syntaxin-1A mobility. Although we observed a slight reduction in mobility, this was not significantly different from the unstimulated condition, suggesting that the hinge-loop deletion mutant lacks the ability to control syntaxin-1A mobility (Fig. 3 B and Fig. 5, H–J). These results were confirmed by sptPALM of syntaxin-1A-mEos2 cotransfected with untagged Munc18-1WT or Munc18-1Δ317-333 (Fig. S4).


The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming
The Munc18-1 domain 3a hinge-loop controls an activity-dependent decrease in syntaxin-1A mobility. DKD-PC12 cells expressing syntaxin-1A-GFP alone or with either Munc18-1WT-mCherry or Munc18-1Δ317-333-mCherry were imaged at 50 Hz (16,000 frames) in either unstimulated or stimulated (2 mM Ba2+) conditions using the uPAINT technique. (A) TIRF image (A1), spt-PALM mean intensity map (A2), and diffusion coefficient map (A3) of a DKD-PC12 cell (detections range from 10−5 to 101 µm2/s) and trajectory maps (A4) calculated from 16,000 images (trajectory color-coding in arbitrary units). (A5 and A6) Nanodomains of syntaxin-1A-GFP from the mean intensity map (outlined) were overlaid with low-mobility and freely diffusing trajectories. Nanodomains preferentially contain low-mobility molecules. Bars: 1 µm; (insets) 22 nm. (B–J) Mean MSD as a function of time, mean distribution of the diffusion coefficients, and the mobile fraction in indicated cells and conditions (n = 12–14 cells for each condition). Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (**, P < 0.01). Mean ± SEM.
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fig5: The Munc18-1 domain 3a hinge-loop controls an activity-dependent decrease in syntaxin-1A mobility. DKD-PC12 cells expressing syntaxin-1A-GFP alone or with either Munc18-1WT-mCherry or Munc18-1Δ317-333-mCherry were imaged at 50 Hz (16,000 frames) in either unstimulated or stimulated (2 mM Ba2+) conditions using the uPAINT technique. (A) TIRF image (A1), spt-PALM mean intensity map (A2), and diffusion coefficient map (A3) of a DKD-PC12 cell (detections range from 10−5 to 101 µm2/s) and trajectory maps (A4) calculated from 16,000 images (trajectory color-coding in arbitrary units). (A5 and A6) Nanodomains of syntaxin-1A-GFP from the mean intensity map (outlined) were overlaid with low-mobility and freely diffusing trajectories. Nanodomains preferentially contain low-mobility molecules. Bars: 1 µm; (insets) 22 nm. (B–J) Mean MSD as a function of time, mean distribution of the diffusion coefficients, and the mobile fraction in indicated cells and conditions (n = 12–14 cells for each condition). Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (**, P < 0.01). Mean ± SEM.
Mentions: We next examined the effect of Munc18-1 expression on syntaxin-1A mobility in DKD-PC12 cells. We first expressed syntaxin-1A-GFP in these cells. TIRF imaging of syntaxin-1A-GFP revealed a relatively homogeneous staining of the plasma membrane with some apparent small puncta (Fig. 5 A1), similar to the pattern observed for Munc18-1 (Fig. 2 A1). To investigate syntaxin-1A-GFP mobility, we used universal point accumulation imaging in nanoscale topography (uPAINT; Giannone et al., 2010), as the C-terminal GFP tag is exposed to the extracellular space. When a low concentration of ATTO 647N–coupled anti-GFP nanobodies (Kubala et al., 2010) were added to the buffer, stochastic binding of the nanobodies to syntaxin-1A-GFP could be detected during live imaging (Giannone et al., 2010). The uPAINT superresolved intensity images revealed syntaxin-1A nanodomains on the plasma membrane (Fig. 5 A2), as previously described (Lang et al., 2001). Tracking of individual molecules and mapping of their trajectories and instantaneous diffusion coefficients also revealed a high level of heterogeneity (Fig. 5, A3 and A4). Most punctate structures were formed by trajectories exhibiting low diffusion coefficients (Fig. 5 A5). Freely moving molecules were less associated with these nanodomains (Fig. 5 A6). The MSD was calculated for the trajectories of individual syntaxin-1A-GFP molecules to assess potential changes in caging in response to Ba2+ stimulation. In DKD-PC12 cells, no significant change was detected in either the MSD or the distribution of diffusion coefficients in response to stimulation (Fig. 5, B and C), as also quantified using the mobile fraction (Fig. 5 D) and area under the MSD curves (Fig. 3 B). Expression of Munc18-1WT-mCherry resulted in a reduced MSD of syntaxin-1A-GFP diffusion in unstimulated cells (Fig. 5, E–G). Moreover, the MSD of syntaxin-1A-GFP was significantly reduced in response to Ba2+ stimulation, demonstrating that the cage size of syntaxin-1A was also reduced during activity (Figs. 3 B and 5 E). Importantly, the distribution of the diffusion coefficients was also altered by stimulation, leading to a concomitant increase in the immobile fraction and a decrease in the mobile fraction (Fig. 5, F and G). We previously demonstrated that the expression of Munc18-1Δ317-333 is able to fully rescue syntaxin-1A transport to the plasma membrane in DKD-PC12 cells (Martin et al., 2013). We therefore asked whether this mutant could also control the activity-dependent change in syntaxin-1A mobility. Although we observed a slight reduction in mobility, this was not significantly different from the unstimulated condition, suggesting that the hinge-loop deletion mutant lacks the ability to control syntaxin-1A mobility (Fig. 3 B and Fig. 5, H–J). These results were confirmed by sptPALM of syntaxin-1A-mEos2 cotransfected with untagged Munc18-1WT or Munc18-1Δ317-333 (Fig. S4).

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