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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 3a domain hinge-loop promotes an activity-dependent increase in Munc18-1 mobility. DKD-PC12 cells expressing Munc18-1WT-mEos2 or Munc18-1Δ317-333-mEos2 were imaged at 50 Hz in either unstimulated or stimulated (2 mM Ba2+) conditions. (A) TIRF image (A1), sptPALM 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) Trajectory color-coding calculated from 16,000 images (in arbitrary units). (A5 and A6) Nanodomains of Munc18-1 from the mean intensity map (outlined) were overlaid with low-mobility and freely diffusing trajectories. Nanodomains preferentially contain low-mobility molecules. Bars: (A1) 1 µm; (A2, inset) 500 nm; (A5) 100 nm. (B–G) Mean MSD as a function of time and mean distribution of the diffusion coefficient and the mobile fraction in indicated cells and conditions (n = 13–15 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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fig2: The Munc18-1 3a domain hinge-loop promotes an activity-dependent increase in Munc18-1 mobility. DKD-PC12 cells expressing Munc18-1WT-mEos2 or Munc18-1Δ317-333-mEos2 were imaged at 50 Hz in either unstimulated or stimulated (2 mM Ba2+) conditions. (A) TIRF image (A1), sptPALM 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) Trajectory color-coding calculated from 16,000 images (in arbitrary units). (A5 and A6) Nanodomains of Munc18-1 from the mean intensity map (outlined) were overlaid with low-mobility and freely diffusing trajectories. Nanodomains preferentially contain low-mobility molecules. Bars: (A1) 1 µm; (A2, inset) 500 nm; (A5) 100 nm. (B–G) Mean MSD as a function of time and mean distribution of the diffusion coefficient and the mobile fraction in indicated cells and conditions (n = 13–15 cells for each condition). Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (**, P < 0.01). Mean ± SEM.

Mentions: That Munc18-1Δ317-333 can rescue vesicle docking but not exocytic fusion events (Martin et al., 2013) suggests that the hinge-loop plays a critical role in priming docked SVs for fusion. To investigate this, we turned to single-molecule imaging of Munc18-1 to measure the changes in mobility patterns associated with SV priming. To generate high-density maps of Munc18-1 localization and mobility at the cell surface, DKD-PC12 cells were transfected with Munc18-1WT fused to photoconvertible mEos2 and imaged by single-particle tracking photoactivated localization microscopy (sptPALM; Manley et al., 2008). We first confirmed that the mEos2 tag did not interfere with the ability of Munc18-1 to rescue neuroexocytosis in DKD-PC12 cells (Fig. S2, A and B). We also checked the expression level of Munc18-1WT-mEos2 in transfected DKD-PC12 cells and found that it was comparable to that of endogenous Munc18-1 in PC12 cells considering a 40–50% transfection efficiency (Fig. S2 C). We then turned to conventional TIRF imaging of Munc18-1WT-mEos2, which produced relatively homogeneous staining with some apparent small puncta (Fig. 2 A1). In contrast, the sptPALM superresolved-intensity image revealed the organization and plasma membrane distribution of Munc18-1WT-mEos2 (Fig. 2, A2 and A3). Tracking individual molecules allowed us to generate maps of their trajectories and instantaneous diffusion coefficients (Nair et al., 2013; Fig. 2 A4), highlighting a high level of heterogeneity in the mobility of Munc18-1WT-mEos2 in unstimulated cells. Indeed, two distinct populations were present, mobile and confined, with the latter representing nanodomains (Fig. 2, A5 and A6). Most punctate structures displayed a relatively restricted radius of diffusion, or “caging,” consistent with previous studies of Munc18-1 and syntaxin-1A mobility in resting neurons (Kavanagh et al., 2014; Fig. 2, A5 and A6).


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 3a domain hinge-loop promotes an activity-dependent increase in Munc18-1 mobility. DKD-PC12 cells expressing Munc18-1WT-mEos2 or Munc18-1Δ317-333-mEos2 were imaged at 50 Hz in either unstimulated or stimulated (2 mM Ba2+) conditions. (A) TIRF image (A1), sptPALM 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) Trajectory color-coding calculated from 16,000 images (in arbitrary units). (A5 and A6) Nanodomains of Munc18-1 from the mean intensity map (outlined) were overlaid with low-mobility and freely diffusing trajectories. Nanodomains preferentially contain low-mobility molecules. Bars: (A1) 1 µm; (A2, inset) 500 nm; (A5) 100 nm. (B–G) Mean MSD as a function of time and mean distribution of the diffusion coefficient and the mobile fraction in indicated cells and conditions (n = 13–15 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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fig2: The Munc18-1 3a domain hinge-loop promotes an activity-dependent increase in Munc18-1 mobility. DKD-PC12 cells expressing Munc18-1WT-mEos2 or Munc18-1Δ317-333-mEos2 were imaged at 50 Hz in either unstimulated or stimulated (2 mM Ba2+) conditions. (A) TIRF image (A1), sptPALM 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) Trajectory color-coding calculated from 16,000 images (in arbitrary units). (A5 and A6) Nanodomains of Munc18-1 from the mean intensity map (outlined) were overlaid with low-mobility and freely diffusing trajectories. Nanodomains preferentially contain low-mobility molecules. Bars: (A1) 1 µm; (A2, inset) 500 nm; (A5) 100 nm. (B–G) Mean MSD as a function of time and mean distribution of the diffusion coefficient and the mobile fraction in indicated cells and conditions (n = 13–15 cells for each condition). Sidak–Bonferroni adjustments were made while performing multiple t test comparisons of mobile fractions (**, P < 0.01). Mean ± SEM.
Mentions: That Munc18-1Δ317-333 can rescue vesicle docking but not exocytic fusion events (Martin et al., 2013) suggests that the hinge-loop plays a critical role in priming docked SVs for fusion. To investigate this, we turned to single-molecule imaging of Munc18-1 to measure the changes in mobility patterns associated with SV priming. To generate high-density maps of Munc18-1 localization and mobility at the cell surface, DKD-PC12 cells were transfected with Munc18-1WT fused to photoconvertible mEos2 and imaged by single-particle tracking photoactivated localization microscopy (sptPALM; Manley et al., 2008). We first confirmed that the mEos2 tag did not interfere with the ability of Munc18-1 to rescue neuroexocytosis in DKD-PC12 cells (Fig. S2, A and B). We also checked the expression level of Munc18-1WT-mEos2 in transfected DKD-PC12 cells and found that it was comparable to that of endogenous Munc18-1 in PC12 cells considering a 40–50% transfection efficiency (Fig. S2 C). We then turned to conventional TIRF imaging of Munc18-1WT-mEos2, which produced relatively homogeneous staining with some apparent small puncta (Fig. 2 A1). In contrast, the sptPALM superresolved-intensity image revealed the organization and plasma membrane distribution of Munc18-1WT-mEos2 (Fig. 2, A2 and A3). Tracking individual molecules allowed us to generate maps of their trajectories and instantaneous diffusion coefficients (Nair et al., 2013; Fig. 2 A4), highlighting a high level of heterogeneity in the mobility of Munc18-1WT-mEos2 in unstimulated cells. Indeed, two distinct populations were present, mobile and confined, with the latter representing nanodomains (Fig. 2, A5 and A6). Most punctate structures displayed a relatively restricted radius of diffusion, or “caging,” consistent with previous studies of Munc18-1 and syntaxin-1A mobility in resting neurons (Kavanagh et al., 2014; Fig. 2, A5 and A6).

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