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

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

The size of Munc18-1 but not Munc18-1Δ317-333 nanodomains is reduced by secretagogue stimulation. Transfected DKD-PC12 cells expressing Munc18-1WT-mEos2 (A–C) or Munc18-1Δ317-333-mEos2 (D–F) were incubated with or without 2 mM BaCl2 for 2 min in buffer A, then fixed in paraformaldehyde before single-molecule localization (SML) imaging and processing. (A and D) Representative low-resolution (green fluorescence TIRF) image and corresponding SML image of unstimulated (top) and stimulated (bottom) Munc18-1WT-mEos2 (A) or Munc18-1Δ317-333-mEos2 (D). Bars: (low-resolution) 2 µm; (enlarged) 200 nm. (B and E) Mean (± SEM) of autocorrelation functions for each region of interest. Three regions of interest were analyzed for each cell, and 10 cells were analyzed for each condition. The experiment was performed three times independently. (C and F) The mean radius (Eq. 1) and the number of molecules per nanodomain (Eq. 2) were obtained from each region of interest by fitting the autocorrelation values. **, P < 0.01, Student’s t test.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: The size of Munc18-1 but not Munc18-1Δ317-333 nanodomains is reduced by secretagogue stimulation. Transfected DKD-PC12 cells expressing Munc18-1WT-mEos2 (A–C) or Munc18-1Δ317-333-mEos2 (D–F) were incubated with or without 2 mM BaCl2 for 2 min in buffer A, then fixed in paraformaldehyde before single-molecule localization (SML) imaging and processing. (A and D) Representative low-resolution (green fluorescence TIRF) image and corresponding SML image of unstimulated (top) and stimulated (bottom) Munc18-1WT-mEos2 (A) or Munc18-1Δ317-333-mEos2 (D). Bars: (low-resolution) 2 µm; (enlarged) 200 nm. (B and E) Mean (± SEM) of autocorrelation functions for each region of interest. Three regions of interest were analyzed for each cell, and 10 cells were analyzed for each condition. The experiment was performed three times independently. (C and F) The mean radius (Eq. 1) and the number of molecules per nanodomain (Eq. 2) were obtained from each region of interest by fitting the autocorrelation values. **, P < 0.01, Student’s t test.

Mentions: Because Munc18-1 is distributed in nanodomains on the plasma membrane (Smyth et al., 2013; Kavanagh et al., 2014), we hypothesized that such an activity-dependent increase in mobility might lead to a reduction in the number of molecules per nanodomain and a concomitant decrease in their size. To assess this, resting or stimulated DKD-PC12 cells expressing either Munc18-1WT-mEos2 or Munc18-1Δ317-333-mEos2 were fixed before PALM imaging (Fig. 4, A–F). To determine the characteristics of the nanodomains of Munc18-1WT-mEos2 and Munc18-1Δ317-333-mEos2, an auto-correlation function was applied, which quantifies the spatial distribution of the proteins (Sengupta et al., 2011; Veatch et al., 2012; Harper et al., 2016). Fitting of the values to Eq. 1 enabled us to obtain parameters of the nanodomains. Munc18-1WT-mEos2 molecules exhibited a range of nanodomain sizes (Fig. 4 A), with a radius of 36.0 ± 1.2 nm (mean ± SEM; Fig. 4 C). Importantly, both the mean nanodomain radius and the calculated number of molecules within Munc18-1WT-mEos2 nanodomains were significantly reduced after stimulation (Fig. 4 C). The number of molecules per nanodomain was relatively low (Fig. 4 C), which is likely caused by incomplete conversion of mEos2 (Sengupta et al., 2011). In contrast, the calculated radius and the number of localizations per nanodomain for Munc18-1Δ317-333-mEos2 were largely unaffected by stimulation (Fig. 4 F). These results are consistent with a role for the domain 3a hinge-loop in triggering the release of previously immobile Munc18-1 from these nanodomains in response to secretagogue stimulation.


The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming
The size of Munc18-1 but not Munc18-1Δ317-333 nanodomains is reduced by secretagogue stimulation. Transfected DKD-PC12 cells expressing Munc18-1WT-mEos2 (A–C) or Munc18-1Δ317-333-mEos2 (D–F) were incubated with or without 2 mM BaCl2 for 2 min in buffer A, then fixed in paraformaldehyde before single-molecule localization (SML) imaging and processing. (A and D) Representative low-resolution (green fluorescence TIRF) image and corresponding SML image of unstimulated (top) and stimulated (bottom) Munc18-1WT-mEos2 (A) or Munc18-1Δ317-333-mEos2 (D). Bars: (low-resolution) 2 µm; (enlarged) 200 nm. (B and E) Mean (± SEM) of autocorrelation functions for each region of interest. Three regions of interest were analyzed for each cell, and 10 cells were analyzed for each condition. The experiment was performed three times independently. (C and F) The mean radius (Eq. 1) and the number of molecules per nanodomain (Eq. 2) were obtained from each region of interest by fitting the autocorrelation values. **, P < 0.01, Student’s t test.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: The size of Munc18-1 but not Munc18-1Δ317-333 nanodomains is reduced by secretagogue stimulation. Transfected DKD-PC12 cells expressing Munc18-1WT-mEos2 (A–C) or Munc18-1Δ317-333-mEos2 (D–F) were incubated with or without 2 mM BaCl2 for 2 min in buffer A, then fixed in paraformaldehyde before single-molecule localization (SML) imaging and processing. (A and D) Representative low-resolution (green fluorescence TIRF) image and corresponding SML image of unstimulated (top) and stimulated (bottom) Munc18-1WT-mEos2 (A) or Munc18-1Δ317-333-mEos2 (D). Bars: (low-resolution) 2 µm; (enlarged) 200 nm. (B and E) Mean (± SEM) of autocorrelation functions for each region of interest. Three regions of interest were analyzed for each cell, and 10 cells were analyzed for each condition. The experiment was performed three times independently. (C and F) The mean radius (Eq. 1) and the number of molecules per nanodomain (Eq. 2) were obtained from each region of interest by fitting the autocorrelation values. **, P < 0.01, Student’s t test.
Mentions: Because Munc18-1 is distributed in nanodomains on the plasma membrane (Smyth et al., 2013; Kavanagh et al., 2014), we hypothesized that such an activity-dependent increase in mobility might lead to a reduction in the number of molecules per nanodomain and a concomitant decrease in their size. To assess this, resting or stimulated DKD-PC12 cells expressing either Munc18-1WT-mEos2 or Munc18-1Δ317-333-mEos2 were fixed before PALM imaging (Fig. 4, A–F). To determine the characteristics of the nanodomains of Munc18-1WT-mEos2 and Munc18-1Δ317-333-mEos2, an auto-correlation function was applied, which quantifies the spatial distribution of the proteins (Sengupta et al., 2011; Veatch et al., 2012; Harper et al., 2016). Fitting of the values to Eq. 1 enabled us to obtain parameters of the nanodomains. Munc18-1WT-mEos2 molecules exhibited a range of nanodomain sizes (Fig. 4 A), with a radius of 36.0 ± 1.2 nm (mean ± SEM; Fig. 4 C). Importantly, both the mean nanodomain radius and the calculated number of molecules within Munc18-1WT-mEos2 nanodomains were significantly reduced after stimulation (Fig. 4 C). The number of molecules per nanodomain was relatively low (Fig. 4 C), which is likely caused by incomplete conversion of mEos2 (Sengupta et al., 2011). In contrast, the calculated radius and the number of localizations per nanodomain for Munc18-1Δ317-333-mEos2 were largely unaffected by stimulation (Fig. 4 F). These results are consistent with a role for the domain 3a hinge-loop in triggering the release of previously immobile Munc18-1 from these nanodomains in response to secretagogue stimulation.

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