Limits...
Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension.

Bretou M, Jouannot O, Fanget I, Pierobon P, Larochette N, Gestraud P, Guillon M, Emiliani V, Gasman S, Desnos C, Lennon-Duménil AM, Darchen F - Mol. Biol. Cell (2014)

Bottom Line: Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood.Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation.We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.

View Article: PubMed Central - PubMed

Affiliation: Université Paris Descartes, Sorbonne Paris Cité, Centre National de la Recherche Scientifique, UMR 8250, 75270 Paris Cedex 06, France Institut National de la Santé et de la Recherche Médicale, U932, Institut Curie, 75005 Paris, France.

Show MeSH

Related in: MedlinePlus

A dual-color TIRFM assay to measure the dynamics of the fusion pore. BON cells expressing both NPY-mRFP and NPY-pHluorin were stimulated with ionomycin and imaged by dual-color TIRFM. (A) Cartoons and time-series images (10 Hz) showing the behavior of pH-insensitive (mRFP, red, top) and pH-sensitive (pHluorin, green, bottom) vesicular content markers upon full-collapse fusion. Fast diffusion of fluorescent markers in the external medium is captured as a fluorescent halo surrounding the SG and leads to complete disappearance of SG fluorescence. In kiss-and-run events (B, C), the pore does not fully enlarge, and NPY-mRFP is slowly (B) or not (C) released; in some cases, the pore reseals (C), leading to reacidification of the SG, as indicated by a decay of the green signal. (D) Mean (±SEM) time course of the fluorescence (arbitrary units) of events associated with (red) or without (blue) a fluorescent halo. (E–G) Cdc42 knockdown increases the fraction of kiss-and-run events (p < 0.0001, Kruskal–Wallis followed by Dunn's test; **p < 0.01; ***p < 0.001). These values were measured in each cell, normalized to the mean value found in control cells, and averaged over the different cells from the same group. The number of analyzed cells is indicated in the bars or above the boxes. NS, nonsignificant. Both effects are rescued by expressing a Cdc42 construct insensitive to siCdc42-A (E, rescue-A) or to siCdc42-C (F, rescue-C). In these experiments, the mean (±SEM) percentage of kiss-and-run events in control cells was 26.6 ± 2.3%. (G) Cdc42 knockdown increased the duration of the pHluorin signal. Cumulative distribution of the pHluorin signal duration measured using an automatic routine. A total of 2746 (control), 1595 (siCdc42-A), 1193 (rescue-A), 727 (siCdc42-C), and 394 (rescue-C) events were automatically analyzed. (H) Expressing a dominant negative Cdc42 construct (mCherry-Cdc42-N17) decreased the fraction of kiss-and-run events, whereas expressing WT Cdc42 had no significant effect. Whiskers in box plots indicate 10th–90th percentiles. Scale bars, 1 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4196869&req=5

Figure 6: A dual-color TIRFM assay to measure the dynamics of the fusion pore. BON cells expressing both NPY-mRFP and NPY-pHluorin were stimulated with ionomycin and imaged by dual-color TIRFM. (A) Cartoons and time-series images (10 Hz) showing the behavior of pH-insensitive (mRFP, red, top) and pH-sensitive (pHluorin, green, bottom) vesicular content markers upon full-collapse fusion. Fast diffusion of fluorescent markers in the external medium is captured as a fluorescent halo surrounding the SG and leads to complete disappearance of SG fluorescence. In kiss-and-run events (B, C), the pore does not fully enlarge, and NPY-mRFP is slowly (B) or not (C) released; in some cases, the pore reseals (C), leading to reacidification of the SG, as indicated by a decay of the green signal. (D) Mean (±SEM) time course of the fluorescence (arbitrary units) of events associated with (red) or without (blue) a fluorescent halo. (E–G) Cdc42 knockdown increases the fraction of kiss-and-run events (p < 0.0001, Kruskal–Wallis followed by Dunn's test; **p < 0.01; ***p < 0.001). These values were measured in each cell, normalized to the mean value found in control cells, and averaged over the different cells from the same group. The number of analyzed cells is indicated in the bars or above the boxes. NS, nonsignificant. Both effects are rescued by expressing a Cdc42 construct insensitive to siCdc42-A (E, rescue-A) or to siCdc42-C (F, rescue-C). In these experiments, the mean (±SEM) percentage of kiss-and-run events in control cells was 26.6 ± 2.3%. (G) Cdc42 knockdown increased the duration of the pHluorin signal. Cumulative distribution of the pHluorin signal duration measured using an automatic routine. A total of 2746 (control), 1595 (siCdc42-A), 1193 (rescue-A), 727 (siCdc42-C), and 394 (rescue-C) events were automatically analyzed. (H) Expressing a dominant negative Cdc42 construct (mCherry-Cdc42-N17) decreased the fraction of kiss-and-run events, whereas expressing WT Cdc42 had no significant effect. Whiskers in box plots indicate 10th–90th percentiles. Scale bars, 1 μm.

Mentions: To elucidate the role of Cdc42 in the secretory process, we first monitored SG trafficking and exocytosis in enterochromaffin BON cells silenced or not for Cdc42. Human BON cells are derived from a pancreatic metastasis of a gut neuroendocrine tumor (Evers et al., 1991). These cells store serotonin and various peptides in SGs and secrete them upon stimulation. SGs were labeled by expressing green fluorescent protein– or monomeric red fluorescent protein–tagged neuropeptide Y (NPY-GFP and NPY-mRFP, respectively) and imaged by total internal reflection fluorescence microscopy (TIRFM). Full-fusion events can be easily detected due to their typical signature in TIRFM: a transient elevation of SG fluorescence is followed by a rapid decay as NPY-GFP diffuses into the external medium, which can be captured as a fluorescent halo (Figure 1A). Complete release is typically achieved in <200 ms (Figure 1A; also see Figure 6, A–D, later in the article). Two silencing RNA duplexes (siRNAs) that significantly reduced Cdc42 levels (Figure 1B) decreased the number of SG full-fusion events (Figure 1, C and D, and Supplemental Videos S1–S3). Coexpression of Cdc42 constructs insensitive to siCdc42-A (rescue-A) or siCdc42-C (rescue-C) restored at least partially the secretory responses, arguing against off-target effects of the siRNAs (Figure 1, C and D). Consistently, overexpression of a dominant negative Cdc42 construct (N17) also reduced the occurrence of full-fusion events (Figure 1E). These observations are in agreement with results obtained in PC12 cells (Gasman et al., 2004; Malacombe et al., 2006; Sato et al., 2012). In our experiments, secretion was triggered by ionomycin, a calcium ionophore that bypasses calcium channels, ruling out the possibility that the observed inhibition of exocytosis resulted from decreased calcium entry. Cdc42 knockdown induced a slight and nonsignificant reduction in the density of SGs in the juxtamembrane area, as measured by TIRFM (Figure 1, F and G), and did not change the colocalization of NPY-mRFP with Rab27a, a marker of SGs (Desnos et al., 2003) on confocal images (Figure 1, H and I), indicating that the biogenesis of SGs and their accumulation at the cell periphery were not significantly impaired.


Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension.

Bretou M, Jouannot O, Fanget I, Pierobon P, Larochette N, Gestraud P, Guillon M, Emiliani V, Gasman S, Desnos C, Lennon-Duménil AM, Darchen F - Mol. Biol. Cell (2014)

A dual-color TIRFM assay to measure the dynamics of the fusion pore. BON cells expressing both NPY-mRFP and NPY-pHluorin were stimulated with ionomycin and imaged by dual-color TIRFM. (A) Cartoons and time-series images (10 Hz) showing the behavior of pH-insensitive (mRFP, red, top) and pH-sensitive (pHluorin, green, bottom) vesicular content markers upon full-collapse fusion. Fast diffusion of fluorescent markers in the external medium is captured as a fluorescent halo surrounding the SG and leads to complete disappearance of SG fluorescence. In kiss-and-run events (B, C), the pore does not fully enlarge, and NPY-mRFP is slowly (B) or not (C) released; in some cases, the pore reseals (C), leading to reacidification of the SG, as indicated by a decay of the green signal. (D) Mean (±SEM) time course of the fluorescence (arbitrary units) of events associated with (red) or without (blue) a fluorescent halo. (E–G) Cdc42 knockdown increases the fraction of kiss-and-run events (p < 0.0001, Kruskal–Wallis followed by Dunn's test; **p < 0.01; ***p < 0.001). These values were measured in each cell, normalized to the mean value found in control cells, and averaged over the different cells from the same group. The number of analyzed cells is indicated in the bars or above the boxes. NS, nonsignificant. Both effects are rescued by expressing a Cdc42 construct insensitive to siCdc42-A (E, rescue-A) or to siCdc42-C (F, rescue-C). In these experiments, the mean (±SEM) percentage of kiss-and-run events in control cells was 26.6 ± 2.3%. (G) Cdc42 knockdown increased the duration of the pHluorin signal. Cumulative distribution of the pHluorin signal duration measured using an automatic routine. A total of 2746 (control), 1595 (siCdc42-A), 1193 (rescue-A), 727 (siCdc42-C), and 394 (rescue-C) events were automatically analyzed. (H) Expressing a dominant negative Cdc42 construct (mCherry-Cdc42-N17) decreased the fraction of kiss-and-run events, whereas expressing WT Cdc42 had no significant effect. Whiskers in box plots indicate 10th–90th percentiles. Scale bars, 1 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4196869&req=5

Figure 6: A dual-color TIRFM assay to measure the dynamics of the fusion pore. BON cells expressing both NPY-mRFP and NPY-pHluorin were stimulated with ionomycin and imaged by dual-color TIRFM. (A) Cartoons and time-series images (10 Hz) showing the behavior of pH-insensitive (mRFP, red, top) and pH-sensitive (pHluorin, green, bottom) vesicular content markers upon full-collapse fusion. Fast diffusion of fluorescent markers in the external medium is captured as a fluorescent halo surrounding the SG and leads to complete disappearance of SG fluorescence. In kiss-and-run events (B, C), the pore does not fully enlarge, and NPY-mRFP is slowly (B) or not (C) released; in some cases, the pore reseals (C), leading to reacidification of the SG, as indicated by a decay of the green signal. (D) Mean (±SEM) time course of the fluorescence (arbitrary units) of events associated with (red) or without (blue) a fluorescent halo. (E–G) Cdc42 knockdown increases the fraction of kiss-and-run events (p < 0.0001, Kruskal–Wallis followed by Dunn's test; **p < 0.01; ***p < 0.001). These values were measured in each cell, normalized to the mean value found in control cells, and averaged over the different cells from the same group. The number of analyzed cells is indicated in the bars or above the boxes. NS, nonsignificant. Both effects are rescued by expressing a Cdc42 construct insensitive to siCdc42-A (E, rescue-A) or to siCdc42-C (F, rescue-C). In these experiments, the mean (±SEM) percentage of kiss-and-run events in control cells was 26.6 ± 2.3%. (G) Cdc42 knockdown increased the duration of the pHluorin signal. Cumulative distribution of the pHluorin signal duration measured using an automatic routine. A total of 2746 (control), 1595 (siCdc42-A), 1193 (rescue-A), 727 (siCdc42-C), and 394 (rescue-C) events were automatically analyzed. (H) Expressing a dominant negative Cdc42 construct (mCherry-Cdc42-N17) decreased the fraction of kiss-and-run events, whereas expressing WT Cdc42 had no significant effect. Whiskers in box plots indicate 10th–90th percentiles. Scale bars, 1 μm.
Mentions: To elucidate the role of Cdc42 in the secretory process, we first monitored SG trafficking and exocytosis in enterochromaffin BON cells silenced or not for Cdc42. Human BON cells are derived from a pancreatic metastasis of a gut neuroendocrine tumor (Evers et al., 1991). These cells store serotonin and various peptides in SGs and secrete them upon stimulation. SGs were labeled by expressing green fluorescent protein– or monomeric red fluorescent protein–tagged neuropeptide Y (NPY-GFP and NPY-mRFP, respectively) and imaged by total internal reflection fluorescence microscopy (TIRFM). Full-fusion events can be easily detected due to their typical signature in TIRFM: a transient elevation of SG fluorescence is followed by a rapid decay as NPY-GFP diffuses into the external medium, which can be captured as a fluorescent halo (Figure 1A). Complete release is typically achieved in <200 ms (Figure 1A; also see Figure 6, A–D, later in the article). Two silencing RNA duplexes (siRNAs) that significantly reduced Cdc42 levels (Figure 1B) decreased the number of SG full-fusion events (Figure 1, C and D, and Supplemental Videos S1–S3). Coexpression of Cdc42 constructs insensitive to siCdc42-A (rescue-A) or siCdc42-C (rescue-C) restored at least partially the secretory responses, arguing against off-target effects of the siRNAs (Figure 1, C and D). Consistently, overexpression of a dominant negative Cdc42 construct (N17) also reduced the occurrence of full-fusion events (Figure 1E). These observations are in agreement with results obtained in PC12 cells (Gasman et al., 2004; Malacombe et al., 2006; Sato et al., 2012). In our experiments, secretion was triggered by ionomycin, a calcium ionophore that bypasses calcium channels, ruling out the possibility that the observed inhibition of exocytosis resulted from decreased calcium entry. Cdc42 knockdown induced a slight and nonsignificant reduction in the density of SGs in the juxtamembrane area, as measured by TIRFM (Figure 1, F and G), and did not change the colocalization of NPY-mRFP with Rab27a, a marker of SGs (Desnos et al., 2003) on confocal images (Figure 1, H and I), indicating that the biogenesis of SGs and their accumulation at the cell periphery were not significantly impaired.

Bottom Line: Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood.Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation.We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.

View Article: PubMed Central - PubMed

Affiliation: Université Paris Descartes, Sorbonne Paris Cité, Centre National de la Recherche Scientifique, UMR 8250, 75270 Paris Cedex 06, France Institut National de la Santé et de la Recherche Médicale, U932, Institut Curie, 75005 Paris, France.

Show MeSH
Related in: MedlinePlus