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Age-dependent preferential dense-core vesicle exocytosis in neuroendocrine cells revealed by newly developed monomeric fluorescent timer protein.

Tsuboi T, Kitaguchi T, Karasawa S, Fukuda M, Miyawaki A - Mol. Biol. Cell (2009)

Bottom Line: Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells.When the vesicles were labeled with mK-GO-tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed.Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro, Tokyo, Japan. takatsuboi@bio.c.utokyo.ac.jp

ABSTRACT
Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO-tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K(+)] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.

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Effect of overexpression or depletion of rabphilin on the dense-core vesicle exocytosis in PC12 cells. (A) Typical TIRF images of plasma membrane-docked green colored- and red colored-NPY-mK-GO vesicles before high-KCl stimulation of scrambled siRNA-transfected control (control), rabphilin-overexpressing (rabphilin), and rabphilin-siRNA-transfected cells (rabphilin-siRNA). The cells were used for experiments in 48 h after transfection. Bar, 10 μm. (B) Stacked bar chart represents the percentage of the number of plasma membrane-docked green, yellow, and red vesicles in control, rabphilin-, and rabphilin-siRNA–transfected cells (n = 35 cells in each). (C) Effect of rabphilin siRNA on expression of rabphilin in PC12 cells. PC12 cells were transfected with rabphilin siRNA (right) or a scrambled-siRNA control (left). Cell lysates were prepared and subjected to 12.5% SDS-PAGE followed by immunoblotting with anti-rabphilin and anti-actin antibodies. The positions of the molecular mass markers (×10−3) are shown on the left. The results shown are representative of three independent experiments with similar results. (D) The density of plasma membrane-docked vesicles was determined by counting the vesicles in each image (n = 31 cells in each). (E). Histograms represent the mean number of plasma membrane-docked NPY-mK-GO vesicles in control (left), rabphilin overexpressing (center), or rabphilin siRNA-transfected cells (right) before (closed bars) and after (open bars) high-KCl stimulation. (F) The effect of expression of rabphilin, or rabphilin siRNA on the percentage of released vesicles. Note that overexpression of rabphilin induced exocytotic events from all colors of NPY-mK-GO vesicles. *p < 0.05 and ***p < 0.001, respectively, in comparison with the control. The results are means ± SE.
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Figure 4: Effect of overexpression or depletion of rabphilin on the dense-core vesicle exocytosis in PC12 cells. (A) Typical TIRF images of plasma membrane-docked green colored- and red colored-NPY-mK-GO vesicles before high-KCl stimulation of scrambled siRNA-transfected control (control), rabphilin-overexpressing (rabphilin), and rabphilin-siRNA-transfected cells (rabphilin-siRNA). The cells were used for experiments in 48 h after transfection. Bar, 10 μm. (B) Stacked bar chart represents the percentage of the number of plasma membrane-docked green, yellow, and red vesicles in control, rabphilin-, and rabphilin-siRNA–transfected cells (n = 35 cells in each). (C) Effect of rabphilin siRNA on expression of rabphilin in PC12 cells. PC12 cells were transfected with rabphilin siRNA (right) or a scrambled-siRNA control (left). Cell lysates were prepared and subjected to 12.5% SDS-PAGE followed by immunoblotting with anti-rabphilin and anti-actin antibodies. The positions of the molecular mass markers (×10−3) are shown on the left. The results shown are representative of three independent experiments with similar results. (D) The density of plasma membrane-docked vesicles was determined by counting the vesicles in each image (n = 31 cells in each). (E). Histograms represent the mean number of plasma membrane-docked NPY-mK-GO vesicles in control (left), rabphilin overexpressing (center), or rabphilin siRNA-transfected cells (right) before (closed bars) and after (open bars) high-KCl stimulation. (F) The effect of expression of rabphilin, or rabphilin siRNA on the percentage of released vesicles. Note that overexpression of rabphilin induced exocytotic events from all colors of NPY-mK-GO vesicles. *p < 0.05 and ***p < 0.001, respectively, in comparison with the control. The results are means ± SE.

Mentions: Overexpression of rabphilin increased plasma membrane-docked vesicles (Figure 4, A and D), especially red vesicles (Figure 4B), whereas knockdown of endogenous rabphilin by siRNA (Figure 4C) decreased the number of plasma membrane-docked vesicles revealed by TIRF microscope (Figure 4, A and D). However, it is possible that overexpression of rabphilin affects the distribution of green- and red-colored vesicles inside of the cells. To check this possibility, we observed the vesicle color under the confocal microscope. As we expected, no differences in the population of green- and red-colored vesicle were observed in the overexpression or depletion of rabphilin (Supplemental Figure 3, A–C).


Age-dependent preferential dense-core vesicle exocytosis in neuroendocrine cells revealed by newly developed monomeric fluorescent timer protein.

Tsuboi T, Kitaguchi T, Karasawa S, Fukuda M, Miyawaki A - Mol. Biol. Cell (2009)

Effect of overexpression or depletion of rabphilin on the dense-core vesicle exocytosis in PC12 cells. (A) Typical TIRF images of plasma membrane-docked green colored- and red colored-NPY-mK-GO vesicles before high-KCl stimulation of scrambled siRNA-transfected control (control), rabphilin-overexpressing (rabphilin), and rabphilin-siRNA-transfected cells (rabphilin-siRNA). The cells were used for experiments in 48 h after transfection. Bar, 10 μm. (B) Stacked bar chart represents the percentage of the number of plasma membrane-docked green, yellow, and red vesicles in control, rabphilin-, and rabphilin-siRNA–transfected cells (n = 35 cells in each). (C) Effect of rabphilin siRNA on expression of rabphilin in PC12 cells. PC12 cells were transfected with rabphilin siRNA (right) or a scrambled-siRNA control (left). Cell lysates were prepared and subjected to 12.5% SDS-PAGE followed by immunoblotting with anti-rabphilin and anti-actin antibodies. The positions of the molecular mass markers (×10−3) are shown on the left. The results shown are representative of three independent experiments with similar results. (D) The density of plasma membrane-docked vesicles was determined by counting the vesicles in each image (n = 31 cells in each). (E). Histograms represent the mean number of plasma membrane-docked NPY-mK-GO vesicles in control (left), rabphilin overexpressing (center), or rabphilin siRNA-transfected cells (right) before (closed bars) and after (open bars) high-KCl stimulation. (F) The effect of expression of rabphilin, or rabphilin siRNA on the percentage of released vesicles. Note that overexpression of rabphilin induced exocytotic events from all colors of NPY-mK-GO vesicles. *p < 0.05 and ***p < 0.001, respectively, in comparison with the control. The results are means ± SE.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2801723&req=5

Figure 4: Effect of overexpression or depletion of rabphilin on the dense-core vesicle exocytosis in PC12 cells. (A) Typical TIRF images of plasma membrane-docked green colored- and red colored-NPY-mK-GO vesicles before high-KCl stimulation of scrambled siRNA-transfected control (control), rabphilin-overexpressing (rabphilin), and rabphilin-siRNA-transfected cells (rabphilin-siRNA). The cells were used for experiments in 48 h after transfection. Bar, 10 μm. (B) Stacked bar chart represents the percentage of the number of plasma membrane-docked green, yellow, and red vesicles in control, rabphilin-, and rabphilin-siRNA–transfected cells (n = 35 cells in each). (C) Effect of rabphilin siRNA on expression of rabphilin in PC12 cells. PC12 cells were transfected with rabphilin siRNA (right) or a scrambled-siRNA control (left). Cell lysates were prepared and subjected to 12.5% SDS-PAGE followed by immunoblotting with anti-rabphilin and anti-actin antibodies. The positions of the molecular mass markers (×10−3) are shown on the left. The results shown are representative of three independent experiments with similar results. (D) The density of plasma membrane-docked vesicles was determined by counting the vesicles in each image (n = 31 cells in each). (E). Histograms represent the mean number of plasma membrane-docked NPY-mK-GO vesicles in control (left), rabphilin overexpressing (center), or rabphilin siRNA-transfected cells (right) before (closed bars) and after (open bars) high-KCl stimulation. (F) The effect of expression of rabphilin, or rabphilin siRNA on the percentage of released vesicles. Note that overexpression of rabphilin induced exocytotic events from all colors of NPY-mK-GO vesicles. *p < 0.05 and ***p < 0.001, respectively, in comparison with the control. The results are means ± SE.
Mentions: Overexpression of rabphilin increased plasma membrane-docked vesicles (Figure 4, A and D), especially red vesicles (Figure 4B), whereas knockdown of endogenous rabphilin by siRNA (Figure 4C) decreased the number of plasma membrane-docked vesicles revealed by TIRF microscope (Figure 4, A and D). However, it is possible that overexpression of rabphilin affects the distribution of green- and red-colored vesicles inside of the cells. To check this possibility, we observed the vesicle color under the confocal microscope. As we expected, no differences in the population of green- and red-colored vesicle were observed in the overexpression or depletion of rabphilin (Supplemental Figure 3, A–C).

Bottom Line: Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells.When the vesicles were labeled with mK-GO-tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed.Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro, Tokyo, Japan. takatsuboi@bio.c.utokyo.ac.jp

ABSTRACT
Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO-tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K(+)] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.

Show MeSH
Related in: MedlinePlus