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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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Exocytotic dynamics of dense-core vesicle cargoes NPY and tPA. Five sequential dual color TIRF images show the behavior of single green colored- (A), yellow colored- (C), and red-colored NPY-mK-GO (E) vesicles after applying 70 mM KCl to the cell. The cells were used for experiments in 48 h after transfection. The vesicle position before exocytosis is outlined by a circle. Bars, 1 μm. Fluorescence intensity trances are shown of vesicles containing both green colored- (B), yellow colored- (D), and red colored-NPY-mK-GO vesicles (F). (G) A histogram represents the mean number of plasma membrane-docked NPY-mK-GO vesicles (left; n = 31 cells) or tPA-mK-GO (right; n = 28 cells) vesicles per cell before (closed bars) and after (open bars) high-KCl stimulation. (H) Percentage of the number of NPY- or tPA-mKO release events during 5-min stimulation to the number of plasma membrane-docked vesicle before stimulation. Note that red colored-NPY- or tPA-mK-GO vesicles showed only few exocytotic events. The results are means ± SE.
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Figure 3: Exocytotic dynamics of dense-core vesicle cargoes NPY and tPA. Five sequential dual color TIRF images show the behavior of single green colored- (A), yellow colored- (C), and red-colored NPY-mK-GO (E) vesicles after applying 70 mM KCl to the cell. The cells were used for experiments in 48 h after transfection. The vesicle position before exocytosis is outlined by a circle. Bars, 1 μm. Fluorescence intensity trances are shown of vesicles containing both green colored- (B), yellow colored- (D), and red colored-NPY-mK-GO vesicles (F). (G) A histogram represents the mean number of plasma membrane-docked NPY-mK-GO vesicles (left; n = 31 cells) or tPA-mK-GO (right; n = 28 cells) vesicles per cell before (closed bars) and after (open bars) high-KCl stimulation. (H) Percentage of the number of NPY- or tPA-mKO release events during 5-min stimulation to the number of plasma membrane-docked vesicle before stimulation. Note that red colored-NPY- or tPA-mK-GO vesicles showed only few exocytotic events. The results are means ± SE.

Mentions: Dense-core vesicles showed green, yellow, and red fluorescence and each florescence was implicated as its age: new, intermediate, and old, respectively. To analyze the single vesicle release behavior related to its age, we monitored the dynamics of single exocytotic events within ∼100 nm beneath the plasma membrane by TIRF microscopy. High-KCl stimulation (70 mM) caused newly synthesized NPY-mK-GO-labeled vesicles (i.e., green vesicles) to brighten and suddenly spread during release of the fluorescent peptide, with an identical time course in older (i.e., yellow or red) vesicles (Figure 3, A–F). Consistently, tPA-mK-GO–labeled green, yellow, and red vesicles showed similar exocytotic dynamics (data not shown). These data indicated that overexpression of NPY- or tPA-mK-GO in the PC12 cells did not disturb their normal secretory behavior and that the kinetics of new and old vesicle exocytosis were identical.


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)

Exocytotic dynamics of dense-core vesicle cargoes NPY and tPA. Five sequential dual color TIRF images show the behavior of single green colored- (A), yellow colored- (C), and red-colored NPY-mK-GO (E) vesicles after applying 70 mM KCl to the cell. The cells were used for experiments in 48 h after transfection. The vesicle position before exocytosis is outlined by a circle. Bars, 1 μm. Fluorescence intensity trances are shown of vesicles containing both green colored- (B), yellow colored- (D), and red colored-NPY-mK-GO vesicles (F). (G) A histogram represents the mean number of plasma membrane-docked NPY-mK-GO vesicles (left; n = 31 cells) or tPA-mK-GO (right; n = 28 cells) vesicles per cell before (closed bars) and after (open bars) high-KCl stimulation. (H) Percentage of the number of NPY- or tPA-mKO release events during 5-min stimulation to the number of plasma membrane-docked vesicle before stimulation. Note that red colored-NPY- or tPA-mK-GO vesicles showed only few exocytotic events. The results are means ± SE.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801723&req=5

Figure 3: Exocytotic dynamics of dense-core vesicle cargoes NPY and tPA. Five sequential dual color TIRF images show the behavior of single green colored- (A), yellow colored- (C), and red-colored NPY-mK-GO (E) vesicles after applying 70 mM KCl to the cell. The cells were used for experiments in 48 h after transfection. The vesicle position before exocytosis is outlined by a circle. Bars, 1 μm. Fluorescence intensity trances are shown of vesicles containing both green colored- (B), yellow colored- (D), and red colored-NPY-mK-GO vesicles (F). (G) A histogram represents the mean number of plasma membrane-docked NPY-mK-GO vesicles (left; n = 31 cells) or tPA-mK-GO (right; n = 28 cells) vesicles per cell before (closed bars) and after (open bars) high-KCl stimulation. (H) Percentage of the number of NPY- or tPA-mKO release events during 5-min stimulation to the number of plasma membrane-docked vesicle before stimulation. Note that red colored-NPY- or tPA-mK-GO vesicles showed only few exocytotic events. The results are means ± SE.
Mentions: Dense-core vesicles showed green, yellow, and red fluorescence and each florescence was implicated as its age: new, intermediate, and old, respectively. To analyze the single vesicle release behavior related to its age, we monitored the dynamics of single exocytotic events within ∼100 nm beneath the plasma membrane by TIRF microscopy. High-KCl stimulation (70 mM) caused newly synthesized NPY-mK-GO-labeled vesicles (i.e., green vesicles) to brighten and suddenly spread during release of the fluorescent peptide, with an identical time course in older (i.e., yellow or red) vesicles (Figure 3, A–F). Consistently, tPA-mK-GO–labeled green, yellow, and red vesicles showed similar exocytotic dynamics (data not shown). These data indicated that overexpression of NPY- or tPA-mK-GO in the PC12 cells did not disturb their normal secretory behavior and that the kinetics of new and old vesicle exocytosis were identical.

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