Limits...
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.

Show MeSH

Related in: MedlinePlus

Proposed model for Slp4-a/rabphilin–dependent preferential dense-core vesicle exocytosis in neuroendocrine cells. Rabphilin and Slp4-a promote docking of dense-core vesicles to the plasma membrane through interaction with SNAP-25 (Tsuboi and Fukuda, 2005; Tsuboi et al., 2007) and Munc18-1/syntaxin-1a complex (Tsuboi and Fukuda, 2006b), respectively. Dense-core vesicles docked to the plasma membrane by the rabphilin/SNAP-25 complex (blue bars) undergo preferential exocytosis (presumably corresponds to the readily releasable pool), whereas dense-core vesicles docked to the plasma membrane by the Slp4-a/Munc18-1/sytnaxin-1a complex (red bars) do not undergo exocytosis (corresponds to the reserve pool). The molecular switch between readily releasable pool and reserve pool is currently unknown. Green-, yellow-, and red-colored vesicles correspond to newly synthesized vesicles, middle-aged vesicles, and old vesicles, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2801723&req=5

Figure 6: Proposed model for Slp4-a/rabphilin–dependent preferential dense-core vesicle exocytosis in neuroendocrine cells. Rabphilin and Slp4-a promote docking of dense-core vesicles to the plasma membrane through interaction with SNAP-25 (Tsuboi and Fukuda, 2005; Tsuboi et al., 2007) and Munc18-1/syntaxin-1a complex (Tsuboi and Fukuda, 2006b), respectively. Dense-core vesicles docked to the plasma membrane by the rabphilin/SNAP-25 complex (blue bars) undergo preferential exocytosis (presumably corresponds to the readily releasable pool), whereas dense-core vesicles docked to the plasma membrane by the Slp4-a/Munc18-1/sytnaxin-1a complex (red bars) do not undergo exocytosis (corresponds to the reserve pool). The molecular switch between readily releasable pool and reserve pool is currently unknown. Green-, yellow-, and red-colored vesicles correspond to newly synthesized vesicles, middle-aged vesicles, and old vesicles, respectively.

Mentions: Based on our present findings, we propose following model for preferential exocytosis in neuroendocrine cells (Figure 6): 1) rabphilin is targeted to Rab27A on the newly synthesized dense-core vesicles; 2) rabphilin-attached dense-core vesicle is then transported to the plasma membrane; and 3) incidentally, rabphilin directly binds to SNAP-25 in the plasma membrane. The resulting Rab27A–rabphilin–SNAP-25 complex links the dense-core vesicles to the plasma membrane and enhances exocytotic events (Tsuboi and Fukuda, 2005). This route presumably corresponds to the “readily releasable pool” for the dense-core vesicle exocytosis. However, in some cases, rabphilin-attached dense-core vesicles cannot bind to SNAP-25. This vesicle will follow different route: 4) rabphilin detaches from Rab27A on the middle-aged dense-core vesicles; 5) Slp4-a, other Rab27A effector, occupies Rab27A on the dense-core vesicles, instead of rabphilin; and 6) Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with closed-form syntaxin-1a, which does not contribute to SNARE complex formation, on the plasma membrane. The resulting quadripartite protein complex (i.e., Rab27A-Slp4-a-Munc18-1-syntaxin-1a) forms a tight complex between the dense-core vesicle and plasma membrane and inhibits high-KCl-induced exocytosis (Tsuboi and Fukuda, 2006b). This route presumably corresponds to the “reserve pool” for the dense-core vesicle exocytosis. Because the vesicle could not detach from the plasma membrane, the vesicles bound to Slp4-a subsequently become old. Although disassembly of the quadripartite protein complex must be necessary for the dense-core vesicle exocytosis, when and how the quadripartite protein complex disassembles remains completely unknown (Figure 6).


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)

Proposed model for Slp4-a/rabphilin–dependent preferential dense-core vesicle exocytosis in neuroendocrine cells. Rabphilin and Slp4-a promote docking of dense-core vesicles to the plasma membrane through interaction with SNAP-25 (Tsuboi and Fukuda, 2005; Tsuboi et al., 2007) and Munc18-1/syntaxin-1a complex (Tsuboi and Fukuda, 2006b), respectively. Dense-core vesicles docked to the plasma membrane by the rabphilin/SNAP-25 complex (blue bars) undergo preferential exocytosis (presumably corresponds to the readily releasable pool), whereas dense-core vesicles docked to the plasma membrane by the Slp4-a/Munc18-1/sytnaxin-1a complex (red bars) do not undergo exocytosis (corresponds to the reserve pool). The molecular switch between readily releasable pool and reserve pool is currently unknown. Green-, yellow-, and red-colored vesicles correspond to newly synthesized vesicles, middle-aged vesicles, and old vesicles, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Proposed model for Slp4-a/rabphilin–dependent preferential dense-core vesicle exocytosis in neuroendocrine cells. Rabphilin and Slp4-a promote docking of dense-core vesicles to the plasma membrane through interaction with SNAP-25 (Tsuboi and Fukuda, 2005; Tsuboi et al., 2007) and Munc18-1/syntaxin-1a complex (Tsuboi and Fukuda, 2006b), respectively. Dense-core vesicles docked to the plasma membrane by the rabphilin/SNAP-25 complex (blue bars) undergo preferential exocytosis (presumably corresponds to the readily releasable pool), whereas dense-core vesicles docked to the plasma membrane by the Slp4-a/Munc18-1/sytnaxin-1a complex (red bars) do not undergo exocytosis (corresponds to the reserve pool). The molecular switch between readily releasable pool and reserve pool is currently unknown. Green-, yellow-, and red-colored vesicles correspond to newly synthesized vesicles, middle-aged vesicles, and old vesicles, respectively.
Mentions: Based on our present findings, we propose following model for preferential exocytosis in neuroendocrine cells (Figure 6): 1) rabphilin is targeted to Rab27A on the newly synthesized dense-core vesicles; 2) rabphilin-attached dense-core vesicle is then transported to the plasma membrane; and 3) incidentally, rabphilin directly binds to SNAP-25 in the plasma membrane. The resulting Rab27A–rabphilin–SNAP-25 complex links the dense-core vesicles to the plasma membrane and enhances exocytotic events (Tsuboi and Fukuda, 2005). This route presumably corresponds to the “readily releasable pool” for the dense-core vesicle exocytosis. However, in some cases, rabphilin-attached dense-core vesicles cannot bind to SNAP-25. This vesicle will follow different route: 4) rabphilin detaches from Rab27A on the middle-aged dense-core vesicles; 5) Slp4-a, other Rab27A effector, occupies Rab27A on the dense-core vesicles, instead of rabphilin; and 6) Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with closed-form syntaxin-1a, which does not contribute to SNARE complex formation, on the plasma membrane. The resulting quadripartite protein complex (i.e., Rab27A-Slp4-a-Munc18-1-syntaxin-1a) forms a tight complex between the dense-core vesicle and plasma membrane and inhibits high-KCl-induced exocytosis (Tsuboi and Fukuda, 2006b). This route presumably corresponds to the “reserve pool” for the dense-core vesicle exocytosis. Because the vesicle could not detach from the plasma membrane, the vesicles bound to Slp4-a subsequently become old. Although disassembly of the quadripartite protein complex must be necessary for the dense-core vesicle exocytosis, when and how the quadripartite protein complex disassembles remains completely unknown (Figure 6).

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