Analysis of exocytosis with adenovirus coexpression vector in salivary gland cells

唾液腺细胞中腺病毒共表达载体的胞吐作用分析

• 批准号: 15591973
• 负责人: TAKUMA Taishin
• 金额: $ 2.24万
• 依托单位: Health Sciences University of Hokkaido
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591973/
• 关键词: SNARE hypothesis; VAMP-2; VAMP-7; human growth hormone; GFP; VAMP-8; PC12; exocytosis

Various combinations of SNARE proteins play an essential role in diverse intracellular membrane fusion processes, including exocytosis. Tetanus neurotoxin-insensitive VAMP (TI-VAMPNAMP-7) has been reported to function in some types of regulated and constitutive exocytosis, although VAMP-7 is also supposed to act in the vesicle trafficking from the late endosomes to lysosomes. In addition, the role of VAMP-2 in constitutive exocytosis remains to be elucidated. Here we evaluated the role of VAMP-2 and VAMP-7 in the exocytic pathways of PC12 cells and HSY cells by using human growth hormone (hGH) tagged (hGH-GFP) or not with C-terminal GFP for visualization and measurement of exocytosis. Exocytosis of hGH and hGH-GFP from PC12 cells, but not from HSY cells, was stimulated by 1 itM ionomycin, indicating that the exocytosis from PC12 and HSY cells was regulated and constitutive, respectively. In PC12 cells hGH-GFP was mainly transported to the tips of neural processes and was colocalized with chromogranin A, a marker of large dense-score granules. The granule containing hGH well overlapped with VAMP-2-GFP, but did so scarcely with VAMP-7-GFP. On the other hand, cytoplasmic vesicles containing hGH clearly overlapped with VAMP-7-GFP, but not with VAMP-2-GFP, in HSY cells. However, when the vesicle transport from the trans Golgi network to the plasma membrane was arrested by incubation at 20 ーC for 2 h and then released by warming up to 37 ーC, VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH-GFP was colocalized with LAMP-1, a marker of the late endosome and lysosome, in HSY cells. These results suggest that 1) VAMP-2 can be a major v-SNARE in both regulated and constitutive exocytosis ; and 2) VAMP-7 is involved in the constitutive exocytosis from HSY cells by acting as a slow v-SNARE.

SNARE蛋白的多种组合在多种细胞膜融合过程中发挥重要作用，包括胞吐作用。据报道，破伤风神经毒素不敏感的VAMP （TI-VAMPNAMP-7）在某些类型的调节和组成性胞吐中起作用，尽管VAMP-7也被认为在从晚期内体到溶酶体的囊泡运输中起作用。此外，VAMP-2在构成性胞吐中的作用仍有待阐明。在这里，我们利用人类生长激素（hGH）标记（hGH-GFP）或不标记c端GFP来可视化和测量分泌，评估VAMP-2和VAMP-7在PC12细胞和HSY细胞的胞外通路中的作用。1 itM离子霉素刺激PC12细胞分泌hGH和hGH- gfp，而HSY细胞不分泌hGH- gfp，表明PC12和HSY细胞的分泌分别受到调节和组成。在PC12细胞中，hGH-GFP主要被转运到神经过程的尖端，并与大密度颗粒的标记物铬粒蛋白A共定位。含有hGH的颗粒与VAMP-2-GFP有很好的重叠，但与VAMP-7-GFP几乎没有重叠。另一方面，HSY细胞中含有hGH的细胞质囊泡与VAMP-7-GFP明显重叠，而与VAMP-2-GFP不重叠。然而，当囊泡从反高尔基网络转运到质膜时，在20ºC孵卵2小时，然后加热到37ºC释放，VAMP-2-GFP和hGH明显地在小细胞质囊泡中共同定位。在HSY细胞中，VAMP-7-GFP和hGH-GFP都没有与LAMP-1（晚期内核体和溶酶体的标记物）共定位。这些结果表明：1)VAMP-2可能是调节和构成性胞吐的主要v-SNARE；2) VAMP-7作为慢v-SNARE参与HSY细胞的组成性胞吐。