Role of VAMP8/endobrevin in Constitutive Exocytotic Pathway in HeLa Cells
Role of VAMP8/endobrevin in Constitutive Exocytotic Pathway in HeLa Cells
复制标题
DOI：
10.1247/csf.09013
复制
发表时间：
2009-01-01
影响因子：
1.5
通讯作者：
Takuma, Taishin
中科院分区：
文献类型：
--
作者：
Okayama, Miki;Arakawa, Toshiya;Takuma, Taishin
为了评估 VAMP8/endobrevin 在组成型胞吐作用中的作用，我们通过全内反射荧光显微镜 (TIRF-M) 检查了 VAMP8 和人生长激素（均带有 GFP 标记）的胞吐途径。人 GH-GFP 和 VAMP8-GFP 在 HeLa 细胞的小圆形囊泡和细长管状囊泡中表达相似，并且大部分在细胞的外围区域被胞吐。 VAMP8-GFP 给出 2 种类型的胞吐图像：突发型和非突发型。突发型显示出峰值荧光强度的急剧瞬时增加和发生胞吐作用的活动窗口中平均强度的缓慢下降，如在“完全融合”类型的胞吐作用中观察到的。非爆发型显示与质膜的相对持久的融合，几乎没有 VAMP8-GFP 转移到质膜，如在所谓的“接吻并运行”型胞吐作用中观察到的。内源性 VAMP8 和 hGH-GFP 至少部分共定位在相同的囊泡上。然而，hGH-GFP 和 CLuc（一种夜光鲤分泌的荧光素酶）的组成型胞吐作用是正常的，即使 VAMP8 和 VAMP3 的 siRNA 大幅降低了它们的蛋白质。这些结果表明，VAMP8 对于组成型胞吐作用不是必需的，尽管它可以参与胞吐作用。
To evaluate the role of VAMP8/endobrevin in constitutive exocytosis, we have examined the exocytotic pathways of VAMP8 and human growth hormone, both GFP-tagged, by total internal reflection fluorescence microscopy (TIRF-M). Human GH-GFP and VAMP8-GFP were similarly expressed in small round vesicles and elongated tubular vesicles in HeLa cells, and were mostly exocytosed at the peripheral area of the cells. VAMP8-GFP gave 2 types of exocytotic images: a burst type and a non-burst type. The burst type showed a sharp transient increase in the peak fluorescence intensity and a much slower decrease in the average intensity in the active windows, where exocytosis took place, as observed in the "full-fusion" type of exocytosis. The non-burst type showed a relatively long-lasting fusion to the plasma membrane with little transfer of VAMP8-GFP to the plasma membrane, as observed in the so-called "kiss-and-run" type of exocytosis. Endogenous VAMP8 and hGH-GFP were colocalized on the same vesicles at least in part. However, the constitutive exocytosis of hGH-GFP and CLuc, a secreted luciferase from Cypridina noctiluca, was normal, even when siRNAs for VAMP8 and VAMP3 robustly decreased their proteins. These results suggest that VAMP8 is not essential for constitutive exocytosis, although it can be involved in the exocytosis.