Okayama Miki, Arakawa Toshiya, Tanimura Akihiko, Mizoguchi Itaru, Tajima Yoshifumi, Takuma Taishin
Department of Orthodontics, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan.
Cell Struct Funct. 2009;34(2):115-25. doi: 10.1247/csf.09013. Epub 2009 Sep 8.
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.
为了评估VAMP8/内体膜泡蛋白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）研究了VAMP8和绿色荧光蛋白标记的人生长激素的胞吐途径。人生长激素-绿色荧光蛋白（hGH-GFP）和VAMP8-绿色荧光蛋白（VAMP8-GFP）在HeLa细胞的小圆形囊泡和细长管状囊泡中表达相似，并且大多在细胞周边区域进行胞吐。VAMP8-GFP产生两种类型的胞吐图像：爆发型和非爆发型。爆发型在发生胞吐作用的活跃窗口中显示峰值荧光强度急剧短暂增加，而平均强度下降则慢得多，这与“完全融合”型胞吐作用中观察到的情况一样。非爆发型显示与质膜的融合持续时间相对较长，VAMP8-GFP向质膜的转移很少，这与所谓的“亲吻-逃离”型胞吐作用中观察到的情况一样。内源性VAMP8和hGH-GFP至少部分共定位于相同的囊泡上。然而，即使针对VAMP8和VAMP3的小干扰RNA（siRNA）显著降低了它们的蛋白质水平，hGH-GFP和来自海萤的分泌型荧光素酶CLuc的组成型胞吐作用仍正常。这些结果表明，VAMP8虽然可以参与胞吐作用，但对于组成型胞吐作用并非必不可少。
