Lin C-C, Huang C-C, Lin K-H, Cheng K-H, Yang D-M, Tsai Y-S, Ong R-Y, Huang Y-N, Kao L-S
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, ROC.
J Cell Physiol. 2007 May;211(2):316-26. doi: 10.1002/jcp.20938.
Rab3A is a small G protein in the Rab3 subfamily, and is thought to act at late stage of exocytosis. However, the detailed mechanism of its action is not completely understood. To study the role of Rab3A in exocytosis, we used a total internal reflection fluorescence microscope to examine the fluorescence changes of EGFP-Rab3A-labeled and NPY-EGFP-labeled vesicles in PC12 cells upon stimulation. The fluorescence of EGFP-Rab3A-labeled and NPY-EGFP-labeled vesicles decreased while showing different patterns. The NPY-EGFP-labeled vesicles that exocytosed showed a transient fluorescence increase before NPY-EGFP fluorescence disappearance, which represents fusion and NPY release. This transient increase was diminished in cells that co-expressed the GDP-bound Rab3A mutant. The fluorescence of EGFP-Rab3A-labeled vesicles dispersed before disappearance, which represents the dissociation of Rab3A from the vesicles. The dispersion was not found in GTP-bound Rab3A mutant-labeled vesicles. Interestingly, EGFP-Rab3A F59S, a mutant unable to bind rabphilin, dissociates slower from the vesicles than wild type Rab3A and caused a slower release of NPY-EGFP. The results provide direct evidence to support the hypothesis that GTP hydrolysis and rabphilin are involved in Rab3A dissociation from the vesicles and the occurrence of exocytosis.
Rab3A是Rab3亚家族中的一种小G蛋白，被认为在胞吐作用的后期发挥作用。然而，其详细的作用机制尚未完全清楚。为了研究Rab3A在胞吐作用中的作用，我们使用全内反射荧光显微镜来检测PC12细胞中经EGFP-Rab3A标记和NPY-EGFP标记的囊泡在受到刺激时的荧光变化。经EGFP-Rab3A标记和NPY-EGFP标记的囊泡的荧光下降，但呈现出不同的模式。发生胞吐作用的NPY-EGFP标记的囊泡在NPY-EGFP荧光消失前显示出短暂的荧光增加，这代表融合和NPY释放。在共表达结合GDP的Rab3A突变体的细胞中，这种短暂增加减弱。经EGFP-Rab3A标记的囊泡的荧光在消失前分散，这代表Rab3A从囊泡上解离。在结合GTP的Rab3A突变体标记的囊泡中未发现这种分散现象。有趣的是，不能结合rabphilin的突变体EGFP-Rab3A F59S从囊泡上解离的速度比野生型Rab3A慢，并导致NPY-EGFP的释放变慢。这些结果提供了直接证据，支持GTP水解和rabphilin参与Rab3A从囊泡上解离以及胞吐作用发生这一假说。
