Ying Ming, Grimmer Stine, Iversen Tore-Geir, Van Deurs Bo, Sandvig Kirsten
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Traffic. 2003 Nov;4(11):772-84. doi: 10.1034/j.1600-0854.2003.00134.x.
Recent work from our laboratory demonstrated that increased cellular cholesterol content affects the structure of the Golgi apparatus. We have now investigated the functional consequences of the cholesterol-induced vesiculation of the Golgi apparatus and the role of actin for these changes. The results showed that cholesterol-induced vesiculation and dispersion of the Golgi apparatus is a reversible process and that reversal can be inhibited by cytochalasin D, an actin-disrupting reagent. Furthermore, electron microscopy revealed that jasplakinolide, which stabilizes actin filaments, prevented the dispersion, but not the vesiculation of the Golgi cisternae. Importantly, the different Golgi markers seemed to be separated even after vesiculation. To investigate whether transport through the different steps of the exocytic pathway was affected in cholesterol-treated cells, we visualized ER to plasma membrane transport by using ts045-VSVG-GFP. In COS-1 cells expressing ts045-VSVG-GFP increased cholesterol levels did not affect transport of VSVG into the vesiculated Golgi apparatus. However, increased levels of cholesterol resulted in retention of the nascent G protein in vesicles with the TGN-marker TGN46. Biotinylation of cell surface molecules to quantify arrival of VSVG at the plasma membrane confirmed that cholesterol treatment inhibited export of the VSVG protein. In conclusion, the data show that transport of VSVG into/through a vesiculated Golgi is feasible, but that cholesterol loading inhibits exit of VSVG from the vesicles containing TGN markers. Furthermore, the data illustrate the importance of actin filaments for Golgi structure.
我们实验室最近的研究表明，细胞内胆固醇含量增加会影响高尔基体的结构。我们现在研究了胆固醇诱导的高尔基体囊泡化的功能后果以及肌动蛋白在这些变化中的作用。结果表明，胆固醇诱导的高尔基体囊泡化和分散是一个可逆过程，并且这种逆转可以被细胞松弛素D（一种破坏肌动蛋白的试剂）抑制。此外，电子显微镜显示，稳定肌动蛋白丝的茉莉酸内酯可防止高尔基体池的分散，但不能防止其囊泡化。重要的是，即使在囊泡化后，不同的高尔基体标记物似乎也被分开了。为了研究在胆固醇处理的细胞中，通过外排途径不同步骤的运输是否受到影响，我们使用ts045-VSVG-GFP可视化了从内质网到质膜的运输。在表达ts045-VSVG-GFP的COS-1细胞中，胆固醇水平升高并不影响VSVG进入囊泡化高尔基体的运输。然而，胆固醇水平的升高导致新生G蛋白与TGN标记物TGN46一起保留在囊泡中。对细胞表面分子进行生物素化以量化VSVG到达质膜的情况，证实胆固醇处理抑制了VSVG蛋白的输出。总之，数据表明VSVG进入/通过囊泡化高尔基体的运输是可行，但胆固醇加载会抑制VSVG从含有TGN标记物的囊泡中排出。此外，数据说明了肌动蛋白丝对高尔基体结构的重要性。
