Langhans Markus, Hawes Chris, Hillmer Stefan, Hummel Eric, Robinson David G
Department of Cell Biology, Heidelberg Institute for Plant Sciences, University of Heidelberg, 69120 Heidelberg, Germany.
Plant Physiol. 2007 Oct;145(2):527-38. doi: 10.1104/pp.107.104919. Epub 2007 Aug 17.
Brefeldin A (BFA) treatment stops secretion and leads to the resorption of much of the Golgi apparatus into the endoplasmic reticulum. This effect is reversible upon washing out the drug, providing a situation for studying Golgi biogenesis. In this investigation Golgi regeneration in synchronized tobacco BY-2 cells was followed by electron microscopy and by the immunofluorescence detection of ARF1, which localizes to the rims of Golgi cisternae and serves as an indicator of COPI vesiculation. Beginning as clusters of vesicles that are COPI positive, mini-Golgi stacks first become recognizable 60 min after BFA washout. They continue to increase in terms of numbers and length of cisternae for a further 90 min before overshooting the size of control Golgi stacks. As a result, increasing numbers of dividing Golgi stacks were observed 120 min after BFA washout. BFA-regeneration experiments performed on cells treated with BFA (10 microg mL(-1)) for only short periods (30-45 min) showed that the formation of ER-Golgi hybrid structures, once initiated by BFA treatment, is an irreversible process, the further incorporation of Golgi membranes into the ER continuing during a subsequent drug washout. Application of the protein kinase A inhibitor H-89, which effectively blocks the reassembly of the Golgi apparatus in mammalian cells, also prevented stack regeneration in BY-2 cells, but only at very high, almost toxic concentrations (>200 microm). Our data suggest that under normal conditions mitosis-related Golgi stack duplication may likely occur via cisternal growth followed by fission.
布雷菲德菌素A（BFA）处理会停止分泌，并导致大部分高尔基体重新吸收到内质网中。洗去药物后，这种效应是可逆的，为研究高尔基体生物发生提供了一种情况。在这项研究中，通过电子显微镜和对定位于高尔基体潴泡边缘并作为COPI囊泡化指标的ARF1进行免疫荧光检测，追踪了同步化烟草BY-2细胞中的高尔基体再生过程。小型高尔基体堆叠最初是作为COPI阳性的囊泡簇出现的，在洗去BFA后60分钟首次变得可识别。在超过对照高尔基体堆叠的大小之前，它们的数量和潴泡长度在接下来的90分钟内继续增加。结果，在洗去BFA后120分钟观察到分裂的高尔基体堆叠数量增加。对仅短期（30 - 45分钟）用BFA（10微克/毫升）处理的细胞进行的BFA再生实验表明，一旦由BFA处理引发，内质网-高尔基体混合结构的形成是一个不可逆的过程，在随后的药物洗脱过程中，高尔基体膜继续进一步并入内质网。蛋白激酶A抑制剂H-89的应用有效地阻断了哺乳动物细胞中高尔基体的重新组装，也阻止了BY-2细胞中的堆叠再生，但仅在非常高的、几乎有毒的浓度（>200微摩尔）下才有效。我们的数据表明，在正常条件下，与有丝分裂相关的高尔基体堆叠复制可能通过潴泡生长随后分裂发生。
