Lucocq J, Berger E, Hug C
Department of Anatomy and Physiology, University of Dundee, United Kingdom.
J Struct Biol. 1995 Nov-Dec;115(3):318-30. doi: 10.1006/jsbi.1995.1056.
Using stereology and immunoelectron microscopy we examined the pathway of Golgi cluster formation during treatment with the phosphatase inhibitor okadaic acid. During the first hour the Golgi stack of suspension HeLa cells lost 90% of its membrane without appreciable reduction in the number of cisternae. During this time clusters of tubules and vesicles (Golgi clusters) appeared and these contained only a fraction of the Golgi membrane present in untreated cells. Despite the overall reduction in membrane the total amount of immunolabeling for galactosyltransferase over the Golgi clusters of a typical cell was maintained, indicating that galactosyltransferase had been retained in Golgi membranes. The observation that, after 40 min okadaic acid treatment, labeling density for galactosyltransferase within trans Golgi cisternae increased 1.6-fold (n = 3, CE 10%) suggests that membrane loss from trans cisternae was selective. Careful evaluation of immunolabeled clusters showed that most of the galactosyltransferase labeling was located over complex tubular profiles and not vesicular profiles. Tubular structures were also observed during disassembly and these were found both connected to disassembling cisternae and within forming Golgi clusters, indicating that they were intermediates in cluster formation. We also investigated the role of vesicular transport in cluster formation. During disassembly we found no accumulation of COP-coated buds and vesicles over Golgi membrane. However, aluminium fluoride, previously found to arrest transport in the Golgi stack, completely inhibited membrane depletion and stack disassembly. Taken together, our results indicate that during Golgi cluster formation, membrane leaves the Golgi but galactosyltransferase is retained within a tubular reticulum which is a direct descendant of trans-Golgi cisternae. Membrane depletion may require ongoing vesicular transport and we postulate that it arises because of an imbalance in membrane traffic into and out of the Golgi apparatus.
我们利用体视学和免疫电子显微镜技术，研究了用磷酸酶抑制剂冈田酸处理期间高尔基体簇形成的途径。在最初的一小时内，悬浮培养的HeLa细胞的高尔基体堆叠失去了90%的膜，但潴泡数量没有明显减少。在此期间，出现了小管和小泡簇（高尔基体簇），这些簇仅包含未处理细胞中高尔基体膜的一部分。尽管膜总量减少，但典型细胞的高尔基体簇上半乳糖基转移酶的免疫标记总量保持不变，这表明半乳糖基转移酶保留在了高尔基体膜中。在冈田酸处理40分钟后，反式高尔基体潴泡内半乳糖基转移酶的标记密度增加了1.6倍（n = 3，置信区间10%），这一观察结果表明反式潴泡的膜丢失是选择性的。对免疫标记簇的仔细评估表明，大部分半乳糖基转移酶标记位于复杂的管状结构上，而非小泡结构上。在高尔基体解体过程中也观察到了管状结构，它们既与正在解体的潴泡相连，也存在于正在形成的高尔基体簇内，这表明它们是簇形成过程中的中间体。我们还研究了小泡运输在簇形成中的作用。在高尔基体解体过程中，我们未在高尔基体膜上发现COP包被的芽和小泡的积累。然而，先前发现能阻止高尔基体堆叠内运输的氟化铝，完全抑制了膜的消耗和堆叠的解体。综上所述，我们的结果表明，在高尔基体簇形成过程中，膜离开高尔基体，但半乳糖基转移酶保留在管状网状结构内，该结构是反式高尔基体潴泡的直接衍生物。膜的消耗可能需要持续的小泡运输，我们推测这是由于进出高尔基体的膜运输失衡所致。
