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Vol. 15, Issue 4, 1843-1852, April 2004
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Department of Cell Biology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, Connecticut 06520-8002
Submitted July 2, 2003;
Revised January 7, 2004;
Accepted January 9, 2004
Monitoring Editor: Benjamin Glick
Early in mitosis, the mammalian Golgi apparatus disassembles, and fluorescence microscopy reveals Golgi clusters and an extensive, nonresolvable haze that either represents scattered vesicles or a merged endoplasmic reticulum (ER)-Golgi compartment. To help decide between these alternatives, we have carried out a combined microscopic and pharmacological analysis, by using a BS-C-1 cell line stably coexpressing ER and Golgi markers. Video fluorescence microscopy showed that these two organelles were morphologically distinguishable at all stages of mitosis, and photobleaching experiments showed that diffusion of the Golgi marker was unaffected by the presence of the ER. Fragmentation of the ER by using filipin III completely blocked diffusion of the ER marker but had no effect on the Golgi marker, unless it was first relocated to the ER by using brefeldin A. The Golgi haze was also studied using BODIPY ceramide. Its diffusion was slower in mitotic Golgi than in mitotic ER, but similar to that of a Golgi enzyme marker in the mitotic Golgi haze or in Golgi vesicles generated by ilimaquinone. Together, these results support the idea that the Golgi and the ER remain separate during mitosis and strongly suggest that Golgi markers move by vesicle diffusion, as opposed to lateral diffusion in continuous membranes.
Abbreviations used: BFA, brefeldin A; CFP, cyan fluorescent protein; EM, electron microscopy; ER, endoplasmic reticulum; FRAP, fluorescence recovery after photobleaching; GalNAc-T2, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2; GalT, 
1,4 galactosyltransferase; GFP, green fluorescent protein; IQ, ilimaquinone; MGC, mitotic Golgi cluster; YFP, yellow fluorescent protein.
* Corresponding author. E-mail address: graham.warren{at}yale.edu.
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