Abstract
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.
Highlights
Brefeldin A (BFA) treatment stops secretion and leads to the resorption of much of the Golgi apparatus into the endoplasmic reticulum
The breakdown and reassembly of the Golgi apparatus during mitosis in animal cells closely resemble the situation in interphase cells when brefeldin A (BFA) is added and washed out, since this drug is well known to prevent the activation of Arf1 (Jackson and Casanova, 2000), and leads both in plants and animals to a redistribution of a significant portion of the Golgi apparatus into the endoplasmic reticulum (ER) (Klausner et al, 1992; Nebenfuhr et al, 2002)
In preliminary time-course experiments where samples from synchronized BY-2 cultures were processed for conventional electron microscopy, we determined that a 2-h treatment with BFA (10 mg mL21) was required to eliminate completely all traces of Golgi membranes
Summary
Brefeldin A (BFA) treatment stops secretion and leads to the resorption of much of the Golgi apparatus into the endoplasmic reticulum. The breakdown and reassembly of the Golgi apparatus during mitosis in animal cells closely resemble the situation in interphase cells when brefeldin A (BFA) is added and washed out, since this drug is well known to prevent the activation of Arf (Jackson and Casanova, 2000), and leads both in plants and animals to a redistribution of a significant portion of the Golgi apparatus into the ER (Klausner et al, 1992; Nebenfuhr et al, 2002) This provides the legitimation for several recent studies on Golgi biogenesis in mammalian cells, where de novo regeneration has been followed in cells recovering from treatment with BFA and H-89, a protein kinase inhibitor that blocks Sar recruitment and prevents COPII vesicle formation (Aridor and Balch, 2000; Puri and Linstedt, 2003; Bejarano et al, 2006). As monitored by the visualization of mannosidase I-GFP (a cislocated glycoprotein-processing enzyme) and sialyltransferase (ST)-GFP (a trans-Golgi marker), the Golgi apparatus recovers fully within 2 to 3 h of washing out the BFA (Ritzenthaler et al, 2002; Saint-Jore et al, 2002), but a detailed investigation into the mechanism of de novo Golgi stack regeneration in BY-2 cells has not been performed
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