Golgi Regeneration after Brefeldin A Treatment in BY-2 Cells Entails Stack Enlargement and Cisternal Growth followed by Division

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 mg mL 21 ) 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 mM). Our data suggest that under normal conditions mitosis-related Golgi stack duplication may likely occur via cisternal growth followed by fission.

Routes to the Tonoplast: The Sorting of Tonoplast Transporters in Arabidopsis Mesophyll Protoplasts

“…In fact, cotransformation of ap-3 b protoplasts with constructs for GFP-SUC4 and another cis-Golgi marker, the Arabidopsis v-SNARE MEMBRIN12 (MEMB12) (Uemura et al, 2004), which carries an mCherry-fusion in the Wave127R construct published by Geldner et al (2009), showed the same colocalization (see Supplemental Figure 8A online). Moreover, at higher magnifications, the labeled structures were clearly donut shaped, which is typical for Golgi stacks but not for the TGN (see Supplemental Figure 8B online; Langhans et al, 2007). Finally, when we coexpressed GFP-SUC4 with the Wave25R construct , which harbors the Golgi and TGN-localized GTPase RabD1 (Pinheiro et al, 2009), we observed red fluorescence mostly in the immediate vicinity of the green fluorescent, GFP-SUC4-labeled Golgi (see Supplemental Figure 9 online).…”

Vesicles versus Tubes: Is Endoplasmic Reticulum-Golgi Transport in Plants Fundamentally Different from Other Eukaryotes?

“…An obvious experiment where COPII vesicles should be seen is in the reformation of Golgi after brefeldin A (BFA)-induced reabsorption into the ER. One such study on tobacco Bright Yellow2 (BY2) cells reported buds on the ER surfaces, which were infrequent, and tubular vesicular clusters, representing the earliest observable stage of stack regeneration cells (Langhans et al, 2007). These clusters immunolabeled for COPI coat components but not for COPII proteins.…”

Analysis of the Golgi Apparatus in Arabidopsis Seed Coat Cells during Polarized Secretion of Pectin-Rich Mucilage

“…The nature of such developmental signals has yet to be elucidated. The mechanism of Golgi doubling in these cells is also not known, but, in plants, Golgi fission is considered to be the mostly likely mechanism (Bosabalidis, 1985;Craig and Staehelin, 1988;Hirose and Komamine, 1989;Langhans et al, 2007). However, de novo production of Golgi stacks (Langhans et al, 2007) could be involved as well.…”