Live-Cell Imaging of Dual-Labeled Golgi Stacks in Tobacco BY-2 Cells Reveals Similar Behaviors for Different Cisternae during Movement and Brefeldin A Treatment

Abstract

In plant cells, the Golgi apparatus consists of numerous stacks that, in turn, are composed of several flattened cisternae with a clear cis-to-trans polarity. During normal functioning within living cells, this unusual organelle displays a wide range of dynamic behaviors such as whole stack motility, constant membrane flux through the cisternae, and Golgi enzyme recycling through the ER. In order to further investigate various aspects of Golgi stack dynamics and integrity, we co-expressed pairs of established Golgi markers in tobacco BY-2 cells to distinguish sub-compartments of the Golgi during monensin treatments, movement, and brefeldin A (BFA)-induced disassembly. A combination of cis and trans markers revealed that Golgi stacks remain intact as they move through the cytoplasm. The Golgi stack orientation during these movements showed a slight preference for the cis side moving ahead, but trans cisternae were also found at the leading edge. During BFA treatments, the different sub-compartments of about half of the observed stacks fused with the ER sequentially; however, no consistent order could be detected. In contrast, the ionophore monensin resulted in swelling of trans cisternae while medial and particularly cis cisternae were mostly unaffected. Our results thus demonstrate a remarkable equivalence of the different cisternae with respect to movement and BFA-induced fusion with the ER. In addition, we propose that a combination of dual-label fluorescence microscopy and drug treatments can provide a simple alternative approach to the determination of protein localization to specific Golgi sub-compartments.

Formation and Maintenance of the Golgi Apparatus in Plant Cells

“…In addition, one of the matrix protein, AtCASP, did not fully relocate to the ER but localized to small punctate structures colocalizing with ERES (Schoberer et al, 2010). On the other hand, we and other have shown that both cis-and trans-Golgi cisternae collapse almost at the same time in tobacco BY-2 cultured cells (Ito et al, 2012;Madison and Nebenführ, 2011). Moreover, we have demonstrated that AtCASP relocalizes to the ER almost completely by BFA treatment (Ito et al, 2012).…”

“…Scale bar, 5 mm. go forward and the stacks sometimes roll to change the leading side (Madison and Nebenführ, 2011). Microtubules, the other major cytoskeletal system, is not required for long-range movement of the plant Golgi stacks, because the treatment by microtubule disrupting drugs such as nocodazole, colchicine, and propyzamide does not have significant effects (Nebenführ et al, 1999).…”

“…The plant Golgi stacks keep intact structure during their rapid movement and never break down while traveling (Madison and Nebenführ, 2011). The best candidate that keeps the structural integrity is the Golgi matrix proteins.…”

cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells

“…They also observed the behavior of ATCASP, which did not fully distribute to the ER by BFA treatment. Madison and Nebenführ (2011) also examined the effect of BFA on Golgi proteins in BY-2 cells. Both cis - and trans -cisternae fused with the ER upon BFA treatment, but no significant difference was found for different cisternae.…”

Loss of Arabidopsis β-COP Function Affects Golgi Structure, Plant Growth and Tolerance to Salt Stress

“…Healthy seedlings were selected and the cotyledons were analyzed by the abaxial side on the confocal microscope. Markers expressed using this system were ManI-YFP (Madison and Nebenführ, 2011), TIP1.1-GFP (Gattolin et al, 2011), and GFP-AGP4 (Martinière et al, 2012).…”