Dynamic Response of Prevacuolar Compartments to Brefeldin A in Plant Cells

Abstract

Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs) in the secretory pathway. Using transgenic tobacco (Nicotiana tabacum) Bright-Yellow-2 (BY-2) cells expressing membrane-anchored yellow fluorescent protein (YFP) reporters marking Golgi or PVCs, we have recently demonstrated that PVCs are mobile multivesicular bodies defined by vacuolar sorting receptor proteins. Here, we demonstrate that Golgi and PVCs have different sensitivity in response to brefeldin A (BFA) treatment in living tobacco BY-2 cells. BFA at low concentrations (5-10 mg mL 21 ) induced YFPmarked Golgi stacks to form both endoplasmic reticulum-Golgi hybrid structures and BFA-induced aggregates, but had little effect on YFP-marked PVCs in transgenic BY-2 cells at both confocal and immunogold electron microscopy levels. However, BFA at high concentrations (50-100 mg mL 21 ) caused both YFP-marked Golgi stacks and PVCs to form aggregates in a dose-and timedependent manner. Normal Golgi or PVC signals can be recovered upon removal of BFA from the culture media. Confocal immunofluorescence and immunogold electron microscopy studies with specific organelle markers further demonstrate that the PVC aggregates are distinct, but physically associated, with Golgi aggregates in BFA-treated cells and that PVCs might lose their internal vesicle structures at high BFA concentration. In addition, vacuolar sorting receptor-marked PVCs in root-tip cells of tobacco, pea (Pisum sativum), mung bean (Vigna radiata), and Arabidopsis (Arabidopsis thaliana) upon BFA treatment are also induced to form similar aggregates. Thus, we have demonstrated that the effects of BFA are not limited to endoplasmic reticulum and Golgi, but extend to PVC in the endomembrane system, which might provide a quick tool for distinguishing Golgi from PVC for its identification and characterization, as well as a possible new tool in studying PVC-mediated protein traffic in plant cells.

Rice SCAMP1 Defines Clathrin-Coated,trans-Golgi–Located Tubular-Vesicular Structures as an Early Endosome in Tobacco BY-2 Cells

“…In plant cells, wortmannin also blocks the trafficking of some vacuolar proteins (Matsuoka et al, 1995;Di Sansebastiano et al, 1998) and prevents the recycling of VSR proteins from the PVC to the Golgi apparatus (daSilva et al, 2005(daSilva et al, , 2006. Morphologically, wortmannin induces the vacuolation of BP-80-labeled PVCs/MVBs in BY-2 cells (Tse et al, 2004;Lo and Jiang, 2006), and it seems that such a vacuolation is a general response to wortmannin treatment in various plant cell types and species (Miao et al, 2006;Tse et al, 2006). Wortmannin inhibits the formation of the internal vesicles in multivesicular endosomes in both mammalian and plant cells and causes these organelles to enlarge significantly (Fernandez-Borja et al, 1999;Bright et al, 2001;Tse et al, 2004).…”

Vesicle Dynamics during Plant Cell Cytokinesis Reveals Distinct Developmental Phases

“…5). In tobacco cells used at low concentration (50 mM), the ARF-GEF inhibitor brefeldin A (BFA) blocks secretory vesicle trafficking (Grebe et al, 2003;Dhonukshe et al, 2006;Tse et al, 2006;Reichardt et al, 2007;Lam et al, 2009) and induces the formation of a hybrid endoplasmic reticulum (ER)-Golgi compartment as well as a BFA compartment consisting of a mixture of the TGN vesicles and endosomal vesicles. However, it does not affect the late endosomal compartment labeled with YFP-ARA7 (Ueda et al, 2004;Dhonukshe et al, 2006; Fig.…”

FRIENDLY Regulates Mitochondrial Distribution, Fusion, and Quality Control in Arabidopsis

“…Sections of the true leaves of 14-d-old Arabidopsis seedlings were highpressure frozen at the Biotechnology and Biological Sciences Research Council Oxford Brookes High Pressure Freezing Facility using a Baltec HPF010 apparatus and freeze substituted in the presence of 2% (w/v) osmium using an AFS apparatus (Leica) as described by Tse et al (2006). After freeze substitution, sections were embedded, sectioned, and observed by TEM according to Osterrieder et al (2010).…”