Three-Dimensional Reconstruction of Tubular Structure of Vacuolar Membrane Throughout Mitosis in Living Tobacco Cells

Abstract

;Plant vacuoles are the largest of organelles, performing various functions in cellular metabolism, morphogenesis and cell division. Dynamic changes in vacuoles during mitosis were studied by monitoring tubular structure of vacuolar membrane (TVM) in living transgenic tobacco BY-2 cells stably expressing a GFP-AtVam3p fusion protein (BY-GV). Comprehensive images of the complicated TVM configurations were obtained by reconstructing three-dimensional (3-D) surface structures from sequential confocal sections, using newly developed software, SSR (stereo-structure reconstructor). Using the surface modeling technique, we succeeded for the first time in clarifying the development process of TVMs and the topological relationship between TVMs and large vacuoles. TVMs, initially organized from large vacuoles, elongated to encircle the spindle at metaphase. Subsequently, the TVMs invaded the equatorial region from anaphase to telophase, and then they were divided to the two daughter cells by the cell plate at cytokinesis. When the daughter nuclei were separating from the cell plate, some TVMs enlarged to form large vacuoles near the division site. Spatial analysis revealed that from anaphase until cytokinesis, TVMs connected the two large vacuoles and functioned as a route for inter-vacuolar transport. Furthermore, the experiments using the inhibitor for actin microfilaments indicated that the microfilaments were indispensable for the development and the maintenance of TVMs.

Increase in Invaginated Vacuolar Membrane Structure Caused by Plant Cell Expansion by Genotoxic Stress Induced by DNA Double-Strand Breaks

“…Thus, zeocin induced cell expansion in BY-2 cells. We then observed the vacuole morphology using a BY-2 cell line (BY-GV) that expresses a vacuolar membrane protein, AtVam3p/SYP22 fused with GFP (Kutsuna et al 2003) under the same condition. Many cytoplasmic strands (also called transvacuolar strands) were generated in the vacuolar lumen in spite of cell expansion in the BY-GV cells cultured in the medium adding 50 μM zeocin (Fig.…”

“…Bright Yellow 2) cultured cell line, BY-GV cell line and BY-GF cell line were cultured by rotary shaking at 130 rpm at 27 C in the dark and maintained using the method of Kumagai-Sano et al (2006). The BY-GV line is expressed GFPAtVam3p/SYP22 fusion protein (Kutsuna et al 2003). The BY-GF line expresses the second actinbinding domain (ABD2) of the Arabidopsis AtFim1 protein fused with GFP (Sano et al 2005).…”

ArabidopsisProtein Disulfide Isomerase-5 Inhibits Cysteine Proteases during Trafficking to Vacuoles before Programmed Cell Death of the Endothelium in Developing Seeds

“…Plant vacuoles are highly dynamic organelles that can undergo rapid changes in form and volume. The best-studied dynamics relate to the changes in vacuolar architecture associated with the cell cycle (Marty, 1999;Kutsuna et al, 2003;Seguí-Simarro and Staehelin, 2006). For example, in Arabidopsis apical meristem cells, as the cells progress from prometaphase to early telophase, the lytic vacuolar system becomes highly tubular and fragments, and its surface area decreases by ;50% and its volume by ;80%.…”

The Dynamic Changes of Tonoplasts in Guard Cells Are Important for Stomatal Movement inVicia faba

“…Many studies support this hypothesis. TVMs also were found in many dividing or growing cells, such as the cultured evacuolated oat (Avena sativa) mesophyll protoplasts (Newell et al, 1998), tobacco mitotic BY-2 cells (Kutsuna and Hasezawa, 2002;Kutsuna et al, 2003), germinating Arabidopsis pollen (Hicks et al, 2004), and dividing Allium mother guard cells (Palevitz and Okane, 1981). As the plant cell enlargement after cytokinesis was mainly from the expansion of vacuolar volume, TVMs in these cells might also take a role in such processes by serving as a reservoir of tonoplasts for the enlargement of vacuolar volume.…”