Journal Article

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

Natsumaro Kutsuna, Fumi Kumagai, Masa H. Sato and Seiichiro Hasezawa

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 44, issue 10, pages 1045-1054
Published in print October 2003 | ISSN: 0032-0781
Published online October 2003 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcg124
Three-Dimensional Reconstruction of Tubular Structure of Vacuolar Membrane Throughout Mitosis in Living Tobacco Cells

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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.

Keywords: Keywords: 3-D reconstruction — AtVam3p — GFP — Mitosis — Tobacco BY-2 cells — Vacuole.; Abbreviations: 3-D, three-dimensional; BA, bistheonellide A; BY-GV, BY-2 cell line expressing GFP-AtVam3p fusion protein; CLSM, confocal laser scanning microscopy; GFP, green fluorescent protein; MF, microfilament; MT, microtubule; SSR, stereo-structure reconstructor; TVM, tubular structure of vacuolar membrane; VM, vacuolar membrane.

Journal Article.  6527 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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