Abstract
In higher plants, vacuoles increase their volume according to cell enlargement and become large vacuoles that occupy most of the cell volume. To observe the vacuolar dynamics in living cells, we have already established a transgenic BY-2 cell-line expressing a GFP-AtVam3p fusion protein (BY-GV), and have also developed 3-D reconstruction software, named REANT. These methods allowed us to reveal unique vacuolar structures including TVM (tubular structure of vacuolar membrane) in mitotic cell and vacuolar networks developed in elongating miniprotoplasts. In this study, vacuolar movements were quantified to explore the physical mechanism of the formation and development of these vacuolar structures. We developed analyzing software tools for time-sequential series of fluorescent images of vacuolar membrane and bright-field images. Frequency analysis using discrete wavelet transform enabled us to evaluate vacuolar movements without image segmentation. Vacuolar movements were regulated by spatio-temporal factors: cell cycle progression, vacuolar structures and actin microfilaments.
