134 T. Sano et al.
vacuolar morphogenesis and maintenance, as well as in cytoplasmic strand
formation during cell cycle progression (Higaki et al. 2006).
5
Vacuolar Dynamics and Functions in Plant Cell Division
Vacuoles constitute the largest of compartments and multifunctional or-
ganelles of plant cells (Wink 1993). During plant cell morphogenesis, vacuoles
act in combination with the cell wall to generate cell turgor, in addition to
their space filling properties within cells (Marty 1999). Recently, GFP fu-
sion with theγ-tonoplast intrinsic protein (γ-TIP) or the vacuolar syntaxin-
related AtVam3 protein demonstrated the detailed structure of the vacuolar
membrane (Hawes et al. 2001; Saito et al. 2002; Uemura et al. 2002; Kutsuna
and Hasezawa 2002). Establishment of a transgenic tobacco BY-2 cell line ex-
pressing a GFP-AtVam3 fusion protein (BY-GV) allowed us to follow vacuolar
dynamics during cell cycle progression (Fig. 3, Kutsuna et al. 2003). Whereas
a single, large vacuole with a rather simple shape was identified from the G1
to G2 phase, compartmented and tubular structures of the vacuolar mem-
brane (TVMs) were found to encircle the mitotic spindle in mitosis, then to
be cut off by the cell plate at cytokinesis. Although the TVMs were recog-
nized as small ellipses on each optical section, a 3-D reconstruction, using our
originally developed software, REANT (reconstructor andanalyzer ofthree-
dimensional structure) together with photobleaching analysis, revealed the
topological connection between TVMs and the two large vacuoles (Fig. 3, Kut-
suna et al. 2003). The REANT permitted quantification of the 3-D structures,
including volume and surface area (Kutsuna and Hasezawa 2005). Following
cytokinesis, the TVMs returned to form large vacuoles by expansion of their
diameters and fusion with each other. Therefore, vacuolar structures changed
cyclically between TVMs and large vacuoles during cell cycle progression,
and the regeneration of the large vacuoles was not from the simple swelling
of small vacuoles.
6
Conclusions
Herein, we have demonstrated the dynamics of the cytoskeleton and vac-
uoles during cell cycle progression. By establishing transgenic cell lines in
whichthesestructurescouldbevisualizedwewereabletoperformtime-
sequential observations of their structural changes in living cells during cell
cycle progression. As a further analysis, by developing the original software
package designated as REANT, the precise 3-D organizations of the vac-
uole could be understood. A new area of research, therefore, will be the