|Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
We have observed CMT reorganization by immunofluorescence microscopy, and reported that MTs were nucleated from daughter nuclear surfaces prior to the CMTs (Kumagai and Hasezawa, 2001). However, we could not precisely define the time-course of events because of the rapid transition from phragmoplasts to CMTs. To observe such rapidly occurring events, we used the green fluorescent protein (GFP) and α-tubulin fusion protein as probes of MTs in living cells (Kumagai et al., 2001). In addition, we attempted to use the dye, FM4-64, to visualize vacuoles in living cells (Kutsuna and Hasezawa, 2002). This dye, first taken up into the lipid bilayer of plasma membranes and then finally incorporated into vacuolar membranes via the endocytotic pathway, unexpectedly enables us to observe dynamic vacuolar movements during cell cycle transition. We also confirmed the dynamic changes of VM structures by the observation using transgenic BY-2 cells expressing GFP-AtVam3p fusion protein (BY-GV). Furthermore, by using transgenic BY-2 cells that stably express a GFP-tubulin fusion protein (BY-GT16, Kumagai et al. 2001), we could study the relationship between the dynamics of vacuoles and microtubules.
By combining these methods, we are therefore able to discuss the mechanisms by which daughter cells prepare the machinery for cell expansion in the proper directions. It is known that after their formation, CMTs are stabilized into the direction appropriate for the cell type, geometry, and circumstance. In this study, we also concentrate on the dynamic behavior of MTs during M/G_1 transition, and leave the details of how CMTs, once formed, are stabilized into proper directions.