Cytoskeletal and Vacuolar Dynamics During Plant Cell Division 127
cell line that can be highly synchronized, it is the main cell line used for plant
cell cycle studies (Nagata et al. 1992). Synchronization in BY-2 cells is usually
performed using aphidicolin, an inhibitor of DNA polymerase (Nagata and
Kumagai 1999). Incubation of fully grown (usually 7-day-old) tobacco BY-2
cells for 24 hinfreshmediumwith 5 mg/L aphidicolin arrests the cell cycle at
the border of the G1/S phase, and subsequent washing of the cell culture with
fresh medium restarts the cell cycle from the S phase. After appropriate treat-
ment, 8 to 9 h after aphidicolin release, 60 to 70 %ofthecellscanbeobserved
in mitosis. Sequential treatment with and subsequent release from propyza-
mide, a reversible inhibitor of microtubule polymerization that arrests the cell
cycle at pro-metaphase and collects the cells at this stage, results in more than
95 % of cells being at mitosis (Nagata and Kumagai 1999). Using these proper-
ties, the dynamics of cytoskeletons and vacuolar structures during cell cycle
progression could be studied and are described below.
3
Microtubule Dynamics and Functions of MT-Associated Proteins
in Plant Cell Division
3.1
Microtubule Dynamics During Cell Cycle Progression
The structure and organization of microtubules in higher plant cells have
been studied mainly by immuno-fluorescence microscopy and microinjec-
tion of fluorescent-labeled tubulin (Zhang et al. 1990; Hasezawa et al. 1991;
Vantard et al. 2000; Hasezawa and Kumagai 2002). The four characteristic
structures of MTs that have to date been identified during cell cycle pro-
gression are as follows: (i) Cortical MTs (CMTs), which align transversely
to the orientation of cell elongation, are observed from G1 through S to
theG2phase.TheCMTisconsideredtobecriticalforthedetermination
of plant cell shape, since CMT arrays determine the direction of the newly
formed cellulose microfibrils in the primary cell walls (Ledbetter and Porter
1963; Giddings and Staehelin 1991; Baskin 2001; Wasteneys 2004); (ii) The
preprophase band (PPB) is a bundle MT structure on the cell surface that re-
places the gradually disappearing CMTs in the late G2 phase. As the PPB also
disappears during mitosis, and the future cell plate fuses at the position where
the PPB existed previously, the PPB is thought to leave some sort of mem-
ory to indicate the position of the future cell division plane (Mineyuki 1999);
(iii) the mitotic spindle then segregates the chromosomes at mitosis; and sub-
sequently (iv) a microtubule structure called the phragmoplast forms the cell
plate.
To gain further insight into the structural changes in these different MT
configurations, the development and observation of a living cell system was