126 T. Sano et al.
However, because only static images could be obtained from these immuno-
fluorescence microscopy studies, due to the need for chemical fixation, the
bases of the structural changes in MTs during cell cycle progression could not
be unequivocally demonstrated. Similarly, although microinjection allowed
time-sequential observations in living cells, more detailed analyses were often
limited because of the brevity of the observation period due to diffusion of the
fluorescence and the applicability of the method to limited types of plant cells.
In contrast, the more recent fluorescent protein techniques allow the vi-
sualization of several intra-cellular structures in living cell systems (Hawes
et al. 2001). The primary advantage of these techniques is that they allow
time-sequential observations. Through the use of fluorescent proteins, the
dynamics of the intra-cellular structures were observed and the sequential
changes in MT configurations during cell cycle progression were clarified
(Hasezawa et al. 2000; Kumagai et al. 2001). The second advantage of these
techniques is that they preserve the cellular structures by avoiding the need
for chemical fixation. To observe sensitive structures against chemical fixa-
tion such as actin microfilaments (MFs), these techniques could show detailed
structures (Sano et al. 2005). Furthermore, as chemical fixation largely abol-
ished membrane structures, fluorescent protein techniques will provide new
insight in observing membrane organelles, such as vacuoles, ER and Golgi-
derived vesicles (Hawes et al. 2001).
Herein, we present an overview of recent progress in our understanding
of the dynamic changes in intra-cellular structures during cell cycle progres-
sion, as revealed by visualization of these structures by fluorescent proteins.
In particular, focus is placed on MTs, MFs and the vacuolar membrane (VM)
which we have analyzed using transgenic tobacco BY-2 cell lines. Starting with
an introduction to the tobacco BY-2 system, the dynamics of MTs and the in-
volvement of MT-associated proteins in cell cycle progression are described.
Subsequently, results from detailed observations of MFs and VM and their
dynamics are discussed in combination with our approach towards the devel-
opment of 3-D reconstruction software.
2
The Tobacco BY-2 System
Here, we first introduce the tobacco BY-2 cell line as the original cell line
of the transgenic lines described below. Of the numerous plant cell lines
available, the tobacco BY-2 cell line is perhaps the most useful for basic
plant cellular and molecular biologystudies because of its rapid growth
rate (Nagata et al. 1992; Nagata 2004). In addition, the establishment of an
Agrobacterium-mediated transformation method (An et al. 1985) has allowed
chimeric proteins, such as fusions with fluorescent proteins, to be quite easily
expressed in the cell line. Furthermore, as the BY-2 cell line is the only plant