Transgenic BY-2 cells stably expressing a GFP (green fluorescent protein)-tubulin fusion protein (BY-GT16) were subcultured in a modified Linsmaier and Skoog medium. The BY-GT16 cells could be synchronized by aphidicolin and the dynamics of their microtubules (MTs) were monitored by the confocal laser scanning microscopy (CLSM). We have succeeded in investigating the mode of reorganization of cortical MTs at the M/G1 interface. The cortical MTs were initially organized in the perinuclear regions and then they elongated to reach the cell cortex, forming the bright spots there. Subsequently, the first cortical MTs rapidly elongated from the spots and they were oriented parallel to the long axis towards the distal end of the cells. Around the time when the tips of the parallel MTs reached the distal end, the formation of transverse cortical MTs followed in the cortex near the division site, as we had previously suggested [Hasezawa and Nagata (1991) Bot. Acta 104: 206, Nagata et al. (1994) Planta 193: 567]. It was confirmed in independent observations that the appearance of the parallel MTs was followed by the appearance of the transverse MTs in each cell. We found that the transverse MTs spread through the whole cell cortex within about 20-30 min, while the parallel MTs disappeared. The significance of these observations on the mode of cortical MT organization is discussed.

The mode of cytokinesis, especially in determining the site of cell division, is not well understood in higher-plant cells. The division site appears to be predicted by the preprophase band of microtubules that develop with the phragmosome, an intracellular structure of the cytoplasm suspending the nucleus and the mitotic apparatus in the center. As the preprophase band disappears during mitosis, it is thought to leave some form of "memory" on the plasma membrane to guide the growth of the new cell plate at cytokinesis. However, the intrinsic nature of this "memory" remains to be clarified. In addition to microtubules, microfilaments also dynamically change forms during cell cycle transition from the late G2 to the early G1 phase. We have studied the relationships between microtubules and microfilaments in tobacco BY-2 cells and transgenic BY-2 cells expressing a fusion protein of green-fluorescent protein and tubulin. At the late G2 phase, microfilaments colocalize with the preprophase band of microtubules. However, an actin-depleted zone which appears at late prometaphase is observed around the chromosomes, especially at metaphase, but also throughout anaphase. To study the functions of the actin-depleted zone, we disrupted the microfilament structures with bistheonellide A, a novel macrolide that depolymerizes microfilaments very rapidly even at low concentrations. The division planes became disorganized when the drug was added to synchronized BY-2 cells before the appearance of the actin-depleted zone. In contrast, the division planes appeared smooth, as in control cells, when the drug was added after the appearance of the actin-depleted zone. These results suggest that the actin-depleted zone may participate in the demarcation of the division site at the final stage of cell division in higher plants.

;Plant vacuoles are the largest of organelles, performing various functions in cellular metabolism, morphogenesis and cell division. Dynamic changes in vacuoles during mitosis were studied by monitoring tubular structure of vacuolar membrane (TVM) in living transgenic tobacco BY-2 cells stably expressing a GFP-AtVam3p fusion protein (BY-GV). Comprehensive images of the complicated TVM configurations were obtained by reconstructing three-dimensional (3-D) surface structures from sequential confocal sections, using newly developed software, SSR (stereo-structure reconstructor). Using the surface modeling technique, we succeeded for the first time in clarifying the development process of TVMs and the topological relationship between TVMs and large vacuoles. TVMs, initially organized from large vacuoles, elongated to encircle the spindle at metaphase. Subsequently, the TVMs invaded the equatorial region from anaphase to telophase, and then they were divided to the two daughter cells by the cell plate at cytokinesis. When the daughter nuclei were separating from the cell plate, some TVMs enlarged to form large vacuoles near the division site. Spatial analysis revealed that from anaphase until cytokinesis, TVMs connected the two large vacuoles and functioned as a route for inter-vacuolar transport. Furthermore, the experiments using the inhibitor for actin microfilaments indicated that the microfilaments were indispensable for the development and the maintenance of TVMs.

The preprophase band (PPB) of microtubules is thought to be involved in deciding the future division site. In this study, we investigated the effects of double PPBs on spindle formation and the directional decision of cytokinesis by using transgenic BY-2 cells expressing green fluorescent protein (GFP)-tubulin. At prophase, most of the cells with double PPBs formed multipolar spindles, whereas all cells with single PPBs formed normal bipolar spindles, clearly implicating the PPB in deciding the spindle poles. At metaphase, however, both cell types possessed the bipolar spindles, indicating the existence of correctional mechanism(s) at prometaphase. From prometaphase to metaphase, the spindles in double PPB cells altered their directions to become oblique to the cell-elongating axis, and these orientations were maintained in the phragmoplast and resulted in the oblique division planes. These oblique cell plates decreased when actin microfilaments were disrupted, and double actin-depleted zones (ADZs) appeared where the double PPBs had existed. These results suggest that the information necessary for proper cytokinesis may be transferred from the PPBs to the ADZs, even in the case of the double PPBs.

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