Fumi Kumagai

Plant cell biology through the window of the highly synchronized tobacco BY-2 cell line

Synchronous cell systems are highly desirable for investigating various aspects of plant cell biology. However, to date, the tobacco BY-2 cell line is the only plant cell line which can be synchronized to high levels. A cell synchrony starting from S phase is obtained after release of BY-2 cells from aphidicolin treatment, while that from M phase is available after release from a sequential treatment of aphidicolin followed by propyzamide. A high level of synchrony is only attained by using rapidly growing tobacco BY-2 cells that propagate ca. 100-fold in a week. Reduced levels of synchrony result if the growth rate becomes lower. Technical notes for maintaining the high growth rate of the tobacco BY-2 cell are described. Using this highly synchronous system it has been possible to demonstrate the cell cycle-dependent oscillation of many genes, such as cyclins, and characterize their role during the cell cycle. Furthermore, this system has facilitated the structural and biochemical analysis of cell cycle specific events such as the development of the phragmoplast and the formation of cortical microtubules. Other potential uses of this highly synchronized cells are also described.

Roles of actin-depleted zone and preprophase band in determining the division site of higher-plant cells, a tobacco BY-2 cell line expressing GFP-tubulin.

Summary.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.

Dynamic changes and the role of the cytoskeleton during the cell cycle in higher plant cells

In higher plant cells microtubules (MTs) show dynamic structural changes during cell cycle progression and play significant roles in cell morphogenesis. The cortical MT (CMT), preprophase band (PPB), and phragmoplast, all of which are plant-specific MT structures, can be observed during interphase, from the late G2 phase to prophase, and from anaphase to telophase, respectively. The CMT controls cell shape, either irreversibly or reversibly, by orientating cellulose microfibril (CMF) deposition in the cell wall; the PPB is involved in determining the site of division; and the phragmoplast forms the cell plate at cytokinesis. The appearance and disappearance of these MT structures during the cell cycle have been extensively studied by immunofluorescence microscopy using highly synchronized tobacco BY-2 cells. Indeed, these studies, together with visualization of MT dynamics in living plant cells using the green fluorescent protein, have revealed much about the modes of MT structural organization, for example, of CMTs at the M/G1 interphase. The microfilaments which also show dynamic changes during the cell cycle, being similar to MTs at particular stages and different at other stages, appear to play roles in supporting MTs. In this article, we summarize our ongoing research and that of related studies of the structure and function of the plant cytoskeleton during cell cycle progression.

γ-Tubulin distribution during cortical microtubule reorganization at the M/G1 interface in tobacco BY-2 cells

Summary Cortical microtubules are considered to regulate the direction of cellulose microfibril deposition. Despite their significant role in determining cell morphology, cortical microtubules completely disappear from the cell cortex during M phase and become reorganized at G 1 phase. The mechanism by which these microtubules become properly formed again is, however, still unclear. We have proposed that the origin of cortical microtubules is on the daughter nuclear surface, but further cortical microtubule reorganization occurs at the cell cortex. Hence it is probable that the locations of microtubule organizing centers (MTOCs) are actively changing. However, the actual MTOC sites of cortical microtubules were not clearly determined. In this paper, we have examined the distribution of γ-tubulin, one of the key molecules of MTOCs in various organisms, during cortical microtubule reorganization using both immunofluorescence and a GFP reporter system. Using a monoclonal antibody (clone G9) that recognizes highly conserved residues in γ-tubulin, γ-tubulin was found to be constitutively expressed and to be clearly localized to microtubule structures, such as the preprophase bands, spindles, and phragmoplasts, specific to each cell cycle stage. This distribution pattern was confirmed by the GFP reporter system. During cortical microtubule reorganization at the M to G 1 transition phase, γ-tubulin first accumulated at the daughter nuclear surfaces, and then seemed to spread onto the cell cortex along with microtubules elongating from the daughter nuclei. Based on the results, it was confirmed that daughter nuclear surfaces acted as origins of cortical microtubules, and that further reorganization occurred on the cell cortex.

Sites of microtubule reorganization in tobacco BY-2 cells during cell-cycle progression

SummaryThe sites of microtubule (MT) reorganization were examined in synchronized tobacco BY-2 cells. The MTs of these cells were completely destroyed by a combined cold and drug treatment at 0 °C with 100 μM propyzamide for 3 h. After the cells were washed and cultured at 30 °C, the reorganization of MTs was observed in detail. Sites for MT reorganization at each stage of the cell cycle were identified on the cell cortex and nuclei, the mitotic apparatus, the nuclei (or the nuclei and cell cortex), and the cell cortex in the S-G2 phase, M phase, M/G1 interface, and g1 phase, respectively. The polypeptide synthesis elongation factor (EF)-1α is co-localized with these sites of MT reorganization. At some stages, microfilaments (MFs) were found to be involved in the reorganization of MTs. Based on these results, the mode of MT reorganization during cell cycle progression is discussed.

Development and disintegration of phragmoplasts in living cultured cells of a GFP∷TUA6 transgenic Arabidopsis thaliana plant

Summary. Cultured suspension cells of Arabidopsis thaliana that stably express a green-fluorescent protein–α-tubulin 6 fusion protein were used to follow the development and disintegration of phragmoplasts. The development and disintegration of phragmoplasts in the living cultured cells could be successively observed by detecting the green-fluorescent protein fluorescence of the microtubules. In the early telophase spindle, where two kinetochore groups and two daughter chromosome groups had completely separated from one another, fluorescence appeared in the interzone between the two chromosome groups. The fluorescent region was gradually condensed at the previous equator and increased in fluorescence intensity, and finally it formed the initial phragmoplast. The initial phragmoplast moved from the cell center towards the cell periphery, and it lost fluorescence at its center and became double rings in shape. The expansion orientation of the phragmoplast was not always the same as that of the future new cell wall before it came in contact with the cell wall. The phragmoplast did not usually come in contact with the cell wall simultaneously with its entire length. A portion of the phragmoplast which was earlier in contact with the cell wall disappeared earlier than other portions of the phragmoplast. The duration of contact between any portions of the phragmoplast and the plasma membrane of the cell wall was 15–30 min. The fluorescence intensity of the cytoplasm did not seem to be elevated by the disintegration of the strongly fluorescent phragmoplast.

Dynamic Behavior of Microtubules and Vacuoles at M/G1 Interface Observed in Living Tobacco BY-2 Cells

Plant cells expand mainly by water uptake into vacuoles. Although the turgor pressure of the cells is isotropic, most of the cells elongate anisotropically. This transformation of the isotropic force into the anisotropic growth is achieved by the establishment of “hoops” consisting of cellulose microfibrils (CMFs) in the cell walls. The cells can only elongate perpendicular to the newly organized CMFs, deposited at the innermost layer of the cell wall. The orientation of CMFs has been thought to be regulated by cortical microtubules (CMTs) under the cell cortex; from the observations that they run parallel to the CMFs, disturbance of CMTs resulted in aberrant cell elongation and the mutants with abnormal CMTs showed defects in elongation (for a recent review, see Baskin 2001). During the cell cycle progression, CMTs are observed only during interphase, and are then thoroughly destroyed during M phase. While CMTs are absent for about 2 h in BY-2 cells, are their any regulatory mechanisms to inhibit cell expansion into aberrant directions? And how do the daughter cells restore the next direction of expansion properly at the M/G1 interface?