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.Correspondence and reprints: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba Prefecture 277-8562, Japan.