Displacement of the mitotic apparatuses by centrifugation reveals cortical actin organization during cytokinesis in cultured tobacco BY-2 cells
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In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global–local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global–local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.
KeywordsActin cable Actin-depleted zone Tobacco BY-2 cells Cortical division zone Global–local live imaging microscope Plant cytokinesis
We thank Prof. S. Hasezawa (The University of Tokyo) for providing BY-GF11 cells. We also thank Dr. E. Yokota (University of Hyogo) and Dr. S. Nonaka (National Institute for Basic Biology) for fruitful discussions. We also thank Mr. Katsumoto Umano (Mitani Corporation) and Mr. Kazuyuki Ishiwata (Nikon Instech Co. Ltd.) for their collaboration for the development of the GLIM system. This work was supported by JST SENTAN.
Supplementary Movie S8 Global and local images of a BY-GF11 cell obtained with the GLIM. Time-lapse DIC images (left) and the fluorescence images of cell cortex (right) of a BY-GF11 cell were taken at a 30-sec interval, and the movie plays at 10 frames per second. See Fig. S6 in detail (AVI 2703 KB)
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