Cell Cycle Synchronization and Time-Lapse Imaging of Cytokinetic Tobacco BY-2 Cells.

Abstract

Transgenic tobacco BY-2 cell lines stably expressing fluorescent protein-tagged marker proteins have been used to visualize the dynamic behaviors of cytoskeletons and organelles during plant cell division. Using time-lapse confocal imaging, we recently revealed that the pharmacological disruption of actin filaments results in the abnormal organization of phragmoplast microtubules during the early phase of cytokinesis in cell cycle-synchronized BY-2 cells. Additionally, disrupting the actin filaments shortens the time from cell plate emergence to the accumulation of green fluorescent protein-tagged NACK1 kinesin on the cell plate, suggesting that there are two functionally diverse types of microtubules in the phragmoplast. We herein describe a protocol for the cell cycle synchronization of BY-2 cells and the time-lapse confocal imaging of cytokinesis combined with a treatment with an actin polymerization inhibitor and the visualization of an emerging cell plate with a vital stain. This protocol is useful for examining the dynamic changes in protein localization or the intracellular architecture and the effects of actin disruption during plant cell division.

Plant phenotyping relevance

植物細胞分裂中の細胞板形成や細胞内構造の動態を取得するタイムラプス共焦点 imaging protocol が論文の中心であり、植物細胞状態の画像ベース計測法として収載可能。

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