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

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.

Disruption of actin filaments delays accumulation of cell plate membranes after chromosome separation.

Phragmoplasts, which comprise microtubules, actin filaments, and membrane vesicles, are responsible for cell plate formation and expansion during plant cytokinesis. Our previous research using the actin polymerization inhibitor latrunculin B (LatB) to investigate the role of actin filaments suggested the existence of two types of microtubules: 1) initial microtubules sensitive to LatB but unassociated with NACK1 kinesin and 2) later LatB-insensitive, NACK1-associated microtubules. The organization of initial phragmoplast microtubules might have been disrupted by the LatB treatment; this hypothesis remained unverified, however, as the exact timing of cell plate membrane accumulation could not be determined. In the present study, we further investigated the timing of cell plate formation during LatB treatment. We monitored chromosome separation during anaphase as well as accumulation of FM4-64-stained cell plate membranes in dividing transgenic tobacco BY-2 cells expressing RFP-tagged histone H2B. We observed that LatB treatment prolonged the time between the slowdown of daughter chromosome migration and the accumulation of cell plate membranes. This result suggests that disruption of actin filaments resulted in delayed cell plate formation possibly by perturbation of initial phragmoplast microtubules or cell plate assembly.

Actin Filament Disruption Alters Phragmoplast Microtubule Dynamics during the Initial Phase of Plant Cytokinesis.

Plant growth and development relies on the accurate positioning of the cell plate between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which contains bipolar microtubules that polymerize to form a framework with the plus ends at or near the division site. This allows the transport of Golgi-derived vesicles toward the plus ends to form and expand the cell plate. Actin filaments play important roles in cell plate expansion and guidance in plant cytokinesis at the late phase, but whether they are involved at the early phase is unknown. To investigate this further, we disrupted the actin filaments in cell cycle-synchronized tobacco BY-2 cells with latrunculin B (LatB), an actin polymerization inhibitor. We observed the cells under a transmission electron microscope or a spinning-disk confocal laser scanning microscope. We found that disruption of actin filaments by LatB caused the membrane vesicles at the equatorial plane of the cell plate to be dispersed rather than form clusters as they did in the untreated cells. The midzone constriction of phragmoplast microtubules also was perturbed in LatB-treated cells. The live cell imaging and kymograph analysis showed that disruption of actin filaments also changed the accumulation timing of NACK1 kinesin, which plays a crucial role in cell plate expansion. This suggests that there are two functionally different types of microtubules in the phragmoplast. Together, our results show that actin filaments regulate phragmoplast microtubules at the initial phase of plant cytokinesis.