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Plant and Cell Physiology Advance Access published online on May 3, 2006
Plant and Cell Physiology, doi:10.1093/pcp/pcj056
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Plant and Cell Physiology 2006 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.
Received February 25, 2006
Accepted April 28, 2006

Regular Paper

Actin Microfilaments Regulate Vacuolar Structures and Dynamics: Dual Observation of Actin Microfilaments and Vacuolar Membrane in Living Tobacco BY-2 Cells

Takumi Higaki 1, Natsumaro Kutsuna 1, Emiko Okubo 1, Toshio Sano 1, and Seiichiro Hasezawa 1 *

1 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo

* To whom correspondence should be addressed.
Seiichiro Hasezawa, E-mail: hasezawa{at}k.u-tokyo.ac.jp


  Abstract

Actin microfilaments (MFs) participate many fundamental processes in plant growth and development. Here, we report the co-localization of the actin microfilaments and vacuolar membrane, as visualized by vital vacuolar membrane (VM) staining with FM4-64 in living tobacco BY-2 cells stably expressing GFP-fimbrin (BY-GF11). The MFs were intensively localized on the VM surface and at the periphery of the cytoplasmic strands rather than at their center. The co-localization between MFs and VM was confirmed by the observation using transient expression of RFP-fimbrin in tobacco BY-2 cells stably expressing GFP-AtVam3p (BY-GV7) and BY-2 cells stably expressing {gamma}TIP-GFP fusion protein (BY-GG). Time-lapse imaging revealed dynamic movement of MF structures which were parallel to that of cytoplasmic strands. Disruption of MF structures disorganized cytoplasmic strand structures and produced small spherical vacuoles in the VM-accumulated region. Three-dimensional reconstructions of the vacuolar structures revealed a disconnection of these small spherical vacuoles from the large vacuoles. Real-time observations and quantitative image analyses demonstrated rapid movements of MFs and VM near the cell cortex, which were inhibited by the general myosin ATPase inhibitor, 2,3-butanedion monoxime (BDM). Moreover, both BA and BDM treatment inhibited the reorganization of the cytoplasmic strands and the migration of daughter cell nuclei at the early G1 phase, suggesting a requirement of the acto-myosin system for vacuolar morphogenesis during cell cycle progression. These results suggest that MFs support the vacuolar structures and that the acto-myosin system plays an essential role in vacuolar morphogenesis.

Keywords: actin microfilament; cytoplasmic strand; FM4-64; green fluorescent protein; tobacco BY-2 cell; vacuole.
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