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

Abstract

Actin microfilaments (MFs) participate in many fundamental processes in plant growth and development. Here, we report the co-localization of the actin MF and vacuolar membrane (VM), as visualized by vital VM staining with FM4-64 in living tobacco BY-2 cells stably expressing green fluorescent protein (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 of MFs and VMs was confirmed by the observation made using transient expression of red fluorescent protein (RFP)-fimbrin in tobacco BY-2 cells stably expressing GFP-AtVam3p (BY-GV7) and BY-2 cells stably expressing gamma-tonoplast intrinsic protein (gamma-TIP)-GFP fusion protein (BY-GG). Time-lapse imaging revealed dynamic movement of MF structures which was parallel to that of cytoplasmic strands. Disruption of MF structures disorganized cytoplasmic strand structures and produced small spherical vacuoles in the VM-accumulating 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 VMs near the cell cortex, which were inhibited by the general myosin ATPase inhibitor, 2,3-butanedion monoxime (BDM). Moreover, both bistheonellide A (BA) and BDM treatment inhibited the reorganization of the cytoplasmic strands and the migration of daughter cell nuclei at early G1 phase, suggesting a requirement for 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.

Critical role of actin bundling in plant cell morphogenesis

“…7 On the other hand, actin bundles have been observed near the cell cortex and large vacuolar surface in elongated BY-2 cells, 18,21 Arabidopsis elongated hypocotyls cells is through trafficking of the channels, as proposed in the case of auxin transport carriers, as previously described. The involvement of actin microfilaments in the translocation of water channel aquaporins between the plasma membrane and endocytic vesicles in animal cells is well documented.…”

“…1,18,22 These observations suggest that the dynamic rearrangement of actin bundles is important for cell elongation and also for the maintenance of intracellular trafficking in elongated cells. This assumption is supported by the finding that actin bundles become looser during the rapid turgor movements, seismonastic movements, of Mimosa pudica.…”

Increase in Invaginated Vacuolar Membrane Structure Caused by Plant Cell Expansion by Genotoxic Stress Induced by DNA Double-Strand Breaks

“…The vacuolar structure is regulated by actin filaments (Higaki et al 2006). Especially, the generation of cytoplasmic strands mainly relies on the actin-myosin system (Hoffmann andNebenführ 2004, Thomas et al 2009).…”

Actin bundling in plants

“…Importantly, unbundling of AFs in root hairs, following the injection of antibodies against the lily bundling protein 135-kDa villin, induces broadening and eventual loss of TVSs, indicating that actin bundles are essential for the integrity and continued existence of TVSs [Tominaga et al, 2000]. In agreement with a mechanical support function, actin bundles localize at the periphery of the cytoplasmic strands, rather than at the center [Higaki et al, 2006].…”