Tetsuhiro Asada

Expansion of the Cell Plate in Plant Cytokinesis Requires a Kinesin-like Protein/MAPKKK Complex

The tobacco mitogen-activated protein kinase kinase kinase NPK1 regulates lateral expansion of the cell plate at cytokinesis. Here, we show that the kinesin-like proteins NACK1 and NACK2 act as activators of NPK1. Biochemical analysis suggests that direct binding of NACK1 to NPK1 stimulates kinase activity. NACK1 is accumulated specifically in M phase and colocalized with NPK1 at the phragmoplast equator. Overexpression of a truncated NACK1 protein that lacks the motor domain disrupts NPK1 concentration at the phragmoplast equator and cell plate formation. Incomplete cytokinesis is also observed when expression of NACK1 and NACK2 is repressed by virus-induced gene silencing and in embryonic cells from Arabidopsis mutants in which a NACK1 ortholog is disrupted. Thus, we conclude that expansion of the cell plate requires NACK1/2 to regulate the activity and localization of NPK1.

Molecular motors in higher plants

Until recently, it was difficult to investigate how plants generated intracellular motility. However, the identification and characterization of molecular motors has improved our understanding of the underlying mechanisms involved, and should facilitate new experimental approaches. Cytoplasmic streaming — the most prominent form of intracellular movement in nondividing plant cells — can be explained by the activity of an actin-based motor first purified from lily pollen tubes. Mitosis and cytokinesis involve microtubule-based movement, and experiments now implicate various microtubule-based motors (kinesin-like proteins) in aspects of cell division. Some of these plant motors have unique features and forms of regulation not seen before in other eukaryotes.

Identification of a novel plant-specific kinesin-like protein that is highly expressed in interphase tobacco BY-2 cells

SummaryThrough reverse transcription-polymerase chain reaction and Northern blot analysis, we identified TBK5, a novel plantspecific kinesin-like protein (KLP) that is highly expressed in interphase tobacco BY-2 cells. TBK5 mRNA was present at a high level throughout the growth cycle, even in cells that had entered the 4 stationary phase, where cell proliferation had ceased. However, transcripts for five other tobacco KLPs that we have identified were preferentially expressed in mitotic cells, and either not or only slightly accumulated in cells that had entered the stationary phase. Thus, TBK5 appears to be a KLP whose cellular function most closely relates to the cortical array of microtubules that plays a key role in plant cell morphogenesis. The predicted structure of TBK5 is characterized by a central motor domain that is phylogenetically distant from those of other reported KLPs, coiled-coil domains located on both sides of the motor domain, and a basic C-terminal domain. In addition, TBK5 has a putative neck domain which is closely related to the neck domain of KLPs with C-terminal motor domains, previously shown to control the direction of KLP movement towards the minus ends. Antibodies against truncated TBK5 recognized a polypeptide with a molecular mass of 74 kDa in cytoplasmic extracts of interphase cells, and this polypeptide cosedimented with microtubules assembled in the cytoplasmic extracts. The 74 kDa polypeptide corresponding to TBK5 dissociated from microtubules with high concentrations of NaCl but was not dissociated by MgATP. We hypothesize that TBK5 functions in the regulation of the arrangement of cortical microtubules.

The use of model systems prepared from tobacco BY-2 cells for studies of the plant cytoskeleton

Model systems, such as plasma membrane‐permeabilized cells, isolated phragmoplasts and membrane ghosts, were prepared from tobacco BY‐2 cells and used for studies of the plant cytoskeleton. The use of membrane‐permeabilized cells enabled us to demonstrate the occurrence in the phragmoplast of the translocation of microtubules away from the equatorial plane and led us to the isolation of a kinesin‐like microtubule‐translocating protein from phragmoplasts. Autoradiographic experiments using preparations of isolated phragmoplasts revealed that 1,3‐β‐glucan was synthesized at the equatorial plane of isolated phragmoplasts and xyloglucan was synthesized in the Golgi apparatus. Membrane ghosts from which pre‐existing cortical microtubules had been removed by a cold solution of CaCl2 bound microtubules that had polymerized in vitro, but those from which pre‐existing microtubules had been removed by KCl did not. We used membrane ghosts that had been pretreated with KCl in an attempt to identify a microtubule‐plasma membrane cross‐linking activity in a fraction of a cell extract. We found that a fraction solubilized by KCl from isolated cortical microtubules had such activity.

Division of Shape-Standardized Tobacco Cells Reveals a Limit to the Occurrence of Single-Criterion-Based Selection of the Plane of Symmetric Division

The plane of symmetric plant cell division tends to be selected so that the new cross-wall halving the cell volume has the least possible area, and several cases of such selection are best represented by a recently formulated model which promotes the view that the strength of the least area tendency is the only criterion for selecting the plane. To test this model, the present study examined the divisions of two types of shape-standardized tobacco BY-2 cell, oblate-spheroidal (os) cells prepared from protoplasts and spheri-cylindrical (sc) cells with unusual double-wall structures prepared from plasmolyzed cells. Measurements of cell shape parameters and division angles revealed that both cell types most frequently divide nearly along their short axes. While os cells did not exhibit any other division angle bias, sc cell division was characterized by another bias which made the frequency of longitudinal divisions secondarily high. The geometry of sc cells barely allows the longitudinal cross-walls to have locally minimum areas. Nevertheless, a comparison of detected and hypothetical standard divisions indicates that the frequency of longitudinal sc cell division can be significantly higher than that predicted when the longitudinal cross-walls are assumed to have locally minimum areas smaller than their original areas. These results suggest that, even in isolated plant cell types, the strength of the least area tendency is not the only criterion for selecting the division plane. The possibility that there is another basic, though often hidden, criterion is discussed.

Cell Plate Formation: Knowledge from Studies Using Tobacco BY-2 Cells

In higher plants, cytokinesis is achieved by cell plate formation, which is responsible for the assembly of the new plasma membrane and new wall matrix. In somatic cell types, this process initiates with the generation of an immature cell plate between separating groups of anaphase chromosomes, and proceeds by centrifugal growth of the cell plate. This cytokinetic process contrasts with those in animal, fungal, and most alga cells, where cleavage of parental cytoplasm starts from the cell cortex with inward furrowing of the plasma membrane (Pickett-Heaps 1975; Gunning 1982; Glotzer 2001; Guertin et al. 2002). The outward cleavage characterizing higher plant cytokinesis ensures partitioning of daughter nuclei irrespective of parental cell volume and selected division plane, and thus is thought to contribute to the ability of higher plants to flexibly set the plane of cell division, and thereby generate variable cell arrangements. Much information about the structure and function of the apparatus responsible for this higher plant-specific cytokinesis, namely the phragmoplast, has been accumulated through studies using stamen hair cells of Tradescantia and endosperm cells of Haemanthus (Gunning 1982; Bajer et al. 1987).