Yoshinobu Mineyuki

The Preprophase Band of Microtubules: Its Function as a Cytokinetic Apparatus in Higher Plants

Features, development, and functions of preprophase bands (PPBs) of microtubules (MTs) are reviewed. The PPB is an array of cortical MTs in higher plants that appears in G 2 and prophase and predicts where the cell plate will be inserted (the division site). Experimental obliteration of the PPB causes misplacement of cell plate insertion, suggesting that the PPB is a determinant of the ultimate division site. Its development contains two elementary processes: Broad PPB formation first fixes the axis of division polarity in the cell, and PPB narrowing then defines the precise division site. The PPB disappears at the prophase/prometaphase transition stage, but it leaves information in some yet unidentified form at the division site. This information assists correct insertion of cell plates and maturation of new cell walls after cytokinesis. Several kinds of molecules are reported to occur in PPBs, but their roles are not yet understood. Actin and cyclin-dependent kinase homologs are suggested to be involved in the band narrowing MT, which is essential for PPBs to mature at the division site. Other possible functions of the PPB, such as premitotic nuclear positioning and prophase spindle orientation, are also reviewed.

p 34cdc2 kinase homologue in the preprophase band

SummaryImmunofluorescence microscopy with a monoclonal antibody raised against the PSTAIR sequence, which corresponds to a peptide conserved in the p 34cdc2 protein kinase throughout the phylogenetic scale including higher plants, was used to study the intracellular localization of p 34cdc2 during the cell cycle in onion root tip cells. Although p 34cdc2 was evenly distributed in the cytoplasm throughout the cell cycle, a more intense staining was observed in the cortical region, where the preprophase band of microtubules (MTs) was located. Double staining with the PSTAIR and plant tubulin antibodies showed that the width of p 34cdc2 band was narrower than that of MT band. These data raise the interesting question regarding the possible role of p 34cdc2 protein kinase in determining the division site in plant cells.

Formation of Highly Twisted Ribbons in a Carboxymethylcellulase Gene-Disrupted Strain of a Cellulose-Producing Bacterium

Cellulases are enzymes that normally digest cellulose; however, some are known to play essential roles in cellulose biosynthesis. Although some endogenous cellulases of plants and cellulose-producing bacteria are reportedly involved in cellulose production, their functions in cellulose production are unknown. In this study, we demonstrated that disruption of the cellulase (carboxymethylcellulase) gene causes irregular packing of de novo-synthesized fibrils in Gluconacetobacter xylinus, a cellulose-producing bacterium. Cellulose production was remarkably reduced and small amounts of particulate material were accumulated in the culture of a cmcax-disrupted G. xylinus strain (F2-2). The particulate material was shown to contain cellulose by both solid-state (13)C nuclear magnetic resonance analysis and Fourier transform infrared spectroscopy analysis. Electron microscopy revealed that the cellulose fibrils produced by the F2-2 cells were highly twisted compared with those produced by control cells. This hypertwisting of the fibrils may reduce cellulose synthesis in the F2-2 strains.

Dynamic changes in the actin cytoskeleton during the high-fluence rate response of theMougeotia chloroplast

SummarySince photo-induced orientation movement of a single, ribbon-shaped chloroplast in each cell of the filamentous green algaMougeotia is inhibited in the presence of cytochalasin B, actin is thought to be involved in the process of chloroplast movements. However, this possibility remains to be proved. A specific class of cytoplasmic filaments, which emerge from the advancing front of the moving chloroplast, can be seen by differential interference contrast (DIC) microscopy. However, no one has yet succeeded in defining the nature of these filaments. We have been able to stain the actin filaments (AFs) associated with the moving chloroplast with fluorescein-conjugated phalloidin (FP) after pre-treatment withm-maleimidobenzoyl N-hydroxysuccinimide ester (MBS). No filamentous structures were observed in cells that had been pre-irradiated with low-fluence rate red light. However, transversely oriented fluorescent filaments appeared at the front edge of the moving chloroplast when it began to rotate under irradiation with high-fluence rate white light. These filaments disappeared after completion of the orientation movement, suggesting the simultaneous appearance of AFs and the orientation movement of the chloroplast. Thick cytoplasmic strands connecting the edge of the chloroplast with the parietal cytoplasm were often seen by DIC microscopy before and after completion of the high-fluence rate orientation movement. These thick cytoplasmic strands could not be stained by FP, but were often stained by 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)), suggesting that they are transvacuolar strands that include endoplasmic reticulum.

Regulation of Actin-Dependent Cytoplasmic Motility by Type II Phytochrome Occurs within Seconds in Vallisneria gigantea Epidermal Cells

The effects of light on actin-dependent cytoplasmic motility in epidermal cells of green leaves of the aquatic angiosperm Vallisneria gigantea were investigated quantitatively using a custom-made dynamic image analyzer. Cytoplasmic motility was measured by monitoring changes in the brightness of individual pixels on digitized images taken sequentially under infrared light. Acceleration and deceleration of cytoplasmic motility were regulated photoreversibly by type II phytochrome(s). This phytochrome-dependent induction of cytoplasmic motility did not occur uniformly in cytoplasm but took place as scattered patches in which no particular organelles, including nucleus, existed. The induction became detectable at 2.5 s after the start of irradiation with pulsed red light. In cells exposed to microbeam irradiation, cytoplasmic motility was induced only in sites in the cytoplasm that were irradiated directly, whereas nonirradiated neighboring areas were unaffected. The effect was short-lived, disappearing within a few minutes, and no signal was transmitted from an irradiated cell to its neighbors. Anti-phytochrome antibody–responsive protein(s) was detectable in the leaf extract by immunoblot and zinc blot analyses and in cryosections of the epidermis by immunocytochemistry. Although the phytochrome-dependent cytoplasmic motility was blocked by exogenously applied latrunculin B or cytochalasins, treatment of the dark-adapted cells with Ca2+-chelating reagents induced the cytoplasmic motility. We have proposed a model for the phytochrome regulation of cytoplasmic motility as one of the earliest responses to a light stimulus.

Plant microtubule studies: past and present

Here, I briefly review historical and morphological aspects of plant microtubule studies in land plants. Microtubules are formed from tubulins, and the polymeric configurations appear as singlet, doublet, and triplet microtubules. Doublet microtubules occur in the axoneme of cilia and flagella, and triplet microtubules occur in the basal bodies and centrosomes. Doublet and triplet microtubules are lost in all angiosperms and some gymnosperms that do not possess flagellated sperm. In land plants with flagellated sperm, centriolar centrosomes transform into basal bodies during spermatogenesis. In flowering plants, however, most male gametes (sperm) are conveyed to eggs without the benefit of cilia or flagella; thus, higher plants lack centriolar centrosome and doublet and triplet microtubules. The loss of centriolar centrosomes from the life cycle of flowering plants may have influenced the evolution of the plant microtubule system. Comparison of mitotic apparatuses in basal land plants and flowering plants illuminates the evolutionary transition from the centriolar microtubule system to the acentriolar microtubule system.

EFFECTS OF CYCLOHEXIMIDE ON PREPROPHASE BANDS AND PROPHASE SPINDLES IN ONION (ALLIUM CEPA L.) ROOT TIP CELLS

SummaryEffects of cycloheximide (CHM) on preprophase bands (PPBs) of microtubules (MTs) and on prophase spindle MTs in root tip cells of onion (Allium cepa L.) were examined. When root tip cells were treated with 36 μM CHM for 0.5–4 h, the population of cells with a PPB did not decrease markedly although the population of mitotic cells and that of prophase cells with a PPB gradually decreased to half of the control root tips. In prophase cells treated with 11 and 36 μM CHM for 2 h, the width of the PPB was 1.4 times broader than that in the prophase PPB without CHM. Electron microscopic observation on the cross section of the PPB showed that the number of MTs and the distance between adjacent MTs in prophase PPBs treated with CHM were similar to those in the early developmental stage of PPBs without CHM. The bipolar spindle, that appeared in late prophase was not seen in prophase cells treated with 11 μM or higher concentrations of CHM for 2 h. In order to examine differences of perinuclear MT arrangement between CHM treated and non-treated prophase cells, arrangement of perinuclear MTs was examined by confocal laser scanning microscopy. In control cells without CHM, MTs appeared on the nuclear surface with several “branched” or “cross over” type MT foci in the cytoplasm when broad PPB formation started. These MT foci were replaced by the “aster” type MT foci, from which several MTs radiated along the nuclear surface. The “aster” type MT foci gradually gathered to form a bipolar spindle. MTs connecting the spindle pole region and the PPB were seen in late prophase. In CHM-treated cells (11-360 μM for 2 h), “branched” and “cross over” type MT foci were prominent, even in prophase cells with well condensed chromosomes. Neither linkages of MTs between the spindle pole region and the PPB nor “aster” type MT foci were seen. These observations showed that CHM prevents the bundling of MTs in the PPB and also inhibits the formation of “aster” type MT foci that is essential for bipolar spindle development.

Plant Cytokinesis: Terminology for Structures and Processes

Plant cytokinesis is orchestrated by a specialized structure, the phragmoplast. The phragmoplast first occurred in representatives of Charophyte algae and then became the main division apparatus in land plants. Major cellular activities, including cytoskeletal dynamics, vesicle trafficking, membrane assembly, and cell wall biosynthesis, cooperate in the phragmoplast under the guidance of a complex signaling network. Furthermore, the phragmoplast combines plant-specific features with the conserved cytokinetic processes of animals, fungi, and protists. As such, the phragmoplast represents a useful system for understanding both plant cell dynamics and the evolution of cytokinesis. We recognize that future research and knowledge transfer into other fields would benefit from standardized terminology. Here, we propose such a lexicon of terminology for specific structures and processes associated with plant cytokinesis.

Meiotic cytokinetic apparatus in the formation of the linear spore tetrads of Conocephalum japonicum (Bryophyta)

Abstract. Most bryophytes produce tetrahedral spore tetrads. However, linear spore tetrads have been reported to occur in Conocephalum japonicum (Thunb.) Grolle. In this study, the distribution of microtubules (MTs) during meiosis in C. japonicum was examined to determine the division pattern resulting in a linear tetrad. Spore mother cells in the pre-meiotic stage were cylindrical with randomly distributed cytoplasmic MTs. In the prophase-metaphase transition, spindle MTs replaced cytoplasmic MTs and a barrel-shaped spindle with two flattened poles developed. Cortical MT arrays were not detectable throughout meiosis. Although a phragmoplast appeared between sister nuclei in telophase-I, it disappeared without expanding to the parental cell wall. Metaphase-II spindles oriented parallel to the long axis of the cell and in tandem to each other resulted in a linear arrangement of telophase nuclei. Radial arrays of MTs developed from the nuclear surfaces and three phragmoplasts appeared among the four nuclei to produce four spores. Two phragmoplasts separating the paired sister nuclei appeared prior to the appearance of a phragmoplast between non-sister nuclei. The MT cycle is basically the same as that reported in meiosis of C. conicum, which produces non-linear tetrads. A morphometric study indicated that the difference in the division pattern between C. conicum and C. japonicum is due to a difference in the shape of spore mother cells. The cylindrical shape of sporocytes of C. japonicum restricts the orientation of spindles and phragmoplasts so that the four resultant spores are arranged linearly.

Isolation of a Protein Interacting with Vfphot1a in Guard Cells of Vicia faba

A recent study has demonstrated that phototropins act as blue light receptors in stomatal guard cells. However, the downstream components responsible for phototropin signaling are largely unknown. In this study, using a yeast two-hybrid system, we isolated a Vicia faba protein that has a high similarity to dynein light chain in the C terminus, which interacts with Vicia faba phototropin 1a (Vfphot1a). Protein-blot and two-hybrid analyses revealed that Vfphot1a interacting protein (VfPIP) bound to the C-terminal region of Vfphot1a but did not bind to Vfphot1b. The interaction between VfPIP and Vfphot was indicated by a pull-down assay. Northern analysis revealed that the transcription level of VfPIP gene was more abundant in guard cells than in other tissues or cell types. The transiently expressed fusion protein of VfPIP-green fluorescent protein was localized on cortical microtubules in Vicia guard cells. Microtubule-depolymerizing herbicides partially inhibited both blue light-dependent H+ pumping in Vicia guard cell protoplasts and stomatal opening in the Vicia epidermis. From these results, we conclude that VfPIP may act as a downstream component of phototropin (Vfphot1a) in blue light signaling in guard cells. The possible role of VfPIP in blue light signaling of guard cells is discussed.