[Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells.

To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells.

[The effect of inhibitors of serinethreonine protein kinases on Arabidopsis thaliana root morphology and microtubules organization in its cells].

The effect of different types of serine/thereonine protein kinases inhibitors (cyclin-dependent, Ca2+-calmodulin-dependent and protein kinase C) on microtubules organization in cells of Arabidopsis thaliana main primary root zones were investigated in vivo. The microtubules in epidermal and cortex cells in the transition and elongation zones as well as microtubules in trichoblasts and atrichoblasts in the differentiation zone showed the greatest sensitivity to protein kinases inhibitors studied. It was established that microtubules in these cell types modified their initial transverse/oblique orientation to a chaotic or longitudinal relative to the major axis of primary root as a result of serinethereonine protein kinases inhibition. The microtubules in cells in root meristematic zone as well as in root hairs were less sensitive to influence of protein kinases inhibitors tested. Alterations of microtubules orientation in the cells in primary root zones under the influence of serinethereonine protein kinases inhibitors led to further disturbances in growth and differentiation processes. It was assumed that phosphorylation of microtubules proteins, especially tubulin, might be involved in the regulation of these processes.

[The effect of okadaic acid on root morphology of Arabidopsis thaliana and microtubule organization in its cells].

To investigate the functional role of protein hyperphosphorylation in plant cells the general morphology of Arabidopsis thaliana primary roots and structural-functional property of cortical microtubules were studied after treatment with okadaic acid, specific inhibitor of protein phosphatases PPI and PP2A. It has been estimated that okadaic acid affects microtubule organization in a different manner depending from the type of the cells and functional zones of the primary root. It was found that the microtubules in epidermis and cortex cells of distal elongation zone which depolymerized after inhibitor treatment were the most sensitive to 0, 1, 1 and 10 nM okadaic acid concentrations. In trichoblasts, antichoblasts and cortex cells of differentiation zone treatment with okadaic acid caused the microtubules stabilization. Okadaic acid influences were particularly evident in root hair morphology, root hairs swelling and branching as a result of abnormal microtubules orientation observed. According to the data obtained, we can suggest that induction of protein hyperphosphorylation as a result of protein phosphatase inhibition plays crucial key in plant cell microtubule organization.

Xyloglucan endotransglycosylase/hydrolase (XTH) is encoded by a multi-gene family in the primitive vascular land plant Selaginella kraussiana.

Xyloglucan endotransglycosylase/hydrolases (XTHs) are enzymes that cleave and rejoin xyloglucan chains. To trace the evolutionary origin of XTHs, we used Selaginella kraussiana, a representative of the most primitive land plants (Lycopodiophyta). A Southern blot with a digoxigenin-labeled probe, designed on the conserved catalytic site of XTHs, indicated nine genes. The presence of at least seven functional XTHs was detected by isoelectric focusing (IEF) followed by overlaying the gel with a XET-test paper. Together, these results indicate that XTHs are encoded by a multi-gene family that originated during or even before the colonization of land by plants.

Cellulose orientation determines mechanical anisotropy in onion epidermis cell walls.

The role of cellulose microfibril orientation in determining cell wall mechanical anisotropy and in the control of the wall plastic versus elastic properties was studied in the adaxial epidermis of onion bulb scales using the constant-load (creep) test. The mean or net cellulose orientation in the outer periclinal wall of the epidermis was parallel to the long axis of the cells. In vitro cell wall extensibility was 30-90% higher in the direction perpendicular to the net microfibril orientation than parallel to it. This was the case for the size of the initial deformation occurring just after the load application and for the rate of time-dependent creep. Loading/unloading experiments confirmed the presence of a real irreversible component in cell wall extension. The plastic component of the time-dependent deformation was higher perpendicular to the net cellulose orientation than parallel to it. An acid buffer (pH 4.5) increased the creep rate by 25-30% but this response was not related to cellulose orientation. The present data provide direct evidence that the net orientation of cellulose microfibrils confers mechanical anisotropy to the walls of seed plants, a characteristic that may be relevant to understanding anisotropic cell growth.

Regulation of cell length in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid: a matter of apoplastic reactions.

Treatment of the Arabidopsis thaliana root with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) immediately imposes a reduced maximal cell length beyond which further elongation is blocked. Here, we investigated possible apoplastic reactions involved in the inhibition of cell elongation. Five-day-old Arabidopsis seedlings were transferred to a growth medium supplemented with ACC and the effect on root cell length was recorded after 3 h of treatment. Altered characteristics in the apoplast of the nonelongating cells in the ACC-treated root, such as 'reactive oxygen species' (ROS) production and callose deposition, were detected using specific fluorochromes. The presence of functional hydroxyproline-rich glycoproteins (HRGPs) and the crosslinking of these cell-wall proteins are essential in limiting cell elongation. The ROS that drive the oxidative crosslinking of HRGPs, accumulate in the apoplast of cells in the zone where cell elongation stops. In the same cells, callose is deposited in the cell wall. The final cell length in the Arabidopsis root treated for a short period with ACC is determined in the zone of fast elongation. Both HRGPs crosslinking by ROS and callose deposition in the cell wall of this zone are suggested as causes for the reduced cell elongation.

Bromodeoxyuridine DNA fiber technology in plants: replication origins and DNA synthesis in tobacco BY-2 cells under prolonged treatment with aphidicolin.

Newly synthesized DNA can be observed on chromosomes or extended chromatin fibers after incorporation and immunodetection of bromodeoxyuridine. This technique, frequently used in animal cells, was adapted for use in BY-2 cells. For the first time, the origins of replication in plant cells could be visualized and monitored on DNA fibers without the use of radioactive traces. The replicon size for BY-2 cells was estimated to be 12.9 microm; and the fork rate, 1.17 microm/h. These values are comparable to those reported for tomato and mustard cells. Furthermore, the data confirm our previous observation that DNA synthesis is not totally blocked by aphidicolin. Bromodeoxyuridine incorporation into DNA was obvious from 24 h onwards after treatment with aphidicolin.

Immunological evidence for the presence of plant homologues of the actin- related protein Arp3 in tobacco and maize: subcellular localization to actin-enriched pit fields and emerging root hairs.

The actin-nucleating and -organizing Arp2/3 protein complex is well known to be conserved throughout the eukaryotic kingdom. For higher plants, however, only limited evidence is available for the presence of the Arp2/3 complex so far. Using heterologous antibodies against the Dictyostelium discoideum and Schizosaccharomyces pombe proteins and a bovine peptide, we found immunological evidence for the presence of Arp3 homologues in plants. First, proteins with a molecular mass of about 47-50 kDa were clearly recognized in extracts of both a dicotyledonous plant (tobacco) and a monocotyledonous plant (maize) in immunoblots with the anti-Arp3 antibodies. Second, immunolocalization with these Arp3 antibodies was performed on different plant cells, selected for their diverse actin organizations and functions. On isolated plasma membrane ghosts derived from tobacco leaf protoplasts, a putative Arp3 was localized along cortical actin filaments. In the inner cortex of maize roots, Arp3 was localized to actin-rich plasmodesmata and pit fields and to multivesicular bodies in the cytoplasm. During root hair formation, distinct site-specific localization was found at the protruding apical plasma membrane portions of these tip-growing cells.

Xyloglucan endotransglucosylase action is high in the root elongation zone and in the trichoblasts of all vascular plants from Selaginella to Zea mays.

The endotransglucosylase action of the enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was localized in the roots of diverse vascular plants: club-mosses (lycopodiophytes), ferns, gymnosperms, monocots, and dicots. High action was always found in the epidermis cell wall of the elongation zone and in trichoblasts in the differentiation zone. Clearly XTH and its action in root development evolved before the evolutionary divergence of ferns and seed plants and also of the lycopodiophytes and euphyllophytes.

Giant mitochondria are a response to low oxygen pressure in cells of tobacco (Nicotiana tabacum L.).

Low oxygen pressure induces fast and reversible formation of giant mitochondria in cells of Nicotiana tabacum. These can have unusual shapes, attain a length of 80 microm and even form a reticulum. In contrast to animal cells, there is no such response to chemically induced oxidative stress.

Plant mitochondria move on F-actin, but their positioning in the cortical cytoplasm depends on both F-actin and microtubules.

Mitochondrion movement and positioning was studied in elongating cultured cells of tobacco (Nicotiana tabacum L.), containing mitochondria-localized green fluorescent protein. In these cells mitochondria are either actively moving in strands of cytoplasm transversing or bordering the vacuole, or immobile positioned in the cortical layer of cytoplasm. Depletion of the cell's ATP stock with the uncoupling agent DNP shows that the movement is much more energy demanding than the positioning. The active movement is F-actin based. It is inhibited by the actin filament disrupting drug latrunculin B, the myosin ATPase inhibitor 2,3-butanedione 2-monoxime and the sulphydryl-modifying agent N-ethylmaleimide. The microtubule disrupting drug oryzalin did not affect the movement of mitochondria itself, but it slightly stimulated the recruitment of cytoplasmic strands, along which mitochondria travel. The immobile mitochondria are often positioned along parallel lines, transverse or oblique to the cell axis, in the cortical cytoplasm of elongated cells. This positioning is mainly microtubule based. After complete disruption of the F-actin, the mitochondria parked themselves into conspicuous parallel arrays transverse or oblique to the cell axis or clustered around chloroplasts and around patches and strands of endoplasmic reticulum. Oryzalin inhibited all positioning of the mitochondria in parallel arrays.

Root hair initiation is coupled to a highly localized increase of xyloglucan endotransglycosylase action in Arabidopsis roots.

Root hairs are formed by two separate processes: initiation and subsequent tip growth. Root hair initiation is always accompanied by a highly localized increase in xyloglucan endotransglycosylase (XET) action at the site of future bulge formation, where the trichoblast locally loosens its cell wall. This suggests an important role of XET in the first stages of root hair initiation. The tip of growing root hairs is not marked by localized high XET action. Experiments in which root hair initiation was modulated and observations on root hair mutants support this view. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid shifts both root hair initiation and the local increase in XET action toward the root tip. On the other hand, roots treated with the ethylene inhibitor aminoethoxyvinyl-glycine, as well as roots of mutants affected in root hair initiation (rhl1, rhd6-1, and axr2-1) revealed no localized increases of XET action at all and consequently did not initiate root hairs. Disruption of actin and microtubules did not prevent the localized increase in XET action. Also, the temporal and spatial pattern of action as the specific pH dependence suggest that different isoforms of XET act in different processes of root development.

Ion fluxes, auxin and the induction of elongation growth in Nicotiana tabacum cells.

Immobilized cultured tobacco cells become polarized upon the addition of naphthalene-1-acetic acid and start to elongate from an initial spherical shape. The question as to how a diffuse-growing cell forms a polar axis is addressed here with approaches successfully applied to the study of tip growth. With two kinds of vibrating probes the electric current flow and proton fluxes were mapped around such elongating cells. No consistent polar pattern of ion fluxes, which is typical for actively tip-growing cells, was detected. Therefore, other signals must provide the positional information needed for polar axis formation. Furthermore, neither a specific pattern of intracellular Ca(2+) concentration nor a polar distribution of putative ion-channel antagonist-binding sites were found in elongating tobacco cells. Auxin flux, on the other hand, was found to be important as TIBA, an inhibitor of polar auxin transport, clearly inhibited elongation in a concentration-dependent way. Cross-linking of arabinogalactan-proteins with the beta-Yariv reagent also resulted in inhibition of elongation. A model is proposed for the induction of polar growth where localized auxin efflux starts a signal cascade that triggers molecules that reorient microtubules. These then guide cellulose deposition in the cell wall, which in turn alters cell wall mechanics and leads to elongation. In this scheme, arabinogalactan-proteins are not causal agents but are probably important regulators of growth and survival of the cell.

A comparison of F-actin labeling methods for light microscopy in different plant specimens: multiple techniques supplement each other.

Different detection methods for F-actin labeling were compared on a range of plant specimens: cultured cells, whole organ mounts and sectioned material. For cultured cells, microinjection of labeled phalloidin yielded the most detailed picture but careful permeation methods come close, while immunocytochemical methods always gave relatively poor detail, especially on the level of the fine filaments. For whole organ mounts and sectioned material, permeation methods and immunolocalization are the methods of choice, however never reaching the level of resolution of permeation methods in single cells. It is clear that there is no general and universal good method and multiple techniques are needed, especially when working with different specimens and with different aims.

Phosphatidylinositol 3-kinase activity is required for the expression of glial fibrillary acidic protein upon cAMP-dependent induction of differentiation in rat C6 glioma.

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed upon maturation of astrocytes and upregulated during reactive astrogliosis. Its expression is modulated by several growth factors and hormones. Although an upregulation of intracellular cAMP is required for the induction of GFAP expression in astrocytes, little information is available on other downstream factors of the signal transduction pathways involved in the regulation of its expression. In this communication, we identified phosphatidylinositol 3-kinase (PI 3-K) as a necessary enzyme for GFAP expression in rat C6 glioma cells. Use of the specific PI 3-K inhibitors wortmannin and LY294002 and transfection of C6 cells with a dominant negative PI 3-K construct, resulting in a decrease of the enzymatic activity of PI 3-K, inhibited the cAMP-dependent expression of GFAP. Furthermore, confocal laser scanning microscopy demonstrated that inhibition of the PI 3-K activity by LY294002 or wortmannin concomitant with induction of differentiation changes the cellular distribution leading to a pericentrosomal localization of GFAP and an altered cell shape lacking process formation. We conclude that the expression and cellular distribution of GFAP is mediated through a PI 3-K-dependent mechanism.

Identification of the tumor metastasis suppressor Nm23-H1/Nm23-R1 as a constituent of the centrosome.

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event.

Nucleoside diphosphate kinase beta (Nm23-R1/NDPKbeta) is associated with intermediate filaments and becomes upregulated upon cAMP-induced differentiation of rat C6 glioma.

Nucleoside diphosphate kinases (Nm23/NDPK) are enzymes functional in cell proliferation, differentiation, development, tumor progression, and metastasis. Nevertheless, no consensus exists about the molecular mechanism by which Nm23/NDPK isoforms exert their role in these processes. We investigated the expression of the rat Nm23-R1/NDPKbeta and Nm23-R2/NDPKalpha isoforms, homologues of the human Nm23-H1/NDPK A and Nm23-H2/NDPK B proteins, respectively, upon cAMP-induced differentiation of rat C6 glioma cells and demonstrated a differential interaction with intermediate filaments. Semiquantitative RT-PCR, immunoblotting, and flow cytometry showed a constitutive expression of both Nm23 isoforms. After induction of differentiation in C6 cells with cAMP analogs or isoproterenol, a dose-dependent 2- and 2.5-fold upregulation of the Nm23-R1 mRNA and protein, respectively, was observed. In contrast, the expression of Nm23-R2 remained unchanged. Localization of both isoforms with confocal laser scanning microscopy demonstrated a punctate reticular staining pattern for both Nm23 isoforms in the cytosol and processes of the cells which was particularly intense in the perinuclear region. In addition, while Nm23-R2 was colocalized and coimmunoprecipitated with vimentin in nondifferentiated cells, both isoforms were associated with GFAP in differentiated cells. The significance of these findings in relation to a possible function of Nm23 isoforms in cell proliferation, differentiation, and tumor-associated mechanisms is discussed.