Disruption of microtubular cytoskeleton induced by cryptogein, an elicitor of hypersensitive response in tobacco cells.

The dynamics of microtubular cytoskeleton were studied in tobacco (Nicotiana tabacum cv Xanthi) cells in response to two different plant defense elicitors: cryptogein, a protein secreted by Phytophthora cryptogea and oligogalacturonides (OGs), derived from the plant cell wall. In tobacco plants cryptogein triggers a hypersensitive-like response and induces systemic resistance against a broad spectrum of pathogens, whereas OGs induce defense responses, but fail to trigger cell death. The comparison of the microtubule (MT) dynamics in response to cryptogein and OGs in tobacco cells indicates that MTs appear unaffected in OG-treated cells, whereas cryptogein treatment caused a rapid and severe disruption of microtubular network. When hyperstabilized by the MT depolymerization inhibitor, taxol, the MT network was still disrupted by cryptogein treatment. On the other hand, the MT-depolymerizing agent oryzalin and cryptogein had different and complementary effects. In addition to MT destabilization, cryptogein induced the death of tobacco cells, whereas OG-treated cells did not die. We demonstrated that MT destabilization and cell death induced by cryptogein depend on calcium influx and that MT destabilization occurs independently of active oxygen species production. The molecular basis of cryptogein-induced MT disruption and its potential significance with respect to cell death are discussed.

Proteolysis of microtubule associated protein 2 and sensitivity of pancreatic tumours to docetaxel.

We have studied the state of microtubule associated protein 2 (MAP2) in the pancreatic ductal adenocarcinomas P03 and P02 (sensitive and refractory to docetaxel respectively) since they express the corresponding mRNA and MAP2-related peptides. Immunohistochemical localization showed that in tumour P03 the MAP2-related peptides are highly expressed and confined to the epithelial malignant cells while in P02 the Intensity of the immunostaining is lower. However, anti alpha-tubulin staining followed a similar pattern suggesting that the net amount of macromolecular structures in the sensitive tumour is higher than in the refractory one. This may explain its higher sensitivity to docetaxel, because tubulin assembled into microtubules is the target of the drug. We found that protein extracts from both tumours differed in their proteolytic activity on rat brain MAP2. Since the proteolysis pattern obtained was similar to the one produced by Cathepsin D, we studied the effect of MAP2 proteolysed by this enzyme on microtubule formation in vitro. Proteolysis was found to increase the tendency of tubulin to assemble into macromolecular structures (microtubules and aggregates) in the presence of docetaxel. This suggests that in vivo proteolysis of MAP2 might increase microtubule alterations and potentiate the antitumour effect of docetaxel.

Plant microtubule-associated proteins (MAPs) affect microtubule nucleation and growth at plant nuclei and mammalian centrosomes.

In this study, we investigated the effect of plant microtubule-associated proteins (MAPs) on microtubule nucleation and growth in vitro. Since it has recently been demonstrated that plant nuclear surface acts as a microtubule-organizing center (MTOC), we tested the effects of plant MAPs using a nucleus-mediated microtubule nucleation assay. Nuclei were isolated from interphase tobacco BY-2 cells, and MAPs were isolated from tobacco BY-2 cells at different stages of the cell cycle. The effects of tobacco MAPs on microtubule nucleation at mammalian centrosomes were also analyzed. Under our experimental conditions, both interphase and mitotic tobacco MAPs promoted microtubule assembly around tobacco nuclei and at mammalian centrosomes below the critical tubulin concentration for spontaneous assembly. Interphase tobacco MAPs increase the mean length of nucleated microtubules in proportion to its molar ratio to tubulin. In contrast, mitotic tobacco MAPs do not induce nucleus- and centrosome-mediated nucleation of microtubules in a dose-dependent manner. Both MAP-fractions possessed microtubule bundling activity. The implications of these plant MAP properties on microtubule nucleation in living cells are discussed.

Isolated Plant Nuclei Nucleate Microtubule Assembly: The Nuclear Surface in Higher Plants Has Centrosome-like Activity.

In most eukaryotic cells, microtubules (MTs) are assembled at identified nucleating sites, such as centrosomes or spindle pole bodies. Higher plant cells do not possess such centrosome-like structures. Thus, the fundamental issues of where and how the intracellular plant MTs are nucleated remain highly debatable. A large body of evidence indicates that plant MTs emerge from the nuclear periphery. In this study, we developed an in vitro assay in which isolated maize nuclei nucleate MT assembly at a tubulin concentration (14 [mu]M of neurotubulin) that is not efficient for spontaneous MT assembly. No MT-stabilizing agents, such as taxol or dimethyl sulfoxide, were used. Our model provides evidence that the nuclear surface functions as a MT-nucleating site in higher plant cells. A monoclonal antibody raised against a pericentriolar antigen immunostained the surface of isolated nuclei, and a 100-kD polypeptide in 4 M urea-treated nuclear extracts was detected.

Higher plant microtubule-associated proteins: in vitro functional assays.

Microtubule-associated proteins (MAPs) can account for the assembly and stabilization of microtubules at low tubulin concentration, for their ability to interact with other microtubules and/or cytoskeletal polymers or organelles and also for regulating microtubule anchoring and bundling properties. The data concerning higher plant MAPs remain limited so far to a few examples. Motor MAPs such as dynein or kinesin remain poorly documented in plants and are not to be discussed here. In this manuscript, the attention is focused on structural MAPs which co-assemble with tubulin during microtubule assembly. Using taxol, we developed an assay where higher plant microtubules were induced to self-assemble in a cytosolic extract of maize cultured cells and could be used as a native matrix for the isolation of putative higher plant MAPs. Seven polypeptides with molecular masses ranging between 60-125 kDa were found in this MAP-enriched fraction. These putative plant MAPs were shown to co-assemble with pig brain tubulin through two cycles of temperature-dependent assembly-disassembly. They were able to initiate and promote MAP-free tubulin assembly under conditions of non-efficient self-assembly and induced bundling of both plant and neural microtubules. One of these polypeptides (83 kDa) was found to be immunologically related to neural tau, suggesting the presence of common epitopes between neural and plant MAPs. Such epitopes may be present at the microtubule-binding domains, as the higher plant MAPs co-assemble with brain tubulin. Plant microtubules exhibit an important in situ bundling activity, as in cortical or pre-prophase band arrays, or during the drastic reorganization of the cytoskeleton during mitosis induction.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody, raised against mammalian centrosomes and screened by recognition of plant microtubule organizing centers, identifies a pericentriolar component in different cell types.

We have used monoclonal antibodies raised against isolated native calf thymus centrosomes to probe the structure and composition of the pericentriolar material. To distinguish prospective antibodies as specific to conserved elements of this material, we screened clones by their identification of microtubule organizing centers (MTOCs) in different animal and plant cells. Among the clonal antibodies that reacted with MTOCs in both plant and mammalian cells, we describe one (mAb 6C6) that was found to immunostain centrosomes in a variety of bovine and human cells. In cycling cells this signal persisted through the entire cell cycle. Microscopy showed that the mAb 6C6 antigen was a component of the pericentriolar material and this was confirmed by biochemical analysis of centrosomes. Using immunoblot analysis of protein fractions derived from purified components of centrosomes, we have characterized the mAb 6C6 antigen as a 180 kDa polypeptide. We conclude that we have identified a protein component permanently associated with the pericentriolar material. Surprisingly, monoclonal antibody 6C6 also stained other mitotic organelles in mammalian cells, in a cell-cycle-dependent manner. During prometaphase and metaphase the antibody stained both centrosomes and kinetochores. At the onset of anaphase the kinetochore-specific staining dissociated from chromosomes and was subsequently redistributed onto a newly characterized organelle, the telophase disc while the centrosomal stain remained intact. It is not known if the 180 kDa centrosomal protein itself redistributes during mitosis, or if the pattern observed represents other antigens with shared epitopes. The pericentriolar material is thought to be composed of conserved elements, which appeared very early during the evolution of eukaryotes. Our results strongly suggest that mAb 6C6 identifies one of these elements.

Characterization of maize microtubule-associated proteins, one of which is immunologically related to tau.

Microtubule-associated proteins (MAPs) are identified as proteins that copurify with tubulin, promote tubulin assembly, and bind to microtubules in vitro. Higher plant MAPs remain mostly unknown. One example of non-tubulin carrot proteins, which bind to neural microtubules and induce bundling, has been reported so far [Cyr, R. J., & Palewitz, B. A. (1989) Planta 177, 245-260]. Using taxol, we developed an assay where higher plant microtubules were induced to self-assemble in cytosolic extracts of maize cultured cells and were used as the native matrix to isolate putative plant MAPs. Several polypeptides with an apparent molecular masses between 170 and 32 kDa copolymerized with maize microtubules. These putative maize MAPs also coassembled with pig brain tubulin through two cycles of temperature-dependent assembly-disassembly. They were able to initiate and promote MAP-free tubulin assembly under conditions of nonefficient self-assembly and induced bundling of both plant and neural microtubules. One of these proteins, of about 83 kDa, cross-reacted with affinity-purified antibodies against rat brain tau proteins, suggesting the presence of common epitope(s) between neural tau and maize proteins. This homology might concern the tubulin-binding domain, as plant and neural tubulins are highly conserved and the plant polypeptides coassembled with brain tubulin.