MAP65/Ase1 promote microtubule flexibility.

Microtubules (MTs) are dynamic cytoskeletal elements involved in numerous cellular processes. Although they are highly rigid polymers with a persistence length of 1-8 mm, they may exhibit a curved shape at a scale of few micrometers within cells, depending on their biological functions. However, how MT flexural rigidity in cells is regulated remains poorly understood. Here we ask whether MT-associated proteins (MAPs) could locally control the mechanical properties of MTs. We show that two major cross-linkers of the conserved MAP65/PRC1/Ase1 family drastically decrease MT rigidity. Their MT-binding domain mediates this effect. Remarkably, the softening effect of MAP65 observed on single MTs is maintained when MTs are cross-linked. By reconstituting physical collisions between growing MTs/MT bundles, we further show that the decrease in MT stiffness induced by MAP65 proteins is responsible for the sharp bending deformations observed in cells when they coalign at a steep angle to create bundles. Taken together, these data provide new insights into how MAP65, by modifying MT mechanical properties, may regulate the formation of complex MT arrays.

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.

Cell cycle regulation of the microtubular cytoskeleton.

The microtubular element of the plant cytoskeleton undergoes dramatic architectural changes in the course of the cell cycle, specifically at the entry into and exit from mitosis. These changes underlie the acquisition of specialized properties and functions involved, for example, in the equal segregation of chromosomes and the correct positioning and formation of the new cell wall. Here we review some of the molecular mechanisms by which the dynamics and the organization of microtubules are regulated and suggest how these mechanisms may be under the control of cell cycle events.

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.

The aphid transmission factor of cauliflower mosaic virus forms a stable complex with microtubules in both insect and plant cells.

We analyzed the distribution of the cauliflower mosaic virus (CaMV) aphid transmission factor (ATF), produced via a baculovirus recombinant, within Sf9 insect cells. Immunogold labeling revealed that the ATF colocalizes with an atypical cytoskeletal network. Detailed observation by electron microscopy demonstrated that this network was composed of microtubules decorated with paracrystalline formations, characteristic of the CaMV ATF. A derivative mutant of the ATF, unable to self-assemble into paracrystals, was also analyzed. This mutant formed a net-like structure, with a mesh of four nanometers, tightly sheathing microtubules. Both the ATF- and the derivative mutant-microtubule complexes were highly stable. They resisted dilution-, cold-, and calcium-induced microtubule disassembly as well as a combination of all three for over 6 hr. CaMV ATF cosedimented with microtubules and, surprisingly, it bound to Taxol-stabilized microtubules at high ionic strength, thus suggesting an atypical interaction when compared with that usually described for microtubule-binding proteins. Using immunofluorescence double labeling we also demonstrated that the CaMV ATF colocalizes with the microtubule network when expressed in plant cells.

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.

A microtubule-associated protein in maize is expressed during phytochrome-induced cell elongation.

Plants can adapt their shape to environmental stimuli. This response is mediated by the reorganization of cortical microtubules, a unique element of the cytoskeleton. However, the molecular base of this response has remained obscure so far. In an attempt to solve this problem, signal-dependent changes in the pattern of microtubule-binding proteins were analysed during coleoptile elongation in maize, that is, under the control of the plant photoreceptor phytochrome. Two putative MAPs of 100 kDa (P100) and 50 kDa apparent molecular weights were identified in cytosolic extracts from non-elongating and elongating cells. Both proteins co-assembled with endogenous tubulin, bound to neurotubules and were immunologically related to the neural MAP tau: the P100 protein, depending on the physiological situation, was manifest as a double band and was always found to be heat-stable. In contrast, the 50 kDa MAP was heat-stable only for particular tissues and physiological treatments. The P100 protein was present in all tissues, however in a reduced amount in elongating coleoptiles. The 50 kDa MAP was expressed exclusively upon induction of phytochrome-dependent cell elongation. As shown by immunofluorescence double-staining, an epitope shared by both proteins colocalized with cortical microtubules in situ, but exclusively in elongating cells. In non-elongating cells, only the nuclei were stained. Partially purified nuclei from elongating cells were enriched in P100, whereas the 50 kDa MAP became enriched in a partially purified plasma membrane fraction.

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.

Characterization of a 100-kDa heat-stable microtubule-associated protein from higher plants.

In higher-plant cells, the different cell-cycle-dependent microtubule arrays are involved in a wide range of activities including chromosome segregation, cell-plate formation and cellulose microfibril distribution and orientation. A wealth of data, obtained using animal cells, has indicated that the differential stability and function of microtubules during cell-cycle and/or differentiation could be primarily regulated by selective microtubule-associated proteins (MAP). Compared to animal MAP, our knowledge of plant MAP is so far very limited. In this study, we have identified a maize heat-stable protein with apparent molecular mass 100 kDa (P-100) which binds to taxol-stabilized neurotubules and copolymerizes in vitro with purified neural tubulin. Moreover, P-100 cross-reacts with affinity-purified tau antibodies like a maize 83-kDa putative MAP described previously [Vantard, M., Schellenbaum, P., Fellous, A. & Lambert, A. M. (1991) Biochemistry 30, 9334-9340]. Polyclonal antibodies directed against P-100 were obtained and indicated that this protein is found in diverse higher-plant cultured cells suggesting the ubiquitous nature of this protein. P-100 can be phosphorylated in vitro by protein kinases present in a maize cytosol extract. Together, our data suggest that P-100 could be a higher plant MAP.

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.

Incorporation of Paramecium axonemal tubulin into higher plant cells reveals functional sites of microtubule assembly.

Incorporation of Paramecium axonemal tubulin into lysed endosperm cells of the higher plant Haemanthus enabled us to identify sites of microtubule assembly. This exogenous Paramecium tubulin could be traced by specific antibodies that do not stain endogenous plant microtubules. Intracellular copolymerization of protozoan and higher plant tubulins gave rise to hybrid polymers that were visualized by immunofluorescence and by immunoelectron microscopy. The addition of exogenous tubulin revealed many free ends of endogenous microtubules that were competent to assemble ciliate tubulin. The functional roles of the nuclear surface and the equatorial region of the phragmoplast as plant microtubule-organizing centers, which were revealed by the intense incorporation of exogenous tubulin, are discussed. These data shed light on the present debate on higher plant microtubule organizing centers.

Amino acid composition and proteolytic generated domains of higher plant tubulin.

The molecular architecture of tubulin from higher plant remains unknown. In this report we have made an attempt to identify higher plant tubulin domains using total and limited proteolysis of Haemanthus endosperm tubulin. The tubulin was previously purified and characterized (Picquot and Lambert 1988). The amino acid composition revealed a high content of basic residues, such as arginine and lysine. Tubulin domains were probed by tryptic and chymotryptic cleavage and analyzed by immunoblotting using specific monoclonal antibodies against alpha or beta subunits. These data shed light on specific properties of the higher plant tubulin.

Microtubule dynamics determine chromosome lagging and transport of acentric fragments.

The general direction of transport of spindle inclusions including acentric chromosome fragments during mitosis in endosperm of the higher plants Haemanthus is predictable and stage-dependent. Their segregation is random and they are usually eliminated from the spindle. This transport is superimposed on normal chromosome segregation. Thus, there are 2 superimposed mitotic transports: one which distributes kinetochores and the other which distributes spindle inclusions. The functional relation of these 2 transports to each other is not well understood. However, due to this 'non-kinetochore transport,' fragments may persist a few consecutive divisions before being permanently eliminated from the nucleus. Malfunction of kinetochores of any chromosome, resulting in the loss of their anchorage within the spindle, subjects them to 'non-kinetochore' transport and nearly certain, permanent elimination from the spindle. Additionally, experimental evidence presented here demonstrates that rapid polymerization (elongation) of microtubules may desynchronize anaphase and cause lagging of whole chromosomes. This may be one more, previously unconsidered, factor which may cause the malfunction of the kinetochore fiber and consequent elimination of one or a few chromosomes from the spindle.

Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus.

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.

Aster-like microtubule centers establish spindle polarity during interphase - Mitosis transition in higher plant cells.

Transformation of interphase microtubular cytoskeleton into initial mitotic spindle in early prophase and the reverse process in telophase were analysed with immunofluorescence techniques in endosperm cells of higher plants, Haemanthus Katherinae Bak. and Clivia nobilis Lindl. We have identified aster-like centers as intermediate basic microtubular structures directly involved in the reorganization of microtubules arrays both at the onset of mitosis and during telophase-interphase transition. These transitory microtubule converging centers determine spindle polarity in early prophase, they are replaced by diffuse poles during metaphase, and form again in anaphasetelophase. We conclude that rearrangement of microtubules during interphase-mitosis involves three superimposed processes: microtubule assembly/disassembly, active transport and reorientation of microtubules, changes in microtubule properties reflected in their lateral interaction during spindle development.