Root contraction in hyacinth IV. Orientation of cellulose microfibrils in radial longitudinal and transverse cell walls

Summary Cellulose microfibrils (MFs) were visualized on the inner surface of root cortex cell walls ofHyacinthus orientalis L. using a replica technique. Microfibril orientation was determined in radial longitudinal and transverse cell walls of the root tip, uncontracted, contracting, and fully contracted regions of the root. In longitudinal walls, the innermost MFs were ordered and parallel to one another and were oriented transversely, axially or obliquely, depending upon the developmental stage of the region. In transverse walls MFs in a single layer formed crisscross or ordered parallel arrays, depending upon the region. Parallel arrays were oriented either parallel, perpendicular, or oblique to the radius of the root. Inner walls of certain cells in the contracting region had MFs which appeared interrupted over their lengths. In general, these findings parallel earlier immunofluorescence and electron microscopic observations of changing cortical microtubule (MT) orientation accompanying root contraction. The major exception to MT-MF congruence occurred in cells of the actively contracting region. In middle and outer cell layers, MFs appeared short and partially obscured, while MTs in these cells occurred in conspicuous laterally aggregated strands parallel to one another over the length of the cells or were absent. This alteration in MF-MT parallelism may be related to the reorientation in cell growth occurring in the contractile zone or to the collapse of specific cells during the process of root contraction.Abbreviations MF microfibril - MT microtubule     

Root contraction in hyacinth III. Orientation of cortical microtubules visualized by immunofluorescence microscopy

Summary Cortical microtubules (MTs) were visualized in root cortex cells ofHyacinthus orientalis L. using immunofluorescence techniques. Cellular MT orientation was determined adjacent to radial longitudinal and transverse walls of root tip, uncontracted, contracting, and fully contracted regions. As seen in longitudinal views, MTs formed parallel, apparently helical arrays which were oriented transversely, axially or obliquely depending upon the region. Transverse sectional views showed that MTs adjacent to transverse cell walls formed a variety of patterns which varied with developmental stage and cell location. Microtubules were oriented in crisscross or parallel arrays. The parallel arrays were oriented either parallel, perpendicular or oblique to the radius of the root. There was an apparent temporal progression in MT reorientation from outer cortical to inner cortical cell layers. A resultant progression of reoriented cell growth could account for root contraction. These findings corroborate earlier electron microscopic observations of changing MT orientation accompanying root contraction, and provide cytological evidence to test mathematical and biophysical models of the mechanics of cell expansion.Abbreviations MT microtubule - MF microfibril - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Resistance of Rosa microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin

The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24°C with an apparent inhibition constant (K _i) of 1.4·10^-4 M for rose tubulin and an apparent K _i=8.8·10^-7 M for brain tubulin. The binding of [³H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2–3 h to reach equilibrium at 24°C, and was not affected by vinblastine sulfate. The binding of [³H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7·10² M^-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24°C. The values for brain tubulin were K=2.46·10⁶ M^-1 and r=0.45 at 37°C. The binding of [³H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.Abbreviations MT microtubule - TC tubulin-colchicine complex     

A mycorrhizal fungus changes microtubule orientation in tobacco root cells

Summary Cortical cells of mycorrhizal roots undergo drastic morphological changes, such as vacuole fragmentation, nucleus migration, and deposition of cell wall components at the plant-fungus interface. We hypothesized that the cytoskeleton is involved in these mechanisms leading to cell reorganization. We subjected longitudinal, meristem to basal zone, sections of uninfectedNicotiana tabacum roots to immunofluorescence methods to identify the microtubular (MT) structures associated with root cells. Similar sections were obtained from tobacco roots grown in the presence ofGigaspora margarita, an arbuscular mycorrhizal fungus which penetrates the root via the epidermal cells, but mostly develops in the inner cortical cells. While the usual MT structures were found in uninfected roots (e.g., MTs involved in mitosis in the meristem and cortical hoops in differentiated parenchyma cells), an increase in complexity of MT structures was observed in infected tissues. At least three new systems were identified: (i) MTs running along large intracellular hyphae, (ii) MTs linking hyphae, (iii) MTs binding the hyphae to the host nucleus. The experiments show that mycorrhizal infection causes reorganization of root MTs, suggesting their involvement in the drastic morphological changes shown by the cortical cells.     

Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells ofArabidopsis thaliana transformed with green-fluorescent-protein gene constructGFP-MBD

Summary Arabidopsis thaliana plants were transformed withGFPMBD (J. Marc etal., Plant Cell 10: 1927–1939, 1998) under the control of a constitutive (35S) or copper-inducible promoter. GFP specific fluorescence distributions, levels, and persistence were determined and found to vary with age, tissue type, transgenic line, and individual plant. With the exception of an increased frequency of abnormal roots of 35SGFP-MBD plants grown on kanamycincontaining media, expression of GFP-MBD does not appear to affect plant phenotype. The number of leaves, branches, bolts, and siliques as well as overall height, leaf size, and seed set are similar between wild-type and transgenic plants as is the rate of root growth. Thus, we conclude that the transgenic plants can serve as a living model system in which the dynamic behavior of microtubules can be visualized. Confocal microscopy was used to simultaneously monitor growth and microtubule behavior within individual cells as they passed through the elongation zone of the Arabidopsis root. Generally, microtubules reoriented from transverse to oblique or longitudinal orientations as growth declined. Microtubule reorientation initiated at the ends of the cell did not necessarily occur simultaneously in adjacent neighboring cells and did not involve complete disintegration and repolymerization of microtubule arrays. Although growth rates correlated with microtubule reorientation, the wo processes were not tightly coupled in terms of their temporal relationships, suggesting that other factor(s) may be involved in regulating both events. Additionally, microtubule orientation was more efined in cells whose growth was accelerating and less stringent in cells whose growth was decelerating, indicating that microtubuleorienting factor(s) may be sensitive to growth acceleration, rather than growth per se.     

Organogenesis in Graptopetalum paraguayense E. Walther: shifts in orientation of cortical microtubule arrays are associated with periclinal divisions

The interior of a new lateral organ, such as a leaf, arises from the products of periclinal divisions of sub-epidermal cells. The biophysical basis of the elongation of such a new axis is transverse (hoop) reinforcement of the cells by cellulose in the primary walls. This structural polarity is associated with transverse alignment of cortical microtubules. We have brought the histological and biophysical views together by showing that the new, periclinal, divisions are a prerequisite for a corresponding change in the orientation of the microtubular array in the daughter cells. Investigation of this relationship required development of criteria for assessing the predominant orientation of a microtubule array in a single section of known orientation. By obtaining information about the predominant orientation of microtubule arrays in the sub-epidermal cells, we were able to study structural polarity shifts which occurred as a detached leaf of Graptopetalum produced a new shoot. During organogenesis, the new polarity is seen only in cells which have divided periclinally. Following single periclinal divisions, cells are seen with microtubules in the old or new orientation or in a mixture of different orientations. Cells with more than one orientation of microtubules are probably at intermediate stages in the shift to the new polarity. Among cells which have undergone two consecutive periclinal divisions, the old polarity is no longer seen, all cells having high frequencies of microtubules in the new orientation. Such cells are either polarized in the new direction or nonpolarized. The shifts in polarity of the cells in the interior anticipate the appearance of the first leaf primordia. However, contrary to the expectations from the histological view of organogenesis, these shifts do not dominate the process. Concurrent polarity changes in the epidermis appear at least as important.     

Role of the microtubule cytoskeleton in alternating changes in cellulose-microfibril orientation in the coenocytic green alga,Chaetomorpha moniligera

The functions of the microtubule (MT) cytoskeleton in changing the orientation of microfibrils (MFs) in the cell walls of the coenocytic green alga Chaetomorpha moniligera Kjellman were investigated by electron microscopy. The cortical MT cytoskeleton in Chaetomorpha was comprised of longitudinally oriented MTs. Cellulose MFs, however, alternately changed their orientation longitudinally and transversely to form crisscross MF textures. Microtubules were parallel to longitudinally oriented MFs but never to those that were transversely oriented. The average density of MTs during the formation of longitudinally oriented MFs was 216 per 50 m of wall and that of transversely oriented MFs 170/50 m. To determine exactly the MT-density dependency of each MF orientation, changes in MF orientation were examined by changing MT density after treating and removing amiprophos-methyl (APM). Microtubules were reduced in number by a half (100/50 m) after 2 h and by 3/4 (50/50 m) after 3 h of treatment with APM (3 mM). This reduction was caused by the disappearance of alternating MTs. Microtubules retained this density (50/ 50 m) up to 6 h, and then gradually disappeared within 24 h. Microfibril orientation in the innermost cell wall was transverse after treatment with APM for 2 h but was helicoidal after 6 h. Polymerization of MTs occurred in the longitudinal direction following the removal of APM after treatment for 48 h. Microtubule density rose to about 100/50 m and 200/50 m after 6 h and 24 h, respectively. The orientation of MTs changed from helicoidal to transverse and transverse to longitudinal after 6 h and 24 h, respectively. When APM was removed prior to formation of the helicoidal texture, longitudinally oriented MFs appeared within 6 h. There is thus an alternating cycle of formation of longitudinally and transversely oriented MFs within a 12-h period. Formation of transversely oriented MFs as a result of APM treatment started in the middle of a cell as hoops which then extended in the apical and basal directions. Formation of longitudinally oriented MFs as a result of the removal of APM started from the apical end and proceeded toward the base. It follows from these results that: (1) the point of formation of longitudinally oriented MFs differs from that for transversely oriented MFs, (2) MF orientation in each case depends on a separately functioning mechanism, (3) MT density changes rhythmically to trigger a switch for crisscross orientation of MFs.Abbreviations APM amiprophos-methyl - MF microfibril - MT microtubule - TC terminal complex We thank Dr. K. Okuda for making helpful discussion and Miss. T. Matsuki for assistance with replica preparation.     

Multiple tubulin forms in ciliated protozoan Tetrahymena and Paramecium species

Summary. Tetrahymena and Paramecium species are widely used representatives of the phylum Ciliata. Ciliates are particularly suitable model organisms for studying the functional heterogeneity of tubulins, since they provide a wide range of different microtubular structures in a single cell. Sequencing projects of the genomes of members of these two genera are in progress. Nearly all members of the tubulin superfamily (α-, β-, γ-, δ-, ɛ-, η-, θ-, ι-, and κ-tubulins) have been identified in Paramecium tetraurelia. In Tetrahymena spp., the functional consequences of different posttranslational tubulin modifications (acetylation, tyrosination and detyrosination, phosphorylation, glutamylation, and glycylation) have been studied by different approaches. These model organisms provide the opportunity to determine the function of tubulins found in ciliates, as well as in humans, but absent in some other model organisms. They also give us an opportunity to explore the mechanisms underlying microtubule diversity. Here we review current knowledge concerning the diversity of microtubular structures, tubulin genes, and posttranslational modifications in Tetrahymena and Paramecium species. Correspondence and reprints: Institute of Molecular Genetics, Czech Academy of Sciences, Vídeňská 1083, 14220 Prague, Czech Republic.     

Changes in microtubule packing during the stretching of an extensible microtubule bundle in the ciliate Nassula

Summary The cytopharyngeal sheath in the ciliate Nassula is a long hollow tube-shaped microtubule bundle that forms part of a large feeding organelle called the cytopharyngeal basket. During the initial stages of ingestion of algal filaments by Nassula the sheath is stretched, becomes approximately elliptical in cross-section, and its external cross-sectional perimeter increases by a factor of about two. The mean circumferential centre-to-centre spacing of radially oriented rows of sheath tubules increases from 57 to 137nm during stretching but sheath thickness and the radial spacing of sheath tubules do not change appreciably. It is suggested that extensible circumferentially oriented intertubule links and relatively inextensible radial links may define the anisometric mechanical properties of this particular microtubule bundle which are related to its cytoskeletal role. The possibility that extensible links resist stretching elastically and provide the restoring forces for return of the sheath to its former shape and dimensions after stretching is considered.Supported by the Science Research Council, U.K. (Grant nos. B/RG/5894.5 and GR/A/0875.8)     

Variations in the number of centrioles,the number of microtubule organizing centers 1 and the percentage of mitotic abnormalities inPhysarum polycephalum amoebae

Summary Several, stable amoebal strains which differ phenotypically from the diploid parental amoebal strain have been obtained in the MyxomycetePhysarum polycephalum. They were detected using their flagellation pattern as a discriminating parameter. This approach is valid since the number of flagella by phase contrast microscopy correlates with the number of anterior centrioles obtained using three-dimensional reconstructions of the nucleo-flagellar complexes from serial thin sections. The complexity of the structures of the various nucleo-flagellar complexes suggests that in these strains the duplication time of centrioles is not strictly regulated as it is in haploid amoebae. In agreement with this hypothesis, several pro-centrioles were observed in interphase amoebae. Although the anterior centrioles are linked to the mtoc 1 during interphase, the number of mtoc 1 cannot regulate the number of centrioles since some strains possess two mtoc 1 but only one pair of centrioles. Neither the number of centrioles nor the number of mtoc 1 are related to ploidy. Stable strains with one (all haploid strains), two (some diploid strains) and three (some diploid strains) mtoc 1 have been observed. Thus each mtoc 1 is duplicated once per cell cycle implying that it must possess some information which plays a role in the morphogenesis of the new mtoc 1. Except in one case, the number of mitotic abnormalities increases exponentially with the number of mtoc 1. This observation suggests that the mtoc 1 could correspond to the interphase state of the mitotic center.     

Coordination of cell proliferation and cell expansion in the control of leaf size in Arabidopsis thaliana

Size is an important parameter in the characterization of organ morphology and function. To understand the mechanisms that control leaf size, we previously isolated a number of Arabidopsis thaliana mutants with altered leaf size. Because leaf morphogenesis depends on determinate cell proliferation, the size of a mature leaf is controlled by variation in cell size and number. Therefore, leaf-size mutants should be classified according to the effects of the mutations on the cell number and/or size. A group of mutants represented by angustifolia3/grf-interacting factor1 and aintegumenta exhibits an intriguing cellular phenotype termed compensation: when the leaf cell number is decreased due to the mutation, the leaf cell size increases, leading to compensation in leaf area. Several lines of genetic evidence suggest that compensation is probably not a result of the uncoupling of cell division from cell growth. Rather, the evidence suggests an organ-wide mechanism that coordinates cell proliferation with cell expansion during leaf development. Our results provide a key, novel concept that explains how leaf size is controlled at the organ level.     

Changes in phosphatase activity associated with cell wall defects in Chlamydomonas reinhardi

Experiments were performed to isolate mutants lacking alkaline phosphatase in Chlamydomonas reinhardi. Mutants with null enzyme activity were obtained. A cytological study of these mutants however revealed cell wall defects, suggesting that the loss of phosphatase activity in these strains is not due to the inactivation of the corresponding phosphatase structural gene but rather to the leakage of this enzyme as a consequence of the cell wall abnormality. Incidentally, this finding provides the basis of a convenient method for selecting easily cell wall mutants of Chlamydomonas.Chercheur qualifié du Fonds National Belge de la Recherche Scientifique.     

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one induces cell cycle arrest and apoptosis in HeLa cells by preventing microtubule polymerization

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) is known as a specific inhibitor of soluble guanylyl cyclase (sGC). Previously, however, ODQ was reported to induce cell death via sGC-dependent and sGC-independent means in a variety of cell types. The aim of this study was to investigate the mechanism by which ODQ induces cell death in HeLa cells.Treatment of HeLa cells with ODQ induced a concentration-dependent decrease in cell viability over the range from 10 to 100 μM. DNA fragmentation and fluorescence-activated cell sorting analysis using annexin V and propidium iodide staining revealed that ODQ triggered apoptosis at concentrations of 50 and 100 μM within 24 to 48 h. The addition of 8-Br-cGMP in the presence of ODQ failed to rescue HeLa cells from death, suggesting that the inhibition of sGC was not responsible for the pro-apoptotic action of ODQ. ODQ arrested the cell cycle at the G2/M phase and caused disassembly of the microtubule network. This process was reversed by dithiothreitol. In addition, ODQ was shown to inhibit the polymerization of purified tubulin, and this was also prevented by dithiothreitol. These results indicate that ODQ inhibits microtubule assembly by direct oxidation of tubulin, induces cell cycle arrest at the G2/M phase, and triggers apoptosis in HeLa cells.     

Interaction of tubulin with the plasma membrane: tubulin is present in purified plasmalemma and behaves as an integral membrane protein

Using monoclonal antibodies we have studied the interaction of tubulin with the plasma membrane of leaves of Nicotiana sylvestris (Speg. et Comes) and tobacco suspension-culture cells. The results show that isolated plasma membranes contain tightly bound -tubulins. Their association with the plasma membrane is resistent to non-ionic detergent and to low and high ionic strength. Only extraction with sodium dodecyl sulfate is capable of dissociating these cytoskeletal proteins. It is unlikely that this membrane-bound tubulin is present in its polymeric form because electron-microscopical analysis does not reveal the presence of filaments, whereas treatment of membranes with oryzalin (which has been shown to destabilize microtubules in vitro) does not remove the tubulins from isolated plasma membrane. When living cells are treated with oryzalin, the amount of membrane-associated tubulin is drastically reduced, which could mean that its presence is related to in-vivo microtubule dynamics.Abbreviations Mes 2 (N-morpholino) ethane sulfonic acid - NP40 Nonidet P40     

Gravitropism of the primary root of maize: a complex pattern of differential cellular growth in the cortex independent of the microtubular cytoskeleton

The spatio-temporal sequence of cellular growth within the post-mitotic inner and outer cortical tissue of the apex of the primary root of maize (Zea mays L.) was investigated during its orthogravitropic response. In the early phase (0–30 min) of the graviresponse there was a strong inhibition of cell lengthening in the outer cortex at the lower side of the root, whereas lengthening was only slightly impaired in the outer cortex at the upper side. Initially, inhibition of differential cell lengthening was less pronounced in the inner cortex indicating that tissue tensions which, in these circumstances, inevitably develop at the outer-inner cortex interface, might help to drive the onset of the root bending. At later stages of the graviresponse (60 min), when a root curvature had already developed, cells of the inner cortex then exhibited a prominent cell length differential between upper and lower sides, whereas the outer cortex cells had re-established similar lengths. Again, tissue tensions associated with the different patterns of cellular behaviour in the inner and outer cortex tissues, could be of relevance in terminating the root bending. The perception of gravity and the complex tissue-specific growth responses both proceeded normally in roots which were rendered devoid of microtubules by colchicine and oryzalin treatments. The lack of involvement of microtubules in the graviresponse was supported by several other lines of evidence. For instance, although taxol stabilized the cortical microtubules and prevented their re-orientation in post-mitotic cortical cells located at the lower side of gravistimulated roots, root bending developed normally. In contrast, when gravistimulated roots were physically prevented from bending, re-oriented arrays of cortical microtubules were seen in all post-mitotic cortical cells, irrespective of their position within the root.Abbreviations CMTs cortical microtubules - CW Cholodny-Went - FF form factor - MT microtubule The research was supported by a fellowship from the Alexander von Humboldt Stiftung (Bonn, Germany) to F.B. Financial support to AGRAVIS by Deutsche Agentur für Raumfahrtangelegenheiten (DARA, Bonn) and Ministerium für Wissenschaft und Forschung (Düsseldorf) is gratefully acknowledged. IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.     

Relationship between microtubule orientation and patterns of cell expansion in developing cortical cells of Lemna minor roots

In actively growing cortical cells in the elongation zone of Lemna minor L. roots, both longitudinal (radial and tangential) and transverse walls expand in both length and width. The longitudinal walls of the three types of cortical cells in the root (i.e. outer, middle and inner) showed the largest expansion in the longitudinal axis. In contrast, the inner cortical cells exhibited the least expansion in width, whereas the middle cortical cells displayed the largest expansion in width. Thus, the profiles of the expansion of longitudinal walls were characteristic for the three types of cortical cells. In this study, both the orientation of cortical microtubule (MT) arrays and their dynamic reorientation, and the density of cortical MTs, were documented and correlated to the patterns of cell wall expansion. Significantly, transverse arrays of cortical MTs were most prominent in the radial walls of the inner cortical cells, and least so in those of the middle cortical cells. Toward the base of roots, beyond the elongation zone, the orientation of cortical MTs shifted continuously from transverse to oblique and then to longitudinal. In this case, the rate of shift in the orientation of cortical MTs along the root axis was appreciably faster in the middle cortical cells than in the other two types of cortical cells. Interestingly, the continuous change in cortical MT orientation was not confirmed in the transverse walls which showed much smaller two-dimensional expansion than the radial walls. Additionally, the presence of fragmented or shortened cortical MTs rapidly increased concomitantly with the decrease of transversely oriented cortical MTs. This relationship was especially prominent in the transverse walls of the inner cortical cells, which displayed the least expansion among the three types of cortical cells investigated. In the root elongation zone, the density of cortical MTs in the inner cortical cells was about three times higher than that in the other two cortical cell types. These results indicate that in the early stage of cell expansion, the orientation of cortical MTs determines a preferential direction of cell expansion and both the shifting orientation and density of cortical MTs affect the magnitude of expansion in width of the cell wall.     

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves

Cell expansion in dicotyledonous leaves is strongly stimulated by bright white light (WL), at least in part as a result of light-induced acidification of the cell walls. It has been proposed that photosynthetic reactions are required for light-stimulated transport processes across plasma membranes of leaf cells, including proton excretion. The involvement of photosynthesis in growth and wall acidification of primary leaves of bean has been tested by inhibiting photosynthesis in two ways: by reducing chlorophyll content of intact plants with tentoxin (TX) and by treating leaf discs with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exposure to bright WL stimulated growth of intact leaves of TX-treated plants. Discs excised from green as well as from TX-or DCMU-treated leaves also responded by growing faster in WL, as long as exogenous sucrose was supplied to the photosynthetically inhibited tissues. The WL caused acidification of the epidermal surface of intact TX-leaves, but acidification of the incubation medium by mesophyll cells only occurred when photosynthesis was not inhibited. It is concluded that light-stimulated cell enlargement of bean leaves, and the necessary acidification of epidermal cell walls, are mediated by a pigment other than chlorophyll. Light-induced proton excretion by mesophyll cells, on the other hand, may require both a photosynthetic product (or exogenous sugars) and a non-photosynthetic light effect.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1 -dimethylurea - OC osmotic concentration - RL red light - TX tentoxin - WL white light We thank Dr. G.E. Templeton, University of Arkansas, Fayetteville, USA, for initially supplying us with TX, and also Dr. Stephen O. Duke, Southern Weend Science Laboratory, Stoneville, Miss., USA, for suggesting this compound for our experiments. We are grateful to Professor E. Ballio for his generous gift of fusicoccin.     

The N-terminus of PrP is responsible for interacting with tubulin and fCJD related PrP mutants possess stronger inhibitive effect on microtubule assembly in vitro

Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Some evidences demonstrate that PrP may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between PrP and tubulin was confirmed using pull-down assays, immunoprecipitation and ELISA. The interacting regions within PrP with tubulin were mapped in the N-terminus of PrP spanning residues 23-50 and 51-91. PrP octapeptide repeats are critical for the binding activity with tubulin, that the binding activity of PrP with tubulin became stronger along with the number of the octapeptide repeats increased. Microtubule assembly assays, sedimental tests and transmission electron microscopy demonstrated that the full-length PrP (aa 23-231) obviously inhibited the microtubule polymerization processes in vitro, whereas the N- (aa 23-91) and C- (aa 91-231) terminal peptides of PrP did not affect microtubule polymerization. Moreover, the familial Cruetzfeldt Jacob disease (fCJD) related PrP mutants with inserted or deleted octapeptide repeats showed much stronger inhibitive capacities on the microtubule dynamics in vitro than wild-type PrP. Our data highlight a potential role of PrP in regulating the microtubule dynamics in neurons.