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First published online May 20, 2005; 10.1104/pp.104.052456 Plant Physiology 138:654-662 (2005) © 2005 American Society of Plant Biologists Two Microtubule-Associated Proteins of the Arabidopsis MAP65 Family Function Differently on Microtubules1State Key Laboratory of Plant Physiology and Biochemistry, Department of Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing 100094, China (T.M., L.J., H.L., M.Y.); and Section of Plant Biology, University of California, Davis, California 95616 (B.L.)
The organization and dynamics of microtubules are regulated by microtubule-associated proteins, or MAPs. In Arabidopsis (Arabidopsis thaliana), nine genes encode proteins of the evolutionarily conserved MAP65 family. We proposed that different MAP65s might have distinct roles in the interaction with microtubules. In this study, two AtMAP65 proteins, AtMAP65-1 and AtMAP65-6, were chosen to test this hypothesis in vitro. Although both fusion proteins were able to cosediment with microtubules in vitro, different properties on tubulin polymerization and microtubule bundling were observed. AtMAP65-1 was able to promote tubulin polymerization, enhance microtubule nucleation, and decrease the critical concentration for tubulin polymerization. It also induced the formation of large microtubule bundles by forming cross-bridges between microtubules evenly along the whole length of microtubules. In the presence of AtMAP65-1, microtubule bundles were more resistant to cold and dilution treatments. AtMAP65-6, however, demonstrated no activity in promoting tubulin polymerization and stabilizing preformed microtubules. AtMAP65-6 induced microtubules to form a mesh-like network with individual microtubules. Cross-bridge-like interactions were only found at regional sites between microtubules. The microtubule network induced by AtMAP65-6 was more resistant to high concentration of NaCl than the bundles induced by AtMAP65-1. Purified monospecific anti-AtMAP65-6 antibodies revealed that AtMAP65-6 was associated with mitochondria in Arabidopsis cells. It was concluded that these two MAP65 proteins were targeted to distinct sites, thus performing distinct functions in Arabidopsis cells.
Microtubules, polymers of  - and  -tubulin dimers, are dynamic filaments that constantly undergo polymerization and/or depolymerization at both the plus end and the minus end. In higher plants, microtubules are organized into distinct arrays in differentiated cells (Cyr and Palevitz, 1995 ; Wasteneys and Galway, 2003). It is widely accepted that microtubule-associated proteins, or MAPs, play regulatory roles in microtubule organization (Lloyd and Hussey, 2001). However, it is still unclear how plant MAPs may contribute to the formation of different microtubule arrays.
Cortical microtubules are commonly found among interphase cells. Individual microtubules in the cortical array often have close contact with the plasma membrane and various organelles as revealed by electron microscopy (Gunning and Steer, 1996
Tireless quest of plant MAPs has recovered a class of MAPs of approximately 65 kD in both tobacco (Nicotiana tabacum) and carrot (Daucus carota) cells, thus called MAP65 (Jiang and Sonobe, 1993
Evidence of functional divergence of different MAP65 proteins is emerging. In bean epicotyls, the MAP65 protein level positively correlates with the elongation activity, implying a potential role in cell elongation (Sawano et al., 2000
To date, it has been concluded that MAP65 bundles microtubules by forming cross-bridges along neighboring microtubules (Jiang and Sonobe, 1993
Because nine forms of AtMAP65 have sequence identity from 28% to 79% (Hussey et al., 2002 In this study, we have expressed AtMAP65-1 and AtMAP65-6 as fusion proteins in bacteria. Purified fusion proteins have been used to test their interaction with microtubules in vitro. We provide evidence that these two related proteins may regulate microtubule organization in different manners. We also demonstrate that AtMAP65-6 is targeted to mitochondria in Arabidopsis cells.
Fusion Proteins of AtMAP65-1 and AtMAP65-6 Bind to Microtubules in Vitro To obtain proteins for in vitro analysis, cDNA sequences of AtMAP65-1 and AtMAP65-6 were obtained in a cDNA clone (109M12) and by reverse transcription (RT)-PCR, respectively. Purified glutathione S-transferase (GST)-AtMAP65-1 fusion protein appeared in doublets (Fig. 1A). GST-AtMAP65-6 appeared in a major band with a minor band at a lower molecular mass (Fig. 1A). The lower band was a degradation product according to immunoblotting results (Fig. 6A).
When incubated with polymerized porcine brain microtubules, fusion proteins cosedimented with microtubules by centrifugation (Fig. 1, B and C). GST-AtMAP65-6 had lower cosedimentation efficiency than GST-AtMAP65-1. To verify that the cosedimentation was due to the interaction with microtubules, we tested whether the fusion proteins sedimented by themselves. Five micromolar AtMAP65 fusion proteins were centrifuged in the same conditions of cosedimentation experiments in the absence of microtubules. Both MAP65 fusion proteins remained predominantly in the supernatant (Fig. 1, B and C). Therefore, the fusion proteins were most likely active in terms of their binding to microtubules.
Because the fusion proteins bound to microtubules in vitro, we wanted to test whether they would affect microtubule organization. GST-AtMAP65-1 was used to test the microtubule-bundling activity according to earlier reports (Jiang and Sonobe, 1993
GST-AtMAP65-6 was then applied to microtubule suspensions under identical conditions. A different effect was observed. A very dense network was induced (Fig. 2C). Electron microscopic examination by negative staining indicated that microtubules were crisscrossed with each other, and very little if any bundling phenomenon was observed (Fig. 2D). These individual microtubules were brought together by the AtMAP65-6-induced crisscrosses to form a dense mesh-like network (Fig. 2C). To test whether such a dense microtubule network was indeed induced by AtMAP65-6, MAP-free microtubules were examined. In the absence of GST-MAP65-6, these microtubules appeared in single separated filaments (Fig. 2, E and F). Therefore, we concluded that the phenomena of microtubule bundling and microtubule crisscrossing, respectively, were caused by the addition of AtMAP65-1 and AtMAP65-6 fusion proteins. To further test the effect of these MAPs on microtubule organization, we applied different concentrations of NaCl to strip the fusion proteins from the microtubules. Microtubule bundles induced by AtMAP65-1 became scattered in the presence of 100 or 200 mM NaCl (data not shown). When the NaCl concentration was brought to 300 mM or above, no more microtubule bundles were observed. Instead, all microtubules appeared in single individuals (Fig. 2G). The microtubule network induced by AtMAP65-6, however, was able to resist NaCl at concentrations as high as 500 mM (Fig. 2H). Thus, the microtubule network induced by AtMAP65-6 was more stable than the bundles induced by AtMAP65-1.
To reveal how these two MAP65 proteins affect microtubule organization differently, we examined microtubule bundles induced by GST-AtMAP65-1 and microtubule meshes induced by GST-AtMAP65-6 by thin sections. Microtubule bundles induced by GST-AtMAP65-1 were evenly spaced in parallel to each other, and the space between each other was approximately the diameter of microtubules (Fig. 3A). Microtubule bundles containing more than a dozen microtubules were frequently found, and bundles tended to merge with each other to form more complex super bundles (Fig. 3A). Spaced ladder-like cross-bridges with a distance close to the microtubule diameter of 25 nm were found between adjacent microtubules (Fig. 3B). These cross-bridges were identical to the data reported using native MAP65 proteins (Chan et al., 1999
When the AtMAP65-6-induced microtubule meshes were sectioned, we did not observe a field densely occupied by microtubules (Fig. 3, C–F). Instead, a few microtubules occasionally jointed together at a given region, and most microtubules appeared to be scattered in a field (Fig. 3, C and E). When microtubules were jointed together, a space of <15 nm was observed between microtubules, and a few filamentous structures were occasionally present (Fig. 3, D and F). The jointed microtubules splayed out quickly.
Because AtMAP65-1 and AtMAP65-6 fusion proteins induced microtubules into very different configurations, we wondered whether such different phenomena were caused by the effect of these two proteins on microtubule polymerization and nucleation. We examined microtubule polymerization and nucleation activities quantitatively in vitro, by monitoring changes of turbidity of tubulin suspension when the two fusion proteins were added. In a 30-min period, microtubule polymerization, reflected by the absorbance value at 350 nm, reached a steady state in approximately 12 min (Fig. 4A). When AtMAP65-1 fusion was added at 0.5 µM, the absorbance value jumped more than 4-fold at its steady state, indicating that more tubulins were polymerized into microtubules (Fig. 4A). The steady state was reached in approximately 8 min (Fig. 4A). When AtMAP65-1 was added at concentrations of 1, 2, and 4 µM, the steady state of turbidity increased even more, although not as dramatically as the difference between 0 and 0.5 µM (Fig. 4A). To test whether the increase of turbidity was not solely due to the bundling activity as suggested by an earlier report (Smertenko et al., 2004
We also examined the lag time of tubulin assembly, an indicator of microtubule nucleation at different concentrations of AtMAP65-1. In the absence of AtMAP65-1, the lag time was approximately 3 min (Fig. 4C). In the presence of 1 µM and 0.5 µM AtMAP65-1, however, the lag time was reduced to approximately 40 s and 60 s, respectively (Fig. 4C). Therefore, we concluded that the AtMAP65-1 fusion protein was able to promote the nucleation of microtubules. When AtMAP65-6 was tested at identical concentrations added to the tubulin suspension, no significant changes in the polymerization curve were observed among different concentrations (0–2 µM) of the fusion protein (Fig. 4D). Thus, the results suggested that AtMAP65-6 did not bear the activity of promoting microtubule polymerization as AtMAP65-1 did. We also examined the critical concentration (Cc) of tubulin assembly in the absence and presence of GST-AtMAP65-1. The Cc value of our purified tubulin preparation was 11 µM (Fig. 4E). In the presence of the AtMAP65-1 fusion protein at 1 µM, Cc dropped to 5 µM (Fig. 4E). We can conclude that tubulins are able to polymerize at much lower concentrations in the presence of AtMAP65-1 than the pure tubulins.
We further tested whether these two MAP65 proteins were able to stabilize microtubules against depolymerizing challenges. When microtubules, previously polymerized by 20 µM rhodamine-labeled tubulins at 37°C for 30 min, were brought to 10°C for 30 min, these microtubules disassembled (Fig. 5A). In the presence of AtMAP65-1 at 0.5 µM, however, the identical microtubules resisted the cold temperature (Fig. 5B). When AtMAP65-6 was used in a similar test, all microtubules disassembled (Fig. 5C).
To further test the effect of these MAP65 proteins on microtubule stabilization, a dilution test was performed. In the test, microtubules were first assembled in the presence of MAP65 proteins and then diluted by 50x warm buffer solution. Similar to what we had observed in cold treatment, the presence of AtMAP65-1 made microtubules resistant to dilution treatment. Without the addition of MAP65s, most microtubules, if not all, were disassembled in 60 min (Fig. 5D). In the presence of AtMAP65-1, however, induced microtubules bundles resisted the dilution (Fig. 5E). On the contrary, AtMAP65-6 did not have such an effect. Microtubules were still disassembled with the dilution in the presence of AtMAP65-6 (Fig. 5F).
Earlier studies by immunofluorescence and green fluorescence protein tagging indicated that AtMAP65-1 is associated with cortical microtubules (Smertenko et al., 2004 Because the monospecific antibodies detected the native protein, they were used to localize AtMAP65-6 in vivo. In protoplasts isolated from Arabidopsis suspension cells, numerous organelle-like particles were detected (Fig. 7A). These particles were in close contact with cortical microtubules attached to the plasma membrane (Fig. 7, B and C). When control rabbit IgG substituted anti-AtMAP65-6, no signal was detected, while cortical microtubules were still revealed (Fig. 7, D–F).
Because anti-AtMAP65-6 decorated an organelle-like structure, we wanted to determine the identity of the organelle. We tested markers of various organelles (data not shown). Anti-AtMAP65-6 signal showed colocalization with mitochondria, as revealed by the mitochondria-specific marker MitoTracker Red (Fig. 8, A–C). The AtMAP65-6 signal appeared in a ring surrounding the mitochondria (Fig. 7D). Thus, we concluded that AtMAP65-6 was targeted to the mitochondria in Arabidopsis cells.
Our results indicated that two Arabidopsis MAP65 proteins, with 44% sequence identity (Hussey et al., 2002 ), acted differently on microtubules in vitro. In the presence of AtMAP65-1, microtubules formed large, bundles as reported by others. AtMAP65-6 induced a mesh-like network of crisscrosses. While AtMAP65-1 decorated cortical microtubules, AtMAP65-6 decorated mitochondria.
Plant MAP65 proteins have been purified from tobacco BY-2 suspension-cultured cells and carrot suspension cells (Jiang and Sonobe, 1993
Even closely related MAP65 proteins exhibit different activities; it was not surprising that we found AtMAP65-1 and AtMAP65-6 affected microtubule organization differently. It has been suggested that AtMAP65-1 homodimers make the cross-bridges between adjacent microtubules (Smertenko et al., 2004 Our data also indicated that AtMAP65-1 and AtMAP65-6 affected microtubule stability differently. The difference further suggests that they may interact with microtubules in different manners. The continuous ladder-like interaction with microtubules allows microtubules to form a fortress-like structure by AtMAP65-1. Such a structure would certainly be more resistant to depolymerization challenges like cold and dilution treatments than mesh-like network of individual microtubules induced by AtMAP65-6.
Antibodies against tobacco and carrot MAP65 preparations decorate microtubules arrays evenly, which is indistinguishable from microtubule staining (Jiang and Sonobe, 1993
Because AtMAP65-6 behaved differently than AtMAP65-1 in vitro, it was not surprising that AtMAP65-6 demonstrated a different intracellular localization pattern than AtMAP65-1. Our AtMAP65-6-specific antibodies revealed that AtMAP65-6 decorated mitochondria. Earlier scrutinizing pharmacological studies indicated that although plant mitochondria move along F-actin filaments, their positioning in the cortical cytoplasm relies on both F-actin and microtubules (Van Gestel et al., 2002 The remaining task is to determine the localization patterns of six other members of the AtMAP65 family.
The different activities and localization patterns of AtMAP65-1 and AtMAP65-6 suggest that they play distinct roles in plant growth and development. Cortical microtubules are highly dynamic, and undergo reorganization upon receiving internal or external cues (Yuan et al., 1994 While the budding yeast S. cerevisiae as a lower eukaryote has a single gene encoding a MAP65-like protein Ase1p, Arabidopsis as a higher plant contains multiple genes encoding MAP65 proteins. These proteins have been evolved to take on distinct tasks required for multifaceted cellular activities besides anaphase spindle elongation.
AtMAP65-1 and AtMAP65-6 Fusion Protein Preparation AtMAP65-1 and AtMAP65-6 are the protein products of the At5g55230 and At2g01910 genes, respectively. To express fusion proteins of AtMAP65-1 and AtMAP65-6, cDNA sequences were amplified from the EST clone 109M12 for AtMAP65-1, and by RT-PCR for AtMAP65-6. Arabidopsis (Arabidopsis thaliana) total RNA was purified from seedlings by the SV total RNA isolation kit (Promega, Madison, WI). The cDNA sequences, available from the GenBank (accession nos. NM-124905 and NM-126252, respectively), were referenced to design primers. Two primers of MAP65-1 (forward, 5'-GCGTCGACATGGCAGTTACAGATACTG-3' [with the SalI site underlined], and reverse, 5'-TTGCGGCCGCTCATGGTGAAGCTGGAACTT-3' [with the NotI site underlined]) were used to amplify AtMAP65-1 cDNA. The primers used for amplification of AtMAP65-6 cDNA were as follow: MAP65-6 forward, 5'-AACCCGGGAATGCTTATGGAATTAGA-3' (with the SmaI site underlined), and MAP65-6 reverse, 5'-TAAACTCGAGTCAGCCTTGGAGAGG-3' (with the XhoI site underlined). RT-PCR was carried out using Promega kit according to manufacturer's instruction. Amplified sequences were A-tailed and cloned into the pMD18-T vector at corresponding sites designed in the primers (TaKaRa Biotechnology, Dalian, China). cDNA sequences were verified by sequencing.
Full-length cDNAs of AtMAP65-1 and AtMAP65-6 were constructured into the vectors pGEX-4T2 and pGEX 4T1 (Amersham Biosciences, Piscataway, NJ) with SalI and NotI, and SmaI and NotI, respectively. Fusion proteins were expressed in the Escherichia coli strain BL21 (DE3) by induction with 1 mM isopropyl
Porcine brain tubulins were purified according to a published method (Castoldia and Popov, 2003 The binding reaction was performed in 200 µL volume containing 1 µM microtubules and 0.5 and 5 µM MAP65-1 or MAP65-6, respectively, in the PEM buffer (1 mM MgCl2, 1 mM EGTA, and 100 mM PIPES-KOH, pH 6.9) containing 1 mM GTP and 20 µM taxol. After 30 min of incubation at room temperature, samples were centrifuged at 100,000g for 30 min. The supernatants and pellets were separated and brought to equal volumes in the SDS sample buffer. They were then analyzed by SDS-PAGE and visualized by staining the gels with Coomassie Brilliant Blue R-250. Gel scanning was performed on AlphaImager 2200 (Alpha Innotech, San Leandro, CA).
A range of concentrations of AtMAP65-1 or AtMAP65-6 were added to 30 µM tubulin in the PEM buffer with 1 mM GTP. Microtubule polymerization was monitored turbidimetrically by the A350 with a 0.4-cm path quartz cell at 37°C in a DU-640 spectrophotometer (Beckman Coulter, Fullerton, CA).
The Cc for tubulin polymerization was determined according to a published method (Algaier and Himes, 1988 Polymerized microtubules were challenged by the following treatments. For cold treatment, 20 µM NHS-rhodamine-tubulin was polymerized at 37°C for 30 min, with or without 0.5 µM AtMAP65-1 or AtMAP65-6. The preformed microtubules were then incubated at 10°C for 30 min before being fixed with 1% glutaraldehyde for observation. For the dilution challenge, microtubules were polymerized under identical conditions. But the samples were diluted by 50x (v/v) with prewarmed PEM buffer and incubated at 37°C for 30 min prior to being fixed with 1% glutaraldehyde for observation. For the NaCl treatment, microtubules polymerized from 20 µM rhodamine-tubulin were used. Polymerized microtubules were first diluted 10x (v/v) with the PEM buffer plus 20 µM taxol and then centrifuged at 60,000g for 30 min at 37°C. The resulting pellet was resuspended with the PEM buffer of the same volume. AtMAP65-1 or AtMAP65-6 was added to a final concentration of 0.5 µM before the microtubule suspension was incubated at 37°C for 30 min. Final concentrations of NaCl were 100 mM, 200 mM, 300 mM, and 400 mM were for AtMAP65-1-treated microtubules, and 400 mM and 500 mM for AtMAP65-6-treated ones. Each sample was incubated at 37°C for 30 min prior to being fixed for observation.
After being incubated at 37°C for 30 min, microtubule polymerization was terminated using 1% glutaraldehyde. The samples were then examined under a Leica DMR microscope (Wetzlar, Germany) equipped with a 100x oil objective (NA 1.3). Images were collected with an MRC1024 confocal laser scanning microscope equipped with argon-krypton laser (Bio-Rad). Microtubules were either directly observed by negative staining or by thin sectioning. Negative staining was done with saturated uranyl acetate. Microtubules were collected by centrifugation at 100,000g. Microtubule pellets were fixed in 1% glutaraldehyde in PEM buffer, followed by 1% osmium tetroxide, and embedded in the LR White resin. Samples were sectioned and stained with uranyl acetate and lead citrate, then observed under a Hitachi 7500 electron microscope (Hitachi, Tokyo).
The AtMAP65-1 peptide of amino acids 471 to 485, [H]-QEREEEKRRLREQKK-[OH], and the AtMAP65-6 peptide of amino acids 470 to 484, [H]-KPSPRRSSSFRKPNG-[OH], were chosen as antigens to raise specific antibodies. A Cys residue was added to the carboxyl terminus of each peptide for conjugation to the carrier Keyhole limpet hemocyanin. Antisera were produced at a commercial facility (GeneMed Synthesis, South San Francisco). Antibodies against each-peptide were affinity purified by the blot purification method (Tang, 1993
Total proteins were extracted from Arabidopsis suspension cells according a published protocol (Preuss et al., 2003
Protoplasts were prepared enzymatically from Arabidopsis suspension cells with 1% Cellulose RS and 0.1% Pectolyase Y23 (Karland, Cottonwood, AZ) in 0.3 M mannitol solution. Isolated protoplasts were attached to poly-L-lysine-coated slides and fixed with 4% paraformaldehyde as published before (Preuss et al., 2003 All images presented in this article were assembled using the Photoshop 7.0 software package (Adobe Systems, San Jose, CA). Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers NM-124905 for AtMAP65-1 and NM-126252 for AtMAP65-6.
We thank the Arabidopsis Biological Research Center for providing the cDNA clone 109M12. Received August 26, 2004; returned for revision December 28, 2004; accepted March 10, 2005.
1 This work was supported by the National Key Basic Research Project of China and the National Natural Science Foundation of China (grant no. 30128008 to B.L. and grant no. 30170057 to M.Y.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.052456. * Corresponding author; e-mail mingyuan{at}cau.edu.cn; fax 86–10–62733491.
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