From the Department of Biology, University of Ottawa,
Ottawa K1N 6N5, Canada, the § Biotechnology and Molecular
Genetic Department, University of Bremen FB 02, Haferwende 12, D-28357
Bremen, Germany, and the ¶ Département de Biologie du
Développement, Institut Jacques Monod/CNRS, F-75251 Paris,
Cedex 05, France
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ABSTRACT |
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To determine how MAP1a interacts with microtubules we expressed several 6myc-tagged MAP1a fragments in P19 EC and HeLa cells. Confocal immunofluorescence microscopy showed that the fragment consisting of amino acids (aa) 1-281 of MAP1a did not bind while the fragment consisting of aa 1-630 did, indicating that the region of MAP1a between aa 281 and 630 contains a microtubule-binding domain. Deletion of the basic repeats from aa 336-540 did not result in loss of microtubule binding, suggesting that the regions flanking the basic repeats can bind MAP1a to microtubules. These observations were confirmed using an in vitro microtubule binding assay. The levels of acetylation and detyrosination of polymerized microtubules were assessed by quantitative dot blotting in cells expressing MAP1a fragments or MAP2c. Compared with untransfected cells, the polymerized tubulin in cells expressing full-length MAP1a was more acetylated and detyrosinated, but these increases were smaller than those seen in cells expressing MAP2c. Consistent with this, the microtubules in MAP2c expressing cells were more resistant to colchicine than those in cells overexpressing MAP1a. These data implicate aa 281-336 and/or 540-630 of MAP1a in microtubule binding and suggest that MAP1a is less able to stabilize microtubules than MAP2c.
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INTRODUCTION |
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In neurons, microtubules become increasingly resistant to microtubule depolymerizing drugs (1), and less dynamic (2) with time of differentiation. This increasing stability of microtubules is believed to be necessary for neuronal morphogenesis. One of the mechanisms for increasing microtubule stability is the interaction of microtubules with microtubule-associated proteins (MAPs).1
MAP1a and MAP1b are structurally related MAPs which may play complementary roles in regulating microtubule dynamics during neuronal development (3, 4). Inhibition of MAP1b expression in cultured neurons using antisense oligodeoxynucleotides leads to the loss of neurite outgrowth (5). Analyses of MAP1b knockout transgenic mice have demonstrated that MAP1b is involved in normal neuronal development (6, 7). In adult brain, MAP1b remains only in regions where growth and plasticity still occur (8, 9), suggesting it also plays a role in neuronal growth in the adult. The function of MAP1a is less clear. The pattern of MAP1a expression in developing brain suggests that it plays a role in regulating the stability of the neuronal cytoskeleton and, consequently, in the transition of growing neurons to the mature, static state (3). The amino terminus of MAP1b contains a domain consisting of 21 repeats of a KKE motif that has been shown to bind microtubules in vitro (10). MAP1b which has the basic repeat domain removed still binds microtubules, but when the regions flanking both sides of the basic repeat domain are also removed, microtubule binding is lost (10). This demonstrates that the microtubule-binding domain of MAP1b consists of two separate regions. The amino terminus of MAP1a also contains a domain consisting of 11 repeats of the KKE motif. The regions flanking this domain are very similar to those in MAP1b (4), suggesting these regions are involved in microtubule binding.
In this study, we have characterized a microtubule-binding domain of MAP1a by expressing several different 6-myc-tagged fragments of MAP1a in undifferentiated P19 embryonal carcinoma (EC) cells and in HeLa cells. The effects of MAP1a and MAP2c on microtubule dynamics were assessed by measuring the acetylation, detyrosination, and resistance to colchicine-induced depolymerization. Our results show that MAP1a contains a microtubule-binding domain similar to that of MAP1b and that MAP1a increases microtubule stability, but to a lesser extent than MAP2c.
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EXPERIMENTAL PROCEDURES |
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Expression Constructs
Three overlapping cDNA clones spanning the entire mRNA
for MAP1a (4) were used to make all expression constructs (see Fig. 1). pKJ1F-6myc and pPOP (a gift from
Dr. M. McBurney, University of Ottawa) are pUC19-based vectors
containing the constitutively active mouse phosphoglycerate kinase
(PGK) promoter. PKJ1
F-6myc drives the expression of 6 repeats of a
9-amino acid (aa) epitope from the human c-MYC protein. These vectors
were used to express MAP1a fragments epitope tagged at the N terminus.
When necessary sequencing was performed to ensure that all MAP1a
fragments were expressed in-frame with the 6myc tag, confirm ligations,
and determine the location of in-frame stop codons in the cDNA. All
sequencing reactions were performed using the ABI PRISM Dye Terminator
Cycle Sequencing Kit (with Amplitaq DNA polymerase, FS) (Perkin-Elmer). All reactions were run on a ABI model 373A automated sequencer. PGK-MAP2cmyc was constructed by C. Addison in our laboratory.
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PGK-6myc--
This is the unmodified pKJ1F-6myc
vector. It expresses the 6myc tag followed by a 25-aa tail
(NSCSPGDPLVL-ERPPPRYSSDPCRN).
PGK-6mycN1a-1--
A NcoI-HindIII fragment
from clone p19a was blunt-end ligated into
SmaI-SacI cut pKJ1F-6myc. This vector
expresses the 6myc tag followed by 5-aa (NSCSP), followed by aa 1-281
from MAP1a terminating with a 6-aa tail (TDPCRN).
PGK-6mycN1a-2--
A NcoI-ApaI fragment
from clone p19a was blunt-end ligated into
SmaI-SacI cut pKJ1F-6myc. This vector
expresses the 6myc tag, the NSCSP linker, aa 1-630 of MAP1a followed
by a 6-aa tail (ADPCRN).
PGK-6mycN1a-3-- A partial NcoI-BamHI fragment (7800 bp) from PGK-6mycN1a-4 (see below) was blunt-ended and religated. The expressed protein contains the 6myc tag, a 7-aa linker (NSREFLH), aa 1-1310 of MAP1a, and a 2-aa tail (IL).
PGK-6mycN1a-4-- A ClaI-BamHI fragment from PGK-6myc1a (see below) was blunt-end ligated into SmaI cut pPOP. The expressed protein contains the 6myc tag, a 7-aa linker (NSREFLH), aa 1-2016 of MAP1a, and a 14-aa tail (RGSSRVDLQLFMIY).
PGK-6myc1a--
A NcoI-EcoRI fragment from
clone p19a was blunt-end ligated into
SmaI-HindIII cut pKJ1F-6myc. A partial
ApaI fragment (1959 bp) from clone p14 was then ligated into
this vector at the ApaI site. Into this vector a 6070-bp
EcoRI-EcoRV fragment from clone p19 was blunt-end
ligated into the Eco site. Finally a short
oligonucleotide containing a SmaI site
(5'-AATTCCCGGG-3', New England Biolabs) was inserted into the
EcoRI site between the 6myc tag and the MAP1a cDNA to
bring the cDNA for MAP1a in-frame with the 6myc tag. The expressed
protein contains the 6myc tag, a 7-aa linker (NSREFLH), and the
full-length cDNA for MAP1a. The predicted molecular weight for the
tagged protein is based on the predicted molecular weight for the MAP1a
heavy chain as reported in Ref. 4.
PGK-6mycN1a-2BR--
PGK-6myc1a was digested with
XhoI and HaeII to yield two fragments (1514 and
1223 bp). A 1357-bp fragment was PCR amplified from the 1514-bp
fragment using primers PCR1 (5'-CGACGGTATCGATAAGCTATG-3') and PCR2
(5'-GGCCAGCTTGCTCACTGCTGT-3'). A 279-bp fragment was PCR amplified from
the 1223-bp fragment using primers PCR3 (5'-GAGAGAGGTTTGCTGGCTGAA-3') and PCR4 (5'-GTCGGGCCCAGCTCTGCTTCTCTC-3'). The 1357- and 279-bp PCR
products were ligated and then further cut with ClaI and
ApaI. This fragment was ligated into
ClaI-ApaI cut pKJ1
F-6myc. Both strands of the
entire PCR product were then completely sequenced to ensure the
fidelity of the PCR reaction and to check the joint between the two PCR
products. The expressed protein contains the 6myc tag, a 7-aa linker
(NSREFLH), aa 1-335 and 541-631 of MAP1a followed by a 29-aa tail
(GGSTSSRAAATAVENLIPYRENMYLGRLR).
Cell Culture
P19 EC cells (11) and HeLa CCL-2 cells (ATCC) were kept
semiconfluent in -modified Eagle's minimal essential medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Flow Labs), and antibiotics (Life Technologies, Inc.). Cells were grown in a
humidified incubator at 37 °C and 5% CO2 and passed
every 2 days. Taxol (obtained from the National Cancer Institute) was kept as a 10 mM stock in dimethyl sulfoxide at
20 °C.
This stock was then diluted to 10
6 M in
medium to treat cell cultures for 24 h. Colchicine (Sigma) was
kept as a 1 mg/ml stock in dH2O and diluted to 1 µg/ml to treat cells.
Expression of MAP1a Fragments
Expression vectors were introduced into P19 EC and HeLa cells by calcium phosphate-mediated transfection (12). For protein extraction, cells were plated onto 60-mm dishes (Corning) at 1.25 × 105 cells/dish (P19 EC) or 2 × 105 cells/dish (HeLa) in 5 ml of medium (see above). For immunofluorescence microscopy, cells were plated onto 18-mm round coverslips (Corning) at 1.25 × 104 cells/coverslip (P19 EC) or 2 × 104 cells/coverslip (HeLa) in 1 ml of medium. Cells were allowed to settle for 24 h. 40 µg of DNA in 500 µl of 0.25 M CaCl2 was then gently mixed with 500 µl of 2 × BES buffer (50 mM BES, pH 6.86, 280 mM NaCl, 1.5 mM Na2HPO4) to a final volume of 1 ml and allowed to sit for 20 min. The solution of calcium phosphate-DNA precipitate was then gently added to the cells (1 ml for dishes, 200 µl for coverslips) without removing the medium and allowed to sit for 8 h in the incubator. The DNA/media solution was then aspirated and replaced with fresh medium. Cells were incubated a further 48 h before processing.
Primary Antibodies
Anti-MAP1a mouse monoclonal IgG (clone 1A-1, a gift from Dr. R. Vallee, Ref. 13) was diluted 1:1000 for Western blotting and 1:500 for
immunofluorescence microscopy. Anti--tubulin rat monoclonal IgG
(clone YOL 1/34, from Serotech) was diluted 1:15 for immunofluorescence
microscopy. Anti-c-MYC mouse monoclonal IgG (clone 9E10, a gift from
Dr. C. Garner) was used at 1:2 for Western blotting and quantitative
dot blotting and undiluted for immunofluorescence microscopy.
Anti-acetylated
-tubulin monoclonal IgG (clone 6-11B-1 (Sigma)) was
diluted 1:1000 for quantitative dot blotting. Polyclonal
anti-detyrosinated
-tubulin (anti-E, Ref. 14) was provided by Dr. T. MacRae and diluted 1:250 for quantitative dot blotting.
Immunofluorescence Microscopy
Cells plated on glass coverslips were briefly rinsed in PEM (80 mM PIPES, pH 6.8, 5 mM EGTA, 1 mM MgCl2, all from Sigma) and fixed at room temperature by two different protocols.
Precipitation (15)-- 1 h incubation in Zamboni's fixative (14% picric acid (Fisher), 4% paraformaldehyde (J. B. EM Services Inc.) in 0.5 M Na2HPO4, 0.5 M NaH2PO4, pH 7.1) followed by a 3 × 5-min phosphate-buffered saline (PBS, 130 mM NaCl, 5 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) wash and 5-min extraction with 0.5% Triton X-100 (Pierce) in PBS.
Extraction/Fixation (16)-- 2-min pre-extraction with 0.2% Triton X-100 in PEM (80 mM PIPES, pH 6.9, 10 mM EGTA, 5 mM MgSO4) followed by a 10-min fixation with 3.7% formaldehyde (BDH), 0.25% glutaraldehyde (J. B. EM Services Inc.), and 0.5% Triton X-100 in PEM, followed by a 3 × 5-min PEM wash and quenching 3 × 5 min with 1 mg/ml sodium borohydride (BDH) in PBS.
Cells prepared by either fixation procedure were again rinsed 3 × 5 min in PBS. All antibody incubations were for 1 h in PBS at room temperature with 3 × 5-min PBS washes after each incubation. Secondary antibodies used were donkey anti-rat IgG fluorescein isothiocyanate, cross-adsorbed to mouse, diluted 1:150 (Sigma) and donkey anti-mouse IgG carboindocyanine, cross-adsorbed to rat, diluted 1:500 (Jackson). For all labeling, sequential primary and secondary incubations were used. Samples were visualized using either a Leica Upright Confocal Laser Scanning Microscope or a Zeiss Universal epifluorescence microscope equipped with a Hammatsu integrating CCD and Metamorph v2.75 (Universal Imaging). Data was recorded electronically using the tagged image file format with a resolution of 512 lines × 512 columns × 256 gray levels. Confocal image stacks were processed using the simulated fluorescence algorithm which projects highlights and shadows on the image stack, emphasizing depth cues. All images were prepared for publication using Adobe Photoshop v4.01 (Adobe) and Powerpoint 97 (Microsoft).Protein Extraction
Whole cell protein for Western blotting was extracted from 100-mm dishes according to Ref. 17, but using higher concentrations of protease inhibitors in the extraction buffer: 40 µM benzamidine HCl (Sigma), 4 mM p-aminoethylbenzenesulfonyl fluoride (Centrichem Inc.), 1 mM 1,10-phenanthroline (Sigma), 40 µg/ml each of aprotinin, pepstatin A, and leupeptin (all from Sigma).
To obtain extracts for the in vitro assembly assay, cells
were rinsed briefly in cold MAB1 (0.1 M MES, pH 6.4, 2.5 mM EGTA, 0.1 mM EDTA, 0.5 mM
MgCl2, Ref. 18) and then 250-500 µl of extraction buffer
(MAB1 + 4 mM PEFA, 1 mM 1,10-phenanthroline,
and 40 µg/ml each of aprotinin, pepstatin A, and leupeptin) was
added. Cells were immediately scraped into an Eppendorf and sonicated
15 s at 94 watts with a Braun sonicator, then immediately spun for 10 min at 10,000 rpm and 4 °C. The supernatant was removed and stored at 80 °C.
To prepare extracts for analysis of -tubulin modifications, cells
were processed according to Ref. 19. Protein concentrations were
determined using the bicinchoninic acid assay (Pierce) using BSA
(Pierce) as a standard.
In Vitro MAP Binding Assay
Parts of this procedure (see Fig. 2) were derived from the taxol-dependent purification of MAPs described in Ref. 20. 1 ml (approximately 13 mg) of 3 × cycled bovine brain microtubule preparation (21, 22) in MAB2 (0.1 M PIPES, pH 6.4, 1 mM EGTA, 1 mM MgCl2, 4 M glycerol, and 0.1 mM GTP) was brought to 1.8 mM GTP and 20 µM taxol, assembled for 30 min and 37 °C, and then spun 15 min at 36,000 × g and 37 °C (18,000 rpm using a Sorval SS-20 rotor). The pellet was gently resuspended using a 1-ml pipetter in the same volume of MAB2 (+400 mM NaCl, 1.8 mM GTP, and 20 µM taxol). The suspension was then spun for 15 min at 36,000 × g and 37 °C. The pellet was then resuspended in the same buffer and spun through a 15% sucrose cushion in MAB2 (+ 1.8 mM GTP and 20 µM taxol) for 15 min at 36,000 × g and 37 °C. The pellet was then resuspended in MAB1 + 1.8 mM GTP and 20 µM taxol, and spun for 15 min at 36,000 × g and 37 °C. The pellet was then gently resuspended in MAB1 as above and to this suspension an equal volume of protein from transfected P19 EC cells was added, brought to 1.8 mM GTP and 20 µM taxol, and incubated for 15 min and 37 °C. The suspension was then spun 15 min at 36,000 × g and 37 °C. The resulting pellet was resuspended in MAB1 (+1.8 mM GTP and 20 µM taxol) and spun through a 15% sucrose cushion in MAB1 (+1.8 mM GTP and 20 µM taxol) for 15 min at 36,000 × g and 37 °C. The resulting pellet was resuspended in MAB1 (+1.8 mM GTP and 20 µM taxol). Samples were taken throughout the procedure to determine the presence or absence of MAP1a and tubulin by Western blotting. Volumes used to resuspend pellets were adjusted throughout the procedure to account for loss of volume due to sampling. Samples were processed for negative staining transmission electron microscopy from the first assembly step and after each spin through sucrose to ensure that microtubules remained polymerized.
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Electron Microscopy
Samples were fixed 1 min at 37 °C in a equal volume of fixative (4% paraformaldehyde, 0.2% glutaraldehyde in MAB1 or MAB2, depending when the sample was taken). 5 µl of the sample was allowed to settle onto parlodion-coated 400 mesh copper grids (J. B. EM Services Inc.) for 1 min. Grids were then washed with 5 drops of Photoflo solution (2 drops of Photoflo (Kodak) in 100 ml of ddH2O), followed by 5 drops of ddH2O and then stained with 4 drops of 1% uranyl acetate. Samples were then examined using a Philips 201 transmission electron microscope. Images were recorded on KODAK Electron Image Film SO-163 and printed on Ilford multigrade III paper.
SDS-PAGE and Western Blotting
Equal amounts of protein in sample buffer (23) were placed in a boiling water bath for 5 min, loaded onto 12% polyacrylamide gels and separated using the Bio-Rad minigel apparatus. Proteins were electroblotted onto nitrocellulose in 20% methanol and the blots were rinsed in PBS. Immunodetection of Western blots was performed as follows: block for 1 h in 5% skim milk in PBS, 1 h incubation in primary antibody diluted in 2% skim milk in PBS, 1 h incubation in biotinylated horse anti-mouse IgG (Vector) diluted 1:1000 in 2% skim milk in PBS, and 1 h of incubation in biotinylated streptavidin horseradish peroxidase (Amersham) diluted 1:5000 in PBS. A 3 × 5-min PBS wash was done between all antibody incubations (with 2% milk added between the primary and secondary). Antibody binding was detected by enhanced chemiluminescence (ECL) (Amersham) using Hyperfilm-ECL (Amersham). All steps were performed at room temperature.
Quantitative Dot Blotting
Protein samples were diluted in PBS and 200 µl/well was passed through nitrocellulose in a 96-well Minifold apparatus (Schleicher & Schuell) which had been pre-wetted with 200 µl/well of PBS. After the entire sample had been passed through the nitrocellulose by a gentle vacuum, an additional 400 µl of PBS/well was passed through the nitrocellulose. The nitrocellulose was then removed from the apparatus, rinsed briefly in PBS, and then fixed for 10 min in transfer buffer (25 mM Tris, 190 mM glycine, 20% (v/v) MeOH). The blot was then re-equilibrated in PBS and processed as for Western blotting. The resulting films were scanned at 200 dpi with an 8 bit dynamic range using a Hewlett-Packard 4c scanner. The chemiluminescent signal from each dot in the digitized image was quantified using SigmaGel v1.0 (Jandel Scientific). These values were then imported into Excel 97 (Microsoft) and Sigmaplot v4.0 (Jandel Scientific) for analysis. Standard curves for all antibodies were established with bovine brain extract to ensure that signals fell within the linear response of the antibody used.
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RESULTS |
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Expression of MAP1a Fragments in P19 EC and HeLa Cells-- All MAP1a fragments used in this study are presented in Fig. 1. To ensure that all fragments were being expressed correctly, they were analyzed by Western blotting using mAb 9E10 to detect 6myc-tagged proteins. In P19 EC (Fig. 3a, arrows) and HeLa (Fig. 3b, arrows) cells all of the fragments displayed mobilities within 5-10 kDa of their predicted molecular masses, except for PGK-6myc1a, which had an apparent mobility of ~360 kDa (compared with(its predicted size of 312 kDa). However, this is not unexpected since endogenous MAP1a migrates at ~350 kDa and has a predicted molecular mass of 299 kDa (4). The amounts of individual fragments decreased as the size of the fragments increased. The bands seen in all lanes (P19 and HeLa cells) at 118 and 70 kDa are nonspecific gel artifacts that we have observed with a variety of monoclonal antibodies. In HeLa cell extracts the endogenous human c-MYC was detected by the 9E10 antibody (see Fig. 3b, *). Additional bands present between 60 and 30 kDa are degradation products from exogenously expressed MAP1a fragments.
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Detection of MAP1a Fragments with mAb 1A-1-- To see if any of the expressed fragments could be detected with mAb 1A-1, Western blots from transfected P19 EC were probed with mAb 9E10 (Fig. 4a) and mAb 1A-1 (Fig. 4b). In all lanes probed with mAb 1A-1, the endogenous MAP1a could be detected. A second band detected in the extract from PGK-6mycN1a-4 transfected cells displayed mobility identical to the MAP1a fragment present.
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Analysis of 6myc-tagged MAP1a Fragment Binding-- To determine which fragments of MAP1a bound to microtubules, transfected P19 EC and HeLa cells were observed by confocal immunofluorescence microscopy. Cells were fixed by precipitation so that expression of 6myc-tagged MAP1a fragments could be monitored even if they did not bind microtubules. The extraction/fixation method was used to determine if a particular fragment was bound to microtubules.
Cells expressing 6myc prepared by precipitation fixation showed a diffuse 6myc staining in the cytoplasm (Fig. 6, a' and e'). The microtubules in transfected cells showed a normal cytoplasmic interphase organization that appeared identical to that of the untransfected cells in the population (Fig. 6, a and e). Extraction/fixation of cells expressing 6myc showed no 6myc labeling (Fig. 6, b' and f'). Similar results were obtained for cells expressing 6mycN1a-1 (data not shown) showing that the tagged protein did not remain bound to microtubules.
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Taxol Treatment of Transfected P19 EC Cells-- We have previously shown that taxol treatment enhances the detection of low levels of microtubule-bound MAP1a in undifferentiated P19 EC cells by concentrating the microtubule-bound protein (24). To see if the lack of detection of the 6myc tag and 6mycN1a-1 was due to low levels of fragments present after extraction/fixation, transfected P19 EC cells were treated with taxol to induce microtubule bundles.
The microtubules in taxol-treated cells were arrayed in thick bundles running through the cytoplasm (Fig. 8, a and b). Microtubule association of 6myc (Fig. 8a') or 6mycN1a-1 (data not shown) after extraction/fixation of taxol-treated, transfected cells was never observed. As expected, extraction/fixation of cells expressing 6mycN1a-2 showed MYC labeling which colocalized with the microtubule bundles present (Fig. 8, b and b'). Similar results were obtained for 6mycN1a-3, 6mycN1a-4, 6myc1a, and 6mycN1a-2
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In Vitro Microtubule Binding of MAP1a Fragments-- To confirm the microscopic analysis of MAP fragment binding, an assay was devised to test the binding of MAP fragments to assembled microtubules in vitro (see "Experimental Procedures" and Fig. 2). Immunoblot analysis indicated that tubulin was present throughout the procedure (Fig. 9a) and electron microscopy showed this tubulin was assembled into microtubules (Fig. 9, c, d, and e). MAP1a, although at very high levels after the first assembly step (Fig. 9b, lane 1), was mostly removed by the salt washes (Fig. 9b, lanes 2-7). Some MAP1a still remained bound to microtubules at the end of the procedure.
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Colchicine Treatment of Transfected P19 EC Cells-- Transfected P19 EC cells were treated with colchicine to ensure that the pattern of 6myc labeling in cells prepared by extraction/fixation was really due to microtubule association and also to see if the colchicine resistance of microtubules was altered by any of the MAP1a fragments. As a positive control, MAP2c was expressed in P19 EC cells (using PGK-MAP2cmyc, provided by C. Addison). MAP2c is a juvenile form of MAP2 and, like high molecular weight MAP2, can bundle microtubules and render them resistant to drug-induced depolymerization (25).
MAP2cmyc expressing cells showed the presence of thick bundles of microtubules in the cytoplasm (Fig. 11a) and the 6myc labeling was found colocalized with these bundles (Fig. 11a'). After 30 min of colchicine treatment almost all microtubules were completely depolymerized, except for the MAP2cmyc-bundled microtubules that remained polymerized (Fig. 11, b and b'). In contrast, with any of the MAP1a fragments no polymerized microtubules were observed after 30 min of colchicine treatment. Fig. 11, c-d', shows an example of cells transfected with 6mycN1a-2
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Effect of MAP1a Fragments on -Tubulin Modifications--
The
results of the colchicine experiments showed that MAP2c was a stronger
microtubule stabilizer than MAP1a, but did not allow us to determine if
MAP1a could also alter microtubule stability. As another test of the
effect of MAP1a fragments on microtubule dynamics, the extent of
-tubulin acetylation and detyrosination in transfected cultures was
determined by quantitative dot blotting. These post-translational
modifications of tubulin have been shown to be biochemical markers of
neuronal microtubules that have a decreased rate of turnover (26). As a
positive control, levels of
-tubulin acetylation and detyrosination
were also determined in cells expressing MAP2cmyc.
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DISCUSSION |
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Microtubule Binding of 6myc-tagged MAP1a Fragments--
In both
P19 EC and HeLa cells the 6myc tag was expressed, but was not detected
bound to microtubules even in taxol-treated cells. 6mycN1a-1 also did
not bind microtubules, showing that aa 1-281 of MAP1a were not
sufficient for microtubule binding. 6mycN1a-2 did bind to microtubules,
suggesting that aa 282-630 of MAP1a are involved in microtubule
binding. Removal of the basic repeats from 6mycN1a-2, to produce
6mycN1a-2BR, did not prevent its binding to microtubules, indicating
the presence of microtubule-binding domain(s) within aa 281-355 or aa
451-630 or both. Langkopf et al. (4) have reported a region
of protein similarity flanking the basic repeat domain in MAP1a and
MAP1b, and in MAP1b, these flanking domains can bind microtubules in
the absence of the basic repeats (10). The larger fragments of MAP1a,
6mycN1a-3, and 6mycN1a-4, and the full-length MAP1a (6myc1a) also bound
microtubules. The ability of these fragments to bind microtubules
in vitro confirmed the microscopical observations.
Effect of MAP1a Fragments on Colchicine Resistance of Microtubules-- Cells expressing MAP2cmyc displayed bundled arrays of microtubules that were resistant to depolymerization by 30 min of colchicine treatment. This is consistent with the strong microtubule bundling and stabilizing activity of MAP2c (25). In contrast, microtubules in cells expressing MAP1a fragments were less resistant to colchicine. This does not preclude a microtubule stabilizing effect of MAP1a, but indicates that the ability of MAP1a to render microtubules resistant to colchicine is small in comparison to MAP2c.
Effect of MAP1a Fragments on Acetylation and
Detyrosination--
Of all the MAP1a fragments tested, only 6myc1a
showed marked increases in the levels of acetylation and detyrosination
per µg of polymerized tubulin relative to control cells. These
increases were approximately half those seen in cells expressing
MAP2cmyc. This suggests that an increase in the amount of MAP1a bound
to microtubules causes these microtubules to turn over more slowly than
in untransfected cells, but that these effects are weaker than those
seen with MAP2c. On a per molecule basis, MAP1a and MAP2c were almost
equivalent in their ability to affect microtubule turnover; however,
approximately twice as much MAP2c was bound to microtubules compared
with MAP1a. These levels of binding occurred under saturating
conditions, since only a fraction of the available MAP expressed was
found associated with microtubules (data not shown). Pedrotti et
al. (28) have shown that the rates of assembly and disassembly
in vitro are 2-3-fold greater for MAP1a than for MAP2,
which is in agreement with our in vivo data on -tubulin modifications. In addition, they observed that more MAP2 could bind to
the microtubule surface than MAP1a under saturating conditions. This
also is consistent with our in vivo observations on the
relative amounts of these two MAPs bound to microtubules.
Regulation of MAP1a Affinity for Microtubules--
As noted above,
the amount of 6mycN1a-2BR bound per µg of polymerized tubulin was
greater than for any other MAP1a fragment, including 6myc1a. This
higher binding of 6mycN1a-2
BR was not due solely to the higher
expression of this fragment as the proportion of total cellular
6mycN1a-2
BR bound to microtubules was also much greater than for all
other fragments (data not shown). This suggests that the
microtubule-binding regions that flank the basic repeats in MAP1a show
higher affinity for microtubules in the absence of the basic repeats.
The presence of the basic repeats in the other fragments resulted in
"reduced" or normal binding. These observations suggest that the
affinity of MAP1a for microtubules may be modulated by the basic
repeats. This type of cooperatively has already been demonstrated for
tau, in which regions flanking the basic repeats have a strong
microtubule binding activity that is modulated by the presence of the
basic repeats (32).
MAP1a Function-- MAP1a expression in developing brain continually increases during development (3, 30) to become one of the predominant MAPs in the adult brain. As this increase in MAP1a is concomitant with an increase in microtubule stability (1, 2) it has been proposed that MAP1a belongs to the group of MAPs that stabilizes microtubules during the maturation of neurons (33).
However, MAP1a is found in regions of the brain where neuronal growth persists in the adult (30, 34). Also, we have previously shown that during the differentiation of P19 EC neurons, MAP1a is found at its highest levels during the growth phase (24). These studies suggest a growth-related function for MAP1a. Our observations of the effects of MAP1a and MAP2 on acetylation, detyrosination, and colchicine resistance of microtubules shows that MAP1a is weaker than MAP2 in reducing microtubule turnover and in stabilizing microtubules to drug-induced depolymerization. We suggest that the role of MAP1a in the growth of neurons is to render microtubules stable enough to support process outgrowth, but still moderately dynamic so growing neurites remain plastic. The stabilization of microtubules by MAP1a without bundling microtubules may be critical for neuritic plasticity. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. R Vallee (Worcester Foundation, MA) for the 1A-1 antibody, Dr. T. MacRae (Dalhousie University, Nova Scotia) for the anti-E antibody. We also thank Dr. M. McBurney for providing the P19 EC cell line and Dr. M. Tenniswood (and his group at the W. Alton Jones Cell Science Center) for their assistance with the sequencing.
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FOOTNOTES |
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* This work was support by a Natural Sciences and Engineering Research Council Grant (to D. L. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
Ottawa, Dept. of Biology, 30 Marie Curie, Ottawa K1N 6N5, Canada. Tel.:
613-562-5800 (ext. 6340); Fax: ext. 5486; E-mail: dbrown{at}science.uottawa.ca.
1 The abbreviations used are: MAP, microtubule-associated protein; aa, amino acid; EC, embryonal carcinoma; MAB, microtubule assembly buffer; PGK, phosphoglycerate kinase; BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; mAb, monoclonal antibody; PIPES, 1,4-piperazinediethanesulfonic acid; bp, base pair(s); PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; 6myc, six repeats of 9-amino acid epitope from human c-MYC.
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