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Originally published In Press as doi:10.1074/jbc.M412304200 on December 28, 2004

J. Biol. Chem., Vol. 280, Issue 10, 9439-9449, March 11, 2005
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Interaction of Microtubule-associated Protein-2 and p63

A NEW LINK BETWEEN MICROTUBULES AND ROUGH ENDOPLASMIC RETICULUM MEMBRANES IN NEURONS*

Carole Abi Farah{ddagger}§, Dalinda Liazoghli{ddagger}§, Sébastien Perreault{ddagger}, Mylène Desjardins{ddagger}||, Alain Guimont{ddagger}, Angela Anton{ddagger}, Michel Lauzon{ddagger}, Gert Kreibich**, Jacques Paiement{ddagger}, and Nicole Leclerc, Scholar of Fonds de la Recherche en Santé du Québec{ddagger}{ddagger}{ddagger}

From the {ddagger}Département de Pathologie et Biologie Cellulaire, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, Québec H3C 3J7, Canada and the **Department of Cell Biology, New York University School of Medicine, New York, New York 10016

Received for publication, October 29, 2004 , and in revised form, December 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neurons are polarized cells presenting two distinct compartments, dendrites and an axon. Dendrites can be distinguished from the axon by the presence of rough endoplasmic reticulum (RER). The mechanism by which the structure and distribution of the RER is maintained in these cells is poorly understood. In the present study, we investigated the role of the dendritic microtubule-associated protein-2 (MAP2) in the RER membrane positioning by comparing their distribution in brain subcellular fractions and in primary hippocampal cells and by examining the MAP2-microtubule interaction with RER membranes in vitro. Subcellular fractionation of rat brain revealed a high MAP2 content in a subfraction enriched with the endoplasmic reticulum markers ribophorin and p63. Electron microscope morphometry confirmed the enrichment of this subfraction with RER membranes. In cultured hippocampal neurons, MAP2 and p63 were found to concomitantly compartmentalize to the dendritic processes during neuronal differentiation. Protein blot overlays using purified MAP2c protein revealed its interaction with p63, and immunoprecipitation experiments performed in HeLa cells showed that this interaction involves the projection domain of MAP2. In an in vitro reconstitution assay, MAP2-containing microtubules were observed to bind to RER membranes in contrast to microtubules containing tau, the axonal MAP. This binding of MAP2c microtubules was reduced when an anti-p63 antibody was added to the assay. The present results suggest that MAP2 is involved in the association of RER membranes with microtubules and thereby could participate in the differential distribution of RER membranes within a neuron.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neurons are polarized cells that present two distinct compartments, dendrites and an axon. These compartments can be distinguished by their morphology; dendrites are multiple and taper, whereas the axon is unique and its diameter is uniform (1–4). Dendrites and axon also differ by their membranous organelle composition; RER1 is found in the somato-dendritic compartment but not in the axon, and the number of free ribosomes is a lot higher in dendrites than in the axon (1, 2, 5). The cytoskeletal elements are also distinctly distributed in these neuronal compartments; the number of microtubules is higher in dendrites than in the axon, whereas the opposite is noted for neurofilaments (1, 2, 6).

Microtubules are involved in the establishment of cell polarity. Notably, the microtubules of dendrites and axon contain different microtubule-associated proteins (MAPs); the microtubule-associated protein-2 (MAP2) is found in dendrites, whereas tau is present only in the axon (6–8). The contribution of these MAPs to the establishment of neuronal polarity has been well documented. Tau contributes to the axonal differentiation in primary neuronal cultures, whereas MAP2 is involved in the differentiation of minor neurites, the neuronal processes that become dendrites, and in the maintenance of dendrites in adult neurons (9–14). Despite the fact that MAP2 and tau are known to induce microtubule formation in neurons, their precise role in the elaboration of dendrites and axon remains elusive. Each of these proteins presents different isoforms that are generated by alternative splicing. Splicing events occur in the microtubule-binding domain that is confined to three or four imperfect repeated domains of 18 amino acids located in the C terminus and in the projection domain that extends at the surface of the microtubules (14, 15). The latter domain, the projection domain, regulates the spacing between microtubules (16–18). MAP2 and tau share sequence homology in the microtubule-binding domain and in the adjacent proline-rich region located between the projection domain and the microtubule-binding domain (19–21). The presence of a distinct class of MAPs in dendrites and axon suggests that these proteins may have another function than the stabilization of microtubules. Consistent with this hypothesis, microtubule formation by MAP2 and tau is not sufficient to induce process outgrowth in Sf9 cells (17, 18, 22). Indeed, MAP2b promoted the formation of microtubules in these cells, but only 7% of the MAP2b-expressing cells developed processes. Thus, the stabilization of microtubules does not seem to be the sole function of MAP2 in dendritic outgrowth. In recent years, our work and that of others (23–29) showed that MAP2 could also interact with actin microfilaments and neurofilaments. Thus, MAP2 could act as a cytoskeletal integrator in dendrites by linking together the three cytoskeletal elements. This role remains to be characterized.

Besides interacting with the three constituents of the neuronal cytoskeleton, MAP2 can also interact with signaling proteins. For example, MAP2 can interact with the regulatory subunit RII of the cAMP-dependent protein kinase (30). In MAP2 knock-out mice, there is a reduction of total cAMP-dependent protein kinase in dendrites, and the rate of induction of phosphorylated cAMP-response element-binding protein is reduced after forskolin stimulation (31). These events were accompanied by a decrease of dendritic elongation. From these data, one can conclude that MAP2 plays an important role in the polarized distribution of signaling proteins that regulate dendritic differentiation and plasticity.

In a recent study (32), we reported that MAP2 was found in a crude membrane preparation from mouse spinal cord homogenate, suggesting that MAP2 could be associated with membranous organelles. In recent years, families of proteins called CLIPs and Hooks were shown to mediate the interaction between microtubules and membranous organelles (33–36). Most interestingly, CLIP-115 was found to be responsible for the polarized distribution of a membranous organelle exclusively present in dendrites termed the dendritic lamellar bodies (34). Until now, no microtubule-associated protein has been identified that contributes to the dendritic distribution of RER membranes. Our present data indicate that MAP2 could play such a role. Here we report a novel association of MAP2 with RER membranes using subcellular fractionation, electron microscopy immunocytochemistry, and electron microscopy in an in vitro reconstitution assay. Moreover, we showed that this association involves the interaction of the MAP2 projection domain with a 63-kDa nonglycosylated type II integral RER membrane protein, termed p63, that was found to mediate the interaction between RER membranes and microtubules in a previous study (37–39). Taken together, our results suggest that the interaction between MAP2 and p63 might contribute to the preferential distribution of RER membranes in the dendritic processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of a Fraction Enriched in Rough Microsomes (RM) Using Sucrose Gradient—Animals were purchased from Charles River Breeding Laboratories (Montreal, Quebec, Canada). The use of animals and all surgical procedures described in this article were carried out according to The Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. Brains were dissected from 20 Sprague-Dawley adult rats, and the protocol described previously by Lavoie et al. (40) was used to separate rough microsomes and Golgi elements from total microsomes. A schematic of the protocol is shown in Fig. 1A. Briefly, total microsomes were isolated by differential centrifugation, and ER and Golgi elements were subsequently purified by ultracentrifugation in a sucrose step gradient.



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  		FIG. 1.
Distribution of MAP2 in subcellular fractions prepared from adult rat brain. A, schematic representation of the subcellular fractionation procedure. A step gradient of sucrose was used to separate both rough microsomes and Golgi derivatives (I1 and I2) from total membrane extract (P). B, immunoblot analysis of the subcellular fractions obtained from adult rat brain. Fractions obtained following subcellular fractionation were electrophoresed on a 7.5% polyacrylamide gel (30 µg/lane) and transferred to a nitrocellulose membrane as described under "Materials and Methods." Antibodies directed against MAP2 (HM2), the plasma membrane marker Na-K-ATPase, the endoplasmic reticulum markers, ribophorin and P63, the mitochondrial marker porin (VDAC), the Golgi marker mannosidase II, and the cytoskeletal marker tubulin were used. C, comparison of MAP2 and tau distribution in the RM subfraction. A monoclonal antibody directed against tau was used (clone tau 5). E, cytoplasmic extract; N, nuclear fraction; P, total membrane extract; S, cytosolic fraction; I, interface; ML, mitochondria and lysosomes; PS, microsomes and cytosol; RM, rough microsomes.

 
Immunoblot Analyses—Protein assay was performed (Bio-Rad kit). Equal amounts of proteins were loaded in each lane and electrophoresed in a 7.5% polyacrylamide gel. Following separation, proteins were electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose strips were incubated with the primary antibodies for 90 min at room temperature. They were washed with phosphate-buffered saline (PBS) and then incubated with the peroxidase-conjugated secondary antibodies. Membranes were again washed and then revealed by chemiluminescence (Amersham Biosciences). The following primary antibodies were used: the monoclonal antibody anti-MAP2 (clone HM2, Sigma), the monoclonal antibody anti-tau (clone Tau-5, Oncogene Research Products, San Diego), the monoclonal antibody anti-{alpha}-tubulin (clone DM 1A, Sigma), a polyclonal antibody against ribophorin II, a polyclonal and a monoclonal antibody against p63 (kindly provided by Dr. H. P. Hauri, University of Basel, Switzerland), a polyclonal antibody against Porin (Oncogene Research Products, San Diego), a polyclonal antibody against mannosidase II (kindly provided by Dr. M. G. Farquhar, University of California, San Diego), and a polyclonal against NaK-ATPase (kindly provided by Dr. D. Fambrough, The Johns Hopkins University, Baltimore).

Electron Microscopy—Microsomes isolated from brain were fixed using 2.5% glutaraldehyde, recovered onto Millipore membranes by the random filtration technique of Baudhuin et al. (41), and processed for electron microscopy as described previously (40).

Pre-embedding Electron Microscope Immunocytochemistry—Immunolocalization of HM2 was modified from that used previously by Dominguez et al. (42). 200 µg of the RM fraction were resuspended in 10% normal goat serum/saline (0.9% NaCl, 10 mM Tris-HCl, pH 7.4) solution containing the primary antibody. The monoclonal HM2 antibody was used at a concentration of 1:200. The incubation was allowed to proceed overnight at 4 °C. RM were then fixed for 30 min at 37 °C in 0.05% glutaraldehyde solution and recovered onto Millipore membranes by the filtration technique of Baudhuin et al. (41). Membranes were then washed with saline solution, blocked in 1% ovalbumin/PBS solution for 30 min at room temperature, and incubated with the anti-mouse IgG-colloidal gold solution (Sigma, 1:10) diluted in 0.02% polyethylene glycol/saline for 60 min. Following the washing steps, membranes were fixed with 2.5% glutaraldehyde at 4 °C overnight and processed for electron microscopy as described above.

Primary Hippocampal Cultures—Primary embryonic hippocampal cultures were prepared from18-day-old rat fetuses as described previously (43). After removal of meninges, hippocampi were treated with trypsin (0.25% at 37 °C for 15 min), then washed in Hanks' balanced solution, and dissociated by several passages through a constricted Pasteur pipette. The cells were then plated on glass coverslips coated with polylysine. Cells were plated at 200,000 cells per 60-mm Petri dishes. Then after 4 h to allow the attachment of the cells to the substrate, the hippocampal cells were transferred either in a serum-free B-27-supplemented neurobasal medium or in N2-supplemented medium.

Immunofluorescence—Neurons were fixed in 4% paraformaldehyde/PBS for 45 min. The cells were then permeabilized with 0.2% Triton X-100 in PBS for 5 min. The MAP2 protein was revealed using a monoclonal antibody directed against MAP2 (clone HM2, dilution 1:200) purchased from Sigma or the rabbit polyclonal anti-MAP2 (1: 2000) (kindly provided by Dr. Richard Vallée, Columbia University, New York). The endoplasmic reticulum was revealed using the A1/59 monoclonal anti-p63 (1:100) (kindly provided by Dr. H. P. Hauri, University of Basel, Switzerland). To visualize tau protein, a rabbit polyclonal anti-tau antibody was used at a concentration of 1:750 (kindly provided by Dr. Virginia M-Y Lee, University of Pennsylvania, Philadelphia). Microtubules were revealed by using a rat polyclonal antibody directed against {alpha}-tubulin (Abcam, Cambridge, UK). We used the following secondary antibodies: a donkey anti-mouse conjugated to fluorescein isothiocyanate (dilution 1:100), a donkey anti-rabbit conjugated to rhodamine (1:500); and an Alexa Fluor 647 anti-rat (1:400) or an AMCA anti-rat (1:300). All these secondary antibodies were purchased from Jackson ImmunoResearch (Bio/Cam, Mississauga, Ontario, Canada). These antibodies were diluted in 5% bovine serum albumin/PBS. Incubations were carried out at room temperature for 1 h. After three washes in PBS, the coverslips were mounted in polyvinyl alcohol (Calbiochem). Fluorescently labeled cells were visualized with a Leica TCS-SP1 confocal microscope using x63 or x100 objectives or a Zeiss Axioplant fluorescence microscope using x20 or x40 objectives.

GFP-MAP2 Fusion Proteins—Rat MAP2c cDNA were inserted in the expression vector pEGFP-C1 (Clontech). The polylinker of this expression vector was modified, and three cloning sites, BamHI, ScaI, and NotI were inserted by annealing of the oligonucleotides on 5'-TCGAGGGATCCAGTACTGCGGCCGCTTAATTAA-3' and 5'-CCCTAGGTCATGACGCCGGCGAATTAATTCTAG-3'. Then the annealed sequence was ligated into the vector pEGFP-C1 at the XhoI and BamHI restriction sites. Then the full-length cDNA coding for MAP2c was excised, with BamHI and NotI restriction enzymes, from the BacPAK-HIS2 vector described previously (17). The GFP tag was fused to the MAP2c sequence at its N terminus. Deleted mutants corresponding to the projection domain of MAP2c (Proc: nucleotides 1–444) and to the microtubule-binding domain (Mt: nucleotides 442–1551) were generated. These mutants were inserted in the vector pEGFP-C1. The pEGFP-C1 Tau4R plasmid was kindly provided by Dr. Ken Kosik (44).

Transfection of Primary Hippocampal Neurons—After 7 days in culture, hippocampal neurons were transfected using a modified calcium phosphate transfection protocol (45). Briefly, neurons were transferred in a 6-well plate. The calcium and DNA precipitate was generated 30 min before transfection by mixing dropwise 4 µg of Qiagen-purified DNA in 60 µl of 250 mM CaCl2 per well with an equal volume of 2x HBS (274 mM NaCl, 10 mM KCl, 1.4 mM Na2HPO4, 15 mM glucose, 42 mM HEPES, pH 7.07). Cells were incubated with transfection precipitate for 30 min at 37 °C and 5% CO2. Following incubation, cells were washed three times with Hanks' balanced solution supplemented with 10 mM HEPES and retransferred in the original Petri dishes containing the glial monolayer and the N2 serum-free medium. Protein expression was allowed to proceed for 24 h. The cells were then fixed and processed for immunofluorescence.

MAP2c and Tau Purification from Sf9 Cells—Sf9 cells were purchased from the American Type Culture Collection (ATCC CRL 1711; Manassas, VA). Cells were grown in Grace's medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT) as a monolayer at 27 °C. For protein purification, Sf9 cells were grown as a suspension to obtain a final concentration of 1.5 x 106 cells/200 ml total media volume and were then infected with MAP2c or tau viral stocks (17, 22). Infection was allowed to proceed for 48–72 h before the cells were centrifuged at 1000 x g. The cell pellet was kept at -80 °C until protein purification. Protein purification was performed using the boiling preparation method as described previously (46).

Overlay Assay—A fraction enriched in proteins of RM was prepared from rat liver as described previously (40). RM proteins from rat liver were separated on a 7.5% polyacrylamide gel. Following separation, proteins were electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose membranes were then incubated for 1 h at room temperature in the overlay buffer: 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 3% non-fat milk, 0.1% Tween. The membrane was then incubated, overnight at 4 °C, in 500 µl of the overlay buffer plus 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride containing 10 µg of the purified MAP2c. The following day, after two washes in TBS/Tween (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20), the membranes were incubated for 1 h 30 min in the overlay buffer containing 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride with the monoclonal anti-MAP2 clone HM2 (1:1000) to detect the interactions of MAP2 with RM proteins. This was followed by an incubation with a horseradish peroxidase-conjugated mouse antibody (Jackson ImmunoResearch). To reveal proteins, an ECL detection kit was used (Pierce) according to the manufacturer's instructions.

Co-immunoprecipitation—Adult or immature embryonic (E19) or neonatal (P0) Sprague-Dawley rat brain were dissected and homogenized in the immunoprecipitation (IP) buffer: 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM EGTA, 5 mM EDTA, 5 mM Na3VO4,50mM NaF, 1 mM DTT, 1% IGEPAL and a mixture of protease inhibitors (Roche Diagnostics). The homogenate was sonicated for 5 s at an amplitude of 6% and then centrifuged for 20 min at 13,500 x g. The supernatant was used for the co-immunoprecipitation experiments. The monoclonal anti-MAP2 (clone HM2) or the polyclonal anti-p63 antibody was added to 1 ml of the supernatant and incubated overnight on a rocking platform at 4 °C. 100 µl of protein A-Sepharose beads were then added to the immunoprecipitates, and incubation was allowed to proceed for1hat4 °C. After a short spin at 14,000 x g at 4 °C, the supernatant was removed, and the beads were washed six times with 1 ml of cold IP buffer. Finally, the beads were resuspended in 50 µl of loading buffer and boiled for 5 min. The protein A-Sepharose was removed by centrifugation at 12,000 x g at room temperature. The samples were analyzed by SDS-PAGE using 12% polyacrylamide gel.

Cell Culture and Protein Expression—HeLa cells (ATCC CRL CCL-2, Manassas, VA) were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO2 incubator. For transfection, HeLa cells were plated in 35-mm Petri dishes and grown overnight to ~80% confluency. Cells were then transiently transfected using the PolyFect transfection reagent (Qiagen, Mississauga, Ontario, Canada) according to the manufacturer's instructions. Briefly, a mixture of 1.5 µg of plasmid DNA, 15 µl of PolyFect reagent, and 100 µl of DMEM were incubated for 10 min at room temperature, and 600 µl of cell culture media was then added to the complex. Cells were washed with PBS and subsequently incubated with the DNA-PolyFect complex. The expression was allowed to proceed for 24 h at 37 °C. The following day, after two washes with cold PBS, transfected HeLa cells were scraped into 1 ml of IP buffer and incubated on ice for 1 h to lyse the cells. The lysate was sonicated for 5 s at an amplitude of 6%, passed 10 times through a 25-gauge needle, and then centrifuged at 14,000 x g for 20 min at 4 °C. The resulting supernatant was used to perform co-immunoprecipitation as described above. To immunoprecipitate the full-length GFP-MAP2c fusion protein and the truncated form GFP-Proc, either the monoclonal anti-MAP2 clone HM2 (1:250) or the monoclonal anti-GFP antibody (Roche Diagnostics) was used. To immunoprecipitate GFP-Mt protein or the GFP-tau 4R protein, the monoclonal anti-GFP antibody (Roche Diagnostics) was used at a concentration of 1:100.

Negative Staining for Electron Microscopy—Tubulin (2 mg/ml) was allowed to polymerize in the presence of MAP2c or tau protein (0.5 mg/ml) in the G-PEM buffer for 35 min at 37 °C. Microtubules were then added to a freshly prepared nuclear fraction from rat liver, and the incubation was allowed to proceed for 30 min. To monitor the presence of microtubules in this preparation, 5 µl of the supernatant were placed on a carbon-coated Formvar-supported EM grid. After an incubation of 30 s, the grid was rinsed with distilled water and stained with 1% (w/v) uranyl acetate for 30 s. Samples were visualized with a Zeiss CM 902 transmission electron microscope.

Preparation of Nuclear Fractions—Nuclear fractions were prepared from rat liver homogenates (1:2, w/v) in 0.25 M sucrose, 0.05 M Tris-HCl, pH 7.5, 0.025 M KCl, and 0.005 M MgCl2 (0.25 M sucrose-TKM) using the procedure of Blobel and Potter (47). The isolated nuclei were resuspended in cold 0.25 M sucrose-TKM and centrifuged for 10 min at 1000 x g. Nuclei from 10 ml of homogenate were resuspended by gentle stirring with a glass rod in 1.2 ml of G-PEM buffer (80 mM PIPES, pH 6.9, 1 mM MgCl2, and 1 mM EGTA to which 1 mM GTP was added prior to use). To detect p63 protein, the nuclear fraction was treated with DNase according to a protocol described previously (48) to get an enrichment of nuclear membranes.

In Vitro Microtubule-Membrane Reconstitution—An in vitro microtubule-membrane reconstitution assay was modified from that described previously (49). Bovine brain tubulin protein was purchased from Cytoskeleton (Denver, CO). Tubulin (2 mg/ml) was allowed to polymerize in the presence of MAP2c or tau protein (0.5 mg/ml) in the G-PEM buffer for 35 min at 37 °C. Freshly prepared nuclear fraction was resuspended in the G-PEM buffer to which 5 mM MgCl2, 1 mM GTP, 2 mM ATP, and a mixture of protease inhibitors (Roche Diagnostics) were added prior to use. 50 µl of this fraction was added to the polymerized microtubules, and incubation was allowed to proceed for another 30 min at 37 °C. The nuclei were then centrifuged at 1000 x g for 5 min at 37 °C. The supernatant was discarded, and the pellet was fixed using 2.5% glutaraldehyde and 1% sucrose in the G-PEM buffer. Fixation was performed at 37 °C for 30 min, and the samples were then processed for electron microscopy as described above. In two sets of experiments, the polyclonal anti-p63 antibody (1:50) was added to 50 µl of the nuclear preparation and incubated at room temperature for 30 min. This preparation was then incubated with the polymerized microtubules.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The Distribution of MAP2 in Subcellular Fractions of Rat Brain—In a previous study, we reported that high molecular weight MAP2 proteins (HMW MAP2) were present in a crude membrane fraction isolated from mouse brain homogenates (32). In the present study, MAP2 distribution was studied using subcellular fractions from rat brain. MAP2 distribution was examined in adult rat brain subfractions prepared by using a fractionation protocol designed to purify rat liver ER (40) and summarized in Fig. 1A. MAP2 distribution was compared with that of various markers for different cellular organelles as follows: Na-K-ATPase, a plasma membrane marker; ribophorin and p63, markers of the endoplasmic reticulum; mannosidase II, a Golgi marker; and voltage-dependent anion channel (VDAC), a mitochondrial marker (Fig. 1B). The cytoskeletal marker tubulin was also used to characterize the subfractions. HMW MAP2 was found in the total microsomal fraction (P) as shown in Fig. 1B. Furthermore, HMW MAP2 was found enriched in a subfraction containing p63 and ribophorin, two proteins mainly found in the RER compartment (37, 50–53). Based on the enrichment with these two markers, this subfraction was named RM for rough microsomes (Fig. 1B). This subfraction also contained mitochondrial membranes as revealed by the anti-VDAC antibody. MAP2 is known to interact with mitochondria (54–56). However, it seemed unlikely that MAP2 in the RM subfraction was associated only with mitochondria because: 1) the subfraction I3 presented an amount of VDAC similar to that of the RM subfraction, but the amount of MAP2 in this subfraction was much lower than that in the RM subfraction; and 2) electron microscopy morphometry analysis confirmed that this subfraction contained very few mitochondria (see Fig. 2). Taken together, these data suggested that HMW MAP2 isoforms could be associated with RER membranes.



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  		FIG. 2.
Electron microscopy of rat brain RM. A and B, electron micrographs of the RM subfraction. A, arrowheads point to ribosomes on the membranes of rough microsomes. The arrows point to smooth microsomes. The ovals in A and B surround ribosomes associated with membrane-free filamentous structures. B, rough microsomes reveal different morphologies: tubular (1), oval (2), dilated (3), cup-shaped (4), and with a spiral alignment of ribosomes (5). C and D, electron microscope immunocytochemistry of MAP2 in the RM subfraction. The monoclonal anti-MAP2 antibody HM2 (1:200) was revealed using a secondary anti-mouse antibody conjugated to 10-nm colloidal gold particles. Immunogold labeling of MAP2 was found associated with smooth membranes (SM) as shown in C and with rough ER membranes (RM) shown in D. Scale bars: A and B, 1 µm; C, 500 nm, and D, 250 nm.

 
Because RER is mainly found in neuronal cell bodies and dendrites and MAP2 is a somato-dendritic MAP (8), it seems reasonable to consider an association between this MAP and RER. In this case, the RM subfraction was expected to contain little or no tau, the MAP found in the axon (8). Fig. 1C illustrates that RM was highly enriched in MAP2 but in contrast contained very little tau.

Electron microscopy of the RM subfraction confirmed the presence of rough microsomes (Fig. 2, A and B). Furthermore, morphometric analysis indicated that 51% of the vesicles in this subfraction presented ribosomes attached to the membrane. The microsomes in RM were heterogeneous in shape and size (Fig. 2, A and B) and were often observed in association with membrane-free filaments with associated ribosomes (Fig. 2, A and B, ovals). A small amount of tubulin was detected by immunoblotting (Fig. 1B), but no microtubule was observed in the RM subfraction (Fig. 2, A and B). Presumably, they were depolymerized at low temperature (4 °C) during fractionation. Electron microscope immunocytochemistry was carried out to reveal the association of MAP2 with membranes in the RM subfraction (Fig. 2, C and D). A quantitative analysis of the gold particles found on membranes was performed to demonstrate the preferential association of MAP2 with rough membranes (Table I). Results from three different sets of experiments revealed that 88.6% of MAP2 immunogold labeling associated with membranes was present on rough membranes.


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  		TABLE I
Quantification of HMW MAP2 immunogold in the RM subfraction The RM subfraction purified from rat brain was labeled by immunogold using anti-MAP2 antibody (clone HM2). The distribution of the gold particles was analyzed when HM2 antibody was omitted (-HM2) and when HM2 antibody was added (+HM2). The numbers represent the mean and the S.E. of three sets of distinct experiments. An analysis of variance (Tukey-Kramer multiple comparisons test) test was performed.

 
Somato-dendritic Compartmentalization of p63 and MAP2 in Primary Hippocampal Cultures—In primary hippocampal cultures, MAP2 is segregated to the somato-dendritic compartment during establishment of neuronal polarity (6–8). Similarly, in mature neurons, RER is also found in the cell body and dendrites (1, 2). We examined whether MAP2 and RER concomitantly segregate to the somato-dendritic compartment in hippocampal neurons during their development. After 1 day in culture, the hippocampal neurons are polarized cells presenting 3–4 short minor neurites that will differentiate to become dendrites, and a long thin neurite that develops into the axon (1–3). All these neurites terminate in a growth cone, a motile structure presenting a rich actin network at the periphery and bundles of microtubules at the center (57). After 10 days in culture, the dendrites and axon are differentiated, and the synaptic contacts are established. MAP2 is found in both minor neurites and axon in the first days in hippocampal cultures and then becomes compartmentalized to the dendrites (58). To examine whether the compartmentalization of MAP2 and RER occurs concomitantly in hippocampal neurons, the distribution of p63 was examined before (1-day-old cultures) and after (7-day-old cultures) MAP2 becomes concentrated in the somatodendritic compartment. To demonstrate clearly that RER membranes become enriched in the somato-dendritic compartment during neuronal differentiation, a polyclonal antibody directed against tau was used to distinguish dendritic and axonal processes. Thus, hippocampal neurons were double-labeled with a polyclonal antibody directed against tau and either with the anti-MAP2 antibody HM2, which recognizes both the high and low molecular weight isoforms of MAP2 expressed in these neurons, or a monoclonal antibody directed against p63 (Fig. 3). Tubulin staining was used to visualize both dendritic and axonal processes (Fig. 3). In 1-day-old primary hippocampal neurons, MAP2 and tau were present in all neuronal compartments as indicated by the tubulin staining. Similarly, p63 was also found in all neuronal compartments. In 7-day-old hippocampal neurons, MAP2 and p63 were concentrated in the dendritic processes but were barely evident in the axon as indicated by their poor co-localization with tau staining (Fig. 3). Thus, as reported previously for the RER protein, ribophorin, p63 becomes enriched in the somato-dendritic compartment during neuronal differentiation (51). These results show that MAP2 and the RER membranes are co-segregated in the somato-dendritic compartment during hippocampal cell differentiation.



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  		FIG. 3.
The somato-dendritic compartmentalization of MAP2 and p63 coincides in cultured hippocampal neurons. The distribution of MAP2, tau, p63, and tubulin is shown in 1- and 7-day-old cultured hippocampal neurons. The polyclonal anti-MAP2 antibody and the polyclonal anti-tau were revealed by using a donkey secondary anti-rabbit conjugated to fluorescein isothiocyanate. The monoclonal anti-p63 antibody was revealed by using a donkey secondary antimouse antibody conjugated to rhodamine. The rat polyclonal anti-tubulin antibody was revealed by using a secondary anti-rat conjugated to aminomethylcoumarin. Tubulin staining was used to visualize both dendritic and axonal processes. In 1-day-old neuronal cultures, MAP2, p63, and tau were present in all processes, whereas in 7-day-old neuronal cultures MAP2 and p63 were enriched in dendrites and tau in the axon. Scale bar, 50 µm.

 
We next determined the effects of overexpression of MAP2c on the distribution of microtubules and RER in primary hippocampal neurons. MAP2c overexpression causes the reorganization of microtubules into thin or thick bundles (18). Therefore, if RER is tightly associated with microtubules, it should undergo a reorganization similar to that of microtubules under MAP2c overexpression. Seven-day-old neurons were transfected with a GFP vector containing cDNA of LMW MAP2, MAP2c. GFP-MAP2c was mostly found in dendrites in 7-day-old control hippocampal neurons (Fig. 4, A and A'). A similar distribution was noted for RER in these neurons. Seven-day-old control neurons transfected with GFP vector revealed no discernible difference in RER distribution compared with control cells (Fig. 4, B and B'). Overexpressing MAP2c produced no important changes in the structure of the dendrites and axon of 7-day-old hippocampal neurons (data not shown), even though in some of the transfected cells, several thin extensions emerged from the cell body (Fig. 4D). A similar phenotype has been described previously in Sf9 cells expressing MAP2c (17, 18). However, transfection with GFP-MAP2c led to a reorganization of the microtubule network in the neuronal cytoplasm (Fig. 4, C'' and D''). In most of the MAP2c-transfected neurons, large microtubule bundles were noted in the cell body (Fig. 4C''). In these cells, RER staining was found along these bundles (Fig. 4, C, C', and C'', arrows). In the MAP2c-transfected neurons harboring multiple thin extensions, very thin microtubule bundles were randomly distributed in the cell body, and an important reorganization of RER staining was noted in the perikaryon (Fig. 4, D, D', and D'', arrowhead in the insets) along these bundles (Fig. 4, D, D', and D''). In both types of MAP2c-transfected neurons, GFP-MAP2c and RER distribution were closely associated with the MAP2c-containing microtubule bundles (Fig. 4, C, C', and C'' and D, D', and D''). The concomitant reorganization of both microtubules and RER observed in MAP2c-overexpressing neurons showed that RER and microtubules are intimately associated in hippocampal cells. However, the above results did not demonstrate whether MAP2c was directly involved in the association of RER with microtubules.



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  		FIG. 4.
Overexpression of GFP-MAP2c fusion protein in hippocampal neurons. A and A' show the distribution of MAP2 (HM2 antibody) and the RER, respectively, in 7-day-old cultured hippocampal neurons. The polyclonal antibody against ribophorin II was used to stain the RER. This antibody was revealed using a secondary anti-rabbit conjugated to rhodamine. Insets show a higher magnification of the cell body. B and B', 7-day-old neurons were transfected with the GFP protein alone as a control. Protein expression was allowed to proceed for 24 h before the cells were fixed and processed for immunofluorescence. Insets show that the GFP protein does not induce a reorganization of the RER membranes. C, C', and C'' show the distribution of the GFP-MAP2c, ribophorin, and tubulin staining in a transfected neuron presenting thick microtubule bundles. In these cells, RER staining is found on these bundles (arrows). D, D', and D'' show the distribution of the GFP-MAP2c, ribophorin, and tubulin staining in a transfected neuron presenting multiple thin extensions. In these cells, a reorganization of the RER was noted in the perikaryon (arrowhead in the inset) and along the thin microtubule bundles. Scale bar for all figures except C, C', and C'' is 20 µm. Scale bar for C, C', and C'' is 8 µm. Scale bar for the insets is 4 µm.

 
Interaction of MAP2 with the Integral RER Membrane Protein p63—To identify the proteins mediating the association of MAP2 with ER membranes, we performed a Western blot overlay experiment (Fig. 5). The proteins contained in the RM subfraction isolated from adult rat liver were separated on a 12% polyacrylamide gel and transferred to a nitrocellulose membrane. The nitrocellulose membrane was then incubated with MAP2c protein purified from Sf9 cells. The interaction of MAP2c with the proteins contained in the RM subfraction was revealed by using the anti-MAP2 antibody HM2. As expected, MAP2 staining was found in a doublet band corresponding to the molecular weight of tubulin. Moreover, a MAP2 immunoreactive band was also observed at an apparent molecular mass of 63 kDa. As shown in Fig. 5, this band reacted with an anti-p63 antibody indicating that p63 could be involved in the association of MAP2 with RER membranes. To demonstrate further this interaction, we tested whether p63 could co-immunoprecipitate with MAP2 in a homogenate prepared from adult rat brain (Fig. 6A). The anti-MAP2 antibody HM2 was used to immunoprecipitate MAP2. As shown in Fig. 6A, a band immunoreactive to the anti-p63 antibody was present in the MAP2 immunoprecipitate. In the brain homogenate, two bands were revealed by the polyclonal anti-p63 antibody, one band at 63 kDa and one band of slightly higher molecular weight. The monoclonal anti-p63 antibody only revealed the lower band at 63 kDa. In the MAP2 immunoprecipitate, only the band at 63 kDa was revealed either using the monoclonal or the polyclonal antibody. We also tested whether the LMW MAP2 isoforms could interact with p63 in immature brain. Brain homogenate was prepared from embryonic (E19) rat or newborn (P0) rat (data not shown), and immunoprecipitation of either MAP2 or p63 was performed. As noted for HMW MAP2 isoforms, in the LMW MAP2 immunoprecipitate, p63 protein was also present (Fig. 6B). Most notably, MAP2c could be immunoprecipitated by using the anti-p63 antibody. From these results, one could conclude that the domain of MAP2 interacting with p63 was located in the peptide sequence common to HMW and LMW MAP2 isoforms.



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  		FIG. 5.
The interaction of MAP2c and p63 in an overlay assay. An overlay assay using MAP2c purified from Sf9 cells was carried out to show the interaction of MAP2 with ER proteins from a rough microsomal fraction prepared from rat liver. The overlay experiment was performed as described under "Materials and Methods." Lane 1, no MAP2c protein was added to the overlay buffer. Lane 2, the membrane was incubated with purified MAP2c protein for 1 h 30 min. Lane 3, the membrane of lane 2 was stained with an anti-p63 polyclonal antibody.

 



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  		FIG. 6.
Co-immunoprecipitation of the MAP2-p63 complex from adult and embryonic (E19) rat brain homogenate. A, the interaction of MAP2 with p63 in adult rat brain was confirmed by co-immunoprecipitation. MAP2 was immunoprecipitated from an adult rat brain homogenate as described under "Materials and Methods." The monoclonal anti-MAP2 HM2 (1:250) was used to immunoprecipitate the MAP2-p63 complex. The nitrocellulose membrane was then revealed with the monoclonal anti-MAP2 HM2 (1:1000) and then with the polyclonal anti-p63 (1:5000). The calnexin was used as a negative control. Note that the polyclonal antibody anti-calnexin recognizes the calnexin only in the adult rat brain homogenate. B, the interaction of MAP2 with p63 in embryonic rat brain was confirmed by co-immunoprecipitation. Either MAP2 or p63 was immunoprecipitated from a embryonic rat brain homogenate by using the monoclonal anti-MAP2 HM2 (1:250) or the polyclonal antibody directed against p63 (1:500). The Western blot was then revealed with the monoclonal anti-MAP2 HM2 (1:1000) and then with the monoclonal anti-p63 (1:250). The precision plus protein standards from Bio-Rad were used for accurate protein sizes.

 
MAP2 proteins have two main domains, the projection domain and the microtubule-binding domain located at the N and C termini, respectively. A construct corresponding to either the projection domain (Proc) or the microtubule-binding domain (Mt) of the LMW MAP2 isoform MAP2c was generated and fused to a GFP tag. To determine which of these domains interacted with p63, they were expressed in HeLa cells, a cell line expressing p63 but not MAP2, and their interaction with p63 was examined by co-immunoprecipitation. When MAP2 immunoprecipitation was performed on HeLa cells expressing full-length MAP2c, p63 was found in the immunoprecipitate (Fig. 7). In HeLa cells, one band of 63 kDa was revealed with both the monoclonal and polyclonal antibody directed against p63 as reported previously (37, 38). However, when immunoprecipitation was performed on HeLa cells expressing fulllength tau or the GFP alone, no p63 was detected in either tau or GFP immunoprecipitate. Most notably, when Proc was immunoprecipitated from HeLa cells with the anti-MAP2 antibody HM2, p63 was found in the immunoprecipitate (Fig. 7). However, p63 was absent from the Mt immunoprecipitate, indicating that the MAP2 interacting peptidic sequence with p63 is located within the first 150 amino acids of MAP2 isoforms.



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  		FIG. 7.
Interaction of the projection domain of MAP2 with p63. HeLa cells were transfected with an expression vector containing either GFP alone, GFP-tau, GFP-MAP2c, GFP-Proc, a deleted form of MAP2c corresponding to its projection domain, or GFP-Mt, a deleted form of MAP2c corresponding to its microtubule domain. 24 h after transfection, HeLa cells were lysed, and co-immunoprecipitation experiments were carried out with an antibody directed against GFP. Our data showed that the binding peptidic sequence of MAP2 to p63 is located in the projection domain of MAP2c. Indeed, the anti-GFP antibody was able to co-immunoprecipitate either GFP-MAP2c and p63 or GFP-Proc and p63 but not GFP-Mt and p63. The GFP antibody was unable to co-immunoprecipitate either p63 and GFP-Tau or p63 and GFP alone. The precision plus protein standards from Bio-Rad were used for accurate protein sizes.

 
In Vitro Reconstitution of the Microtubule-MAP2-ER Complexes—To show that MAP2 can act as a linker between the ER and microtubules, an in vitro membrane-microtubule reconstitution assay was developed. Briefly, ER membranes were incubated with microtubules containing MAP2, and the membrane-bound microtubules were co-sedimented by centrifugation. The association of microtubules with membranes was then examined by electron microscopy. Because both microtubules and ER membranes co-sediment at 100,000 x g, we decided to use a nuclear preparation as a source of RER membranes. There are two main advantages to this preparation. 1) RER membranes are with nuclei (59), and therefore, the results generated with nuclei are of relevance to the mechanism of interaction between MAP2 microtubules and RER. 2) Nuclei sediment at low speed (1000 x g) in contrast to microtubules that sediment at high speed (100,000 x g). Thus, the only way microtubules can co-sediment with nuclei in this assay is through an association with the nuclei. A fraction of rat liver nuclei prepared using the Blobel and Potter procedure (47) was used because it is devoid of MAP2 and tau. Moreover, no tubulin was detected in this fraction by immunoblotting (Fig. 8A). Finally, the presence of p63 in this nuclear preparation was confirmed by immunoblotting (Fig. 8B).



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  		FIG. 8.
In vitro reconstitution of the ER-MAP2-microtubule complexes. A, the nuclear fraction was examined by Western blotting for its content in tubulin. No endogenous tubulin was detected in this fraction. B, the presence of p63 in the nuclear fraction was confirmed by Western blot by using the polyclonal anti-p63 antibody. As noted in brain homogenate, in a subfraction enriched in rough membranes isolated from rat liver, the polyclonal antibody anti-p63 revealed two bands. However, in a nuclear fraction isolated from rat liver and treated with DNase to concentrate the membranes, only the band at 63 kDa was present. C, the microtubule polymerizing activity of MAP2c and tau was determined as described under "Materials and Methods." Western blotting shows similar amounts of tubulin in the MAP2c and in the tau pellets, indicating that these proteins have similar capacities to polymerize microtubules. D, electron micrograph of MAP2c pre-polymerized microtubules visualized by negative staining. E, electron micrograph of tau pre-polymerized microtubules visualized by negative staining. F, electron micrograph illustrating that tau pre-polymerized microtubules were intact after incubation with nuclei. Scale bar for D–F is 0.25 µm

 
The pre-polymerized microtubules used in the present assay were composed of pure bovine brain tubulin and MAP2c protein purified from Sf9 cells. As a negative control, microtubules were pre-polymerized using the axonal MAP tau also purified from Sf9 cells. We tested the microtubule polymerizing activity of MAP2c and tau to eliminate the possibility that any difference in the number of microtubules bound to nuclei could be caused by a difference of tau and MAP2c capacity to form microtubules under our experimental conditions. To this purpose, MAP2c (0.5 mg/ml) or tau (0.5 mg/ml) was incubated with pure bovine brain tubulin (2 mg/ml) in the G-PEM buffer. Following an incubation of 30 min at 37 °C and a high speed centrifugation (100, 000 x g), the pellet and the supernatant were analyzed by Western blotting for their content in tubulin, MAP2c, and tau. As shown in Fig. 8C, similar amounts of tubulin were found in the MAP2c and the tau pellets, indicating that these proteins have a similar tubulin polymerizing activity in the present conditions. Finally, microtubule formation by MAP2c and tau was examined at the electron microscopic level by negative staining. Individual and bundles of microtubules were observed in both MAP2c- and tau-microtubule preparations (Fig. 8, D and E). Nuclei were incubated with exogenous microtubules that were pre-polymerized either in the presence of MAP2c or tau. Following the incubation, nuclei were pelleted at a centrifugation speed of 1000 x g. Nuclei were then resuspended in fresh buffer and processed for electron microscopy. All incubations of nuclei with microtubules were performed at 37 °C because microtubules depolymerize at lower temperature. No microtubule was observed in association with either the control nuclei or nuclei incubated in the presence of tau-prepolymerized microtubules (Fig. 9, A and B). To eliminate the possibility that the tau-containing microtubules depolymerized during the incubation with the nuclei, the presence of microtubules was monitored at the electron microscopic level by negative staining (Fig. 8F). However, when nuclei were incubated with MAP2c pre-polymerized microtubules, microtubules were observed as small cylinders with a constant diameter (~25 nm in diameter) sectioned in different orientations on their surface (Fig. 9C, arrows). Thus the above experiments showed that MAP2c- but not tau pre-polymerized microtubules could associate with nuclei, and this association was necessary for microtubules to co-sediment at 1000 x g. These results were reproduced in five different series of experiments.



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  		FIG. 9.
Electron micrographs of the in vitro reconstitution of the ER-MAP2-microtubule complexes. A, nuclei (N) are shown after incubation in the presence of exogenously added tau pre-polymerized microtubules. Under these conditions, no microtubules were observed in association with the nuclei. A higher magnification of the region outlined by a rectangle in A is shown in B. Arrowheads point to ribosomes on the outer nuclear membrane (ONM). C, micrographs showing high power electron microscopy of the surface of nuclei after incubation in the presence of exogenously added MAP2c pre-polymerized microtubules. Arrows point to cross-sections and oblique sections of microtubules located within 25 nm of outer nuclear membrane. Scale bars: A is 2 µm; B is 1 µm; and C is 500 nm.

 
A quantitative analysis was carried out to evaluate the number of MAP2c-containing microtubules associated with the rat liver nuclei. Microtubules located within 25 nm of the outer nuclear membrane were included in the analysis, because this is considered a physiologically relevant distance for microtubule interaction based on measurement of spacing between microtubules in bundles induced by MAP2c and tau in Sf9 cells (17, 60). Most of the microtubules (>80%) were organized in bundles of two or more microtubules. The number of microtubule bundles per nucleus was quantified on 19 nuclei from four different sets of experiments for a total of 76 nuclei. On average, 2.26 ± 0.25 microtubule bundles were found associated with one nucleus. These results indicate that MAP2c might be involved in the interaction of RER membranes with microtubules in vivo. In the last two sets of experiments, an additional experimental condition was added to examine the contribution of p63 in the interaction of MAP2-containing microtubules with nuclei. To do this, the polyclonal anti-p63 antibody was added to the nuclei preparation 30 min before their incubation with the MAP2c-microtubules. In these two sets of experiments, anti-p63 antibodies were found to be able to reduce MAP2-microtubule binding to the nuclei. Quantification revealed 2 ± 0.26 bundles of MAP2c-microtubules associated with one nucleus in the control experiments, whereas in presence of the anti-p63 antibody, 0.94 ± 0.14 bundle was attached to one nucleus. This indicated that p63 contributes to the association of MAP2c containing microtubules with nuclei.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we describe a novel type of ER-microtubule interaction that is mediated by the dendritic MAP, MAP2. A subfraction enriched in rough ER microsomes was isolated from rat brain as indicated by its high content in the two RER proteins, ribophorin and p63, as well as by an electron microscope morphometric analysis using the ribosome as a morphological marker for RER. An important amount of MAP2 was detected in the RM subfraction by immunoblotting. In primary hippocampal neurons, MAP2 and p63 were co-segregated to the somato-dendritic compartment during neuronal differentiation. An overlay assay and co-immunoprecipitation experiments indicated that the association of MAP2 with RER membranes involved p63. The interacting domain of MAP2 with p63 was found to be located in the first 150 residues of the MAP2 projection domain. By using an in vitro reconstitution assay, MAP2 was shown to mediate the association between microtubules and RER membranes. Most notably, this association was significantly reduced in the presence of an anti-p63 antibody. Collectively, our results point to a role for MAP2 and p63 in the distribution and structural maintenance of RER in neurons.

Previous studies have shown that the ER membranes are associated with microtubules (61–65). This association could be either dynamic for trafficking of ER membranes or stable for the positioning of these membranes within a cell (61, 65, 66). Depolymerization of microtubules using the drug nocodazole affects both the trafficking and positioning of ER membranes (62–64, 67). The ER movement along microtubules has been well documented (61, 64, 67, 68). The sliding of ER membranes along microtubules seems to be driven by motor proteins such as kinesin and dynein (69–73)

Moreover, it was shown that ER could be moved within a cell by its attachment to the microtubule-plus end (67). However, when the expression of either dynein or kinesin is suppressed within a neuron, the association of membranous organelles including the ER with microtubules is not completely eliminated, indicating that other proteins are involved in this association (74, 75). These proteins would be involved in the positioning of membranous organelles along microtubules and thereby would allow the maintenance of the structure of these organelles. The maintenance of membrane structure is particularly important in post-mitotic cells such as mature neurons, where ER membranes are found along the length of dendrites and axon. Recently, the relative proportion of dynamic and static ER was determined within hippocampal neurons. It was found that only a small ER subcompartment is dynamic in mature hippocampal neurons (70). Proteins mediating a stable interaction between microtubules and membranous organelles could play a role in the maintenance of membrane structure and distribution.

Cytoplasmic linker proteins (CLIPs), for example, are known to establish a link between microtubules and membranous organelles. As such, CLIP-170 has been reported to mediate the interaction of endocytic carrier vesicles to microtubules (33). Furthermore, CLIP-115 was shown to be responsible for the polarized distribution of the dendritic lamellar bodies in neurons (34, 35). Recently, a new class of proteins termed CLASPs (CLIP-associated proteins) was identified (76). These proteins bind CLIPs and microtubules and have a microtubule-stabilizing effect. A family of proteins named Hooks also mediates the interaction between microtubules and membrane organelles. More specifically, Hook3 links the Golgi membranes to microtubules (36). Furthermore, p63 was shown to be involved in the interaction of ER with microtubules in a non-neuronal cell line (37–39). Because p63 is an integral membrane protein, it was termed a CLIMP (cytoskeleton-linking membrane protein).

To our knowledge, no dendritic cytosolic linker protein has been identified so far that mediates the interaction between the ER and microtubules in neurons. Our results show that MAP2 plays such a role. However, MAP2 is a cytosolic linker protein that has to be classified in a category of its own for two reasons. 1) The microtubule-binding domain of MAP2 has no sequence homology with that of CLIPs, CLASPs, and Hooks. 2) All the linker proteins identified so far bind to the distal end of microtubules, whereas MAP2 binds to microtubules along their length (77).

Our studies suggest that the association of MAP2 with RER membranes involves p63. The exact function of p63 remains to be clarified. However, convincing data were generated in COS cells showing its potential role in the positioning of the RER along microtubules. The overexpression of p63 in COS cells induced an important rearrangement of the ER from a punctate/reticular pattern to a tubular one (38). This was accompanied by the bundling of microtubules. The cytoplasmic domain of p63 was shown to be responsible for its microtubule bundling activity (38). In neurons, the highly polarized distribution of the RER in the somato-dendritic compartment might require more complex molecular interactions between RER membranes and microtubules than in nonpolarized cells. In this context, the direct binding of p63 to microtubules would not be enough to determine the positioning of RER in neurons. Thus, the selective interaction of MAP2- but not tau-containing microtubules with p63 would contribute to the concentration of RER membranes in the somato-dendritic compartment.

Previous studies showed that microtubule motor proteins are involved in the positioning of ER membranes. The present results indicate that structural microtubule-associated proteins could also contribute to the distribution of ER membranes within a neuron. The activity of these proteins might vary to allow dendritic growth and remodeling.


   FOOTNOTES
 
* This work was supported by a National Sciences and Engineering Research Council of Canada grant and by the Canadian Institute of Health Research Grants MOP-53218 (to N. L.) and MOP-44022 (to J. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

Recipient of a studentship from the Centre de Recherche en Sciences Neurologiques. Back

|| Recipient of a studentship from the National Sciences and Engineering Research Council of Canada. Back

{ddagger}{ddagger} To whom correspondence should be addressed. Tel.: 514-343-5657; Fax: 514-343-5755; E-mail: nicole.leclerc{at}umontreal.ca.

1 The abbreviations used are: RER, rough endoplasmic reticulum; ER, endoplasmic reticulum; MAPs, microtubule-associated proteins; HMW, high molecular weight; LMW, low molecular weight; GFP, green fluorescent protein; VDAC, voltage-dependent anion channel; RM, rough microsomes; PIPES, 1,4-piperazinediethanesulfonic acid; DTT, dithiothreitol; PBS, phosphate-buffered saline; Proc, projection domain; Mt, microtubule-binding domain; IP, immunoprecipitation; CLIPs, cytoplasmic linker proteins. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Hans-Peter Hauri for providing the anti-p63 antibodies, Dr. M. G. Farquhar for the anti-mannosidase II antibody, and Dr. D. Fambrough for the NaK-ATPase antibody. The polyclonal anti-MAP2 antibody and the polyclonal anti-tau antibody were generously provided by Dr. Richard Vallée and Dr. Virginia M-Y Lee, respectively. The monoclonal antibody E7 directed against {beta}-tubulin, developed by Michael Klymkowsky, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, National Institutes of Health, and maintained by the Department of Biological Sciences, University of Iowa, Iowa City. We also thank Jean Léveillé and Annie Vallée for their excellent technical support and Diane Gingras for helpful discussion.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
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 DISCUSSION
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