the control of microtubule stability in vitro and in ... · map1b promoted microtubule formation at...

The Control of Microtubule Stability In Vitroand in Transfected Cells by MAP1B

and SCG10

Percy Bondallaz,1,2 Anne Barbier,1 Sophia Soehrman,1 Gabriele Grenningloh,1,2

and Beat M. Riederer1,2*

1Departement de Biologie Cellulaire et de Morphologie, University of Lausanne,1005 Lausanne, Switzerland

2Center for Psychiatric Neuroscience, Department of Psychiatry-CHUV,University of Lausanne, 1008 Prilly, Switzerland

In neurons, the regulation of microtubules plays an important role for neurite out-growth, axonal elongation, and growth cone steering. SCG10 family proteins arethe only known neuronal proteins that have a strong destabilizing effect, arehighly enriched in growth cones and are thought to play an important role duringaxonal elongation. MAP1B, a microtubule-stabilizing protein, is found in growthcones as well, therefore it was important to test their effect on microtubules in thepresence of both proteins. We used recombinant proteins in microtubule assemblyassays and in transfected COS-7 cells to analyze their combined effects in vitroand in living cells, respectively. Individually, both proteins showed their expectedactivities in microtubule stabilization and destruction respectively. In MAP1B/SCG10 double-transfected cells, MAP1B could not protect microtubules fromSCG10-induced disassembly in most cells, in particular not in cells that containedhigh levels of SCG10. This suggests that SCG10 is more potent to destabilizemicrotubules than MAP1B to rescue them. In microtubule assembly assays,MAP1B promoted microtubule formation at a ratio of 1 MAP1B per 70 tubulindimers while a ratio of 1 SCG10 per two tubulin dimers was needed to destroymicrotubules. In addition to its known binding to tubulin dimers, SCG10 bindsalso to purified microtubules in growth cones of dorsal root ganglion neurons inculture. In conclusion, neuronal microtubules are regulated by antagonistic effectsof MAP1B and SCG10 and a fine tuning of the balance of these proteins may becritical for the regulation of microtubule dynamics in growth cones. Cell Motil.Cytoskeleton 63:681–695, 2006. ' 2006 Wiley-Liss, Inc.

Key words: assembly; growth cone; neurites; MAPs; stathmin; microtubules

INTRODUCTION

Growing neurites and their highly motile tips de-pend on continual rearrangements of the major cytoske-letal elements, actin filaments and microtubules, under-lying axon guidance during outgrowth and regeneration.The microtubule polarity is characterized by a moreactive plus-end oriented towards the distal part of theaxon and the growth cone [Mitchison and Kirschner,1984]. The dynamic state of microtubules has beenshown to be important for neurite elongation, growthcone turning and phosphorylation of microtubule-associ-

*Correspondence to: Dr. B.M. Riederer, DBCM, Universite de Lau-

sanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland.

E-mail: [email protected]

Contract grant sponsor: Swiss National Research Foundation; Con-

tract grant number: 3100-067201.01; Contract grant number: 3100-

61600.00; Contract grant number: 3100A0-104258.

Received 30 March 2006; Accepted 26 June 2006

Published online 27 September 2006 in Wiley InterScience (www.

interscience.wiley.com).

DOI: 10.1002/cm.20154

' 2006 Wiley-Liss, Inc.

Cell Motility and the Cytoskeleton 63:681–695 (2006)

ated protein 1B (MAP1B) [Mack et al., 2000]. Molecularmechanisms involved in neurite pathfinding could beexplained by a selective stabilization or destabilizationof the microtubule cytoskeleton, such as complementaryactions of proteins that disassemble and rescue microtu-bules respectively. Several cellular factors regulatemicrotubule dynamics such as microtubule-associatedproteins (MAPs), CLIP-170, APC-binding protein EB1and the stathmin family [as reviewed by Mori and Morii,2002; Gordon-Weeks, 2004; Grenningloh et al., 2004;Folker et al., 2005]. A variety of MAPs, such as tau andhigh molecular weight proteins MAP1 and MAP2, are ofspecial importance for the formation of axonal and den-dritic processes. MAP1B is a major cytoskeletal proteinand essential to promote microtubule assembly duringdevelopment [Bloom et al., 1985; Riederer et al., 1986;Gonzalez-Billault et al., 2005]. In contrast to MAP1B,SCG10 has a microtubule destabilizing effect. It is a pro-tein of the stathmin family and promotes neurite elonga-tion in stably transfected neuronal cells [Riederer et al.,1997]. There is recent evidence that blocking its functioninhibits growth cone advance [Suh et al., 2004]. Bothproteins are considered to be major growth-associatedproteins and essential during growth processes. Sinceboth proteins are present in growth cones and importantfor axonal development it was of interest to test theircombined effects on microtubule stability.

MAP1B, also known as MAP1.2, MAP1x orMAP5, is expressed in the central and peripheral nervoussystem (CNS and PNS). It is composed of 2463 aminoacids, with a microtubule binding domain (MBD) in theN-terminal part [Noble et al., 1989]. The protein is de-velopmentally regulated [Riederer et al., 1986; Schoen-feld et al., 1989] and abundant in growing processes. Itdecreases in concentration with development [Bloomet al., 1985; Riederer et al., 1986; Schoenfeld et al.,1989]. During embryonal and early postnatal CNS devel-opment, MAP1B is highly concentrated in axonal pro-cesses [Schoenfeld et al., 1989] and is involved in nerveregeneration in CNS and PNS [Bush et al., 1996a;Ramon-Cueto and Avila, 1999; Ma et al., 2000] as wellas in CNS areas with plasticity potential [Nothias et al.,1996]. Its function is to increase microtubule assemblyand axogenesis [Riederer et al., 1986; Pedrotti and Islam,1995; Gordon-Weeks and Fischer, 2000]. Phosphoryla-tion may reduce its microtubule-stabilizing effects[Gordon-Weeks et al., 1993] and influence a directionalgrowth [Mack et al., 2000] with a gradient of phospho-rylated MAP1B towards the tip of growing axons [Boyneet al., 1995; Bush et al., 1996b; Goold et al., 1999].

A group of proteins, including SCG10, a memberof the stathmin family, are able to destabilize microtu-bules [Belmont and Mitchison, 1996; Riederer et al.,1997]. SCG10 is a membrane associated neuronal pro-

tein composed of 179 amino acids [as reviewed by Gren-ningloh et al., 2004]. It is associated with membranousvesicles targeted to the tips of growing processes[DiPaolo et al., 1997a, b; Lutjens et al., 2000]. Largeexpression of the protein occurs during development andis diminished over 3 weeks postnatally. In the adultbrain, high levels of mRNAs are found in the hippocam-pus [Himi et al., 1994]. SCG10 plays a role in neuriteoutgrowth, inducing strongly enhanced neurite elonga-tion in response to nerve growth factor [Riederer et al.,1997]. Its activity is known to be negatively regulated byphosphorylation of certain serine residues, involving sev-eral protein kinases like protein kinase A (PKA), mito-gen-activated protein kinase (MAPK) or cyclin-depend-ent kinase (CDK) [Antonsson et al., 1998]. Several inter-actions with other intracellular proteins have beendescribed, for example with RGSZ1 [Nixon et al., 2002]and stress-activated protein kinase JNK3 [Neidhart et al.,2001]. The mechanism by which SCG10 destabilizesmicrotubules is not yet clear, but, like stathmin, it hasthe capacity to sequester tubulin dimers. Its close se-quence similarity to stathmin suggests that it may alsohave a catastrophe-promoting activity. This may beessential for the regulation of growth cone microtubulesand determine growth cone advance and stearing.

Here we have tested how SCG10 and MAP1B reg-ulate the microtubule cytoskeleton at the molecularlevel. Since both proteins are present in axonal growthcones, it was crucial to determine a possible antagonisticrole of these proteins in the regulation of microtubule dy-namics. Changes in the balance between the oppositeeffects of the two proteins were tested by using variousconcentrations of SCG10 versus recombinant MAP1B atendogenous concentration levels in assembly and elec-tron microscopy assays. Furthermore, we have used im-munocytochemical detection of the microtubule cyto-skeleton in transfected cells and in vivo localization ofthe two proteins in cell lines and in neuronal primaryculture.

MATERIALS AND METHODS

DNA Constructs

The full coding sequence of mouse MAP1B [Nobleet al., 1989; Edelman et al., 1996] was used to constructtruncated forms of MAP1B by restricting the total cDNAsequences 1-1404 and 1-981, to obtain MAP1B1404 andMAP1B981 including the flanking regions of the MBD.All DNA manipulations were conducted as previouslydescribed [Sambrook et al., 1989]. Full-length and frag-ment cDNAs sequences were introduced into a bacterialexpression vector, pQE31. First, a SalI restriction sitewas introduced by PCR in place of the initiation codon.

682 Bondallaz et al.

The new sequence of MAP1B, restricted by SalI/DraI,was subcloned in frame in the pQE31 vector digested byHindIII, blunt ended by Klenow fragment and restrictedby SalI. pQE31 vector is designed to insert a 6 His-pep-tide at the N-terminus of the amino-acid sequence. ThecDNA sequences of two truncated fragments of MAP1Bwere also subcloned in frame in pQE31 vector digestedby HindIII, blunt ended by Klenow fragment and re-stricted by SalI. The first MAP1B truncated fragment(named MAP1B1404) was obtained by NdeI restriction,blunt ending by Klenow fragment and SalI digestion andencoded for a 157 kDa protein. The second MAP1Btruncated fragment (named MAP1B981) was obtained byAlw44I restriction, blunt ending by Klenow fragmentand SalI digestion, and encoded for a 112 kDa protein.The two truncated cDNA sequences of MAP1B werealso subcloned into the mammalian expression vector,pcDNA3.1/myc-His, designed to introduce a myc-Histag at the C-terminus of the amino-acid sequence byusing the same restriction sites. In these constructs, theinitiation codons were restored by site-directed mutagen-esis using the QuickChange kit and the sequences wereverified by sequencing the insert. The SCG10 constructand production of recombinant protein has been des-cribed previously [Antonsson et al., 1997].

Expression in Escherichia coli and Purificationon Ni-Gel Column

In pQE31 vector, the expression of recombinantproteins is driven by the phage T5 promoter and two lacoperator sequences. The expression was performed in E.coli strain M15 [pREP4]. The culture was grown at 378Cuntil OD600 nm was equal to 0.6, then the expression ofrecombinant protein was induced by the addition of1 mM isopropyl-b-D-thiogalactoside and the culture wascontinued at 228C for 16 h in order to overexpress therecombinant proteins with minimal formation of degra-dation products. The cells were harvested by centrifuga-tion at 5000g for 15 min. Then, the cells were resus-pended into 20 mM Tris-HCl pH 8.8 and 100 mM NaCland lysed for 30 min at room temperature by addition of2 mg/ml of lysozyme in the presence of protease inhibi-tor cocktail and 1 lM pepstatin. After sonication, thelysis was continued with 2.5 lg/ml DNAse I and 1 lg/mlRNAse A. The proteic solution was then centrifuged at12,000g, 15 min at 48C. The pellet was incubated 30 minat room temperature in 20 mM Tris-HCl pH 8.8, 100mM NaCl in the presence of 0.1% (v/v) Triton X-100,protease inhibitor cocktail and 1 lM pepstatin. After asecond step of centrifugation at 12,000g, 15 min at 48C,the pellet was finally incubated for 16 h at 48C in dena-turing conditions in 6 M guanidine-HCl pH 7.8/10 mMTris/100 mM NaH2PO4. The cell lysate was then puri-fied by affinity chromatography with Ni2þ-gel column.

After centrifugation at 12,000g, 15 min at 48C, the super-natant was loaded onto a Ni2þ-NTA agarose column(Qiagen), previously equilibrated with buffer B (8 MUrea/100 mM NaH2PO4/10 mM Tris-HCl pH 8.0). Thegel was extensively washed with buffer B. When OD280 nm

was equal to 0.05, the gel was washed with the samebuffer at pH 6.3. The bound material was eluted bydecreasing the pH of the same buffer to pH 4.0. The puri-fied fractions were immediately extensively dialyzed at48C against 0.1 M MES pH 6.4, 0.5 M NaCl, 1 mMdithiotreitol (DTT) containing 6 M urea. Dialysis wascontinued in the presence of decreasing concentration ofurea and finally in a buffer without urea until a bufferwithout urea was reached in the sample. The protein con-centrations were determined by a Bradford assay. Thefull-length MAP1B cDNA was too large to be producedin bacterial cells therefore two truncated forms wereexpressed. Large scale production of recombinant pro-teins was performed at 228C, which enabled 1–2 mg ofMAP1B fragments per liter of culture to be obtained.

Tubulin and Microtubule Purification

Microtubule proteins were prepared from freshporcine brain. Starting material was hundred grams ofbrain which were homogenized in 100 ml buffer A (0.1 MMES/1 mM EGTA/0.5 mM MgCl2/1 mM AEBSF, pH6.4) containing 0.5 M sucrose and ATP 1 mM. TheMAPs-enriched microtubules fraction was isolated bytwo cycles of temperature-dependent disassembly or as-sembly followed by either cold (28C, 60,000g) or warm(308C, 150,000g) centrifugations as previously described[Shelanski et al., 1973]. Volumes and centrifugationcycles were adapted accordingly. For assembly, 1 mMGTP and 30% glycerol were added to buffer A. Forthe purification of tubulin, MAPs-enriched microtubuleswere depolymerized on ice and resuspended at a concen-tration of 40 mg/ml in buffer PEM (80 mM Pipes/1 mMEGTA/4 mM MgCl2, pH 6.85). Tubulin was separatedfrom MAPs by an ion exchange chromatography using a5-ml Whatman P11 phosphocellulose column pre-equili-brated with buffer PEM as previously described [Wein-garten et al., 1973]. The microtubules were resuspendedin PEM þ 1 mg/ml GTP, and were loaded onto the col-umn. Tubulin was eluted without binding to phosphocel-lulose, while MAPs were eluted with buffer PEM þ 1 MNaCl. Protein concentration was determined by Bio-Radprotein assay with bovine serum albumin as standard andtubulin assembly was tested using a light scatteringassay.

In Vitro Microtubule Assembly andSedimentation Assay

Two mg/ml of purified tubulin, or 1 mg/ml ofmicrotubules, in PEM (80 mM Pipes/1 mM EGTA/4

MAP1B, SCG10, and Microtubule Stability 683

mM MgCl2, pH 6.85) buffer containing 1 mg/ml of GTP,were assembled at 378C in the absence or in presence of10, 30, 100, and 300 lg/ml recombinant MAP1B frag-ments in 0.1 M MES buffer pH 6.4, 0.5 M NaCl, 1 mMDTT. Polymerization was monitored for 30 min by meas-uring changes in turbidity at 350 nm in an Uvikon XSspectrophotometer fitted with a temperature-controlledcompartment. After the in vitro assembly of tubulin(2 mg/ml) or microtubules i.e. tubulin and endogenousMAPs (1 mg/ml) at 378C during 30 min, in absence or inpresence of recombinant MAP1B981, MAP1B1404 orSCG10. Fifty microliters of samples were placed on100 ll 10% glycerol in buffer PEM (for tubulin experi-ments) or 40% glycerol in buffer PEM (for microtubuleexperiments) and centrifuged (26,000g, 30 min, 308C).Soluble and polymerized tubulin, with associated MAP1Band/or SCG10, were visualized on SDS-PAGE 3.6–15%gradient gel by Coomassie blue staining. This allowed tomeasure the effect of microtubule formation. Soluble andpellet fractions with associated recombinant MAP1B frag-ments were analyzed by ECLTM Western-blotting (Amer-sham Pharmacia Biotech) using the polyclonal antibodyanti-MAP1B at a dilution of 1:50,000 and peroxidase la-beled secondary antibody. Coomassie blue staining en-abled the measurement of the tubulin contents, whileauto-radiographs of immunoblots were used for densito-metric evaluation of the MAP1B content.

Antibody Production and Characterization

Antibodies against recombinant MAP1B N-termi-nal fragments (amino acid sequence 1-1404: MAP1B1404

or amino acid sequence 1-981: MAP1B981) were pro-duced in adult New Zealand White rabbits by injecting30 lg proteins and Specol as adjuvant. After a month,fortnightly injections of antigen and Specol were given,until a satisfactory antibody titer was reached. Thespecificity of the sera was tested on brain homogenates,PC12 cells and purified recombinant proteins and dem-onstrated high sensitivity (1:3000–1:100,000 diluted)and showed on Western blots no crossreactivity withother proteins nor with the MAP1B light chain. The anti-body against SCG10 has been previously characterizedand recognizes SCG10 independent of its phosphoryla-tion state [Antonsson et al., 1998]. Experimental pro-cedures including animals were authorized by the localveterinary office.

Immunoelectron Microscopy

Ten ll of the samples of assembled microtubulesuspensions were taken directly from the spectrophotom-eter cuvettes after 90 min of polymerization and fixedfor 10 min by addition of 5 ll 3% glutaraldehyde inPBS. Samples were applied to carbon-coated grids, nega-

tively stained for 30 min with 3% uranyl acetate in 10%ethanol and subsequently examined in the Zeiss EM10Celectron microscope (EM). Microtubules (0.75 mg/ml)were assembled in absence or presence of different con-centrations of SCG10 (0.1 lM, 0.3 lM, 1 lM, 3 lMSCG10), after incubation at 378C for 25 min the assem-bly was stopped by adding pre-warmed glutaraldehyde(at a final concentration of 0.5%). After 10 min, drops offixed microtubules were placed on 1% Formvar coatedcopper grids and placed on a heated plate (308C) for afew seconds. The grids were rinsed with PBS and pre-pared for immunological detection, by blocking with 5%FCS in PBS for 10 min. Grids were incubated with anti-SCG10 (dilution 1:100), and/or anti-MAP1B (AA6) dilu-tion 1:10 in PBS with 1% FCS for 60 min at room tem-perature. After several rinses with PBS, the grids werethen exposed to 12 nm colloidal gold-affinity pure goatanti-rabbit IgG (1:10 diluted, Jackson Immunoresearch)and 6 nm colloidal gold-affinity pure goat anti-mouseIgG (1:20 diluted, Jackson ImmunoResearch) in PBSand 1% FCS for 30 min. After several rinses with PBS,final wash was performed with distilled water and gridswere counterstained with 3% uranyl acetate in 10% etha-nol for 30 min. The grids were examined with a ZeissEM10C electron microscope.

Expression in Mammalian Cell Lines

COS-7 cells were grown at 378C, under 5% CO2 inDMEM/F12 medium supplemented with 10% heat-inac-tivated fetal calf serum, 2 mM l-glutamine, penicillin(100 lg/ml) and streptomycin (100 lg/ml) (Gibco).Three hundred nanograms of pcDNA3.1/myc-His vectorscontaining the MAP1B981 or MAP1B1404 cDNA sequen-ces were transfected into COS-7 cells using the FuGENEreagent (Roche). Forty eight hours after transfection, thecells were treated 30 min in the presence of 0, 1, 3, or10 lM colcemid (Sigma), a microtubule destabilizingagent. To identify the relation of MAP1B, SCG10 andmicrotubules, COS-7 cells were double-transfected byusing FuGENE and then processed as described above.Cell cultures were then processed for immunofluores-cence as described below.

Immunofluorescence

COS-7 cells were fixed 30 min in a 4% formalde-hyde solution in sulfate-buffer pH 7.45 (90 mM Na2SO4/30 mM K2SO4/580 lM MgCl2/250 lM CaCl2/1 mMNaH2PO4). Tubulin was visualized with the mouse mo-noclonal antibody Tu9B (diluted 1:10) [Caceres et al.,1984] and the Oregon green-linked goat anti-mouse anti-body (Jackson ImmunoResearch) at a dilution of 1:200or rat monoclonal antibody anti-alpha tubulin (YL1/2)(diluted 1:100) (Oxford Biotechnology Limited) and the


indocarbocyanine Cy3-linked donkey anti-rat anti-body (1:500) (Jackson ImmunoResearch), recombinantMAP1B fragments (MAP1B1404 or MAP1B981) weredetected with polyclonal antibodies anti-MAP1B (BR17and BR18), that were raised against the two MAP1Bfragments by using Specol as adjuvant by fortnightlyinjections of 30 lg of antigen. Antibodies were used at adilution of 1:3000 followed by an incubation with Cy3-linked goat anti-rabbit secondary antibody (1:200)(Jackson Immuno Research) or a Alexa 488-linkedgoat anti-rabbit secondary antibody (1:200) (MolecularProbes). Recombinant MAP1B981 was also visualizedwith the mouse monoclonal anti-His (C-terminal) anti-body at a dilution of 1:100 (Invitrogen) and Oregongreen-linked goat anti-mouse antibody (1:200). Further-more, recombinant MAP1B1404 fragment was also vi-sualized with the mouse monoclonal anti-myc antibodyat a dilution of 1:30 (Invitrogen) and Oregon green-linked goat anti-mouse antibody (1:200), to confirm suc-cessful transfection. Cells were inspected with a confocalmicroscope (Leica).

In three series of experiments, 100 transfected andnon-transfected cells respectively, which had beenexposed to colcemid were analyzed for the presence andabsence of microtubules. Numbers were expressed inpercentage of microtubule containing cells.

Double-transfected cells were triple-labeled withall primary antibodies together in antibody buffer (3%BSA/5% FCS/5% NHS/0.3% Triton X-100, and TBS)for 2 h, followed several rinses and incubation for 45min with fluorescent secondary antibodies in antibodybuffer. The polyclonal antibody for MAP1B (BR18) wasused at 1:3000 dilution and visualized with Cy5 (blue)(Jackson Immuno Research) at 1:100 dilution. Microtu-bules are identified with a rat monoclonal anti-tyrosi-nated tubulin (YL1/2) at 1:100 followed by Cy3 (red) at1:500. SCG10 was detected with an anti-EE at 1:8000dilution and Alexa 488 (green) at 1:200 dilution.

The DRG neurons were cultured as described [Bar-akat and Droz, 1989] and fixed according to an extrac-tion/fixation protocol [Lee and Rook, 1992], except thatwe used paraformaldehyde instead of glutaraldehydebecause the latter destroyed the SCG10 immunoreactiv-ity. They were then double-labeled with a rat monoclo-nal anti-tyrosinated tubulin (YL1/2) at 1:100 and rabbitpolyclonal antibody directed against SCG10 [Antonssonet al., 1998]. The latter was then detected by immunoflu-orescence using an Alexa 488-conjugated secondaryantibody (green), and anti-tubulin with a Cy3-conjugatedsecondary antibody (red). Rat hippocampal neurons werecultured according to Brewer et al., 1993 and triple-la-beled using the same antibodies as above plus anti-MAP1B detected with Cy5-cnjugated secondary anti-body (blue).

RESULTS

MAP1B981 and MAP1B1404 Promote MicrotubuleAssembly In Vitro

In the sedimentation assay, the proportion ofassembled microtubules (tubulin and MAPs) in the pelletwas increased when assembly was performed in the pres-ence of MAP1B1404 or MAP1B981 fragment (Figs. 1aand 1b). Microtubule pellets, assembled without MAP1Bpresent, contained about 20% of the total amount oftubulin. In the presence of 1 lM of MAP1B1404 andMAP1B981, the amount of tubulin in the pellet increasedto 58% and 63%, respectively. Experiments performedwith purified tubulin have shown that, in the absence ofMAP1B and other MAPs, the tubulin content in the pel-let was nearly 20%, while the addition of 1 lM ofrecombinant MAP1B fragments in solution increasedthis percentage to 38% and 40% for MAP1B1404 andMAP1B981 respectively. In both assays, either withmicrotubule proteins or with pure tubulin only, MAP1B

induced an increase in microtubule formation. The re-

combinant fragments of MAP1B showed similar func-

tional properties in tubulin polymerization as MAP1B

isolated from brain. In addition to the sedimentation

assay, the formation of microtubules was corroborated

by electron microscopy. From the same samples, just

before loading onto the glycerol cushion for the sedi-

mentation assay, small aliquots were taken to test the

quality of microtubules by electron microscopy. Results

confirmed that microtubules were present in samples

containing MAP1B fragments.MAP1B promoted microtubule assembly. Microtu-

bule proteins, i.e. tubulin and MAPs, from porcine brainor pure tubulin (Figs. 2a and 2b) were incubated in thepresence of 1 mg/ml GTP with defined amounts of puri-fied recombinant MAP1B1404 or MAP1B981 (0.4 and1 lM). At the start of the assay, the assembly wasinduced by changing the temperature from 4 to 378C(time 0) and OD was monitored. In the absence ofrecombinant MAP1B no polymerization of tubulin andonly little polymerization of microtubule proteinsoccurred, whereas in the presence of any MAP1B frag-ment a rapid assembly was observed. For both recombi-nant proteins increasing concentration of MAP1B had adose-dependent effect on microtubule formation. At aconcentration of 0.75 mg/ml of microtubules (tubulinfraction with other MAPs present), the addition of equalamounts of MAP1B1404 or MAP1B981 induced similarmicrotubule assembly rates. After 30 min, MAP1B1404,at concentrations of 0.4 and 1 lM, induced a 3.9- and7.3-fold increase respectively compared to the microtu-bule assembly without recombinant protein. Similar val-ues were obtained with MAP1B981 at the same concen-trations. The assembly rate of pure tubulin was also


increased in the presence of MAP1B fragments. At aconcentration of 0.4 lM, MAP1B981 increased the tubu-lin assembly by a factor of 3.5, while MAP1B1404

increased the assembly by a factor of 5.5. This suggeststhat MAP1B1404 is more potent than MAP1B981 inducingmicrotubule assembly. At higher concentrations of re-combinant MAP1B, the larger fragment also resulted inan increased microtubule assembly rate when tubulin ortubulin with endogenous MAPs were used.

MAP1B981 and MAP1B1404 Have a MicrotubuleStabilizing Activity on TransfectedNonneuronal Cells

The microtubule stabilizing effect of MAP1B wastested with tubulin, MAP1B fragments and different con-centrations of colcemid (Fig. 2c). In the assembly assay,0, 3, and 6 lM colcemid were added to 1.5 mg/ml tubu-lin containing 1 lM of MAP1B fragments (MAP1B1404

or MAP1B981). Microtubule assembly was monitoredover a period of 30 min. The presence of 3 and 6 lM col-

cemid reduced the microtubule assembly rate by 50% 68% and 67% 6 9% respectively. However, in thepresence of 1 lM MAP1B fragments MAP1B981 orMAP1B1404, addition of 3 lM colcemid had no effect onthe assembly rate. A concentration of 6 lM colcemidresulted in a reduction of only 10 and 25% respectivelyfor the two fragments. This clearly demonstrated themicrotubule-stabilizing effect of MAP1B fragments andfurthermore suggests some protective role of MAP1B forlabile microtubules.

After transfection of cells with MAP1B constructs,a culture period of 48 h and fixation, double labelingwith polyclonal and monoclonal antibodies for tubulin,MAP1B, His-tag or myc-tag (Figs. 2d and 2e) showedclear differences in microtubule preservation in trans-fected and non-transfected cells. Transfection rate wasbetween 35 and 40% for MAP1B981 and 50% forMAP1B1404 and a densitometrical analysis demonstratedan overexpression of MAP1B-fragments up to 2% of thetotal protein content, while the tubulin was estimated tobe 3.5% of total protein content and corresponds to a

Fig. 1. Sedimentation assay and microtubule formation in the pres-

ence or absence of MAP1B1404 (a) or MAP1B981 (b) shown by West-

ern blots and electron microscopy. After centrifugation, soluble and

polymerized tubulin with associated MAP1B fragments were visual-

ized on 3.6–15% SDS-polyacrylamide gradient gel stained with Coo-

massie Brilliant Blue. The presence of MAP1B fragments was deter-

mined by Western blots with rabbit polyclonal anti-MAP1B981 anti-

body (BR18) and peroxidase labeled secondary antibody by ECLTM.

Densitometric analysis of the gels and blots was performed to estimate

the repartition of tubulin and MAP1B into soluble or pellet fraction.

The values are the mean of 3 experiments. Microscopy (310,000) was

performed after in vitro polymerization of tubulin (1.5 mg/ml) per-

formed at 378C during 90 min in the presence of 30 lg/ml of

MAP1B1404 or MAP1B981.


precedent study [Takemura et al., 1992]. The transfec-tion efficiency may play some role, however, bothMAP1B fragments retained microtubule binding activity.After 48 h incubation after transfection, COS-7 cells

were treated with colcemid. Increasing concentrations ofcolcemid were added (0, 1, 3, and 10 lM), and in allconditions yielded an increased number of cells with adestroyed microtubule network in non-transfected cells.

Fig. 2. MAP1B assembly assays and COS-7 cell transfection in the

presence of colcemid. MAP1B increases microtubule assembly and

reduces the microtubule disrupting effect of colcemid. Assembly of

porcine microtubules (Tubulin þ MAPs) at a concentration of 1 mg/

ml (a) or 2 mg/ml (b) in the absence (open circle) or presence of

0.4 lM (dark triangle) and 1 lM (dark square) MAP1B1404. The as-

sembly rate is measured in changes of absorbance at 350 nm with a

spectrophotometer. The values are the mean of three experiments. (c)Resistance to depolymerization induced by MAP1B when tubulin po-

lymerization occurred in the presence of colcemid. Two milligram/

milliliter of tubulin was assembled in absence (rhombus) or in the

presence of 1 lM MAP1B981 (circle) and MAP1B1404 (triangle). As-

sembly occurred in absence (black marks) or in presence of 3 lM col-

cemid (white symbols) or 6 lM colcemid (white symbols and dotted

lines). Preservation of microtubules in MAP1B1404 transfected COS-7

cells treated with colcemid (d–g) and double labeling with MAP1B1404

and tubulin. COS-7 cells were transfected using the FuGENE re-

agent (Roche) with the expression vectors pcDNA3.1/myc-His A-

MAP1B1404 cDNAs. Forty eight hours after transfection a set was

directly stained for tubulin (d) and MAP1B (e), a second set of cells

were treated for 30 min with 3 lM colcemid prior to staining for tubulin

(f) and MAP1B (g). Confocal microscopy (363) of MAP1B1404- trans-

fected cells. Tubulin was labeled with monoclonal antibody Tu9B,

MAP1B was detected with anti-MAP1B981 rabbit polyclonal antibody.

Note that in colcemid-treated cells (f) the microtubules have disap-

peared in the non-transfected cells. Scale bar 30 lm.


At 3 lM colcemid exposure for 30 min, 73% 6 10% (inthree separate experiments and cell count of 100 cellsper experiment) of the non-transfected cells lacked anintact microtubule network. In contrast, transfected cellshad a significant amount of intact microtubules (Figs. 2fand 2g). 82% 6 16% of MAP1B981-transfected cells and75% 6 8% of MAP1B1404-transfected cells showed an

intact microtubule network. When the concentration ofcolcemid was increased to 10 lM, the percentage oftransfected cells showing intact microtubules decreasedbut still remained at high levels (67% 6 26% and 59%6 15% respectively with MAP1B981 and MAP1B1404).Our results showed that transfected cells, which ex-pressed truncated fragments of MAP1B (MAP1B1404

Fig. 3. Microtubule assembly assay with MAP1B and SCG10 com-

petition followed by sedimentation assay. Light scattering (a and b)and sedimentation assay (c) were used to test microtubule stability in

vitro. (a) 25 lM purified tubulin (open circle), together with 0.3 lMwith MAP1B (open square), or together with 13 lM SCG10 (dark

circle) or with 0.3 lM MAP1B and 13 lM SCG10 combined (dark

square), were assembled over a period of 20 min. (b) 25 lM purified

tubulin (open circle), together with 0.3 lM with MAP1B (open

square), or together with 8 lM SCG10 (dark circle) or with 0.3 lMMAP1B and 8 lM SCG10 combined (dark square), were assembled

over a period of 20 min. Absorbance of light scattering was measured

at 350 nm. A small difference in SCG10 concentration is able to shift

the balance between MAP1B and SCG10 effects on microtubules.

These results are confirmed by a sedimentation assay (c), 50 ll ofsamples from the polymerization assay were layed on 100 ll 10%glycerol- (for tubulin experiments) or 40% glycerol-PEM buffer (for

microtubule experiments) and centrifuged at 26,000g for 30 min

(308C). Soluble and polymerized tubulin fractions were visualized on

SDS-PAGE 3.6–15% gradient gel by Coomassie blue staining.


and MAP1B981), have a higher resistance to the microtu-bule destabilizing effect of colcemid.

Stoichiometric Change of SCG10 to Tubulin CanOvercome MAP1B Stabilizing Effect

The former microtubule assembly assays showedthat the proportions of MAP1B needed to stabilizemicrotubules and to protect against the depolymerizingeffects of colcemid is a ratio of 1 MAP1B to 70 tubulindimers corresponding to the physiological concentra-tion of MAP1B. At that concentration, the efficacy ofMAP1B to stabilize microtubules showed a clear differ-ence depending in the relative concentration of themicrotubule destabilizing protein SCG10 (Fig. 3). AtSCG10 concentrations of 8 lM, MAP1B still promotedmicrotubule polymerization. When SCG10 concentrationwas increased to 13 lM, microtubule assembly wasblocked independently of the presence of MAP1B andthe rate of polymerized microtubules remained at a level

near that of tubulin alone. This means that a ratio of 1SCG10 molecule per two tubulin dimers, correspondingto a concentration of 13 lM SCG10, was sufficient todestroy the integrity of microtubules, while a ratio of 1SCG10 molecule per three tubulin dimers, i.e. 8 lMSCG10, was not enough to overcome the MAP1B stabi-lizing effect. The sedimentation assay confirmed that asufficient dose SCG10 induced a destabilization onMAP1B-protected microtubules and brought to the levelof tubulin.

MAP1B and SCG10 Are Both Localized onMicrotubules In Vitro, in Transfected Cellsand in Neuronal Growth Cones

Different concentrations of SCG10 were added to0.75 mg/ml microtubules and assembly was induced bychanging the temperature from 4 to 378C. After 25 min,microtubules were fixed with glutaraldehyde (0.5% finalconcentration) and placed on EM grids. SCG10 was found

Fig. 4. Immunogold localization of SCG10 and MAP1B on microtu-

bules. Microtubules (0.75 mg/ml) were assembled without additional

SCG10 present (a). Immunolabeling demonstrates the presence of en-

dogenous SCG10. Microtubules (0.75 mg/ml) were assembled in the

presence of exogenous SCG10 at a concentration of 0.1 lM (b) or 1lM SCG10 (c). The presence of SCG10 on immobilized microtubules

is shown by using a rabbit anti-SCG10 and 12 nm colloidal gold con-

jugated anti-rabbit secondary antibody. (d,f) Immobilized microtu-

bules on grids were also used for localization of MAP1B using mono-

clonal antibody AA6 and 6 nm colloidal gold-conjugated anti-mouse

secondary antibody alone (d) and together with anti-SCG10 12 nm

gold particles (e). (f) In control samples, primary antibodies were

omitted and colloidal gold conjugated secondary antibodies were

used.


Fig.5.

DoubletransfectionandtriplelabelingofCOS-7

cells(a–d)andim

munolabelingofgrowth

cones

(e–g)

andprimarycultures(h).COS-7

cellswereco-transfectedwithSCG10andMAP1B981

totestmicrotubulestability.TriplelabelingforMAP1B(bluein

a),tubulin(red

inb)andSCG10(green

inc)

andcomposite(d).Scalebar

25lm

.LocalizationofSCG10alongmicrotubulesin

agrowth

cones

ofprimarydorsalrootgangliacultures(e–g).Dorsalrootgangliacellsweresimultaneouslyextractedandfixed

andthen

double-labeled

witharatmonoclonalanti-tyrosinated

tubulinYL1/2

(red

ine)

andarabbitpolyclonal

antibodydirectedagainstSCG10(green

inf,g:composite).Non-extractedSCG10could

bedetectedalongthemicrotubulesin

thegrowth

coneanddownthelength

ofthe

filopodia.Ahippocampalneuronin

culture

was

fixed

accordingto

astandardprotocolandadditionally

labeled

forMAP1Bin

blue(h:composite).NotethepunctateMAP1Bstainingin

thethreetimes

magnified

insert(arrow).

to be localized on these microtubules under the differentconditions (without additional SCG10, with 0.1 lMSCG10 and 1 lM SCG10 added to microtubules; Figs.4a–4c). Microtubules in panel a indicate that endogenousSCG10 was present in isolated microtubules since 63.3%of gold particles were localized on or close to microtu-bules (in a range of 20 nm). While in the presence of 0.1lM SCG10 (Fig. 4b) a similar staining could be observedand 60.9% of gold particles were counted along themicrotubules, in presence of 1 lM SCG10 (Fig. 4c) only29.1% immunogold particles of 12 nm diameter were onmicrotubules. This reduction in SCG10 binding to micro-tubules might be explained by the presence of a largeexcess of exogenous SCG10 that attracts more gold par-ticles. Based on the similar results obtained between thecondition where no SCG10 was added to the microtubulesand 0.1 lM SCG10 was added, one may speculate thatSCG10 associates with microtubules at a sub-micromolarlevel below 0.1 lM. When anti-MAP1B and smallergold-particles were used, 93.6% of immuno-staining wasassociated to microtubules (Fig. 4d). This indicates thatmost MAP1B is attached to assembled microtubules andonly few molecules may not be bound. Immunostainingof microtubules with anti-MAP1B and anti-SCG10 withdifferent diameter colloidal gold labeled secondary anti-bodies revealed the same proportion (Fig. 4e). Further-more a large proportion of MAP1B and SCG10 stainingwas found at the same places (Fig. 4e). Without the pres-ence of primary antibodies rarely gold particles wereobserved (Fig. 4f), thus underlining the microtubule-bind-ing character of MAP1B and the specificity of the micro-tubule staining with SCG10 and MAP1B antibodies.

In MAP1B and SCG10 co-transfected COS-7,microtubule stability was severely changed according tothe presence or absence of MAP1B or SCG10. In the ab-sence of SCG10, MAP1B was found along an elaboratedmicrotubule skeleton (data not shown). In many cellsexpressing SCG10 and MAP1B, microtubules had disap-peared, while a few microtubules could be rescued andwere also characterized by the presence of MAP1Blocated along the microtubules (Figs. 5a–5d). This showsthat SCG10 is able to destroy the microtubule network inthe presence of MAP1B, and that the stabilizing activityof MAP1B is not sufficient to completely counteract thiseffect.

To visualize a possible association of SCG10 withmicrotubules in growth cones, we used chick dorsal rootganglion neurons in culture which are known for theirlarge growth cones. Normally SCG10 shows a densestaining filling out the entire central domain of thegrowth cone [data not shown, see Grenningloh et al.,2004]. Therefore, in order to detect SCG10 that mightbind along the microtubules, we used fixation conditionsin which cells were simultaneously extracted. A typical

growth cone, which was stained for tubulin (red) andSCG10 (green), is shown in Figures 5e–5g. SCG10showed indeed a staining pattern along microtubulesindicating that this procedure removed the vesicle-asso-ciated and/or soluble SCG10 and unmasked microtu-bule-bound SCG10. The remaining SCG10 was not onlyfound along the microtubules in the central domain ofthe growth cone but also on the microtubules thatextended into the filopodia. The distribution of SCG10,MAP1B, and tubulin was also analyzed in primary ratneurons (Fig. 5h), where both proteins were present ingrowth cones. Besides its enrichment in the growth cone,SCG10 was found in the Golgi complex near theunstained nucleus and along neurites. MAP1B was foundespecially in the larger neurites.

DISCUSSION

Here we have compared the combinatorial effect oftwo neuronal proteins with opposite effects on microtu-bule stability. Individually, both fragments of MAP1B(MAP1B981 and MAP1B1404) promoted microtubule as-sembly in vitro and had a microtubule stabilizing activityin transfected non-neuronal cells, which can be com-pared with that of MAP1B. This confirms previousresults [Takemura et al., 1992]. The microtubule-stabi-lizing effect could be overcome by a change in theratio between tubulin and the microtubule-destabilizingprotein SCG10. Moreover, we could show that, likeMAP1B, also SCG10 bound to purified microtubules aswell as to microtubules in growth cones. These resultssuggest that a tight control in the balance of these stabi-lizing and destabilizing proteins play an important rolein the regulation of microtubule dynamics in the growthcones of developing neurons.

Similar studies in Xenopus oocytes have previ-ously shown that microtubule growth and stability arecontrolled by opposing effects of stabilizing and destabi-lizing influences of several proteins such as XMAP215,tau and XKCM1 [Becker et al., 2003]. Therefore, onemay find different pairs of proteins that interact withmicrotubules and influence their stability depending onthe cell type, subcellular compartment and developmen-tal state.

The correct development of the nervous systemdepends on the ability of neuronal process to find theirway along complex paths and to establish appropriateconnections. The tips of these structures are highlymotile, and pharmacological manipulations using drugssuch as taxol or nocodazole have shown that treatedmicrotubules loose their dynamic properties, thus lead-ing to reduced axonal elongation. Therefore, it is ofprime importance to understand how the dynamic insta-bility of growth cone microtubules is involved at a mo-


lecular level, and how the regulation of the stochasticprocess of assembly and disassembly of tubulin hetero-dimers at microtubule plus-ends occurs. Proteins such asSCG10 and MAP1B have a function in the control ofmicrotubule dynamics during neurite growth and theirphosphorylation states have a modulating effect onmicrotubules. In this study, we have defined the antago-nistic role of recombinant SCG10 and MAP1B proteinsin the control of microtubule stability under several con-ditions and in different assays. Since it is known thattheir state of phosphorylation may influence their activ-ities, we have used recombinant proteins produced inE. coli as they are non-phosphorylated.

MAP1B Stabilizing Function on Microtubules

First, to investigate the function of MAP1B, weused two truncated MAP1B forms that showed similarfunctions to MAP1B isolated from brain. The shear sizeof MAP1B makes it a difficult protein to work with invitro systems. Therefore, two truncated fragments ofMAP1B, which contain the MBD and C-terminal flank-ing sequences of varied length, were used in this study tooptimize the expression system. The two truncated re-combinant MAP1B fragments, obtained by restriction ofthe full-length MAP1B cDNA, were expressed in E. coliM15[pREP4] strain. These MAP1B constructs containedthe MBD and downstream sequences presenting severalpotential phosphorylation sites [Noble et al., 1989], butnot the MAP1 light chain actin filament binding domainwhich corresponds to the C-terminal part of MAP1B[Togel et al., 1998]. MAP1B is a long, filamentous mole-cule with a small spherical portion at one end, forminglong cross-bridges between microtubules in vitro and inneurons [Sato-Yoshitake et al., 1989] and known to havea strong stabilizing effect on microtubules. Our resultsdemonstrate that both recombinant MAP1B1404 andMAP1B981 recovered the ability of MAP1B to promotemicrotubule assembly, as described previously [Pedrottiand Islam, 1995; Pedrotti et al., 1996], from newborn ratbrain MAP1B [Riederer et al., 1986] and from porcinebrain MAP1B [Vandecandelaere et al., 1996]. Both frag-ments contained the MBD, yet the assembly rate wasincreased nearly by twofold for the larger fragment.Therefore, the additional C-terminal sequence followingthe MBD in MAP1B1404 must have additional assemblyproperties. However, this seems to be contradictory withprevious results showing that a N-truncated form ofMAP1B looses its function in neurite outgrowth [Uchida,2003] and may need additional studies. The increasedrate of microtubule assembly and the reduced disruptingeffect of colcemid on microtubules demonstrate thatMAP1B plays not only a role in the initiation and rate ofmicrotubule assembly but is also crucial in the stabiliza-

tion of microtubules under microtubule destabilizingconditions. This further consolidates that not only MAP2and tau show these properties [Drechsel et al., 1992;Kowalski and Williams, 1993], but also MAP1B is ableto protect microtubules against disassembly.

SCG10 Activity Can Counteract theEffect of MAP1B

Microtubule assembly and sedimentation assayswith MAP1B and SCG10 in competition have shownthat SCG10 in the lM range can inhibit microtubulepolymerization and induce depolymerization in the pres-ence of MAP1B in vitro. It is of importance to note thatsmall differences in SCG10 concentration between 8 and13 lM, in the presence of a physiological concentrationof MAP1B, can shift the balance from microtubule sta-bility towards disassembly. This indicates that microtu-bule assembly versus disassembly is highly susceptibleto small changes in the concentration of this destabiliz-ing factor. When SCG10 was used alone it completelyblocked microtubule assembly at a concentration of13 lM, which corresponds to a ratio of 1 SCG10 mole-cule to 2 tubulin heterodimers. This is consistent with itstubulin sequestering activity by forming a ternary 2TScomplex [Charbaut et al., 2001]. At this concentration,MAP1B addition could not counteract the blockingeffect of SCG10, probably because there was no avail-able free tubulin for polymerization. The reduction ofSCG10 to 8 lM corresponding to a SCG10:tubulin ratioof 1:3 and thus leaving free tubulin dimers available forpolymerization allowed MAP1B to stabilize microtu-bules despite the presence of SCG10. MAP1B at a ratioof 1:70 stabilized microtubules and therefore the amountof MAP1B molecule needed to stabilize microtubuleswas considerably lower than that of SCG10.

Transfection experiments in intact cells indicatethat SCG10 overexpression can depolymerize cellularmicrotubules and that co-expression of MAP1B cannotprevent the SCG10-induced disruption of the microtu-bule network. This activity of SCG10 might be related toother microtubule-directed activities of the protein, inaddition to sequestration. Consistent with a direct effecton microtubule, we could show that SCG10 binds topurified microtubules and that it is also associated withcellular microtubules in growth cones. It will be impor-tant to investigate how precisely SCG10 regulates micro-tubule dynamics at the mechanistic level.

Subcellular Localizations of SCG10 andMAP1B in Growth Cones

Double transfection and triple labeling of COScells as well as immunolabeling of growth cones in pri-


mary neuronal cultures showed that both MAP1B andSCG10 were concentrated in the distal axon andgrowth cones, accordingly with previous descriptions[reviewed by Grenningloh et al., 2004; Tint et al.,2005]. These areas contain the more labile tyrosinatedform of tubulin which has not yet been stabilized bypost-translational modifications, resulting in anincreased microtubule instability that may favor direc-tional growth. By extracting vesicle-associated SCG10,which is highly concentrated in the growth cone [Lut-jens et al., 2000], we were able to reveal SCG10binding to tyrosinated microtubules. This is the firstevidence that SCG10 binds directly to microtubules.Moreover, SCG10 was not only found along the micro-tubules in the central domain of the growth cone butalso on microtubules that extend into the filopodia.There is evidence that these microtubules and theirinteraction with actin play a role for growth cone turn-ing. The dynamic state of these microtubules seems tobe crucial for the interaction with actin [reviewed byGordon-Weeks, 2004]. SCG10 may therefore be an im-portant factor for the regulation of microtubule dynam-ics during growth cone turning and axonal pathfinding.In a previous study, we found that blocking SCG10 inthe growth cone strongly affects growth cone behaviorduring axonal elongation. In the absence of SCG10, thegrowth cones stop forward elongation [Suh et al.,2004]. It will be of interest to see whether a role forSCG10 can be defined during growth cone turning andwhether antagonistic effects of MAP1B and SCG10might regulate the highly dynamic behavior of microtu-bules during pathfinding.

Furthermore, a spot-like staining in neurites mayalso suggest that SCG10 and MAP1B could play a rolein neurite branching. Using immunogold localization, wesaw in this study that only a fraction of SCG10 mole-cules present in solution are associated with microtu-bules (up to 0.1 lM). We also localized recombinantMAP1B along microtubules, as one would expect forMAPs, confirming the property of MAP1B binding totubulin in vivo and stabilizing microtubules. However,such binding did not lead to a reorganization of microtu-bules or the formation of bundles, as observed for otherMAPs such as microtubule-associated protein 2 (MAP2)or tau proteins [Takemura et al., 1992]. It needs to beshown whether MAP1B and SCG10 may have a directsynergistic action on microtubules. Some co-localizationof MAP1B and SCG10 on in vitro assembled microtu-bules would suggests a collaborative interaction. Yet, nodirect proof of such an interaction has so far beenreported. It may also be possible that other proteins maycontribute to microtubule dynamics such as tau proteins,with similar functions attributed to MAP1B [Takei et al.,2000].

Regulation of SCG10 and MAP1B Functionin the Cell

We have shown that small variations in the relativeconcentrations of SCG10 to MAP1B are able to inducedramatic changes in microtubule assembly. We assumethat selective activations of these proteins may take placein the regulation of microtubules stability such as duringaxon guidance and growth cone turning. MAP1B structurepresents 33 identified phosphorylation sites [Collins et al.,2005] and a variety of kinases and phosphatases areinvolved in the regulation of MAP1B function [Ulloaet al., 1993a, b; Garcia-Perez et al., 1998; Lucas et al.,1998; Goold et al., 1999; Gong et al., 2000]. SCG10 activ-ity decreases with an increasing phosphorylation state.The four identified sites on SCG10 are phosphorylated byseveral kinases such as PKA, MAPK and CDK5 whichare highly expressed in neurons. As previously describedfor MAP2 [Sanchez et al., 2000], phosphorylation ofMAP1B seems to play a key role in the modulation ofMAP1B function for dendritic, axonal or growth conegrowth and turning [Mack et al., 2000]. We suggest thatthis results of a fine tuning in the activations of microtu-bules-associated proteins: selective action of MAP1B onmicrotubules in one side of the growth cone could protectthem from the activity of others microtubule-directed pro-teins like SCG10 or CLIPs [Mimori-Kiyosue and Tsukita,2003], and thus allow the microtubule bundles to be stabi-lized in the direction of the turn. Moreoever, certain neu-ropathologies present an imbalance in the proportionbetween kinases and phosphatases, producing modifica-tions of the phosphorylation state of proteins associatedwith the cytoskeleton such as MAP2 [as reviewed by San-chez et al., 2000]. To further understand the role of thephosphorylation state, it would be interesting to testMAP1B mutations in the phosphorylation sites near theMBD and measure differences in microtubule assemblyand stabilizing properties when single phosphorylationsites are modified. During axonal navigation, intracellularsignaling cascades take place in the growth cone betweenenvironmental cues and proteins that regulate the microtu-bules dynamics to allow the cytoskeleton to give the axonnew orientations. The signaling pathways of Rho GTPaseand glycogen synthase kinase-3b (gsk3b) are known tobe mostly involved. Reelin, a secreted protein that regu-lates brain layer formation during embryonic develop-ment, can induce MAP1B phosphorylation, both in vivoand in vitro, through gsk3 and cdk5 activation [Gonzalez-Billault et al., 2005; Goold and Gordon-Weeks, 2005;Kawauchi et al., 2005]. Similarly, SCG10 is coupled withthe G protein signaling pathway by RGSZ1 [Nixon et al.,2002], which potentially inhibits its microtubule-depoly-merizing activity.

In conclusion, our study could then be useful to es-tablish a link between the activation of intracellular bio-


chemical cascades by external cues and the reorganiza-tion of the cytoskeleton, involving the fine regulation ofmicrotubules dynamics by MAPs. Even slight changes intheir concentrations or activities may have considerableimpact on the stability of the cytoskeleton. This is espe-cially important in growth cones, where decisions haveto be made for choosing the direction of growth. MAP1Band SCG10 may therefore play an important role in regu-lating the dynamics of microtubules during these events.

ACKNOWLEDGMENTS

The authors thank Dorine Savoy, Evelyne Ruchti,Irene Riederer, and Claudine Pfulg for excellent techni-cal help. We also thank Ibtissam Walter-Barakat for helpwith the preparation of the DRG neurons.

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