kineticanalysisoftubulinassemblyinthepresenceofthe...

Kinetic Analysis of Tubulin Assembly in the Presence of theMicrotubule-associated Protein TOGp*

Received for publication, June 13, 2006, and in revised form, November 14, 2006 Published, JBC Papers in Press, December 17, 2006, DOI 10.1074/jbc.M605641200

Claude Bonfils‡1, Nicole Bec‡, Benjamin Lacroix‡, Marie-Cecile Harricane§, and Christian Larroque‡

From the ‡INSERM, EMI 229, CRLC Val d’Aurelle, 34298 Montpellier, France and §Centre de Recherche en BiochimieMacromoleculaire, CNRS, 1919 route de Mende, 34293 Montpellier, France

Themicrotubule-associated protein TOGp, which belongs toa widely distributed protein family from yeasts to humans, ishighly expressed in human tumors and brain tissue. From puri-fied components we have determined the effect of TOGp onthermally induced tubulin association in vitro in the presence of1 mM GTP and 3.4 M glycerol. Physicochemical parametersdescribing the mechanism of tubulin polymerization werededuced from the kinetic curves by application of the classicaltheoretical models of tubulin assembly. We have calculatedfrom the polymerization time curves a range of parameterscharacteristic of nucleation, elongation, or steady state phase. Inaddition, the tubulin subunits turnover atmicrotubule endswasdeduced from tubulinGTPase activity. For comparison, parallelexperiments were conducted with colchicine and taxol, twodrugs active on microtubules and with tau, a structural micro-tubule-associated protein from brain tissue. TOGp, whichdecreases the nucleus size and the tenth time of the reaction (thetime required to produce 10% of the final amount of polymer),shortens the nucleation phase ofmicrotubule assembly. In addi-tion, TOGp favors microtubule formation by increasing theapparent first order rate constant of elongation. Moreover,TOGp increases the total amount of polymer by decreasing thetubulin critical concentration and by inhibiting depolymeriza-tion during the steady state of the reaction.

Microtubules are highly dynamic structures that switchbetween growing and shrinking phases both in vivo and in vitro.These cytoskeleton polymers are necessary for many functionswithin the cell including intracellular transport, motility, mor-phogenesis, and cell division. The intrinsic dynamic instabilityof microtubules is further modified in the cell by numerousprotein factors that favor alternatively elongation, shortening,or anchoring of these polymers. Because the mitotic spindleplays a crucial role in cell division, it has been used for decadesas an important target in cancer chemotherapy. Many tubulinpoisons have been identified, some of them, taxanes and vincaalkaloids, have demonstrated therapeutic value. However, alltubulin poisons are not of clinical utility. This has led to exten-sive efforts to explore other targets that could affect spindle

integrity. A promising approach is to identify the protein regu-lators that modulate tubulin polymerization and to investigatetheir mechanism of action.The dynamic instability of microtubules is controlled in vivo

by several classes of cellular factors including depolymerizingkinesins (MCAK/XKCM1) (1, 2), stathmins (3), and microtu-bule-associated proteins (MAPs).2 This last group is composedof structural MAPs (MAP2, tau) that were first identified inbrain tissue and of a group ofXMAP215-related proteinswhosegeneric member was first characterized in Xenopus eggs (4).TOGp (HUGO gene CKAP5), which is highly expressed intumors and brain (5), is the human homolog of XMAP215.TOGp promotes microtubule assembly both in solution andfrom nucleation centers (6). It was evidenced that this MAPpossesses a high affinity for polymer lattice and that it bindsprotofilaments by its N terminus (7). This protein is involved inmicrotubule aster formation in mammalian mitotic cells (8);moreover, TOGp is required for centrosome integrity and spin-dle pole organization (9).The TOGp family has a wide distribution; it is present from

yeasts to humans. In addition to the human TOGp and to thefrog XMAP215 protein, members of this group have been inde-pendently discovered in Drosophila melanogaster (Msps), inDictyostelium discoideum (DdCP224), and in Arabidopsisthaliana (Mor1) (10, 11, 12). Other forms with more divergentprotein structure were identified in Caenorhabditis elegans(Zyg-9) (13) and in yeasts. Two forms, Dis1 and Alp14, arepresent in fission yeast (14, 15), whereas one member, StuII,was characterized in Saccharomyces cerevisiae (16). This evolu-tionary conserved protein family is implicated in microtubulepolymer assembly and spindle formation.Microtubules are hollow cylindrical aggregates of 25-nm

diameter composed of heterodimers of �- and �-tubulin. Eachof these subunits binds 1 mol of GTP. GTP bound to �-tubulinis not exchanged, whereas �-tubulin-bound GTP is hydrolyzedto GDP soon after tubulin polymerization. A significantamount of the free energy of this hydrolysis goes into themicro-tubule via a conformational change of the tubulin dimer; itsconsequence is to destabilize the structure.Microtubules can spontaneously assemble in vitro from a

solution of purified tubulin in the presence of GTP by a tem-perature jump from 0 to 37 °C. The kinetics of tubulin assemblyare generally interpreted as a two-step nucleation elongation* The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: INSERM EMI 229, CRLC Vald’Aurelle-Paul Lamarque, Rue des Apothicaires, 34298 Montpellier cedex 5,France. Tel.: 33-4-67-61-85-36; Fax: 33-4-67-61-37-87; E-mail: [email protected].

2 The abbreviations used are: MAP, microtubule-associated protein; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Pipes, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 8, pp. 5570 –5581, February 23, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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process. The theoretical interpretation of tubulin polymeriza-tion is based on the actin helical aggregation model (17, 18).However, the polymerization of microtubules is much morecomplex than the assembly of actin filaments and necessitateskinetic as well as thermodynamic considerations (19). Itsmath-ematical analysis requires an infinite set of interrelated differ-ential equations (20). In the case of actin, some approximationswere introduced byWegner and Engel (18), leading to simplifythe process to two inter-related differential equations, whichafter integration give a numerical solution of the polymeriza-tion curves (21, 22). The actin model cannot be directly extrap-olated to tubulin. Microtubule elongation is well documentedboth at the structural andmechanistic levels; in contrast, nucle-ation is still poorly understood, mainly because it is composedof weakly concentrated transient intermediates (23). Voter andErickson (24) introduced a two-dimensional nucleation mech-anism that improves the fitting with the experimental kineticcurves. More recently Flyvbjerg et al. (25) formulated a newassembly model in which the final nucleus is the result of aseries of intermediate aggregates formed by the step by stepaddition of a variable number of tubulin monomers.Quantitative parameters describing the mechanism of tubu-

lin assembly can be deduced from the kinetics by application ofthe theoretical models. It is possible to calculate from thepolymerization time curves a range of physicochemical param-eters characteristic of nucleation, elongation, or steady statephase. In addition, the tubulin subunit turnover at microtubuleends can be deduced from tubulin GTPase activity. In thispaper we have determined the influence of TOGp on thesekinetic parameters. The results showed that this MAP was astrong activator of microtubule production, able to influencevarious steps of the reaction at low concentration. For compar-ison, parallel experiments were conducted with colchicine andtaxol, two microtubule reactive drugs, and with tau, a classicalMAP from brain tissue.

MATERIALS AND METHODS

Tubulin Purification

Tubulin was prepared according to the purification proce-dure described by Williams and Lee (26).

Purification of TOGp

TOGp was isolated from pig brain cytosol. Pig brains wereobtained from the local slaughterhouse and transported to thelaboratory on icewithin 2h after bleeding.Meninges and super-ficial blood vessels were removed from the brains at 4 °C. Twobrains (160 g) were homogenized in 200 ml of PEM buffer (100mM Pipes/NaOH, pH 6.6, 1 mM EGTA, 1 mM MgSO4, 1 �g/mlleupeptin) for 30 s in a Warring blender mixer. The tissue wasfurther disrupted bymeans of a Tenbroeck homogenizer with aTeflon pestle (five strokes on ice). The homogenate was centri-fuged at 125,000� g for 75min at 5 °C, and the supernatant wasrecovered. This fraction was brought to 32% saturation by add-ing progressively solid ammonium sulfate at room tempera-ture. The precipitate was recovered by centrifugation at 5000�g for 20 min. The pellet was resuspended in 250 ml of a H2O/PEM (v/v) mixture. The solution was dialyzed overnight at 4 °Cagainst 4 liters of PEM buffer. A small protein precipitate was

eliminated after centrifugation at 10,000 rpm for 20 min. Theprotein TOGp was purified from the supernatant in two chro-matographic steps.Hydroxyapatite—The column (1.6 � 20 cm) was filled with

Macro-Prep ceramic hydroxyapatite from Bio-Rad and equili-brated with PEM buffer. The column was loaded with thecleared supernatant and rinsed with PEM buffer. The proteinswere eluted with two successive salt concentration gradients.First the KCl concentration was raised from 0 to 2 M in PEMbuffer. Then the PEM buffer was replaced by phosphate buffer(10 mM potassium phosphate, pH 6.8, 1 mM EGTA, 1 mMMgSO4, 1 �g/ml leupeptin), and a second gradient from 10 to600 mM potassium phosphate was applied to the column. Usu-ally, TOGp eluted with �400 mM potassium phosphate. Theprotein fractions were analyzed by Western blotting, and thechromatographic fractions enriched in TOGp were pooled.DEAE-Sepharose—This chromatography was performed in

TEM buffer (Tris/HCl, 20 mM, pH 8.2, 1 mM EGTA, 1 mMMgSO4, 1 �g/ml leupeptin) on a 1 � 10 cm column of DEAE-Sepharose Fast Flow fromAmersham Biosciences. The proteinfraction eluted from the hydroxyapatite column was dialyzedagainst 2 liters of TEMbuffer for 5 h and loaded on the column,and unadsorbed proteins were eliminated by rinsing with theTEM buffer. A KCl concentration gradient from 0 to 0.1 Min TEM buffer was then applied. The protein fractions elutedwith this gradient were immediately stored at �80 °C. The quali-tative composition of each fraction was determined on SDS-PAGE, andWestern blots revealed with anti-TOGp antibodies.

Purification of Protein Tau

We applied the purification procedure described by Cleve-land et al. (27) to the 125 000 g pig brain supernatant.

Antibodies

Rabbit polyclonal anti-TOGp antibodies were prepared aspreviously mentioned (6). Mouse monoclonal anti-tau (clonetau-2) andmouse monoclonal anti-�-tubulin antibodies (cloneTub 2.1) were from Sigma. Immunogold-conjugated goat anti-rabbit IgG (5- and 15-nm particles) were from British BiocellInternational (Cardiff, UK). Fluorescein isothiocyanate-conju-gated goat anti-rabbit antibody andTexas-Red-conjugated goatanti-mouse antibody were from Cappel (MP Biomedicals,Strasbourg, France).

Electrophoresis and Western Blots

Proteins were resolved in denaturing 6% acrylamide gels, in adiscontinuous buffer system, as described by Laemmli (28).Then they were electrotransferred for 1 h at 350 mA on a poly-vinylidene difluoride membrane (Immobilon P fromMillipore,Bedford, MA) in Tris/glycine buffer (48 mM Tris, 39 mM gly-cine, pH 9.2, 1.3 mM SDS and 20% methanol). Protein bandswere stained nonspecifically by Amido Black.Membranes wereblocked overnight in 6% nonfat milk in phosphate-bufferedsaline (PBS) at 4 °C. Theywere subsequently probed for 2 hwitheither anti-TOGp, anti-tubulin, or anti-tau antibodies, diluted1/1000. The blots were rinsed and incubatedwith the appropri-ate secondary antibody (1/2000) conjugated with peroxidase.

TOGp Effect on Tubulin Assembly Kinetics

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Bound antibodies were detected by the enhanced chemilumi-nescence reagent ECL from Amersham Biosciences.

Peptide Sequencing of TOGp Immunoreactive Forms

The protein extract was resolved in a 6% acrylamide gelunder denaturing conditions. After the run, the gel was stainedwith 0.2%Coomassie Blue R250 dissolved in 2% acetic acid, 50%methanol (high performance liquid chromatography grade). Itwas destained in 30% methanol. The 160- and 130-kDapolypeptides were both sequenced. They were excised from thegel and hydrolyzed by trypsin according to Rosenfeld et al. (29).The resulting digest was fractionated using reverse phase chro-matography on C8 then C18 Aquapore (2 � 10 mm) columnsfrom Applied Biosystems (Foster City, CA) and eluted by anacetonitrile gradient in 0.1% trifluoroacetic acid. The eluatewasmonitored by UV spectroscopy (220 nm). Purified peptideswere sequenced on a Procise sequencer from Applied Biosys-tem using the pulsed liquid program.

Protein Identification by Mass Spectrometry

Proteins resolved by polyacrylamide gel electrophoresis wereidentified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Selected proteinspots were excised from the gel and destained. After reductionand alkylation of disulfide bonds, the dried gel pieceswere incu-bated with trypsin. The resulting peptides were extracted, puri-fied with Millipore Zip-Tip C18 columns, and added to the�-cyano-4-hydroxycinnamic acid matrix. MALDI-TOF massspectrometry was performed at the Institut de GenomiqueFonctionnelle (CNRS UPR 2580, Montpellier, France) using aUltraflex apparatus from Bruker Daltonics (Billerica, MA). Thepeptide masses were matched with the theoretical peptidemasses of all proteins in the Swiss-Prot data base using theMASCOT search engine.

Microtubule Assembly Assays

Tubulin polymerization was monitored turbidimetrically at350 nm with a MC2 (Safas, Monaco) spectrophotometerequipped with a thermal-jacketed cuvette holder. The cuvettehad a 10-mm path length and was 2 mm wide internally. Thefinal volume of the sample was 200�l. Experiments were run inPEM buffer, 3.4 M glycerol (25% v/v), 1 mM GTP, tubulinamount was varied from 5 to 20 �M, and MAPs or drugs wereadded in themedium as indicated elsewhere. The reactionmix-ture was prepared at 0 °C, and the reaction was started by plac-ing the cuvette in the spectrophotometer cell compartmentthermostatted at 37 °C.

GTP Hydrolysis Associated with Tubulin Assembly

The GTPase activity was detected by using the PiPer phos-phate assay kit from Molecular Probes (Eugene, OR). Briefly,inorganic phosphate is combined with maltose to give glucose1-phosphate and glucose. Then glucose oxidase converts glu-cose to gluconolactone and H2O2. Finally horseradish peroxi-dase converts Amplex Red to Resorufin in the presence ofhydrogen peroxide. Resorufin can be detected bymeasuring theincrease in fluorescence or absorbance of the solution. Tubulinwas polymerized at 37 °C in the conditions indicated above,

except that the amount ofGTPwas lowered to 0.1mM to dimin-ish the deep red coloration of the blank. The medium wasdivided in ten 50-�l samples that were incubated at 37 °C. Atvarious time intervals the polymerization was stopped by dena-turing the proteins for 5 min in boiling water. The precipitatewas removed by centrifugation, and the supernatant wasmixedwith an equal volume of the kit reagent. The reactionwas devel-oped for 3 h at 37 °C, and the absorbance was measured at 570nm. A dilution range was prepared in the same conditions froma 50�M potassium phosphate solution to standardize the assay.

Kinetic Parameters of Tubulin Assembly

Tubulin assembly is usually described as a sequence of bimo-lecular reactions (for details, see Ref. 20) inwhich the polymer isbuilt by successive additions of basal units of �- and �-tubulindimers; for simplicity, these building blocks are frequentlytermed monomers. The initial bimolecular reactions are ther-modynamically unfavorable (24); small aggregates tend to dis-sociate. Once a certain size, n monomers (commonly named“critical nucleus”), is reached, the addition of the next mono-mer gives a polymer more stable than its precursor. From thisstep the elongation takes place by polymerization of subunitsonto the microtubule ends. The reaction continues until theelongation process is compensated by the release of monomersatmicrotubule ends. At that time the polymer is in simple equi-libriumwith a fixed (critical) concentration of tubulin subunits.As indicated above, we followed the reaction of polymeriza-

tion at 350 nm. It was shown previously (30) that there is a quitelinear relationship between the turbidity and the total amountofmicrotubules. In consequence we considered the absorbanceat 350 nm (A350 nm) as proportionally related to the mass con-centration of tubulin polymer. Information concerningnucleation as well as elongation was drawn from the analysisof the sigmoid kinetics (30, 31). Two distinct parts can be seenon the curves; from time 0 to the first fewminutes there is a lagphase corresponding principally to nucleation, then an expo-nential decay process takes place corresponding to elongation.Nucleation—This phase may be characterized by various

parameters. The determination of the tenth time, t1⁄10 (the timenecessary to produce 10% of the final amount of polymer) is ofcurrent use to estimate the lag time duration. Moreover,according to the theoretical models, we find that two parame-ters can be used to characterize the nucleus size. In this paperwe termed these parameters p and q. The former is defined byFlyvbjerg et al. (25), on the basis of the “scaling” properties ofthe polymerization curves obtained with various amounts oftubulin. From the experimental results, these authors noticedthat the increase in polymer concentration for the earliest timesis proportional to tp. They formulated a theoretical model inwhich the parameter p was indicative of the number of succes-sive steps in the nucleation process. The value of p can be easilydetermined by plotting log(A(t)/A∞) against log t. The secondparameter, q, originates from the theory of helical aggregationof macromolecules of Oosawa and Kasai (17). In the case ofactin polymerization, it was demonstrated that the half-time ofthe reaction (t1⁄2) was proportional to [M0]q, [M0] being the ini-tial monomer concentration. This relationship is valuable withother characteristic times, t1⁄20 or t1⁄10 (24, 21, 25). Parameter q is




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obtained from the log-log plot of the tenth time of polymeriza-tion versus the total amount of monomer. Tobacman and Korn(21) indicate that it is equal to half of the nucleus size (n/2),whereas according to Voter and Erickson (24) it is equal to (n�1)/2. This apparent discrepancy is due in fact to different defi-nitions of the critical nucleus in the two papers. In the former,the nucleus is defined as the first polymer that is itself morestable than its precursor, whereas in the second paper thenucleus is the least stable intermediate in the reaction. Never-theless parameter q gives an objective estimation of the numberof monomers in the critical nucleus.Elongation—Elongation develops after the lag phase follow-

ing a procedure that is strongly similar to a first order chemicalreaction. According to the pioneering work of Johnson andBorisy (30) the elongation reaction rate can be interpreted asthe sum of the rates of polymerization and depolymerization asindicated in the equation dP/dt � �dM/dt � k�[M][E] �k�[E], whereP represents the polymer, [M] is the concentrationof free tubulin, [E] is the concentration of assembly competentmicrotubule ends, k� is an apparent second order associationrate constant corresponding to the sumof the rate constants formonomer addition at the two filament ends, and k� is an appar-ent first order dissociation rate constant corresponding to thesum of the rate constants for monomer dissociation at the twofilament ends. At steady state the reactions of growth andshortening ofmicrotubules are identical. At that time [M] is equalto [M∞], the critical concentration of tubulin. Consequently,k�[M∞][E] � k�[E], and k� � k�[M∞]. It can be determinedthat the critical concentration [M∞] is equal to k�/k� or to 1/K(K is the equilibrium association constant (30)).By replacing k� by its value in the differential equation,

dP/dt� �dM/dt� k�[E]([M]� [M∞]). It can be assumed that[E] is constant during the elongation phase, the expressionreduces to a pseudo-first order rate expression, the productk� [E] can be replaced by a constant termed k, and the factor([M] � [M∞]) represents the concentration of active tubulinnamed [Ma]. After integration, ln[Ma]/[Ma0] � �kt. The ratio[Ma]/[Ma0] is easily accessible from themeasures of the absorb-ance at 350 nm. It is equal to (A∞ � At)/A∞), where At repre-sents the absorbance at a given time, and A∞ is the absorbancemaximum obtained at the plateau of the kinetic curve. By plot-ting ln(1 � At/A∞) as a function of time, the pseudo-first orderrate constant of elongation, kobs, can be determined.

Electron Microscopy

Microtubules were prepared in a spectrophotometer cuvette(200 �l final volume) at 37 °C as indicated above in the micro-tubule assembly assays. Polymers preparedwith native 200-kDaTOGp and control polymers with tubulin alonewere incubatedsimultaneously.First Method—Microtubules were centrifuged at 36,000 � g

for 30 min, the supernatant was discarded, and the pellet wasresuspended in 200 �l of PEM buffer, 25% glycerol, 0.1 mMGTP. Anti-TOGp antibodies (5 �l) were added, and the mix-ture was incubated for 3 h at 30 °C. The antibodies were elimi-nated by centrifugation at 36,000 � g for 30 min at 35 °C. Thepellet was resuspended in 200 �l of PEM/glycerol/GTP bufferand mixed with 5 �l of immunogold-conjugated goat anti-rab-

bit IgG (5- and 15-nm gold-labeled antibodies were used alter-natively). The incubation lasted 2 h at 30 °C. The secondaryantibody was eliminated by centrifugation at 30 °C, and themicrotubules were suspended in 200 �l of fresh buffer andplaced at 30 °C.Second Method—After tubulin aggregation at 37 °C, 4 �l of

anti-TOGpantibodieswere added to the solution, and the incu-bation was continued for 3 h at 30 °C. The secondary antibody(15�l) was then included, and the incubationwas prolonged for2 h. The polymers were separated from tubulin and antibodiesby centrifugation in a 2-ml sucrose gradient (37–60%) for 1 h at180,000 � g in a swinging rotor thermostatted at 30 °C. Micro-tubules were present in the first 0.1-ml fraction at the bottomofthe gradient. Microtubules were diluted in PEM/glycerol/GTPbuffer to a protein concentration of 0.2 mg/ml, deposited ontoFormvar-carbon coated grids, and negatively stained with 2%uranyl acetate. Grids were examined using a Jeol 1200 EX elec-tron microscope at an accelerating voltage of 80 kV.

Immunofluorescence Microscopy

Primary cultures of hypothalamus cells were prepared bymechanoenzymatic dissociation of fetal (day 17) rat hypothal-amus. Cells (106) were plated in 16-mmdiameter culture dishescontaining a 10-mm glass coverslip previously coated withpoly-D-lysine (32). Cultures were maintained at 37 °C in a 95%air, 5% CO2 atmosphere in minimum Eagle’s medium contain-ing 10% Nu serum, 0.6% glucose, 2 mM glutamine, 2.5 units/mlpenicillin-streptomycin adjusted to pH 7.3. Two days afterseeding, the cells on the coverslip were fixed for 10 min in coldmethanol (�20 °C) and gradually rehydrated with PBS. Cellswere then incubated for 60 min with a mixture of anti-TOGprabbit antiserum (1/200) and anti-tubulinmonoclonal antibody(1/200) in PBS containing 1 mg/ml albumin. After washing inPBS, incubationwas carried out in the same solution containingboth fluorescein isothiocyanate-conjugated anti-rabbit anti-bodies and Texas Red-conjugated anti-mouse antibodies.Stained cells were mounted in 0.25% Airvol 205 in PBS, andimages were recorded using a 63� NA objective on a Leicainverted microscope.

RESULTS AND DISCUSSION

Purification of TOGp—The purification procedure isdetailed under “Materials andMethods.” The elution profile ofthe DEAE-Sepharose chromatography, which is the last step ofthe purification, is shown in Fig. 1. TOGp was eluted from thecolumn by increasing the ionic strength of the buffer with 0.1 MKCl (fraction T21 to fraction T32). Several TOGp immunore-active proteins are present in the eluate, as indicated in Fig. 2A.In addition to the 200-kDa TOGp native form, we observe twopolypeptides of, respectively, 160 and 130 kDa. The amino acidsequencing of these polypeptides was performed after trypsinhydrolysis. Two internal peptides were detected in the 160-kDahydrolysate that were identical, respectively, to amino acids213–220 and 1207–1221 in the TOGp sequence. One peptideidentical to amino acids 213–220 was identified in the hydrol-ysate of the 130-kDa subform. This result confirms our previ-ous observation (6) that the 130- and 160-kDa fragments wereproduced by the proteolytic degradation of TOGp. In addition,




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because the anti-TOGp antibodies are reactive against the cen-tral part of the protein (amino acids 844–1230), it can be con-cluded that the 160- and 130-kDa forms correspond to theN-terminal moiety of TOGp. During our purification assays wehave always detected these two subforms; whatever the purifi-cation procedure that we employed, there was a progressivehydrolysis of the native protein in the 160-kDa and then in the130-kDa subform. A similar splitting was reported by Shirasu-Hiza et al. (33) during the purification of the TOGp-relatedprotein XMAP215.To decrease the proteolytic degradation of TOGp through-

out our purification procedure, we included various proteaseinhibitors within the buffers. We added phenylmethylsulfonylfluoride, pepstatin A, leupeptin, aprotinin, and MG115, a pro-teasome inhibitor. We noticed that leupeptin (1 �g/ml) wasable to slightly delay the degradation process, although it didnot stop totally this phenomenon.After unspecific protein staining of the blot (Fig. 2B), we see

that the TOGp fractions eluted from the DEAE-Sepharose col-umn are contaminated by various proteins. The two main con-taminants have apparentmolecularmasses of 40 and 90 kDa. Bymass spectrometrywe have identified dynamin (score 173, pep-tides matched 42) and glutamine synthetase (score 75, peptidesmatched 11) in the two protein spots. Dynamin (34) is aGTPaseinvolved in endocytosis. It has been viewed in the past as a

mechanochemical enzyme thatpinches vesicles from the plasmamembrane, but more recently it hasbeen proposed as a classical regula-tor that recruits effectors of endocy-tosis. Glutamine synthetase (35) is aprotein of the vertebrate nervoussystem that plays a central role inthe detoxification of brain ammoniaand in the metabolic regulation ofthe neurotransmitter glutamate.We tried to eliminate these lowmolecular components by gel filtra-tion on a Sephacryl S300 column.We finally suppressed this step,which decreased dramatically theyield of the preparation withoutimproving significantly the purity ofTOGp.The ability of TOGp to catalyze

tubulinpolymerizationwasmeasuredin the fractions eluted fromtheDEAEcolumn (Fig. 1). The biological func-tion of TOGp will be investigatedmore thoroughly in thenext chapters.We report here the influence of iden-tical aliquotsof theDEAEfractionsonthe in vitro polymerization of a givenamount of tubulin at 37 °C. The val-ues of the pseudo-first order rate con-stant ofmicrotubule elongation (kobs)are indicated in the figure. Fractions19 and 36 can be considered as con-

FIGURE 1. Purification of TOGp by ion exchange chromatography onDEAE-Sepharose column. The experiment was performed in TEM buffer(Tris/HCl, 20 mM, pH 8.2, 1 mM EGTA, 1 mM Mg SO4, 1 �g/ml leupeptin). Pro-teins bound to the column were eluted with a 0 – 0.1 M KCl gradient in TEMbuffer. The absorbance at 280 nm is indicated in arbitrary units (solid line). Theapparent first order rate constant of microtubule elongation catalyzed by theeluate was determined as follows. A dialyzed sample (65 �l) of each fractionwas incubated at 37 °C with 20 �M tubulin in PEM buffer, 3.4 M glycerol, 5 mM

Mg SO4, 1 mM GTP. The polymer formation was recorded at 350 nm for 30 min,and the kobs, expressed in min�1, was calculated from the kinetics. The con-centration of the TOGp subforms was deduced from the densitometric anal-ysis of the Western blots and from the total protein content of each chro-matographic fraction. The difference in protein amount between fraction T22and T20 is considered to represent the total amount of TOGp in fraction T22.This value was used to standardize the measure. The 130-, 160-, and 200-kDaTOGp subform concentrations were estimated in each fraction.

FIGURE 2. Electrophoretic pattern of protein fractions eluted from the DEAE-Sepharose column by a 0–0. 1 M

KCl gradient in TEM buffer. A, Western blots revealed by anti-TOGp polyclonal antibodies. B, electrophoresistransferred on a polyvinylidene difluoride membrane stained with Amido Black. Lane 1, standard molecular massproteins. Lane 2, 125,000 � g pig brain supernatant (400 �g). Lanes 3–24, DEAE-eluted fractions (100 �l each); thefraction numbers are identical to those indicated in Fig. 1. Fractions T21–T23, T24–T25, and T26–T29, which contain,respectively, the 130- and 160-kDa forms, a mixture of the three TOGp variants, and the native 200-kDa subform, arebrought together. For simplification they are termed, respectively, TOGp mix2, TOGp mix3, and TOGp 200.




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trols, since they are TOGp-free and contain only the protein con-taminants of the preparation. From the figure we see that the rateconstant increases in the fractions containing theTOGp immuno-reactiveproteins.Thekobs reachesmaximalvalues in fractionsT22

and T23 and in fractions T26 andT27. These fractions correspond,respectively, to the peak value of the130 and 160 TOGp polypeptides andto themaximumvalueof the200-kDanative form. In addition we observedthat the stimulationof tubulinpolym-erization by these fractions could betotally suppressed by anti-TOGpantibodies (Fig. 3).The DEAE column chromatogra-

phy leads to a partial resolution of theimmunoreactive TOGp polypep-tides. The 160- and 130-kDa formsare collected in fractions T21 to T23,fractions T24 and T25 contain amix-tureof the 200, 160, and130 isoforms,and the 200-kDa native form is prin-cipally eluted in fractions T26 toT29. These chromatographic frac-tions were pooled according to theircomposition. For simplification, inthe next part of this paper they willbe termed, respectively, TOGpmix2, TOGp mix3, and TOGp 200,as indicated in Fig. 2. Although wecould not totally eliminate someprotein contaminants from ourpreparation, it is important to notethat the biological function ofTOGp is preserved. Moreover, the130- and 160-kDa cannot be simplyconsidered as degraded side prod-ucts of the purification since theypossess a significant enzymaticactivity on tubulin assembly.Effect of TOGp on Tubulin Poly-

merization; Effect of Tubulin Concen-tration—We first asked if TOGp tar-gets nucleation, elongation, or bothsteps in tubulin polymerization. Toanswer this question we determinedthe critical concentration of tubulinM∞, and the nucleus size parametersp and q, as defined above, in the pres-ence of purified TOGp. In theseinvestigations we used the DEAE-pu-rified fraction TOGp 200 (T26 toT29). Parallel experiments were per-formedwith two drugs, taxol and col-chicine, known for their oppositeeffect on tubulin polymerization.The variation of turbidity as a

function of time was recorded withvarious tubulin concentrations (Fig. 4). The initial tubulin con-centration was plotted versus the absorbance maximum at 350nm. There is a linear relationship between the two values.When extrapolated to absorbance 0, we can determine the crit-

FIGURE 3. Immuno-inhibition by anti-TOGp antibodies of tubulin assembly induced by the DEAE-Sepha-rose fraction TOGp mix2 (left graph) or TOGp 200 (right graph). Tubulin assembly was recorded continu-ously as a function of time by measuring the increase in absorbance at 350 nm. Tubulin (12 �M) was polymer-ized in PEM buffer (filled triangles); an identical experiment was conducted in the presence of 2 �l of anti-TOGpanti serum (open triangles). Tubulin (12 �M) was polymerized with purified TOGp (30 nM TOGp mix2 or 20 nM

TOGp 200) (filled circle); a similar experiment was conducted in the presence of 2 �l of anti-TOGp anti serum(open circle).

FIGURE 4. Estimation of the tubulin critical concentration. Various amounts of tubulin are polymerized at37 °C as indicated under “Materials and Methods.” The curves of assembly of the tubulin control are shown inthe inset. The absorbance maximum at 350 nm determined at the end of the polymerization is plotted versusthe initial tubulin concentration. The experiment determined successively with 25 nM TOGp 200, 1 �M taxol,and 1 �M colchicine in the presence of 0.5% Me2SO (DMSO, this solvent is required to solubilize taxol andcolchicine). The control with tubulin alone is performed both in the presence or in the absence of 0.5% Me2SO.Tubulin amounts extrapolated through the points to absorbance 0 gives the value of the critical concentration.




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ical concentration of tubulinM∞ on the y axis. This value indi-cates the minimum amount of tubulin necessary to obtainpolymerization. Asmentioned above, it is equal to 1/K (K is theequilibrium association constant). It can be calculated from thefigure that the value of the equilibrium constant is 0.19 � 106M�1 with control tubulin polymerized in 0.5%Me2SO, whereasit is slightly less elevated in the absence of Me2SO (0.1 � 106M�1). Me2SO, which is employed to solubilize taxol and colchi-cine, is known to activate tubulin aggregation (36). To obtaincomparative results, we adjusted the final concentration ofMe2SO to 0.5% in all the experiments. In the presence of 25 nMTOGp, the equilibrium constant K increases to 1.4 � 106 M�1.We noticed that 1 �M taxol has about the same effect; in con-trast, 1�M colchicine decreases the constant to 0.12� 106 M�1.From these results it is obvious that TOGp strongly favorstubulin subunits association.Weobserved a similar influence oftaxol; however, the molar concentration of drug required toobtain a comparable effect is 40-fold more elevated than theconcentration of TOGp.As explained above, we determined the twoparameters p and

q to characterize the nucleus size. The results are indicated inFigs. 5 and 6. There is no clear modification of factor q in the

presence of TOGp; it is only slightlydecreased by 1�M taxol. In contrast,parameter p, which is equal to 4 inthe control tubulin sample, isdivided by two when TOGp wasadded. It is noteworthy that taxolfurther decreases this parameter to1 and that colchicine has no inci-dence on p. In consequence, TOGpseems to influence nucleation bydecreasing the nucleus size. Itshould be noticed that the values ofp and q, which we obtained in theabsence ofMe2SO, are, respectively,close to 5 and 3, as found by Flyvb-jerg et al. (25).Parameters p and q are linked to

the nucleus size; however, in thepolymerization model they can beinterpreted differently. In the caseof actin (17, 21), parameter q isequal to the half-value of the num-ber of monomers included in thenucleus. In the case of tubulin,parameter q is considered by Voterand Erickson (24) as proportional tothe number ofmonomers present inthe first nucleus. In contrast, in thepaper of Flyvbjerg et al. (25), q indi-cates the number of monomersadded at each step of the nucleationphase, whereas p is linked to thenumber of successive steps. In tubu-lin polymerization models, each ofthese parameters was attributed to aspecific dimension of the nucleus.

They are not simply indicative of the number of nucleusmono-mers, as in the case of actin. Moreover, recently (23) it wassuggested that the nucleus should not correspond to a strictlydefined structure but should be an average betweenmany alter-native association pathways. Nevertheless, TOGp decreasessignificantly the value of parameter p and influences themicro-tubule nucleation step. In function of the theoretical model, wecan conclude that TOGp could either decrease the nucleus sizeor simplify the nucleus association process.Effect of TOGp on Tubulin Polymerization; Effect on GTP

Hydrolysis—We followed the liberation of inorganic phosphate(Pi) as a function of time during tubulin assembly. The effect ofTOGp was compared with the effect of colchicine and taxol,which are known to have an opposite influence on tubulinGTPase activity (37, 38, 39). In these experiments we alterna-tively employed the three DEAE-Sepharose-purified fractionsof TOGp, TOGp mix2, TOGp mix3 and TOGp 200.Tubulin dimers bind 2 mol of GTP, one exchangeable in

�-tubulin and the other nonexchangeable in �-tubulin. GTPbound to the exchangeable site became hydrolyzed after incor-poration of the tubulin dimer into the microtubule. Accordingto Carlier and Pantaloni (40), when tubulin was polymerized

FIGURE 5. Nucleus size estimated by parameter p as defined by Flyvbjerg et al. (25). Various amounts oftubulin are polymerized at 37 °C in the conditions described under “Materials and Methods.” The absorbanceat 350 nm (A) was recorded as a function of time (t), and parameter p was determined by plotting log A/Amaxagainst log t. At early times the two variables displayed a straight line of slope p independent of the initialtubulin concentration. The inset shows the plot for tubulin control. Parameter p � S.D. was determined suc-cessively with 25 nM TOGp 200, 1 �M taxol, and 1 �M colchicine in a solution containing 0.5% Me2SO (DMSO).The control with tubulin alone was performed both in the presence and absence of 0.5% Me2SO.




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in vitro, there was a “burst” of Pi liberation accompanyingtubulin polymerization, then the rate of GTP hydrolysisslowed down and reached a stable steady-state rate onlyabout 15–20 min after the reaction was started.We observed that the kinetics of GTP hydrolysis, expressed

as inorganic phosphate (Pi) released, exhibited minimal differ-ences in the presence of the TOGp isoforms or drugs by refer-ence to the tubulin control (data not shown).Wenoticed that Piliberated during the burst (10–12 �M) was roughly equal to theconcentration of tubulin-GTP dimers present at the beginningof the reaction (13.2 �M).We determined the amount of tubulin polymer produced at

the steady state of the reaction by centrifugation at 36,000 � g.The amount of Pi liberated in function of time was expressedversus the final amount of polymer (Fig. 7). The results weobtainedwith colchicine and taxol (Fig. 7A) are in good accord-ancewith those previously published (37, 39). As can be seen onthe ordinate axis, the production of an equivalent quantity ofpolymer in the presence of 4 �M colchicine necessitates 2 timesmore GTP than in the tubulin control sample. In contrast, thehydrolysis of GTP is slightly reduced in the presence of 1 �Mtaxol. The kinetics that we obtained with the three purifiedfractions ofTOGpare situated between those of tubulin controland taxol.We have calculated the reaction rates of GTP hydrol-ysis during the steady state part of the kinetics, from 15 to 45min. The results are indicated in Fig. 7B. With tubulin we seethat the 200-kDa isoform (TOGp 200) significantly slows downGTP hydrolysis. From an energy point of view, it seems that thenative 200-kDa protein renders tubulin polymerization more

economical. We do not see a similar decrease with TOGpmix3and TOGp mix2, which contain the 130- and 160-kDaisoforms.Tubulin dimers could adopt two conformations.When�-tu-

bulin is liganded with GTP, tubulin is in a “straight” conforma-tion, whereas upon GTP hydrolysis, the tubulin dimer tends toadopt a “curved” conformation, favoring depolymerization (41,42). It was deduced that GTP hydrolysis renders the microtu-bule lattice more unstable. During the steady state, when thepolymerization equilibrium is reached, the constant GTPaseactivity reflects the cyclic addition and release of tubulin dimersat the ends of the microtubules (43). In consequence, thedecrease in GTP hydrolysis induced by the 200-kDa TOGp iso-form is indicative that TOGp favors microtubule cohesion andantagonizes depolymerization.We obtained a similar effectwith 1 �M taxol, which is known to inhibit depolymerization.Effect of TOGpAmount onTubulin Polymerization; Compar-

ison with Protein Tau—We have seen in the previous para-graphs that the purified TOGp 200-kDa subform favors thenucleation process as well as the association of tubulin dimerson growing microtubules; moreover, we have evidenced thatthis protein antagonizes depolymerization. To determine theintrinsic activity of the purified TOGp 200-kDa subform ontubulin polymerization, we have compared its concentrationeffect with protein tau, a classic MAP of the nervous system, aswell as with colchicine and taxol. The results are presented inFig. 8. A constant amount of tubulin (15 �M) was incubated inthe presence of increasing amounts of MAP or drug. The poly-mer formation was recorded at 350 nm. The Amax, t1⁄10, and kobswere calculated from the kinetic curves as indicated under“Materials and Methods.”The variation in final polymer amount to a blank containing

tubulin alone is plotted versus the concentration of the effector(Fig. 8A). In these assays we employed, alternatively, TOGp200, TOGpmix3, andTOGpmix2. Fig. 8A clearly indicates thatthe three purified fractions of TOGp increases the total amountof polymer. They are active within the concentration range5–70nMTOGp.We can estimate that the half-maximal effect isobtained with 25 nM protein. In contrast, the half-maximaleffect of tau is roughly 1�M, indicating that the TOGp isoformsare �40 times more active than protein tau. In addition, it isnoteworthy that TOGp mix3 and TOGp 200, which both con-tain the 200-kDa native TOGp isoform, are the most activefractions.The effect of TOGp can be estimated as well from the meas-

urement of the tenth time of the reaction, which is characteris-tic of the nucleation phase. As indicated in Fig. 8B, the threeTOGp fractions, tau, and taxol decrease the tenth time ofpolymerization in a dose-dependent manner, whereas colchi-cine, which induces microtubule depolymerization, has noeffect on this parameter. When the results obtained with theTOGp isoforms and tau are compared, we can see that anequivalent decrease in the tenth time necessitates about 20times more tau than TOGp. Taxol has the greatest influence inlowering the tenth time; however, its half-maximal effect isobtained at higher concentrations (200 nM) than with theTOGp fractions (20–30 nM).

FIGURE 6. Nucleus size estimated by parameter q as defined by Oosawaand Kasai (17). Various amounts of tubulin were polymerized at 37 °C.Parameter q was determined by the log-log plot of the tenth time of thereaction against the initial tubulin concentration. The inset shows the deter-mination of this parameter for tubulin control. Parameter q � S.D. was deter-mined successively with 25 nM TOGp 200, 1 �M taxol, and 1 �M colchicine in asolution containing 0.5% Me2SO (DMSO). The control with tubulin alone isshown both in the presence and absence of 0.5% Me2SO.




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The kobs (Fig. 8C) is strongly increased by TOGp at low con-centrations, indicating that thisMAP takes an active part in theelongation process of microtubules. TOGp 200 and TOGpmix3 fractions are slightly more efficient than the TOGp mix2fraction, which contains principally the 130 and 160 isoforms.Taxol is active at higher doses than TOGp; in contrast, proteintau has a much moderate influence on this parameter.In conclusion, the three TOGp isoforms exert a strong effect

on in vitro tubulin polymerization; an equivalent effect withprotein tau is seen at more elevated concentrations. TOGpaccelerates the nucleation and the elongation processes and

increases the final amount of poly-mer. In addition, we see that thepurified fractions containing the200-kDa TOGp isoform are slightlymore active than the fraction con-taining the two other isoforms. Thethree TOGp-related polypeptidesthat we obtained at the end of ourpurification procedure differ bytheir C-terminal part. Their bio-chemical activity on tubulin polym-erization is very similar, indicatingthat the N-terminal moiety of theprotein plays a fundamental role inthe catalysis of tubulin assembly.Onthe other hand, the C-terminal partmay exert some control on thisactivity, since the native 200-kDaisoform is slightly more active thanthe 160- and 130-kDa polypeptides.It is reported in literature (44)

that half-maximal polymerizationoccurs, respectively, at 0.33 and 2.5�M for MAP2 and tau and thatmicrotubules formed in the pres-ence of theseMAPs contain, respec-tively, 1 mol of MAP2/5 mol oftubulin and 1 mol of tau/4 mol oftubulin. Because MAP2 and tauhave been shown to promote tubu-lin polymerization stoichiometri-cally rather than catalytically, thisprotein group is often considered asa structuralMAP family.Our exper-iments show that TOGp, which isactive at low doses and which acti-vates tubulin polymerization at var-ious steps of the biochemical path-way, is clearly distinct from thisgroup of MAPs.TOGp Localization on Microtu-

bules—By performing electronmicroscopy of immunogold-labeledmicrotubules (Fig. 9), we saw thatTOGp was located both along themicrotubule fibers and at microtu-bule ends.Wemeasured, on a group

of microtubules (n � 186) with visible extremities, the occur-rence of gold spots every 50 nm along the length of the fibers.The first 50-nm fragment was placed at the microtubule ter-mini and the last one 1000 nm away. We found 14 gold-deco-rated ends versus 172 unlabeled ends. On the other hand, wecounted on the microtubule walls 46 gold spots versus 2986unlabeled 50-nm fragments. According to the Fisher’s exacttest, the two groups are significantly different (p� 0.001). It canbe argued that this calculation is impaired by the fact that thepopulation of short microtubules is more elevated than thepopulation of long microtubules of more than 1000 nm in

FIGURE 7. GTPase activity of tubulin associated with microtubule assembly. Tubulin (13.2 �M) was treatedin PEM buffer, 3.4 M glycerol, 5 mM Mg SO4, 0.1 mM GTP. The reaction was performed successively with TOGpmix2, TOGp mix3, TOGp 200 (25 nM each), colchicine (2 and 4 �M), and taxol (1 �M). The mixture was incubatedat 37 °C. At various time intervals a 50-�l aliquot was withdrawn, and the reaction was stopped in boiling water.The precipitated proteins were eliminated by centrifugation, and inorganic phosphate (Pi) was measured inthe supernatant by Amplex Red reagent as indicated under “Materials and Methods.” The amount of polymerproduced at the steady state of the reaction was determined after centrifugation at 36,000 � g of an aliquotincubated for 45 min at 37 °C. A, GTPase activity from 0 to 45 min for control, 4 �M colchicine and 1 �M taxol. Theresults are expressed in �M Pi released/�M concentrations of the final tubulin polymer. The amount of Piliberated during the initial reaction is visible on the ordinate axis. B, GTPase activity during the steady state ofthe reaction (from 15 to 45 min) with various effectors. The results are expressed in �M Pi liberated/�M concen-trations of final polymer/min � S.E. Student’s t test was calculated with the tubulin control as a reference value.NS, not significant.




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length. If the number of gold spots is normalized to 100 micro-tubules, the probability according to the Fisher’s test slightlyincreases to 0.001. Nevertheless, the two estimations suggest ahigher affinity of TOGp for microtubule ends than for micro-tubule walls. To localize TOGp on “native” microtubules, we

double-stained primary cultures of rat hypothalamus cells (Fig.10) with anti-tubulin and anti-TOGp antibodies. The cytoskel-eton is abundant and well developed in the interphasic cyto-plasm of these cells. By immunofluorescence microscopy, wesaw that TOGp was located in a punctuate pattern all along themicrotubules fibers.It was reported by us in a previous study (6) that TOGp co-

localizes with centrosomes and spindles in mitotic cells andthat TOGp co-sediments with taxol-stabilized microtubules invitro. It was later evidenced (7) by the use of cloned truncatedfragments that both full-length and the N-terminal part of

FIGURE 8. Dose effect of TOGp, tau, and drugs on the kinetic parametersof tubulin assembly. The experiments were run in PEM buffer, 3.4 M glycerol,1 mM GTP containing 15 �M tubulin and various amounts of MAPs or drugs.The absorbance at 350 nm (A350 nm) was recorded as a function of time. Thevalues of A350 nm max, t1⁄10, and kobs were calculated from the sigmoid kineticcurves as indicated under “Materials and Methods.” The experiments wereperformed successively in the presence of increasing concentrations of TOGpmix2, TOGp mix3, TOGp 200, protein tau, colchicine, and taxol. The differentparameters are expressed versus the amount of effector on a logarithmicscale. A, the ordinate axis represents the values of the absorbance maximumminus the absorbance maximum with no effector. B and C show, respectively,the values of the tenth time (t1⁄10) and the values of the pseudo-first order rateconstant (kobs) of the reaction.

FIGURE 9. Electron microscopy of negatively stained microtubulesassembled in the presence (a– d) or absence (e) of TOGp 200 by incuba-tion of tubulin with 1 mM GTP for 30 min at 37 °C. Tubulin polymers werefirst treated with anti-TOGp antibodies then with immunogold-labeled anti-rabbit IgG; alternatively, 5 nm (a, c, and d) and 15 nm (b) gold particle-labeledantibodies were used. Samples diluted 1⁄20 in PEM buffer, 3.4 M glycerol, 0.1mM GTP were loaded on the microscope grid and stained with 2% uranylacetate.




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TOGpbind along the length of individual protofilaments with agreat affinity for microtubule ends.Within dividing cells, it wasshown that the major function of TOGp was to maintain cen-trosome integrity by focusing microtubule minus-ends at spin-dle poles (9).Msps protein from Drosophila associates with microtubules

in vitro. In the embryonic division cycles Msps localizes to thecentrosomal region at all mitotic stages and spreads over thespindles during metaphase and anaphase (10). DdCP224 fromD. discoideumwas detected at the centrosome andmoreweaklyalong microtubules throughout the entire cell cycle; further-more, it binds to microtubules in vitro (11). XMAP215 fromXenopus promotes the formation of long microtubules byincreasing the rate of microtubule polymerization, particularlyat the plus end (4, 45). Expression of truncated segments ofXMAP215 in vivo (46) showed that the entire protein partici-pates in microtubule binding.In all species examined so far, TOGp orthologs have been

found onmicrotubules and centrosomes in all stages of the cellcycle (46, 47). Fromour investigations on the kinetics of tubulinassembly, we have shown that TOGp induces a stimulation ofmicrotubule growth and a reduction of depolymerization. Botheffects could be easily explained by the localization of TOGp atmicrotubule ends. The affinity of TOGp for microtubule endsin vitro was previously reported by Spittle et al. (7). Moreover,TOGp-related proteins in other species have been principallydetected in vivo at centrosome and spindle poles, confirming a

function of this protein family on the extremities of tubulinpolymers. Within the cell the location of these MAPs is com-plicated by the presence of interacting proteins. It has beenreported that the attachment of TOGp isoforms tomicrotubuleterminal organelles should involve the participation of othercell components. In this sense, spindle-kinetochore attachmentin fission yeast requires the combined action of two kinesinproteins, KIp5 and KIp6, with Alp14 and Dis1, which are twoMAPs of the TOGp family (48). Moreover, D-TACC, the Dro-sophila form of transforming acid coiled-coil protein, main-tains Msps at centrosomes and helps it to bind to microtubuleminus-ends and plus-ends asmicrotubules grow out of the cen-trosome (49, 50). By electron microscopy of immunogold-la-beled microtubules, we confirmed the affinity of TOGp formicrotubule ends; however, this attachment is not exclusivebecause we detected TOGp molecules on microtubule wallsboth in vitro and in vivo.In conclusion, we have tested the influence of TOGp on

tubulin polymerization in vitro by investigating the kineticparameters of the reaction. Three subforms of TOGp were iso-lated from brain tissue; the native 200-kDa protein and twopolypeptides of 160 and 130 kDa, resulting of the proteolyticsplitting of C-terminal fragments of the protein. The 200-kDaTOGp form has a strong effect on microtubule formation. Itfavors the nucleation phase, increases the association constantof tubulin subunits on elongating microtubules, and antago-nizes depolymerization during the steady state of the reaction.The native 200 kDa aswell as the 160- and 130-kDa polypeptidefragments enhance the total amount of polymer, decrease thetenth time of the reaction, and augment the rate constant ofelongation. However, the purified fraction containing the 200-kDa polypeptide is more efficient than the fraction containingthe hydrolyzed forms.The study of tubulin polymerization under controlled condi-

tions led us to determine a set of kinetic parameters, allowing abetter understanding of TOGp action. This method could beextended in the future to other microtubule protein effectorsadded individually or in conjunction with TOGp. More inter-estingly, these investigations should facilitate a screening ofdrugs targeting the interaction TOGp-microtubule with theaim to uncover new microtubule active drugs.

Acknowledgments—We are grateful to L. Cassimeris (Lehigh Univer-sity, Bethlehem, PA) for many useful comments on the manuscript.We thank J. Derancourt (Centre de Recherche en Biochimie Macro-moleculaire, Montpellier) for peptide sequencing, and P. Jouin andN.Galeotti (Institut de Genomique Fonctionnelle, Montpellier) for massspectrometry analysis. We greatly acknowledge the Veterinarian andthe employees from the Abattoir of Ales (France) for help in pig braincollection. We also thank S. Arancibia (Universite Montpellier II) forproviding cultures of rat hypothalamus cells and J. Piette (EMI229,Montpellier) for critical reading of the manuscript.

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LarroqueClaude Bonfils, Nicole Bec, Benjamin Lacroix, Marie-Cécile Harricane and Christian

Microtubule-associated Protein TOGpKinetic Analysis of Tubulin Assembly in the Presence of the

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