Functional organization of microtubule-associated protein tau ...

THEJOURNAL OF BlOLOClCAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 268, No. 5, Issue of Febmau 15, pp. 3414-3419,1993 Printed in U.S. A.

Functional Organizationof Microtubule-associated Protein Tau IDENTIFICATION OF REGIONS WHICHAFFECT MICROTUBULE GROWTH, NUCLEATION, AND BUNDLE FORMATION IN VITRO* (Received for publication, September 3, 1992)

Roland Brandtand Gloria Lee From the Center for Neurologic Diseases,Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

Tau protein is a microtubule-associatedprotein that i s almost exclusively expressed in the brain and is enriched in the axon. Determination of tau’s sequence has revealed three to four tandem repeats that have been shown to constitute the microtubule binding site. In order to study the functional organization of tau, we prepared a series of truncated tau fragments using an Escherichia coli expression system. We assayed each fragment’s activity in promoting growth of microtubules and in nucleating free microtubules. We found that tau’s ability to nucleate microtubules requires the presence of additional sequence amino-terminalto that required for growth. We demonstrate that tau’s carboxyl and amino termini differentially affect microtubule growth and nucleation. Finally, we show that in vitro microtubulebundle formation occurs when tubulin is assembled in the presence of an amino- and carboxyl-terminally truncated tau protein, whereas almost no bundling is observed in the presence of fulllength tauortau fragments that contain the amino terminus in addition to the repeat domain. We conclude that although the presence of the repeat domain promotes the growth of microtubules, the structural requirements for nucleation activity are more stringent. The differentiation between the growth promoting and nucleation activities on the structural level makes it possible for the two activities to be differentially regulated in vivo.

Microtubules play a role in various cellular functions such as mitosis, intracellular transport, andspecification and maintenance of cell shape. Among the factors thought to regulate these functions are microtubule-associated proteins (MAPs),’ which promote microtubule assembly i n vitro and stabilize microtubules in vivo (1).In brain, threeclasses of MAPs have been identified MAP1, MAP2, and tau protein (for recent reviewsseeRef. 2 and 3). Tau protein has been localized primarily to theaxon i n s i h(4-7) and has been implicated in the development of axonal morphology (8, 9). Tau protein consists of multiple isoforms that are developmentally regulated and are produced, at least in part, by * This work wassupported by a research fellowshipof the Deutsche Forschungsgemeinschaft (to R. B.) and National Institutesof Health Grant GM39300 (to G. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be herebymarked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: MAP, microtubule-associated protein; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction, PMSF, phenylmethylsulfonyl fluoride; PIPES, piperazine-N,N’-bis(2-ethanesulfonic acid).

alternative mRNA splicing (for reviews see Ref. 10 and 11). The carboxyl-terminal half of tau protein is conserved across species and is also homologous to MAPB, whereas the amino terminus is more variable. The carboxyl-terminal half contains the repeat domain, which consists of stretches of 18 residues that areimperfectly repeated three times in the fetal and four times in the adult specific form; the repeats are separated by 13- or 14-residue spacer regions. The repeat domain constitutes the microtubule binding region with a single repeat being sufficient for interacting with tubulin (1216). Synthetic 18-mer copies of a repeat can promote microtubule polymerization, although the molar ratio of peptide required is 2 orders of magnitude higher than that of fulllength tau (12, 13, 15). The adult form of tau promotes assembly of microtubules more actively than fetal forms (1719), probably reflecting a higher binding affinity due to the additional repeat (16). Although it is clear that the repeat domain constitutes the microtubule binding site, it is less clear what roles the aminoand carboxyl-terminal parts of tau play. It has been reported that sequences adjacent to the repeat domain increase the binding affinity to taxol-stabilized microtubules i n vitro (16) and are required for binding of tau to microtubules in cells (20). In addition, evidence exists that regionsbeyond the repeats might affect the formation of microtubule bundles i n vitro and i n vivo (19-23). In order to study the functional organization of tau protein and theeffects of sequences adjacent to therepeats on microtubule assembly, we prepared a series of truncated fragments of tau using a bacterial expression system. We tested the fragments for activity to promote growth of microtubules by length measurements of centrosome-nucleated microtubules and activity to nucleate free microtubules using a newly developed ELISA-based assay and immunofluorescent microscopy. In addition we studied the role of the amino- and carboxyl-terminal parts of tau protein in the formation of microtubule bundles i n vitro using electron microscopy. EXPERIMENTALPROCEDURES

Materials-All reagents, unless otherwise specified, were obtained from Sigma. Construction of Expression Plasmids-Prokaryotic expression plasmids were constructed in PET-3d(24). Inserts for all plasmids except for n fragment and n[1841 wereprepared by polymerase chain reaction (PCR) using pl9tau and p2ltau cDNA (14, 20). PCR primers contained restriction sites and start and stop codons as needed. The inserted sequences are shown in Fig. 1. Plasmids for n fragment and n[184] were constructed by cutting and re-circularizing the plasmid containing n123c; amino acids contributed at the carboxyl terminus of these tau fragments are given in the legend of Fig. 1. Preparation of Tau, Tubulin, and Centrosomes-PET-3d tau plasmids were transformed into Escherichia coli BL21(DEB)pLysScells for expression (24). Cells weregrown, induced, and harvested as

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Functional Organization of Tau described previously (24). Cell extract from 100 mlof culture was prepared by lysis of the cell pellet with 10 ml of 30 mM Tris, 5 mM EDTA, 1 mM PMSF, 0.1% Triton X-100, 1 mM dithiothreitol , 2.5 pg/ml DNase I (Boehringer, Mannheim, FRG), pH 8.0, and subsequent centrifugation at 23,400 X g for 20 min. The supernatant was bound to DE52 (Whatman, Maidstone, United Kingdom); flowthrough was subjected to phosphocellulose chromatography (P11, Whatman) in CB (50 mM K-PIPES, 1 mM EGTA, 0.2 mMMgC12, pH 6.8), containing 1 mM PMSF and 5 mM 2-mercaptoethanol. Tau protein was eluted with CB containing KCl; tau protein eluted between 0.2 and 0.4 M KCl. Protein was dialyzed against BRB80 (80 mM K-PIPES, 1 mM EGTA, 1 mMMgC12, pH 6.8), containing 5 mM 2-mercaptoethanol, and subsequently concentrated using a Centricon 10 Microconcentrator (Amicon, Danvers, MA). The amino-terminal fragments of tau protein (n, n[184]) were prepared in essentially the same manner, except that the DE52 was subsequently washed with CB containing 0.1 M KCl, which wascombined with the flow-through, and theP11 eluates containing0.1-0.4 M KC1 were combined. Tubulin was isolated from calf brain by two assembly-disassembly cycles and phosphocellulose chromatography as described previously (25), applying less than 3 mg of protein/ml of resin. Peak fractions were pooled, brought to 80 mM K-PIPES, 1 mM EGTA, 1 mMMgC12, 0.1 mM GTP, pH 6.8, and stored in small aliquots at -135 “c. Immunoblotting using affinity-purified tau antibody and ‘251-labeled secondary antibodies failed to detect contaminating levels of tau protein in the tubulin preparation. Centrosomes were isolated from Chinese hamster ovary cells as described previously (25). Fractions containing centrosomes were identified by performing regrowth assays in the presence of 0.7 mg/ ml twice cycled microtubule protein; fractions were stored in small aliquots at -135 “C. Centrosome-mediatedMicrotubuleRegrowth Assay-The assay was essentially performed as described previously (25,26). The incubation mixture was prepared at 0 “C and contained, in 50 pl of BRB80, 1 mM GTP, 15 p~ tubulin, centrosomes, and, when specified, 0.3 p M tau fragment. Incubation wasfor 10 min at 37 “C. Reaction was stopped by addition of 200 p1 of 1%(v/v) glutaraldehyde in BRB80, containing 1 mM GTP, at room temperature followed 3 min later by the addition of 1 ml of BRB80. The mixture was sedimented for 15 min at 25,000 X g through a 5-ml cushion of 25% (v/v) glycerol in BRB80 onto polylysine-treated coverslips. The supernatant was aspirated, and the interface washed with 1%Triton X-100. The coverslip was removed and postfixed with methanol at -20 “C for 5 min. Immunofluorescence was performed with DMlA anti-tubulin monoclonal antibody and rhodamine-conjugated goat anti-mouse antibody (Boehringer). Coverslips were mounted in Aquamount (Lerner Laboratories, Pittsburg, PA). Fluorescent microscopy employed a Zeiss Axioskop Neofluar 63X lens. For length measurements, microtubules were photographed and lengths determined by projection of 35-mm negatives onto a digitizing tablet interfaced with a Macintosh computer. For each condition, 100 microtubules were traced; the mean length and standard error calculations were computer-aided. To determine the number of microtubules nucleated per centrosome, microtubules nucleated by 10 randomly chosen centrosomes were counted from the immunofluorescence photographs and averaged. Competitive Enzyme-linked Immunosorbent Assay (ELISA)-The incubation mixture contained, in 50 pl of BRB80, 1 mM GTP, 15p~ tubulin, and tau fragment as specified. Incubation was for 10 min at 37 “C. Reaction was stopped by addition of200 pl of 1% (v/v) glutaraldehyde in BRB80, containing 1 mM GTP, at room temperature followed 3 min later by the addition of 1 ml of BRB80. Polymerized tubulin was pelleted for 30 min at 100,000 X g, and aliquots of the supernatant were assayed using biotinylated tubulin as a competitor. Residual glutaraldehyde was found not to interfere with the assay. Biotinylation was performed as previously described (27) at a ratio of250pgof biotin-N-hydroxysuccinimide (Zymed, San Francisco, CA) per mg of tubulin for 4 hat room temperature. ELISA was conducted on Microtiter plates (Nunc, Kamstrup, Denmark) coated with 100 pllwell 1:5,000 DMlA anti-tubulin monoclonal antibody and evaluated using a biotin-avidin-alkaline phosphatase-system (Vector, Burlingame, CA). The linear range of the ELISA was determined as 0.2-2 pg of tubulin. Determination of Microtubule Number-Microtubule assembly was performed as described above (see “Competitive ELISA”) but in a total volume of 25 pl. The reaction was stopped after 2, 5, 10, and 30 min at 37 ‘C by the addition of 100 plof 1%glutaraldehyde in BRB80, containing 1 mM GTP, at room temperature followed 3 min later by the addition of 500plof BRB80. Microtubules were collected by

sedimenting 1% or 0.1% aliquots, diluted in atotal of 500 p1 of BRBSO, onto polylysine-treated coverslips for 1 h at 25,000 X g. Immunofluorescence and fluorescent microscopy was performed as described above (see “Centrosome-mediated Microtubule Regrowth Assay”). The number of microtubules was determined by counting microtubules fromfive randomly chosen frames (175 X 120 pm); average number per frame and standard error were calculated. Blot Overlay Assay-1 pg of tau protein and fragments of tau were separated by SDS polyacrylamide gel electrophoresis (28) with 15% acrylamide. Electrophoretic transfer onto Immobilon-P (Millipore, Bedford, MA) was carried out in 0.2 M glycine, 250 mM Tris, 20% (v/ v) methanol overnight at constantcurrent (100 mA). Blots were washed and incubated with tubulin (25pg/ml) as previously described (29).Immunodetection was done with DMlA anti-tubulin monoclonal antibody, alkaline-phosphatase-conjugated goat anti-mouse antibody (Promega, Madison, WI), and developed using a 5-bromo-4-chloro-3indolyl-phosphate/nitro blue tetrazolium phosphatase substrate system (Kirkegaard and Perry, Gaithersburg, MD). Electron Microscopy-Microtubule assembly was performed as described above (see “Competitive ELISA”) but in a reaction volume of 100 p l . The reaction was stopped after 10 min at 37 “Cby the addition of 400 p1 of 1%glutaraldehyde in BRB80, containing 1 mM GTP, at room temperature. Microtubules were pelleted either on a polylysinetreated coverslip as described above (see “Centrosome-mediated Microtubule Regrowth Assay”), followed by postfixation for 5 min at -20 “C in methanol or in Beem embedding capsules (Fullam Inc., Latham, NY) for 1h at 100,000 g. Samples were rinsed in cacodylate buffer (0.1 M sodium cacodylate, pH 7.4), stained with 2 mg/ml tannic acid in cacodylate buffer for 5 min, fixed for 20 min with 1%OsOl (Electron Microscopy Sciences, Ft. Washington, PA) in cacodylate buffer containing 0.8% (w/v) potassium ferricyanide (Electron Microscopy Sciences) dehydrated in ethanol series, and embedded in quetol653 (Ted Pella, Redding, CA). Polymerization of the resin was for 24 h at 60 “C. Sections of the sample were stained with lead citrate and viewed by electron microscopy. Other Methods-Protein concentrations were determined by the method of Bradford (30) using bovine serum albumin as a standard. SDS-polyacrylamide gel electrophoresis was performed as previously described (28) and stained with Coomassie Blue. RESULTS

Purification of Tau Fragments Expressed in E. coli-We constructed 10 recombinant plasmids encoding amino- and carboxyl-terminally truncated tau fragments as shown in Fig. 1. The fragments were expressed and purified as described under“Experimental Procedures.” All purified fragments were reactive to polyclonal anti-tau antibody (data not shown) and were pure according to SDS-polyacrylamide gel electrophoresis (Fig. 1).Bands at molecular weights lower than the full-lengthprotein were duetolimited proteolysis during expression, as confirmedby immunoreaction. Microtubule Growth-promoting Activity of Expressed Tau has been reported previously that isolated Fragments-It centrosomesnucleate microtubuleassembly in uitro when

incubated withphosphocellulose-purified tubulin (25). In this assay, the use of centrosomes gives microtubule nucleation a kinetic advantage by anchoring and stabilizing microtubules and provides a population of microtubules whose growth can be studied in uitro. As previously reported, thepresence of tau protein increases both microtubule growth and the average number of microtubules nucleated per centrosome, presumably by reducing microtubule instability (26). We used the centrosome-mediated microtubuleregrowthassay to assess the effect of tau fragments on the promotion of microtubule growth and on centrosome-mediated nucleation activity. Theminimalconcentration of tubulinheterodimers for centrosome-nucleated microtubule growth in the absence of tau was 9.5 p M for our preparation, which is in the range of previous reports (25, 26, 31). Increasing the tubulin concentration led to a linear increase in themean length of centrosome-nucleatedmicrotubulesas measured after 10 min of incubation(datanotshown).Inaddition, when the mean

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FIG. 2. Centrosome-mediated microtubule regrowth of microtubules with different t a u fragments. Regrowth assay was done in the presence of 15 p~ tubulin for 1 0 min at X7 “ C at a tau:tuhulin-ratioof 0.02 except the negative control. Immunofluorescence and analysis was done as described under “Experimental Procedures.” The mean length of 100 microtuhules and standard error are shown.

with the numberof the repeats and the amino-terminal flanking region (residues 1-172) moderately affecting its activity. - 18.4 In addition, the presence of the fragments that contained 14.3 therepeatdomain increased thenumber of microtubules nucleated by the centrosomes (average of 22.0 5 5.6 microFIG. 1. Schematicdiagramand SDS polyacrylamide gel tubules/centrosome); this represented 4-5-fold a increase over electrophoresis of truncated tau fragments expressed in E. that found in the absence of tau or in the presence of the coli. Construction of the plasmids, expression, andpurification were amino terminus (4.0 f 0.8 and 3.8 f 0.7 microtubules/centroperformed as described under “Experimental Procedures.” In the some, respectively). Since the fragments’ activities to promote schematic, first and last amino acid from tau sequence are indicated. nuRepeats are shaded; the repeat domain in three repeat isoform spans microtubule growth and to promote centrosome-mediated residues 198-278. Four-repeat forms are identical to the three-repeat cleation correlated, the increase in the number of microtufragments except for the insertion of31 residues at position 217. The bules percentrosomemightresult from the reduction of inserted sequence is shown by the heauy bar and contains the fourth microtubule instability by tau rather thanfrom a direct effect repeat. Fragments initiating in the middle of the molecule include a of tau on the centrosome. Met-Gly fusion at the aminoterminus; amino-terminal fragments (n, Microtubule Nucleation Activity of Expreswd Fragmentsn[1841) include Arg-Ser-Gly-Cys at the carboxyl terminus. Electroreportedthat MAPS promotethe phoretic separation of nl2Rc (lane I ), n12.7 (lane 2). 12.7~(lane 3 ) , Ithas beenpreviously [154]123c (lane 4 ) , 123 (lane 5), n1234c (lane 6 ) , 1112.74 (lane 7), nucleation of microtubules in vitro (32,33). In order to assess 1234c (lane 8).n (lane 9),and n[184] (lane 1 0 ) was done on a 10- the abilityof tau fragments to nucleate microtubules, we used 20% polyacrylamide-gradient gel; staining was with Coomassie Blue. a n ELISA-based assay to quant,itate the amount of polymerMolecular weight markers are indicated at rixht. As reported previ- ized tubulin in small reaction samples(see“Experimental ously. tau fragments run at higher molecular weight than that preProcedures”).In developing thisassay, we hadfound by dicted from primary sequence data (14). examining the products by immunofluorescent microscopy, thattheappearance of free microtubulescorrelated with length of centrosome-nucleated microtubules was determined measurable microtubule mass (data not shown). In the abduring a time course ranging from 2 to 10 min, the increase sence of tau no microtubule mass was measurable after 30 was linear (data not shown), indicating that the mean length min of incubation and tubulin concentrations up to 80 p M . of the assembled microtubules at 10 min reflects the micro- Since all fragments that contained the repeat domain protubule growth rate. We routinely used 15 p~ tubulin and an moted microtubule growth as measured by the centrosomeincubation timeof 10 min in all assays; under these conditions mediated microtubule regrowth assay (Fig. 2), accumulation centrosome-nucleated microtubules, assembled inthe absence of microtubule mass in the absence of centrosomes is indicaof any tau, were long enough to be measured from immuno- tive of tau’s ability to nucleate free microtubules. The number fluorescence photographs. Pilot experimentsrevealed that tau of free microtubules correlated with the amount of polymerincreasedthemeanlength of microtubules a t tau:tubulin ized tubulin as measured by the ELISA-based assay (datanot ratios up to 0.04 in the presence of 15 p~ tubulin. At higher shown),indicatingthattheELISAdata reflect primarilv tau:tubulin ratios, spontaneous assembly of free, i.e. not cen- nucleation rates rather than growth rates. trosome-nucleated, microtubuleswas observed. Therefore, all When 15 p~ tubulin was incubated for 10 min with increasregrowth assays were carried out a t a tau:tubulin ratioof 0.02 ing amounts of tau protein, an increase in the amount of (tau concentration of 0.3 p ~ ) . polymerized tubulin was observed (Fig.3a). This is consistent When tubulin and centrosomes were incubated with full- with previous results obtained by turbidometric studiesshowlength tau protein (n123c), the mean microtubule length was ing that tau protein promotes tubulin polymerization in a more than 10-fold that found in the absence of tau (Fig. 2). dose-dependent manner (17-19, 34-36). In the presence of n123fragment did not differ significantly in itsactivity, 11123 and n12.14 fragment a similar increase in the amount of whereas 1232 fragment was approximately 30% less active. polymerized tubulin was observed (Fig. 3 c ) . In the presence Fragments that contained four repeats were generally more of the 123c and 1234c fragment no polvmerization of tubulin active compared to the corresponding three-repeat constructs. was observed (Fig.3 e ) . A t high tau:tubulin ratios (>O.l),some T h e n fragment was not active. These results indicate that assembly of microtubules could be detected bv immunofluthe repeat domainis necessary to promote microtubule growth orescent microscopy; however, sinceextensive hundling of

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after 2 min, indicating a lower nucleation activity compared to full-length tau. In the presence of 123c fragment almost no microtubules were observed. Whentubulin was incubated with [154]123c fragment, many microtubules were observed, but the number of microtubules increased more slowly compared to full-length tau, indicating a lower nucleation activity. In order to test whether the region that is additionally 0 required for tau's ability to nucleate microtubules (residues 30 0 10 20 ,001 .01 Time [mln] 154-173), actsindependently from therepeatdomain, we Tau:lubulin prepared an n fragment containing additional residues 164184 (n[184], Fig. 1). n[184] fragmenthadnomicrotubule growth promoting activity as tested by the centrosome-mediated microtubule regrowth assay (data not shown). When coincubated with 123c fragment no tubulin polymerization could be observed (data not shown), indicating thatresidues 154-173 restoretheability to nucleatemicrotubulesonly 30 0 10 20 Time [min] when part of the same molecule that contains the repeat domain. Since tau's ability to nucleate microtubules might require binding of tubulin heterodimers,we tested tubulin bindingby 300. a blot overlay assay (29). When tubulin heterodimers were incubated withimmobilized tau fragments,all fragments containing the repeat domain-bound tubulin (Fig. 4). Interest- 0 ingly, although tubulindid not bind to the n fragment, a slight 0 10 20 30 ,001 .01 binding to n(1841 fragment was detectable (Fig. 4, lone 91, Tlme [minl Tau:tubulln suggesting a weak tubulin-binding site a t positions 164-184. Electron Microscopic Obseruatwn of Assembled Microtubules and Effect of Expressed Fragments on the Formation of Micro30 tubule Bundles-It has been reported previously that microtubules assembled in vitro in the presence of synthetic peptides encoding part of the repeat domain are bundled (22); z 04 the term "bundle" was used to describe the appearance of .1 .001 0 10 20 30 Tau:tubulln Tlme [min] several microtubules being closely attached to each other as FIG.3. Microtubule-nucleation activity of tau fragments. observed by electron microscopy. In order to study theeffect carboxyl termini of tau on bundle formation, The amount of polymerized tubulin in the presence of 15 pM tubulin of the amino and and n123c and n1234c ( a ) , n123 and n1234 (c), 123c and 1 2 3 4 ~( e ) , we prepared a tau fragment containing mainly the repeat and [154]123c (g) was determined as described under "Experimental domain (Fig. 1, fragment 123). This fragment behaved unlike Procedures." Incubation wasfor10 min at 37 'C and tau:tubulin larger fragments that contained the repeat domain and two ratios as indicated. The number of microtubules in the presence of anomalies were observed. 1) Whentubulin was incubated 15 p~ tubulin and n123c ( b ) , n123 ( d ) , 123c (f), and [154]123c ( h ) was determined as described under "Experimental Procedures." with this fragment, a substantial amount of microtubule asTau:tubulin ratio was 0.1 for all fragments and incubation at 37 'C sembly was observed despite the absence of sequences upstream of 173. 2) Free singlemicrotubules were never oba s indicated. Standard error is shown. served, and electron microscopy revealed that the microtumicrotubules was observed under these conditions, it likely is bules were bundled (Fig. 5, a and 6). We speculated that in that this assemblydiffers from tau-specific nucleation of the presence of this fragment, microtubules assembled abnormicrotubules (discussed below). In order to determine whether part or all of the amino-terminalregion is necessary for tau's mally with nucleation occumng through a mechanism other ability to nucleatemicrotubules, we tested an additional con- than thatemployed by the other tau fragments. Inordertoquantitatetheextent of bundleformation, struct which extended 19 residues into the amino-terminal region ([154]123c). In the presenceof this fragmenta similar 1 2 3 4 5 6 7 8 9 1 0 1 1 M , increase comparedto full-length tau in the amount of polym(X10 - 3 ) erized tubulin was observed (Fig. 3g), indicating that a small 9- 43 amino-terminal region is sufficient to restore the ability to nucleate microtubules to 123c fragment. -29 In order to investigate the activityof microtubule nucleation, we determined the numberof microtubules as a function of reaction time (Fig. 3, 6,d , f , and h). Using a tau:tubulin - 18.4 ratio of 0.1, the number of microtubules was maximal after - 14.3 5-10 min. The decrease of microtubulenumber a t longer reaction times is probably due to some dynamic instability FIG.4. Binding of tubulin heterodimersto tau fragmente. 1 under conditions of lowered concentrations of free tubulin, pg each of n123c (&neI ) . n123 ( l o n e 2), [154]123c (&ne 3). 123c since thepolymerization of tubulin, asjudged by ELISA, was (lane 4 ) , 123 ( l o n e 5 ) , n1234c (lane 6 ) , n1234 (&ne 7), 1234c (lone almost complete after 10 min (data not shown). The number8),n[184] (lane 9),n (&neIO). and tubulin as positive control (lane 11)were separated by SDS-polyacrylamide gel electrophoresis, transof microtubulespolymerized in the presence of full-length tau ferred to Immobilon-P, and incubated with tubulin heterodimers. (n123c) was almostmaximal(>95%)after 2 min.When Bound tubulin was detected as described under "Experimental Protubulin was incubated with n123 fragment, the number of cedures." Molecular weight markersare indicated at right. Arrow microtubules was maximal after 5 min, with 60% maximal shows full-length n[184] protein.

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of T a u ment (Fig. 6). In the presenceof n123 fragment, the extentof bundling was similar to t h a t of full-length tau. In all cases, the individual microtubules appeared morphologicallv normal with a diameter of 25 nm, indicating that the 123 fragmentis sufficient to assemble normal microtubules. DISCUSSION

In this report, we demonstrate that tau’s activitv to promote growth of microtubules and its activity to nucleate microtubules are mechanistically distinct and involve different regions of tau. This is consistent with previous studies, which have demonstrated that microtubule assemblv occurs by a condensationpolymerizationmechanismconsisting of distinct nucleation and microtubule growth steps with MAPS affecting both (32,871. Although the repeat domain is required for both steps, the structural requirements for nucleation are more stringent and require additional sequence. Since this region(residues154-173)cannotconferinitiationactivitv alone and cannot complement nucleation activityif not contiguous to the repeat domain, it might either contribute an additional tubulin-binding site, as suggestedby t h e blot overlay result, or berequiredforproteinfolding.Accordingto Chou-Fasman secondary structure prediction (38, 39), residues 168-173 would contribute an additional /$-sheet structure to the protein. The presence of thisregionmightalsohe important for tau’s function in oioo, since the inclusion of residues 164-172 is required for the 123c fragment to localize to microtubules in cells (20). The presence of tau’s carboxyl terminus (residues 308-352 for the three-repeat form) affects microtubule nucleation activity, whereas its effect on microtubule growth is small, if any. The presence of the fourth FIG.5. Electron microscopy of assembled microtubules. Microtuhules were assemhled in the presence of 15 P M tuhulin and 123 repeat in the adult specific form does not change tau’s structural requirements for nucleation and growthof microtubules ( a and b ) , n12:k ( c ) , n123 ( d ) , and [154]12Rc ( a ) . lncuhation was for 10 min at a tau:tuhulin ratio of 0.06 ( a ) and 0.1 ( b - e ) . Microtuhules but confers a higher activity in promoting growth of microwere pelleted eitherontoa coverslip ( a ) or in Reem embedding tubules. capsules (b-a) and prepared for electron microscopy as described The differentiation at the structural level betweentau’s under “Experimental Procedures.” Scale bar is 100 nm. activity to promote microtubule growth and to nucleate mito be crotubulesmakes it possibleforthetwoactivities a b independently regulated. The area required for tau’s abilitv to nucleate microtubules (residues 154-307) is conserved in [154]123c all isoforms and species of tau protein sequenced and is also found in MAPS. In vivo tau is posttranslationallv modified, nl23 and phosphorylation has been shown to affect tau’s activity in vitro(40) and MAP2’s localization in oioo (41). Tau can be nl23c n123c phosphorylated in vitro by various protein kinases (42-48). Interestingly, all phosphorylation sites so far identified are 0 2 4 6 8 0 50 100 Bundled mfcrolubulesp.1 Mcrotubules per bundle located within or close t o t h e region required for promotion FIG. 6. Effect of tau fragments on bundling of microtubules. ofmicrotubulegrowthandnucleation.Since it hasbeen The extent of hundled microtuhules ( a ) and the average numher of shown that phosphorylation affects structural properties of microt.uhules per hundle ( b ) were determined from the electron microscopy results shown in Fig. 5 (h-a). At least 100 microtubules in tau (49), it is possible that phosphorylation induces conformationalchangesthatmightaffecttau’sactivity in wavs cross-sections of microtubule pellet were evaluated. Closely attached by deletions. microtuhules with no detectable intermicrotuhule distance were con- similar to those brought about sidered to he “hundled.” As previously reported(16), the presenceof regions flanking the repeat domain increase hindingto h x o l stabilized micromicrotubules were polymerized in the presenceof fragments, tubules. Increased hinding to microtubules might contribute 5 , to the increased growth-promoting activitv of the constructs fixed, pelleted, and analyzed by electron microscopy (Fig. h-e, and Fig. 6). Since this procedure requires assembly of containing amino-terminal flanking regions and the fourth free microt,ubules prior to analysisof bundle formation, only repeat. However, the effect of amino-terminal deletions on those fragments capableof nucleating free microtubules were tau’s ability to nucleate microtubules is more complex and tested. When microtubules were polymerized in the presence cannot he explained by the binding data; tau-dependent nuof full-length tau (n123c), almost no microtubules in bundled cleation of microtubules may involve steps that are not afformationwereobserved(Fig. 5 c ) . In the presence of 12.7 fected by its binding affinity to microtubules. Ourstudydoesnotassesstau’seffectonthedynamic 5h). fragmentalmost all microtubuleswerebundled(Fig. When tubulin was incubated with[ 1.541123c, both the extent instabilit,y of individual microtubules (50). However, such a by Drechsel ct nl. using of bundle formation as well as the average number of micro- study has been recently completed t,ubules per bundle were reduced in comparison to 123 frag- real-timevideomicroscopy (.51). Thevfindthatat 3 p~

123F7 [,9,;;kF -I ” ” ”

Organization Functional tubulin and 0.3 PM tau, there is essentially no evidence for dynamic instability of microtubules. Since we work at higher tubulin concentrations, this further lessens the catastrophe frequency in our system. Therefore, it is likely that themean microtubule growth rate measured in our assays reflects the elongation rate. Furthermore, it is unlikely that dynamic instability would account for the absence of nucleation activity, since under our conditions, microtubules exhibitnet growth at both ends even in the absence of any MAPs (52). It has been proposed that microtubule stabilization and bundling result from the neutralization of the acidic carboxylterminal region of tubulin, which can be achieved by the binding of MAPs or a variety of other treatments (22). However, it has been reported that MAPs suppress bundling of microtubules in uitro (53, 54). In this report, we show that a fragment lacking the amino and carboxyl termini (fragment 123) induces bundling of microtubules, whereas full-length tau and a fragment that contains the amino-terminal region and the repeat domain (fragment n123) do not. A fragment containing the carboxyl terminus and the repeat domain has intermediate activity. Therefore, the carboxyl- and aminoterminal regions of tau might hinder microtubules from becoming spatially close to one another; the large number of prolines in the amino-terminal region andthe negatively charged amino acids at the extreme ends of tau suggest an extended structurethat protrudes from the microtubule. This is consistent with the observation of tau as aprojection from the microtubule wall (55). Since fragment 123 assembles microtubules despite the absence of residues upstream of 173 and since extensive bundling was observed in thepresence of this fragment, we suggest that these microtubules are assembled by a mode similar to that achieved by non-MAP related conditions which may involve neutralization of the carboxylterminal region of tubulin (22, 53, 54, 56-58). A non-repeatcontaining MAP4 fragment also exhibits similar behavior (23). Although these activities have mainly been described in uitro, we speculate that animportant featureof tau may beto suppress similar activities in uiuo, leaving the nucleation of microtubules to be regulated by tau alone. Acknowledgments-We acknowledge D. Gard for valuable advice a t several stages of this work, especially regarding set-up of the microtubule regrowth assay. We thank S. L. Rook for technical assistance in the preparation and characterization of E. coli expression plasmids and for help in the blot overlay assay. We also thank K. Williams for assistance at the early stages of E. coli tau protein purification. We are grateful to S. Rubenstein (Cambridge Scientific Computing Inc., Cambridge, MA) for creating Macintosh software for us, to P. A. Paskevich for advice on electron microscopy, and to C. T. Morita for help with graphics. Finally, we thank T. Mitchison and M. Kirschner for helpful suggestions and G . Hall for critically reading the manuscript. REFERENCES 1. Amos, L.A., and Amos, W. B. (1991)Molecules ofthe Cytoskeleton,Guilford, New York 2. Matus, A. (1988) Annu. Rev Neurosci. 11,29-44 3. Tucker, R. P. (1990) Brain Res. Rev. 1 6 , 101-120 4. Binder, L. I., Frankfurter, A., and L. I. Fkbhun, L. I. (1985) J. Cell Biol. 101,1371-1378 5. Brion, J. P., Guilleminot, J., Couchie, D., Flament-Durand, J., and Nunez,

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