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Journal of Science 107, 339-344 (1994) 339 Printed in Great Britain © The Company of Biologists Limited 1994 JCS8430

Kinesin and tau bind to distinct sites on

Pankaj K. Marya, Zarrin Syed, Paul E. Fraylich and Peter A. M. Eagles* Department of and Biophysics, The Randall Institute, King’s College, University of London, 26-29 Drury Lane, London WC2B 5RL, UK *Author for correspondence

SUMMARY

We have used a fluorescent derivative of , AF- β-tubulin subunits. These treated microtubules can no kinesin (kinesin conjugated with 5-(iodoacetamido)fluores- longer bind tau, but are able to bind AF-kinesin in the cein), to investigate the binding site of kinesin on micro- presence of AdoPP[NH]P. Finally, AF-kinesin will support tubules and to compare this site with that to which tau the gliding of subtilisin-digested microtubules in the binds. Microtubules saturated with tau will bind AF- presence of ATP at rates comparable to those obtained with kinesin in the presence of the ATP analogue, 5′-[β,γ- non-digested microtubules. These results show directly that imino]triphosphate (AdoPP[NH]P). This shows that there the binding site for kinesin is outside the C-terminal region are distinct binding sites for the two . Further of tubulin that is removed by subtilisin and is distinct from evidence comes from digestion studies where taxol-sta- the binding site of tau. bilised microtubules were treated with subtilisin, resulting in the of C-terminal residues from both the α- and Key words: kinesin, tau,

INTRODUCTION al. (1990) showed that microtubules that have been digested with subtilisin, so that the MAP2 binding site was lost, were Microtubules have on their surface a number of binding sites still capable of binding kinesin. Using the approach of in vitro for associated proteins, and much work is currently aimed at motility assays (von Massow et al., 1989; Heins et al., 1991), identifying domains that might bind microtubule motors like it was found that the binding of MAP2 to microtubules does kinesin or , and domains that might bind other micro- not prevent microtubule gliding due to kinesin. Thus from a tubule-associated proteins (MAPs) like MAP2 and tau. It is variety of evidence, the two sites for binding are distinct. important to elucidate these issues in order to find out, for Regarding the binding site for tau, the cloning of the genes example, whether those microtubules that do not act as tracks for tau has enabled the microtubule binding domain of the for vesicle transport in neurones (Miller et al., 1987) do so to be determined (Himmler et al., 1989; Butner and because their binding sites for a motor are sterically blocked Kirschner, 1991). The domain, in the basic C-terminal part of by other molecules. the molecule, consists of four repeat regions together with a With MAP2, it has been well demonstrated that the C proline-rich sequence (Kanai et al., 1992; Aizawa et al., 1991). termini of α- and β-tubulin are required for its binding to There is considerable similarity in the repeat regions with those microtubules (Serrano et al., 1984; Littauer et al., 1986), and found in MAP2, which has lead to the proposal that on micro- there is evidence that cytoplasmic dynein also utilizes these tubules MAP2 and tau probably occupy a similar site. The regions for interaction (Paschal et al., 1989). For example, sub- corollary of this is that the tau site is distinct from the kinesin tilisin-digested microtubules, which lack the C-terminal binding site; however, the idea has not been proved by direct peptides of tubulin (Serrano et al., 1984; Littauer et al., 1986; experiment. This would be important to test because there are Maccioni et al., 1986) failed to stimulate the ATPase activity good grounds for believing that the line of argument that of cytoplasmic dynein (Paschal et al., 1989). However, the assumes that what is true for the binding of MAP2 is also true issue is complex because other workers, who also used subtil- for the binding of tau may not be entirely valid. isin-digested tubules, showed that such tubules were in fact Firstly, although MAP2 and tau share similarity in their capable of interacting with dynein (Rodionov et al., 1990). repeat regions they have low in the sequence of the The region on α- and β-tubulin to which MAP2 binds is proline-rich region, which is also important for microtubule probably different from the site on β-tubulin to which kinesin binding (Kanai et al., 1992; Aizawa et al., 1991), thus tau and binds (Song and Mandelkow, 1993). This follows from a MAP2 may differ in their binding details. Secondly, the pro- variety of studies. Those of Cowan and colleagues (Lewis et jection domains on tau and MAP2 differ considerably not only al., 1988, 1989; Noble et al., 1989) indicate that the micro- in sequence but also in length (Lewis et al., 1988; Lee et al., tubule binding domain of MAP2 shares no obvious sequence 1988). The steric influence from these regions on the binding homology with kinesin (Yang et al., 1989), and Rodionov et of other molecules in the vicinity has not been clearly 340 P. K. Marya and others addressed. So again we see that conclusions concerning the Preparation of microtubules with saturating amounts of attachment of kinesin and MAP2 to microtubules may not be tau valid if transposed to the situation regarding the attachment of HPLC-purified tau (300 µg/ml) was incubated (20 minutes, 25¡C) kinesin and tau. with microtubules (up to 60 µg/ml) in Pipes buffer supplemented with In view of this, we have examined, by direct experiment, the 10 µM taxol. The mixture was centrifuged on an airfuge (0.17 MPa, issue of whether there is overlap between the binding sites for 30 minutes) and the pellet, containing microtubule-bound tau, was kinesin and tau by using a novel fluorescent derivative of removed and used for analysis. kinesin, AF-kinesin (Marya et al., 1990a,b, 1991; Löffler et al., Binding assay 1992). In summary, we show by experiment that the binding A mixture of HPLC-purified tau and taxol-stabilized microtubules of tau to microtubules does not interfere with their interaction was made up and incubated as above. A 10 µl sample was allowed involving kinesin. to adsorb to a polylysine-coated, number 1, glass coverslip for 10 minutes. A 20 µl sample of 1% BSA was added, left for 30 minutes and then removed. A 10 µl sample of AF-kinesin (10 µg/ml) con- taining 0.1% BSA and 2.5 mM AdoPP[NH]P was added. The kinesin MATERIALS AND METHODS was left to bind for 1 hour, after which unbound protein was washed away with two additions of Pipes buffer plus taxol (10 µM), and the Materials coverslip was fixed in cold methanol. The coverslip was then blocked DEAE-cellulose 52 was from Whatman. Coverslips were purchased with 1% BSA and stained for the presence of tau using an anti-tau 1 from Clay Adams (Gold Seal). Taxol was a gift from Dr M. Suffness monoclonal antibody (1:500) and an anti-mouse (donkey) Texas (National Institute). Horseradish peroxidase-conjugated and Red-linked secondary antibody (1:50). Several washes with PBS Texas Red-linked secondary antibodies were purchased from were carried out subsequent to each antibody incubation. After the Amersham. All other chemicals were from Sigma. Kinesin was final wash, 20 µl PBS with anti-photobleaching reagent (ascorbic isolated from ox spinal nerve roots, conjugated with 5-(iodoac- acid, pH 7.0, 100 mg/ml) was added to the sample, which was then etamido)fluorescein and purified by high-performance liquid chro- covered with a number 0 glass coverslip and observed under matography (Marya et al., 1990a). immunofluorescence microscopy. The procedure was repeated using subtilisin-digested microtubules in place of the non-digested micro- Preparation of tubulin tubules. Tubulin, prepared by recycling (Murphy and Heibsch, 1979), was purified by DEAE-cellulose column chromatography, and - Electrophoresis ized in Pipes buffer (25 mM Pipes, 0.5 mM EGTA, 0.5 mM EDTA, SDS/PAGE was performed by the method of Laemmli (1970) with 5 mM magnesium chloride, 1 mM dithiothreitol, pH 7.0) by the 10% acrylamide gels. The gels were stained with PAGE blue 83 addition of taxol to 10 µM and GTP to 1 mM. Microtubules (0.3 (BDH). Proteins were immunoblotted onto nitrocellulose, which was mg/ml) prepared in this way were stored frozen in liquid N2, and were subsequently blocked in 1% BSA and then reacted with DM1A, then thawed as required. DM1B YL 1/2, or YOL 1/34. Secondary antibodies were conjugated with horseradish peroxidase (Amersham). Microtubule gliding assay This was based on the assay developed by Vale et al. (1985) and Fluorescence microscopy performed as in the method of Marya et al. (1990a). Samples were viewed using a Zeiss Axiomat microscope equipped with epifluorescence optics. Illumination was from an HBO 100 W Preparation of subtilisin-digested microtubules mercury arc via a heat filter. Sets of standard Zeiss fluorescence filters Taxol-stabilized microtubules (200 µg/ml) in Pipes buffer (supple- (BP 450-490, LP 515-565; BP 546, LP 590) were used. A ×50 mented with 10 µM taxol), generated from DEAE-cellulose 52 objective (numerical aperture, 1.0) together with a ×4 intermediate purified tubulin, were digested with subtilisin (60%, w/w) at 37°C for lens were employed for magnification. Areas were viewed under illu- 60 minutes. Proteolysis was halted by the addition of phenylmethyl- mination appropriate for Texas Red or fluorescein fluorophores, and sulfonyl fluoride (10 mM), after which, digested microtubules showed images were photographed using 35 mm Kodak 3200 ASA film at 30 no changes in their appearance, when viewed by video microscopy second exposure times. over a period of 24 hours. Digested samples were prepared for SDS/PAGE, and run on 10% polyacrylamide gels. Preparation of tau RESULTS Crude tau was prepared from bovine brain using the methods of Lindwall and Cole (1984) and Grundke-Iqbal et al. (1986). This gave Digestion of microtubules with subtilisin gave proteolytic a fraction highly enriched in tau. Further purification was by HPLC cleavage patterns similar to those described previously by using an LKB system. A sample of the tau-enriched fraction (1.5 ml) Paschal et al. (1989). Digestion for 1 hour with a was clarified by centrifugation (0.17 MPa, 30 minutes) on a Beckman subtilisin/tubulin ratio of 60% (w/w) removed all the C- airfuge and then applied to a gel-filtration column (TSK G4000-SW; terminal domains of both α- and β-tubulin, and their removal 600 mm length) equilibrated in Pipes buffer with 50 mM NaCl at a caused an increased mobility of the chains as determined by flow rate of 0.5 ml/minute. Fractions were collected (0.5 ml) and SDS/PAGE (Fig. 1 (A) and (D)). Loss of the C-terminal analysed by SDS/PAGE. Immunoblots of gels stained with anti-tau 1 domain of β-tubulin occurred faster than the loss for α-tubulin. monoclonal antibody (Binder et al., 1985) cross-reacted with a peptide No undigested chains could be detected by immunoblotting. of 50 kDa. Fractions containing this peptide were pooled, dialysed α with Pipes buffer and applied to an ion exchange column (TSK After digestion with subtilisin the -chain loses reactivity DEAE-5PW; 75 mm length). Proteins bound to the column were towards the antibody YL 1/2, indicating a loss of the extreme eluted with a linear salt gradient of 0 to 0.5 M NaCl. The column was C-terminal residues where the epitope is located (Wehland et run at a flow rate of 1 ml/minute and 0.5 ml fractions were collected. al., 1984). The pattern of binding for YOL 1/34 was identical Samples were analysed by SDS/PAGE. to that of DM1A (data not shown), which might be expected Microtubule binding of kinesin and tau 341

DM1B DM1A Fig. 1. Immunoblots of microtubule samples after digestion with subtilisin (60%, w/w). Lanes (A) and (B) show samples that were reacted with DM1B. (A) The preparation after reaction with subtilisin; (B) The control, which had an addition of a small amount of the subtilisin-digested Fig. 3. SDS/PAGE of HPLC-purified tau sample, thus protein (arrowhead). The dye front is emphasizing the clear indicated by the arrow. Molecular mass increase in motility of the β subunit after digestion. (C) and (D) standards are shown on the side. Samples reacted with DM1A. (C) The control preparation before digestion; (D) the digested material. (E) and (F) Similar samples as (C) and (D) from the same gel, though they were here reacted with revealed a stoichiometry of tau to tubulin of about 1:5 (molar YL 1/2; (E) the control sample, and (F) the sample after digestion, where no reaction is seen signifying the complete loss of the epitope ratio). In addition, microtubules were added to the supernatant after digestion. and the experiment repeated; a similar ratio was found. This showed that the tau fractions being used contained an excess of binding-competent tau. Fig. 2. Histogram showing velocities for To investigate the ability of kinesin to bind to tau-saturated individual microtubules microtubules, a study was carried out by using the tau-micro- (2-10 µm in length) that tubule complex adsorbed onto a coverslip, and by adding to moved on kinesin- this AF-kinesin together with AdoPP[NH]P (2.5 mM). The coated coverslips. The sample was then fixed, stained with anti-tau antibodies, and microtubules used here observed under immunofluorescence microscopy (Fig. 4). were digested with Microtubules were fluorescent when viewed under Texas subtilisin (60%, w/w, Red and fluorescein channels (Fig. 4B and A), indicating the for 1 hour at 37¡C) and binding of both tau and AF-kinesin to the microtubules. In showed no evidence of control studies, where microtubules were incubated with having intact C termini on either α- or β- purified tau alone (Fig. 4C and D), the microtubules were seen tubulin subunits. (Sample corresponds to lane A, D and F in Fig. 1.) only in the Texas Red channel and had a similar intensity (Fig. Most of the microtubules in the field moved. F, frequency; 4B). Microtubules when incubated with AF-kinesin alone (Fig. V, velocity. 4E and F) were seen only in the fluorescein channel, thus no spill-over of fluorescence signal was occurring. from a consideration of the position, in the tubulin sequence, When the binding study was repeated with tau and AF- of the antibody’s epitope (Breitling and Little, 1986). kinesin using subtilisin-digested microtubules, no signal was When digested microtubules were applied to a kinesin- observed in the Texas Red channel (Fig. 4H) indicating that coated coverslip, they moved (Fig. 2) and rates were compa- tau does not bind to these modified tubules. Microtubules rable to control preparations, which moved at rates of 0.5-0.6 having similar intensity to the controls were clearly visible µm/second (Marya et al., 1990a). Thus, the C-terminal regions under the fluorescein channel, however (Fig. 4G). This clearly of neither α- nor β-tubulin are necessary for the binding or the showed that subtilisin digestion abolishes the ability of micro- activity of kinesin. tubules to bind tau, though they retain their binding potential We next examined the relationship between the binding site for kinesin. for kinesin and that for tau. Bovine tau was purified, and analysis by SDS/PAGE of selected, pooled fractions (Fig. 3) revealed the presence of a single band with a relative molecular DISCUSSION mass around 50 kDa. This species is at the lower end of the molecular mass range for tau proteins (Butner and Kirschner, We have investigated the microtubule binding sites for kinesin 1991; Kanai et al., 1992). Immunoblots of these fractions and tau molecules by using an active fluorescent analogue of showed cross-reactivity of the polypeptide with anti-tau 1 mon- kinesin together with the technique of subtilisin digestion of oclonal antibody. This sample was used in all further experi- microtubules (Serrano et al., 1984; Rodionov et al., 1990; ments. Paschal et al., 1989). Previous studies (e.g. see Paschal et al., In order to obtain microtubules that have all their binding 1989) have demonstrated that under controlled conditions there sites for tau occupied, taxol-stabilized microtubules were can be loss of the extreme C-terminal ends of both α- and β- mixed with a saturating amount of , then incubated tubulin. Rodionov et al. (1990) showed that kinesin could bind and subsequently pelleted. Analysis of the pellet showed that to subtilisin-digested microtubules, but in this work no analysis the purified tau bound to the microtubules, and densitometry was done of the subunit composition of tubulin. We used 342 P. K. Marya and others

Fig. 4. Immunofluorescence pictures of microtubules showing the distribution of AF-kinesin (fluorescein channel, A,C,E,G) and the distribution of anti-tau (Texas Red channel, B,D,F,H). (A) and (B) tau-saturated microtubules that have been incubated with AF-kinesin; (C) and (D) controls without incubation with AF-kinesin; and (E) and (F) control without tau incubation, taken under identical lighting and exposure conditions as those used for A and B. (G) and (H) Subtilisin-digested microtubules that have been saturated with tau and incubated with AF-kinesin under similar conditions used to produce A and B. Bar in A, 10 µm; all prints are at the same magnification. immunoblot analysis with the antibodies DM1A, DM1B, YL that the C terminus of the α subunit was intact at least to 1/2 and YOL 1/34 to monitor the progress of subtilisin residue α 430 (Breitling and Little, 1986). The β subunit, after digestion. We found that the time course for subtilisin digestion, still reacted with DM1B; thus again we can say from digestion of microtubules was very similar to that described by the location of the epitope to this antibody (Breitling and Little, Paschal et al. (1989). The β subunit showed a drop in mobility 1986) that only the residues beyond about β 430 are missing. first, followed closely by the α subunit. Loss of the α subunit’s We are confident therefore that the microtubules that we used reactivity to YL 1/2, the epitope of which is at the extreme C were modified in both α and β subunits, and thus were similar terminus and which incorporates tyrosine (Wehland et al., to those used by Paschal et al. (1989). 1984), was complete after one hour’s digestion; reactivity to When examined by video microscopy, microtubules DM1A and YOL 1/34 was retained after digestion, suggesting digested for 1 hour (60%, w/w) showed no obvious signs of Microtubule binding of kinesin and tau 343 damage. Estimates were made of their lengths and they were from those removed by subtilisin. Studies by Goldsmith et al. not significantly reduced in size compared with untreated (1991) also point to a region away from the extreme C terminus preparations. These microtubules are active in gliding assays of β-tubulin in the binding of dynein. They used a polyclonal (Vale et al., 1985) with kinesin, clearly showing that the C- antiserum against tubulin, NS20, to inhibit sperm motility and terminal ends of neither the α- nor β-tubulin subunits are found that the inhibitory activity could be removed by needed for kinesin activity in this assay. absorbing out those antibodies that reacted with a peptide cor- We next investigated the issue of whether the binding site responding to residues 400-436 of β-tubulin, thus implicating on microtubules for kinesin overlapped with that for tau. Adult this region in the association of flagellar dynein. The antiserum brain tau consists of a complex mixture of six isoforms that also inhibits both anterograde and retrograde motility of can be phosphorylated to produce a range of polypeptides with organelles in squid axoplasm (Johnston et al., 1986, 1987), molecular masses in the region of 50-70 kDa (Cleveland et al., which has lead to the proposition of a fundamental similarity 1977; Lee et al., 1988, 1989). By using HPLC, we purified one between flagellar motility and the fast species that reacts with anti-tau 1 antibody. We do not know generated by kinesin and cytoplasmic dynein (Goldsmith et al., as yet the structure of this particular tau form, but judging from 1991). However, because NS20 is polyclonal it probably its size it probably corresponds to the S4 or S3 forms, or their contains antibodies to other epitopes, which could well com- phosphorylated versions, previously identified in rat brain plicate the issues unless peptide inhibition experiments are also (Kanai et al., 1992). tau isoforms differ in the size of the micro- performed. Further detailed studies are needed to clarify these tubule repeat region and the N terminus, which is involved in important issues. microtubule bundling (Aizawa et al., 1988; Kanai et al., 1992). Although we have provided evidence that tau and kinesin To what extent these differences might modulate the binding molecules do not interfere with each others binding along the of motor proteins like kinesin is unclear at the present time. microtubules, it is still intriguing how kinesin can function as Binding assays with this tau fraction gave a molar ratio of 5:1 a motor molecule without steric hindrance from tau. Kinesin (tubulin/tau), similar to results from other workers (Butner and is believed to move along single microtubule protofilaments Kirschner, 1991; Cleveland et al., 1977; Hirokawa et al., 1988) (Gelles et al., 1988) through an interaction mediated by the β- when microtubules were saturated with tau, so the tubules we tubulin subunit (Song and Mandelkow, 1993). In order to used had their full complement of tau molecules. generate force production there must be strong interactions tau-saturated microtubules were still capable of interacting between kinesin and microtubules. If tau were to sit towards with AF-kinesin when visualized by fluorescence microscopy. the grooves between subunits, then kinesin may function in the The distribution of AF-kinesin was seen directly by viewing in same way that the motor can still function when the fluorescein channel and the arrangement of tau was identi- sits in the grooves formed from subunits fied less directly after labelling tau with a primary antibody, (e.g., see Squire, 1981). anti-tau 1, and a Texas Red-labelled secondary antibody. In Present evidence indicates that tau is a very flexible this study the same tubule can be clearly seen to be decorated molecule (Lichtenberg et al., 1988; Butner and Kirschner, with both kinesin and tau, thus these molecules can associate 1991) that may link together adjacent protofilaments and, with microtubules at the same time and must therefore occupy because of its relatively weak interactions, migrate over the different attachment sites. It might be argued that the effects surface of the microtubule (Butner and Kirschner, 1991). tau that we are seeing are due to the displacement from micro- must therefore be thought of as being a highly dynamic tubules of tau by kinesin. We think this unlikely because the molecule constantly moving around and easily displaced. intensity of the fluorescent signal from tau-labelled micro- These properties would allow it to accommodate itself tubules did not change in the presence of kinesin nor did it admirably to the activities of kinesin. change by varying the amount of kinesin in the preparation. Further support for the idea that kinesin and tau occupy We thank the Medical Research Centre and the Wellcome Trust for different attachment sites comes from the study where tau and support. We also thank Mrs Retnam Rao for technical help, Stephen AF-kinesin were incubated with subtilisin-digested micro- Blose for supplying DM1A and DM1B, Bob Burgoyne for giving YL tubules. Much previous work (Littauer et al., 1986; Serrano et 1/2, John Kilmartin for supplying YOL 1/34, and Roy Burns for donating anti-tau 1 antibody, and for advice on the preparation of tau. al., 1985; Maccioni et al., 1986) has implicated the C-terminal regions of tubulin in the association of tau with microtubules. After subtilisin digestion of microtubules, we demonstrated by fluorescence microscopy that, whereas tau binding was com- REFERENCES pletely abolished, the tubules were still capable of being Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K. and Sakai, decorated by AF-kinesin. We conclude therefore that the H. (1988). Microtubule binding doamin of tau proteins. J. Biol. Chem. 263, binding domains for tau and kinesin must be largely distinct 7703-7707. and that the molecules do not interfere with each others asso- Aizawa, H., Emori, Y., Mori, A., Murofushi, H., Sakai, H. and Suzuki, K. ciation on the microtubule surface. (1991). 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