Competition between Motor Molecules (Kinesin and Cytoplasmic Dynein) and Fibrous Microtubule-associated Proteins in Binding to Microtubules*

In neuronal cells, microtubule-associated proteins (MAPs) can be classified into two distinct groups. One consists of force-producing M A P S , the main components of which are kinesin and cytoplasmic dynein. The other is composed of fibrous M A P S , which include tau and MAP2. Many studies have been performed on the respective groups to understand their structures and func- tions. However, the problem of how the groups interact with each other on microtubules is still unresolved. ’Ib elucidate the interaction between kinesin or cytoplasmic dynein and tau or MAP2, we performed three ex- periments: competition, motility assay, and cosedimentation. To distinguish whether the binding competition is caused by steric hindrance of the projection domains of MAPs or by the competition of the binding sites on microtubules, we used microtubule binding domains of tau and MAP2 as well as native proteins. Our results revealed that kinesin or cytoplasmic dynein and tau or MAP2 compete for almost the same binding domains lo- cated on the carboxyl-terminal side of a- and the amino-terminal side of @-tubulin from the site of subtilisin cleavage. Furthermore, the projection of tau, and probably of MAP2, might inhibit the binding of kinesin or cytoplasmic dynein to microtubules by steric hindrance. These findings will provide a useful step toward under- standing the regulation system of intracellular organelle transport. objective and ICT condenser (Zeiss). The images were filmed with a video camera (Hamamatsu Photonics) and processed with an ARGUS-10 image processor (Hamamatsu Photonics). In real time, the images were recorded on a videocassette recorder (model EVO-9650; Sony) and analyzed directly from a monitor screen. The final magnification on the video screen was x 8,000. We confirmed that more than 95% of the microtubules showed gliding motility at a velocity of about 0.5 pm/s (kinesin) and 1.5 p d s (cytoplasmic dynein). Then we added taupw, tauBs, or “2,s to the assay system by the following three procedures to test the effects of these proteins on the kinesin- or cytoplasmic dynein-induced microtubules motility. First, 7 pl of kinesin or cytoplasmic dynein solution was preadsorbed on a coverslip for 12 min, and then 1 pl of tau or “2 and 1 pl of microtubules was added to the solution (procedure a); tau or MAP2 and microtubules were incubated for 3 min, and then 2 pl of the mixture was added to the solution (procedure b); or the mixture obtained from procedure b was centrifuged further at 100,000 x g for 3 min, and 2 pl of the resuspended pellet was added to the solution (procedure c). Analysis of the images was performed in the same manner as described above. To check the activities of motors, we observed MAPS-free preparation every time aRer MAPS-containing preparation and confirmed that motility was restored to nearly 100%.

It is well known that microtubules are one of the main cytoskeletal elements in all eukaryotic cells and are particularly abundant in neuronal cells. The microtubules in living cells are used for diverse movements (such as organelle transport and cell division) and the maintenance of cell shape. Microtubuleassociated proteins ( M A P S ) ' have been thought to play an important role in mediating these functions of microtubules. In neuronal cells, MAPs can be classified into two main groups. The first group is involved in microtubule-based movements, and they are called force-producing M A P S or motor molecules. Major components of this group are kinesin and cytoplasmic dynein. Kinesin consists of two heavy chains (120-124 kDa) * This work was supported by a special grant-in-aid for scientific research; a grant-in-aid for scientific research from the Japan Ministry of Education, Science, and Culture (to N. H.); and by a grant from the Institute of Physical and Chemical Research (RIKEN) (to N. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The abbreviations used are: M A P @ ) , microtubule-associated proteinb); Pipes, 1,4-piperazinediethanesulfonic acid; PW, purified whole molecule; BS, binding site; AMP-PNP, 5'-adenylyl-@,yimidodiphosphate; PAGE, polyacrylamide gel electrophoresis. and two light chains (62-64 kDa) Kuznetsov et al., 1988). The heavy chains contain binding sites for ATP and microtubules (Gilbert and Sloboda, 1986;Penningroth et al., 1987;Bloom et al., 1988;Ingold et al., 1988;Young et al., 1988;Kuznetsov et al., 1989). Morphological observations suggest that kinesin binds to microtubules by globular domains that contain the heavy chains and that attachment sites for organelles are at the opposite, fan-shaped end of kinesin, where light chains are located Scholey et al., 1989). The role of kinesin was suggested to be that of a motor for anterograde transport because it can produce force toward the plus ends of microtubules in vitro. The rate of kinesininduced microtubule gliding on coverslips was evaluated as 0.3-0.6 @s (Vale et al., 1985a(Vale et al., , 1985b(Vale et al., , 1985c. Kinesin may be responsible for anterograde organelle transport in neurons (Vale et al., 1985a;Hirokawa et al., 1991).
Cytoplasmic dynein consists of two heavy chains (410 kDa) thought to be the site of ATP hydrolysis; three subunits of 74 kDa; and 59-, 57-, 5 5 , and 53-kDa subunits (Lye et al., 1987;Vallee et al., 1988). The role of cytoplasmic dynein was suggested to be that of a motor for retrograde transport because it can produce force toward the minus ends of microtubules in vitro Schnapp and Reese, 1989;Schroer et al., 1989). The rate of cytoplasmic dynein-induced microtubule gliding on coverslips was evaluated as 1-2 pm/s (Lye et al., 1987). Cytoplasmic dynein may be responsible for retrograde organelle transport in neurons (Hirokawa et al., 1990;Vallee and Shpetner, 1990;Vallee, 1991).
A great number of studies have been carried out both on force-producing MAPs and fibrous M A P S . However, little attention has been paid to how these force-producing MAPs and fibrous M A P S interact with each other on microtubules. This is an interesting and important problem when we consider how the microtubule-based motility is regulated. As for the interaction of kinesin and the fibrous MAPs, Mandelkow's group has studied this question (Massow et al., 1989;Heins et al., 1991). They used native kinesin and MAP2, MAP2c or tau, but their approach was restricted to the use of a motility assay. As for the interaction of cytoplasmic dynein and the fibrous MAPs (mainly MAP2), two conflicting results were published. Paschal et al. (1989) reported that cytoplasmic dynein and native MAP2 compete for binding to microtubules and that they share a common or overlapping binding site on the carboxyl termini of tubulin subunits, whereas Rodionov et al. (1990) reported that cytoplasmic dynein as well as kinesin and high molecular weight MAPs do not compete for binding to microtubules and that their binding sites are different. Recently, Lopez and Sheetz (1993) showed steric inhibition of cytoplasmic dyneinand kinesin-induced microtubule motility by native M A P 2 .
To solve this important unsettled question and to elucidate precisely the interaction between kinesin or cytoplasmic dynein (main components of force-producing M A P S ) and tau or MAP2 (main components of fibrous M A P S ) in binding to microtubules, we performed three distinct experiments: competition, motility assay, and cosedimentation. To exclude the possibility of competition being caused by the steric hindrance of projection domains of tau and M A P S , we used their binding domains as well. Our results revealed that kinesin or cytoplasmic dynein and tau or MAP2 compete for almost the same binding domain located on the carboxyl-terminal side of a-and the amino-terminal side of p-tubulin from the point of subtilisin cleavage. Furthermore, the projection of tau and probably that of MAP2 can be considered to inhibit the binding of kinesin or cytoplasmic dynein to microtubules by steric hindrance.

EXPERIMENTAL PROCEDURES
Materials-Taxol was a generous giR of Dr. N. R. Lomax, Drug Synthesis and Chemistry Branch, Developmental Therapeutics Programs, Division of Cancer Treatment, National Cancer Institute. Subtilisin Carlsberg (protease type VIII) was from Sigma. Bovine serum albumin was from Boehringer Mannheim.
Tau was prepared from porcine brain as described previously, with slight modification (Hirokawa et al., 1988a(Hirokawa et al., , 1988b. The ammonium sulfate precipitation was suspended in 1 volume of PEM and then subjected to Superose 6 preparation grade column chromatography (Pharmacia LKB Biotechnology Inc.). The peak fractions were dialyzed against PHEM (50 nm Pipes, 50 m Hepes, pH 7.0,2 m MgCl,, 1 m EDTA) and stored at -80 "C until use. MAP2 was also prepared as described previously (Hirokawa et al., 1988a(Hirokawa et al., , 1988b. Tau and MAP2 purified from the above procedures were designated as taupw and MPw (pw: purified whole molecule), in contrast to the Escherichia coli expressed products tauas and mas binding site), described below.
Kinesin was prepared from porcine brain as described previously, with slight modification (Sato-Yoshitake et al., 1992). The first resuspended solution was incubated for 30 min at room temperature, 10 min at 37 "C, and then centrifuged at 100,000 x g for 50 min at 20 "C. The resulting supernatant was dialyzed against PEM for 10 h at 4 "C instead of using gel filtration. The dialysate was collected, and kinesin was attached again to purified microtubules using 2 nm AMP-PNP and then released using 10 nm ATP. The kinesin-enriched solution was carefully applied to 12 ml of 5 2 0 % sucrose density gradients in PEM buffer and centrifuged at 200,000 x g in an SW 40-Ti rotor (Beckman Instruments) for 14 h at 2 "C. The gradients were collected in 0.5-ml fractions and stored at -80 "C until use. Cytoplasmic dynein was prepared from rat brain. Rat brains (60 g) were briefly homogenized in a Warring blender in 1 volume of extraction buffer (PHEM containing 1 m phenylmethylsulfonyl fluoride, 10 pdml leupeptin, 10 pglml pepstatin A, 10 pglml p-tosyl-L-arginine methyl ester hydrochloride, and 0.5 m dithiothreitol). The homogenate was centrifuged at 95,500 x g for 30 min at 2 "C. The supernatant was recovered and centrifuged at 175,000 x g for 70 min at 2 "C. The supernatant was again recovered, added to 6 m taxol and 0.2 m GTP, and the sample was warmed to 25 "C for 20 min (Amos, 1989). Then the sample was centrifuged at 50,000 x g for 30 min at 20 "C. The supernatant was recovered; added to 10 pv taxol, 10 unitdml hexokinase, and 50 nm glucose; and warmed to 25 "C for 30 min. The sample was centrifuged at 50,000 x g for 30 min at 20 "C again. The pellet was washed twice in extraction buffer containing 50 m KC1 and 5 m taxol.
The second washing solution contained 3 m GTP to remove the contaminating kinesin . After washing, the pellet was resuspended in 3 volumes of extraction buffer containing 100 m KCl, 20 p taxol, and 10 m ATP and incubated for 20 min at 25 "C to dissociate cytoplasmic dynein from microtubules, followed by centrifugation at 190,000 x g for 30 min at 20 "C. The supernatant was concentrated by Centricon-100 (Amicon) at 1,000 x g for 3-4 h at 2 "C. The concentrated sample was carefully applied to 12 ml of 5-20% sucrose density gradients in PHEM buffer containing 50 nm KC1 and centrifuged at 200,000 x g in an SW 40-Ti rotor for 13 h at 2 "C. The gradients were collected in 0.5-ml fractions. The purified cytoplasmic dynein was used within 24 h a h r fractionation because its activity is lost after freezing and thawing (Amos, 1989).
Construction of cDNAs of n u and MAP2 Deletion Mutants Containing Microtubule Binding Domain-Tau deletion mutant (tau,& lel:tauBs) was constructed by the oligonucleotide-directed in vitro mutagenesis system (Amersham Corp.) using the clone pTAU-16 (L4) (Kanai et al., 1989. MAP2 deletion mutant (MAPP-16&L:MAP2BS) was newly constructed according to the method described above (Umeyama et al., 1993). TauBs contains four repeats of microtubule binding domains and the proline-rich region, and M A P 2 B s contains three repeats of microtubule binding domains and the proline-rich region. Each sequence was inserted into PET expression vectors for the transformation into E. coli BL21(DE3) pLYS-S cells.
Expression and Purification of Microtubule Binding Domain of Tau or MAP2 (Tau~s or MAP2&"Transformed E. coli cells were grown in M9ZB with Amp and Chl media in fermenters to an absorbance of 20 at 595 nm. Isopropyl-1-thio-0-D-galadopyranoside (0.4 m) was added to induce tau or MAP2 expression. After reaching an absorbance of 30, the cells were centrifuged, resuspended in PE (100 m Pipes, 1 m EGTA, pH 6.9) with 0.1 m phenylmethylsulfonyl fluoride, and then sonicated on ice 15 times at 30-s intervals using an ultrasonic disruptor (model UD-201, TOMY) at 100 watts. The suspension was clarified by centrifugation at 18,000 x g for 15 min at 4 "C. The supernatant was then heated at 95 "C in boiling water for 5 min and centrifuged at 95,000 x g for 30 min at 4 "C. The resulting supernatant was dialyzed for 12 h against PE. Then the samples were subjected to column chromatography. In the case of tauas, the dialysate was loaded onto P11 equilibrated in PE, eluted with 1 M NaCl in PE, and then fractionated. Mas was loaded onto Bio-Gel A-0.5m (Bio-Rad) equilibrated in PE and fractionated. Peak fractions were detected by the assays of Bradford (1976) using bovine serum albumin as a standard and were collected into one tube. Ammonium sulfate was added to the sample to achieve 50% saturation (to precipitate tauBs) or 100% saturation (for MAP2, , ). The precipitated proteins were collected by centrifugation at 30,000 x g for 30 min at 4 "C, and the pellets were resuspended in PHEM. The samples were dialyzed for 1 h using UH100/25 (Schleicher & Schuell) against PHEM.
Cleauage of Thbulin with Subtilisin-'lko types of subtilisin-cleaved microtubules, (10. and aspa, were prepared by the procedure of Rodionov et al. (1990) and Melki et al. (1991), with some modifications. (10.tubulin indicates the tubulin in which only the 0-subunit was cleaved, and a.p.-tubulin is the one that had both subunits cleaved. Subtilisin treatment was performed at 37 "C for 15 min to obtain (16. (Melki et al., 1991) and for 13-14 h to obtain (1.0. (Rodionov et al., 1990). The resulting microtubules were processed with 10% polyacrylamide gel containing 0.1% sodium lauryl sulfate at pH 9.2 for optimal resolution of (I-and 0-tubulin . Kinesin or Cytoplasmic Dynein and Taupw or TauBs Competition Experiment-Kinesin or cytoplasmic dynein, microtubules and tau (tau, or tauBs) were mixed and incubated in the presence of 20 m taxol for 15 min at room temperature and then for 10 min at 37 "C. The mixture was applied onto a cushion containing 100 pl of 4 M glycerol. The glycerol solution was prepared in PHEM buffer with 2.5 m tripolyphosphate, 0.5 m MgCl,, and 20 m taxol. Centrifugation was performed at 100,000 x g for 40 min at 30 "C to pellet down microtubules and bound tau. The supernatant and the pellets were examined by SDS-polyacrylamide gel electrophoresis (PAGE). Each of the three protein bands was measured with a densitometer (model CS 9000, Shimadzu Corp).
Assay for Kinesinor Cytoplasmic Dynein-induced Gliding of Microtubules in the Presence of Taupw, TauBs, or MAP2,,Motility assay was performed by the method of Vale et al. (1985aVale et al. ( , 1985b and Sato-Yoshitake et al. (1992) with some modification. Kinesin or cytoplasmic dynein solution (7 pl, 25 pglml) with 20 taxol was applied to a glass coverslip and allowed to adsorb for 12 min at room temperature. Taxol-stabilized microtubules (1 pl) were added to the solution. The  (2), 50 p1(3), or 100 pl(4) of taupw (500 pp/ml) or tauBs (340 pglml) was added to the solution. The lines of pellets (PI-P4) in both A and B suggest that microtubulebound kinesin ( I ) and cytoplasmic dynein ( I I ) are reduced compared with that of the control in a dose-dependent manner to tau. In the supernatants, kinesin (1) and cytoplasmic dynein ( I I ) appear clearly in both A and B . Molecular masses are shown in m a . Electrophoresis was performed with 7.5% polyacrylamide gel for A and 12.5% for B.

panel IIB, cytoplasmic dynein and tauBs. Lane Tau is taupw (A) or tauBs ( B ) . Lane C is a control (A and B ) . 10 pl ( I ) , 25 p1
coverslip was applied to a glass slide, and the edges of the coverslip were sealed with nail polish. The microtubules were visualized with an AVEC-DIC system, using a n h o p h o t microscope equipped with a x 100 1.3NA Plan-Neofluar objective and ICT condenser (Zeiss). The images were filmed with a video camera (Hamamatsu Photonics) and processed with an ARGUS-10 image processor (Hamamatsu Photonics). In real time, the images were recorded on a videocassette recorder (model EVO-9650; Sony) and analyzed directly from a monitor screen. The final magnification on the video screen was x 8,000. We confirmed that more than 95% of the microtubules showed gliding motility a t a velocity of about 0.5 pm/s (kinesin) and 1.5 p d s (cytoplasmic dynein). Then we added taupw, tauBs, or " 2 , s to the assay system by the following three procedures to test the effects of these proteins on the kinesin-or cytoplasmic dynein-induced microtubules motility. First, 7 pl of kinesin or cytoplasmic dynein solution was preadsorbed on a coverslip for 12 min, and then 1 pl of tau or "2 and 1 pl of microtubules was added to the solution (procedure a); tau or MAP2 and microtubules were incubated for 3 min, and then 2 pl of the mixture was added to the solution (procedure b); or the mixture obtained from procedure b was centrifuged further a t 100,000 x g for 3 min, and 2 pl of the resuspended pellet was added to the solution (procedure c). Analysis of the images was performed in the same manner as described above. To check the activities of motors, we observed MAPS-free preparation every time aRer MAPS-containing preparation and confirmed that motility was restored to nearly 100%.
Cosedimentation Then 15 111 of 50 pdml bovine serum albumin was loaded onto a lane of each gel. The SDS-PAGE gel was scanned with a densitometer, and the protein concentration was determined from the peak area, using bovine serum albumin as a primary standard.

Competition between Kinesin or Cytoplasmic Dynein and Tau for Binding to Microtubules
Kinesin and cytoplasmic dynein were obtained as described under "Experimental Procedures," and fractions of each molecule were processed on SDS-PAGE (data not shown). The fraction that showed the peak of the concentration of kinesin or cytoplasmic dynein was used for the following experiments.
We performed a competition experiment to test whether kinesin or cytoplasmic dynein and tau compete for binding to microtubules. First, we tested kinesin or cytoplasmic dynein and taupw. Taupw was a native, whole molecule purified from porcine brain (see "Experimental Procedures"). Kinesin or cytoplasmic dynein, tauw, and microtubules were mixed and incubated for 15 min at room temperature and then for 10 min at 37 "C. The binding of kinesin, cytoplasmic dynein, or tau to microtubules had been confirmed by a preliminary experiment. Centrifugation was performed a t 100,000 x g for 40 min to pellet down the microtubules. SDS-PAGE of the resulting supernatants and pellets is presented in Fig. 1, ZA (kinesin) and ZZA (cytoplasmic dynein). It was clear that kinesin or cytoplasmic dynein appeared in the supernatant and that microtubulebound kinesin or cytoplasmic dynein was reduced in amount compared with the control in which the pellet was obtained from a kinesin or cytoplasmic dynein and microtubule mixture without tau and processed in the same manner (lane C).
To exclude the possibility that the competition might result from steric hindrance of the projection of tau, we used t a u~s , which was expressed in E. coli and purified as described under The constructs of the binding sites of tau ( t a u , ) and "2 ("2,s) are presented in addition to the full-length of tau (corresponding to L4; Kanai et al., 1992) and h"2. The thick lines indicate the region of tauBs and W 2 B S . The shaded bows represent the repeats of the microtubule binding site.
"Experimental Procedures." TaUBs contained four repeats of microtubule binding sites and the proline-rich region. The construct of tauBs is shown in Fig. 2. We checked the purity of the products of this construct on SDS-PAGE. TauBs showed a single band of a molecular mass of 38 kDa (SDS-PAGE not shown). A competition experiment was then carried out in the same manner as for taupw. The resulting supernatants and pellets are shown in Fig. 1, ZB (kinesin) and ZZB (cytoplasmic dynein). The bands of kinesin and cytoplasmic dynein also appeared in the supernatants, and microtubule-bound kinesin and cytoplasmic dynein in the pellets were reduced in amount compared with the control.
We performed another experiment to test whether tau directly binds to motors, resulting in the pellets after centrifugation in the previous experiments. Kinesin or cytoplasmic dynein and tau (taupw or tauBs) were mixed and centrifuged as described above. The supernatants and pellets were examined on SDS-PAGE. It was shown from our result that motors and tau appeared in the supernatants, whereas no band is detectable in the pellets (data not shown). This result clearly denies the possibility that tau-motor complexes were contaminants in the competition experiments.
The amounts of microtubule-bound kinesin, cytoplasmic dynein, and tau were further analyzed densitometrically, and the results are summarized in Fig. 3, Z (kinesin), ZZ, (cytoplasmic dynein), ZZZA (taupw), and ZZZB (tauBs). The vertical line represents the relative value of the control ( Z and ZZ) and of the amount of microtubule-bound tau (ZZZ). The figure indicates that the amounts of microtubule-bound kinesin and cytoplasmic dynein were reduced, and microtubule-bound tau was increased by the addition of tau in a dose-dependent manner ( p < 0.01; Jonckheere's test).
Although the difference between taupw and tauBs in this experiment was not statistically significant for either kinesin ( Fig. 31) or cytoplasmic dynein (ZZ), it became quite obvious in the following microtubule gliding experiment.

Effect of Tau on Kinesinor Cytoplasmic Dynein-induced
Microtubule Gliding Kinesin and cytoplasmic dynein are motor molecules. To observe the competition while these proteins are working, we tested the effect of tau on kinesinor cytoplasmic dynein-induced microtubule gliding using a motility assay system. We prepared the samples according to procedures illustrated in Fig. 4 and observed the motility using the AVEC system.

Effect of Tau on Kinesin-induced Microtubule
Gliding-First, we tested the effect of taupw. Taupw and microtubules were added to the kinesin solution (procedure a). The saturation ratio of taupw to microtubules had been determined to be 1:4 (pglpg) in preliminary experiments. Taupw was added to each solution at the ratios to microtubules of 1:4, 2:4, 4:4, and 10:4 (pg/pg). The results of this procedure are summarized in Fig. 5  ZA(a). The vertical line represents the relative values of the number of gliding microtubules to total microtubules. In the control, a preparation of kinesin and microtubules without tau, >95% of microtubules showed motility at a velocity of 0.5 p d s (Fig. 51, column C). The horizontal line represents the mass ratio of taupw to microtubules. It is clear that kinesin-induced microtubule gliding was inhibited by the addition of taupw. However, >90% of microtubules still showed motility in the presence of taupw at 1:4. ' , We conjectured that the weak inhibition in procedure a reflected the small alpount of taupw bound to microtubules. Indeed M A P S are known to attach to glass surfaces under various conditions. We therefore performed another procedure to confirm the binding of taupw to microtubules, as follows. Taupw and microtubules were incubated for 3 min, and then the mixture was added to the kinesin solution which had been preadsorbed on a coverslip (procedure b). The result is shown in Fig.  5ZA(b). The inhibition was more prominent in comparison with procedure a. Motility was blocked to about 35% at the saturation ratio (1:4). Motility was restored at a 2/3 dilution (1:6), whereas it was reduced to about 12% at 4:4.
To exclude the possibility that free taupw, which was not bound to microtubules, would influence microtubule gliding, we carried out another procedure as follows. Taupw and microtubules were incubated for 3 min, and then the mixture was centrifuged further at 100,000 x g for 3 min. The resuspended pellet was added to the kinesin solution (procedure c). The result is summarized in Fig. 5ZA(c). Taum inhibited microtubule motility to around 5% at 1:4. Motility was restored to 13% a t a 2/3 dilution (1:6). In procedure a, motors were preadsorbed on the coverslip, and then MAPs and microtubules were added to the solution. The inhibition of microtubule gliding was weak, presumably reflecting the small amount of M A P S binding to microtubules. In procedure b, to confirm the binding of M A P S to microtubules, M A P S were preincubated with microtubules before adding to the solution. M A P S showed inhibition of microtubule gliding, but we could not observe microtubule bundles. In procedure c, the preincubated M A P S and microtubules were centrifuged to wash out free M A P S . Microtubules formed bundles, and the inhibition of microtubule gliding was prominent. This result argues against the presumption that free M A P S directly interact with motors preventing their association with microtubules.
Secondly, we tested tauBs to exclude the possibility that the inhibition could result from steric hindrance by the projection of tau. The saturation ratio of tauBs was determined to be 1:4 (pglpg). TauBs was added at the same ratio as taupw. Almost the same result was obtained as for taupw by procedure a (data not shown). TauBs and microtubules were incubated for 3 min, and the mixture was added to the solution (procedure b), the result being summarized in Fig. 5ZB(b). Although it was less severe than in the case of taupw (Fig. 5ZA(b)), inhibition was stronger in comparison with procedure a. TauBs was added according to procedure c, the result being summarized in Fig.SZB(c). Motility was restrained to around 5% in 1:4 and restored to 25% at a U3 dilution (1:6).
Effect of Tau on Cytoplasmic Dynein-induced Microtubule Gliding-Tau-or tauBs was added to the cytoplasmic dynein solution in the same manner as kinesin. In the control, >95% of microtubules showed motility a t a velocity of 1.5 p d s (Fig. 511,  column C).
The effects of taupw are summarized in Fig. 5ZZA. It is clear that cytoplasmic dynein-induced microtubule gliding was inhibited by the addition of taupw by procedure a. However, >80% of microtubules still showed motility a t 1:4 ( Fig. 5ZZA(a)). No motility was observed at a saturation ratio (1:4), and only slight motility was restored at a 114 dilution (1:16) by procedure b (Fig. 5ZZA(b) ).
The effects of tauBs are summarized in Fig. 5ZZB. The result was almost the same as for taupw by procedure a (data not shown). When tauBs was added by procedure b (shown in Fig.  5ZZB(b)), inhibition was stronger in comparison with procedure a, although it was less prominent than in the case of taupw ( Fig. 5ZZA(b)). As illustrated in Fig. 5ZZB(c), tauBs inhibited microtubule motility to around 10% at 1:4 by procedure c. Motility was restored to 20% at a 213 dilution.
It is clear that both taupw and tauBs inhibited both kinesinand cytoplasmic dynein-induced microtubule motility in a dosedependent manner. When comparing taupw with tauBs, as illustrated in Fig. 5,ZA(b) and ZB(b) (kinesin), and Fig. 5,ZZA(b) and ZZB(b) (cytoplasmic dynein), the inhibitory effect of taupw is more severe than that of tauBs both on kinesin and cytoplasmic dynein.
Effect of MAP2 on Kinesin-or Cytoplasmic

Dynein-induced Microtubule Gliding
To determine the effect of other fibrous M A P S on kinesin-or cytoplasmic dynein-induced microtubule gliding, we tested MAP2. MAPBBS, prepared by the same method as ~U B S , contained three repeats of microtubule binding sites and the proline-rich region (Fig. 2) and showed a single band of a molecular mass of 26 kDa by SDS-PAGE (data not shown). The saturation ratio of m 2 B S to microtubules was determined to be 1:6 (pglpg). MAp2Bs was added according to procedure c, to the kinesin or cytoplasmic dynein solution at the ratios to microtubules of 1:6,2:6,4:6, and 6:6 (pglpg). As illustrated in Fig. 6, w 2 B s as well as tauBs inhibited kinesin (Fig. 61) and cytoplasmic dynein (6ZZ)-induced microtubule gliding. These results of the motility assay indicate that both tau and MAP2 inhibit both kinesin-and cytoplasmic dynein-induced microtubule gliding in a dose-dependent manner.
Cosedimentation of Kinesin, Cytoplasmic Dynein, Tau, or MAP2 with ap-, a&-, or a,P,-Microtubules Cosedimentation experiments using subtilisin-cleaved microtubules were carried out .to define the binding sites of kinesin and cytoplasmic dynein on microtubules. We prepared @,and a,@,-microtubules as described in "Experimental Procedures." The results of gel electrophoresis clearly indicated that the subtilisin digestion for 15 min shifted only the @-subunit, whereas a 13-14-h digestion shifted both a-and @-subunits (SDS-PAGE not shown). The negatively stained electron micrographs of a@-, a@,-, and a,@.-microtubules showed quite similar morphology (data not shown). We tested whether kinesin or I Kinesin  Fig. 8).

20
The binding ratio of every molecule to ap:afls:asPa can be regarded as 1:(0.8-1.0):(0.6-0.7). induced a&microtubule gliding. The same experiment as described we tested not whether h e s i n or cytoplasmic dynein binds in Fig. 4 was carried out for ~B S by procedure C. panel I Shows the to and induces motility but also whether tau inhibits the effect on kinesin, and panel ZZ, that on cytoplasmic dynein. Each column represents mean * S.E. mas also showed inhibition of kinesin-(1) kinesinor cytoplasmic dynein-induced asps microtubule moand cytoplasmic dynein-(ZZ) induced microtubule gliding.
tility. TaUBs was used to exclude the projection effect. Almost 70% of asp, microtubules moved on the kinesin-coated coverslip cytoplasmic dynein and other M A P S could bind to them. Kine-(shown in Fig. 91, column C ) , and more than 80% of a,& sin, cytoplasmic dynein, taupw, tauBs, MAP2,, or MAP2Bs microtubules moved on the cytoplasmic dynein-coated coverslip was incubated with 150 pg of ap, ape, or asps and then applied (Fig. 911, column C ) . As with a,p,-microtubules preincubated to a 4 M glycerol cushion and centrifuged. Resulting pellets with tauBs, similar inhibition in a dose-dependent manner was . It is suggested that the binding of each molecule to aP or ap. was nearly equal, although the bindings to asps were reduced to some degree. Molecular masses are indicated in thousands. Electrophoresis was with 7.5% or 12.5% polyacrylamide gel. observed with ap-microtubules (kinesin is summarized in Fig.  91, cytoplasmic dynein in Fig. 911).

DISCUSSION
Both Kinesin and Cytoplasmic Dynein Compete with Tau or MAP2 for Binding to Microtubules-% gain insight into the interaction of kinesin or cytoplasmic dynein and fibrous M A P S , we carried out two distinct experiments, one being a competition experiment and the other a motility assay.
The competition experiment revealed that when kinesin or cytoplasmic dynein and tau were incubated with microtubules simultaneously, the amount of microtubule-bound kinesin or cytoplasmic dynein in the pellet was reduced, and microtubulebound tau in the pellet was increased in a dose-dependent manner to tau. Although in the competition experiment the difference between taupw and tauBs was not statistically significant, meaning that the effect of the projection of tau was not clear, the difference between them became very distinct in the microtubule gliding experiment.
Competition between kinesin or cytoplasmic dynein and the fibrous M A P S was also observed in the motility assay experiment. First, we confirmed that kinesin or cytoplasmic dynein induces more than 95% of microtubule gliding without tau or MAP2, and then we added tau or MAP2 to the solution. The dose of tau or "2 ranged from 1/4 to 10 times the saturation ratio. The saturation ratios of taupw and tauBs to tubulin were determined to be 1:4 (pg/pg) and 1:4 (pg/pg) and that of m 2 B S was 1:6 (pg/pg). The molar ratio of taupw to tubulin was 1:4.4, using an average molecular weight of 60,000 for taupw, and t a u~s to tubulin was 1:2.8 using a molecular weight of 38,000 for tauBS. The molar ratio of taupw is in good agreement with our previous study (ranging from 1:3 to 1:6) (Hirokawa et al., 1988a(Hirokawa et al., , 1988b, and the difference in that between taupw and tauBs might be explained by the steric hindrance of the projection of tau. Kinesin and cytoplasmic dynein still induced more than 80% of microtubule gliding in the presence of tau (taupw or t a u~s ) a t the saturation ratio by procedure a. Therefore we performed procedure b to confirm the binding of tau to microtubules. Both taupw and tauBs showed the inhibition of kinesin-or cytoplasmic dynein-induced microtubule gliding in a dose-dependent manner, and the inhibition with taupw was clearly greater than with t a~B s for this procedure (kinesin: Fig. 5 of the effect of steric hindrance of the projection of tau. We could not exclude the possibility that free tau, which was not bound to microtubules, may have some effect on microtubule gliding in procedure b. Thus, in procedure c, we pelleted down microtubule-bound tau and washed out free tau. For this procedure, tau showed almost the same inhibition of kinesin-or cytoplasmic dynein-induced microtubule gliding at the ratio to microtubules from 1:4 to 4:4 (shown in Fig. 5, M(c) and B(c) (kinesin), and 5ZZB(c) (cytoplasmic dynein)). This result argues against the possibility that tau directly interacts with the motor preventing its association with microtubules. The results of the motility assay using taupw or tauBs suggest that kinesin or cytoplasmic dynein and tau compete for the same, or at least overlapping, binding domains on microtubules in a dose-dependent manner to tau. Furthermore, the projection of tau might block the binding of kinesin or cytoplasmic dynein to microtubules by steric hindrance.
It is reasonable to consider that the projection of MAP2 might also block the binding, and therefore we used M 2 B s to test the effect of MAP2 on kinesin and cytoplasmic dyneininduced microtubule gliding. The results suggest that kinesin or cytoplasmic dynein and MAP2 compete for the same binding domain in a dose-dependent manner to "2.
Combined with the results of the competition experiment and motility assay, we are able to conclude that force-producing MAPs such as kinesin and cytoplasmic dynein and fibrous M A P S such as tau and " 2 compete for binding to microtubules on the same, or at least overlapping, domain.

MAP2 Locate on the Same Portion of Both a-and P-Tubulin
"The result of the motility assay using subtilisin-cleaved microtubules (a.Pa) revealed that even if both carboxyl termini of a-and p-tubulin were removed, microtubules retained their ability to bind to kinesin or cytoplasmic dynein and tau. Therefore, it seems to be clear that the binding domains of kinesin or cytoplasmic dynein and tau are shared with at least the aminoterminal side of the subtilisin cleavage site on microtubules. However, the locations of the binding domains of the respective molecules on microtubules are still obscure. 'Ib define the binding domain of kinesin or cytoplasmic dynein and fibrous M A P S more precisely, we carried out a cosedimentation experiment using the subtilisin cleavage points on both a-and p-tubulin on microtubules as reference points. Our results from the cosedimentation experiment revealed that even completely cleaved microtubules (a,&) were able to bind to every molecule: kinesin, cytoplasmic dynein, taupw, tauss, " 2 p w , and MAP~Bs. Moreover, the molar ratio of each molecule that bound to aP:aP.:a.P. was evaluated as being nearly the same, that is, about 1:(0.8-1.0):(0.6-0.7). In this experiment, tauBs showed weak binding in comparison with the other molecules. We do not yet know the exact reason for this, but perhaps the binding affinity of tauBs is different from that of the others. This problem will require further examination.
The molar ratio of aP:aPs:asPs is 1:(0.8-1.0):(0.6-0.7), indicating that there is no substantial difference between ap and aP., which means that the removal of the carboxyl-terminal of P-tubulin had little effect on the binding of kinesin, cytoplasmic dynein, tau, or MAP2 to microtubules. Once a-tubulin was cleaved, the binding was reduced to 60-70% of control. The most likely explanation for this result is that on a-tubulin the binding domain of each molecule lies mainly on the carboxylterminal side of the subtilisin cleavage point, whereas on p-tubulin it lies mainly on the amino-terminal side. As for tau, there is complete agreement with the view of Melki et al. (1991) that the subtilisin cleavage removes its binding domain of tau on a-but not on P-tubulin. The reason that the binding to asp, is not one-half but 60-70% can be explained in the case of tau or MAP2 according to the study of Maccioni et al. (19881, which demonstrated that the binding domain on P-tubulin interacts with tau or MAP2 somewhat more strongly than that on a-tubulin. On these grounds it seems reasonable to assume that the binding domain of kinesin or cytoplasmic dynein lies in almost the same portion as tau or "2, which locates mainly on the carboxyl-terminal side of the subtilisin cleavage site on a-tubulin and mainly on the amino-terminal side on p-tubulin.
The purpose of this study was to elucidate the interaction of force-producing MAPs and fibrous MAPs for binding to microtubules. It can be concluded that kinesin or cytoplasmic dynein and tau or MAP2 compete for almost the same binding domain, which lies mainly on the carboxyl-terminal side on a-and on the amino-terminal side on P-tubulin from the point of the subtilisin cleavage. In addition to competing for the binding domain, the projection of tau, and probably of " 2 , acts as steric hindrance for kinesin and cytoplasmic dynein binding to microtubules.
As for the interaction of kinesin and fibrous M A P S , Mandelkow's group reported that kinesin-induced microtubule motility is not affected by MAPs (Massow et al., 1989;Heins et al., 1991). However, their conclusion was based only on a motility assay system using native M A P S , and their experimental conditions were different from ours. It seems insufficient to investigate the competition between kinesin and fibrous MAPs for binding to microtubules, using only whole molecules. Our conclusion agrees with that of Paschal et al. (1989) in terms of cytoplasmic dynein and MAP2 competing in binding to microtubules on a common domain at the carboxyl-terminal portion of tubulin. As for a comparison with the view of Rodionov et al. (1990), our observations that kinesin or cytoplasmic dynein showed binding to asps and induced gliding are in good agreement. However, concerning the binding of MAP2 to asps, the results are conflicting. This discrepancy may be because of differences in experimental conditions and source of materials. Our conclusion coincides with that of Lopez and Sheetz (1993) that MAP2 inhibits cytoplasmic dynein and kinesin motility by steric hindrance. In the same study, they also reported that tau and the chymotryptic microtubule-binding fragment of MAP2 did not inhibit microtubule gliding using cytoplasmic dynein and kinesin as motors. In contrast, the results of our competi-tion experiment and motility assay indicated that both tau (taupw and tauBs) and MAP2 compete with kinesin and cytoplasmic dynein in binding to microtubules. The main reason for this discrepancy may lie in the differences of the ratio of M A P S and motor molecules used in the two studies. Under their conditions (the ratio of tau or MAP2 to microtubules was 1:7-1:30 (moVmol)) we also did not observe competition, although, as we mentioned in the results, if we raise the ratios of MAPs to microtubules the competition clearly occurs. Therefore, under their conditions, the binding sites of microtubules may not be saturated by MAPs. In the present study, biochemical determinations of the presence of competition between kinesin or cytoplasmic dynein and tau or M A P S , as well as investigation of the location of the binding domain of kinesin or cytoplasmic dynein on microtubules, were also performed in addition to the motility assay. It appears difficult to rule out the possibility of competition between motor molecules and fibrous MAPs for specific sites on microtubules based only on a motility assay.
In this paper, we defined the binding domain of kinesin, cytoplasmic dynein, tau, or MAP2 as a relative position of the subtilisin cleavage site. Although many studies have been carried out on the subtilisin cleavage site on microtubules, establishing that it is located on the carboxyl-terminal portion of both a-and p-tubulin (Sackett et al., 1985), the precise site is still unclear since the results have been so divergent (Serrano et al., 1984;Sackett et al., 1985;Maccioni et al., 1986;Vina et al., 1988;Melki et al., 1991). As for the binding domain of tau and MAP2 on microtubules, it is generally considered that they locate on the carboxyl-terminal portion of both a-and p-tubulin. However, their precise positions are also in disagreement (Littauer et al., 1986;Rivas et al., 1988). Determination of the microtubule binding domain of kinesin and cytoplasmic dynein at the amino acid level will provide useful information about the competition by comparing this region with that of tau and " 2 .
Our findings provide a useful step toward understanding the regulatory system of intracellular organelle transport. Organelle transport on microtubules, which is induced by force-producing M A P S such as kinesin and cytoplasmic dynein, may be regulated by fibrous M A P S such as tau and MAP2 through competition for the same binding domain.