Effect of MAP2, MAP2c, and tau on kinesin-dependent microtubule motility

SUSANNE HEINS, YOUNG-HWA SONG, HOLGER WILLE, ECKHARD MANDELKOW and EVA-MARIA MANDELKOW*

Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Notkestrasse 85, D-2000 Hamburg 52, Germany

* Corresponding author

Summary

By making use of DIC video microscopy to monitor microtubule motility we have studied the effect of several MAPs (MAP2, MAP2c, tau) on microtubule- kinesin interactions and microtubule gliding. Of the three MAPs tested, MAP2 interferes most strongly with kinesin-dependent microtubule motility.

Key words: kinesin, microtubules, motility, MAPs.

Introduction

The transport of vesicles and organelles in nerve cells depends on microtubules and their motor proteins (Allen et al. 1985; Brady et al. 1985; Vale et al. 1985). These cells also contain a variety of microtubule-associated proteins (MAPs) which tightly bind to and stabilize the micro­tubules. Some of the MAPs (e.g. MAP2) are fairly large, have elongated shapes and can protrude for several tens of nanometres from the microtubule surface. The crowding of proteins in the axoplasm poses some problems; for example, how can a motor protein move along a micro­tubule in spite of the MAPs present? What is the influence of MAPs on axonal transport?

The analogue of anterograde axonal transport can be studied in vitro by observing microtubules gliding over a surface covered with the motor protein kinesin, using video-enhanced DIC microscopy (Allen et al. 1985; Vale et al. 1985). The visualization of the microtubules depends not only on the optical parameters and image enhance­ment, but also, because of the small depth of field, on the confinement of the microtubules to a region just above the surface. If the microtubules detach from the surface and diffuse into the solution they become essentially invisible. Thus the image contains information both on microtubule attachment and on motility. It can therefore be used to study factors that affect these two parameters.

We have recently reported on the interference of MAP2 with microtubule attachment and kinesin-dependent motility (von Massow et al. 1989). The experiments (see diagrams in Fig. 1) showed that (1) pure microtubules attach to the surface and are therefore visible, (2) a surface covered with MAP2 prevents the attachment of micro­tubules which are therefore invisible and (3) kinesin attached to the surface binds and moves microtubules. However (4) when the surface is covered with both kinesin and MAP2, the effect of MAP2 dominates, i.e. micro­tubules neither attach nor move, and thus are not visible; (5) when MAP2 is present both on the surface and on microtubules, microtubules attach to the surface again, in contrast to (2), as if the MAP2 molecules coming fromJournal of Cell Science, Supplement 14, 121-124 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

opposite directions interlocked with one another; in this case kinesin cannot induce movement either.

These effects depend on certain concentration ratios between kinesin, MAPs, and microtubules, which we have now studied in more detail. In addition we were interested to determine if other MAPs had effects similar to MAP2. We have now tested two proteins which are homologous with MAP2 (1828 residues) in the C-terminal region, which contains the internal repeats responsible for microtubule binding (Lewis et al. 1988). They are tau (around 400 residues, depending on isoform; Lee et al. 1988; Goedert et al. 1988), which has a much shorter N- terminal domain than MAP2 and shows no homology to it, and MAP2c (467 residues), a juvenile form of MAP2, arising from alternative splicing, which is homologous to MAP2 but lacks most of the N-terminal region (Papan- drikopoulou et al. 1989). The results reported below show that their effects are much less pronounced than those of MAP2 in terms of the above assays, suggesting that only the full length MAP2 contains special regions, presumably in the N-terminal domain, that are responsible for the repulsion of microtubules from the surface or the MAP- 2-MAP2 interlocking.

Materials and methods

Preparation of microtubulesPC-tubulin was prepared as previously described (Mandelkow et al. 1985). MAP-free microtubules were polymerized in assembly buffer (0.1 m PIPES, pH 6.9, Im M each of MgS04, EGTA, DTT, and GTP) and stabilized by 10 /iM taxol.

Preparation of kinesinKinesin was prepared after Kuznetsov and Gelfand (1986) or Vale et al. (1985), with modifications (von Massow et al. 1989; Song, unpublished).

Preparation of MAPsMAP2 and tau were prepared from porcine brain as described (Hagestedt et al. 1989), involving a boiling step, separation on Mono S HPLC (Pharmacia), and their differential solubility in perchloric acid. MAP2c was prepared similarly and separated from MAP2 by gel filtration (Superose 12 column, Pharmacia).

Motility assay and sample preparation Microtubules and their movement by kinesin were observed by DIC video microscopy following the method of Allen et al. (1985). The effects of MAPs on microtubule attachment and gliding were performed as previously described (von Massow et al. 1989). Briefly, the samples are prepared in two steps: (1) coating of the glass surface by pre-incubation with kinesin, MAPs, or both, (2) addition of microtubules, made either from PC-tubulin and stabilized by taxol, or with re-attached purified MAPs. The pre-

121

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Xi_IFig. 1. Diagrams illustrating possible mechanisms of interaction between kinesin, MAP2, and microtubules.(A) Kinesin adsorbed to the glass surface is capable of moving microtubules (arrow), irrespective of whether the added microtubules have attached MAP2 or not (see H, below).(B) Microtubules added without prior adsorption of kinesin or MAP2 attach to the glass and can thus be seen easily by focussing the microscope to the level of the glass surface.(C) MAP2 adsorbed to the glass surface repels microtubules so that they tend to remain in the bulk solution and are difficult to visualize. (D) MAP2 plus kinesin adsorbed to the glass does not allow attachment or motility of MAP-free microtubules.(E) As a control, kinesin adsorbed to the glass together with BSA shows uninhibited motility, i.e. BSA does not interfere with the interaction between microtubules and kinesin. (F) The same result as with BSA is obtained when kinesin and the assembly fragment of MAP2 are adsorbed to the glass, showing that this fragment does not prevent the kinesin-based motility.(G) Similar to (F), but using the projection fragment of MAP2 which leaves the kinesin-induced motility largely unaffected.(H) Kinesin adsorbed to the glass by itself supports motility of MAP2-containing microtubules, provided that no free MAP2 is present on the glass surface; i.e. microtubule-bound MAPs do not interfere with the microtubule-kinesin interaction. This is indicated by the altered configuration of MAP2. (I) Kinesin and MAP2 pre-adsorbed to the glass plus microtubules with attached MAP2. The MAP2 molecules projecting from the glass and from the microtubule become entangled and/or crosslinked and keep the microtubule near the surface, but prevent their gliding. From von Massow et al. 1989.

incubation was typically done by applying a volume of 8/d of kinesin/MAP solution to the coverslip, spread over an area of about 0.4 cm2, and letting the proteins adsorb for about 15min. This area was marked with a felt pen for later identification of the

^boundary. After that, 2 /A of microtubules and ATP were added, and the sample was covered with a 1.5 x 1.5 cm2 coverslip. The 10 fil solution was distributed over the whole area of 2.25 cm2 (thickness of layer about 40 fan), but the pre-adsorbed proteins diffused only very slowly outside the initial area so that the boundary could be identified by the microtubule attachment and

motility criteria described below. All protein concentrations refer to the final 10 ¡A volume.

The upper limit of the surface density of pre-adsorbed proteins (number of particles nm"2) can be estimated by assuming that all particles attach to the surface area so that:

<5 = (V/A)(CW/M) x 6 x 10® ,

and the mean separation between particles is d (nm)=l/V<5 . C„ is the weight concentration in mg ml-1 , M is the molecular weight, V is the applied volume in ml, and A the area in cm2. For example, for a typical solution of kinesin (Mr=360000) with V=0.01ml, A=0.4cm2, Cw=0.05mgml_1 we get &=0.02nm~2 and d~7 nm.

Results

(1) Effect of MAP2/kinesin ratios on microtubule gliding Coating with kinesin alone leads to visible and motile microtubules, irrespective of whether the microtubules are covered with MAPs or not, as previously described (von Massow et al. 1989; see Fig. 1A,H). Pre-adsorption of the surface with MAP2 leads to the repulsion of microtubules which are therefore not visible (Fig. 2; Fig. 1C). The same effect occurs in the presence of kinesin, even at very low molecular ratios of MAP2:kinesin of 0.02:1 (Fig. ID). Thus the repulsion of microtubules by MAP2 dominates over the attraction by kinesin. This can be nicely visualized by pre-incubating two adjacent areas in different ways, one with kinesin only, the other with kinesin and MAPs. Microtubules glide over the surface containing only kinesin; when they cross the boundary into the MAP2- containing region, they lift off and disappear out of focus (Fig. 3).

The repulsion of microtubules by surface-bound MAP2 is routinely observed for MAP2-free microtubules; when the added microtubules are also saturated with MAP2 the behavior is more complex. Repulsion still dominates when the MAP2:kinesin ratio is above 0.15:1. However, between ratios of 0.15:1 and 0.02:1 the microtubules are attached to the surface but do not glide. This can be interpreted as an ‘interlocking’ of MAP2 molecules protruding from the glass and microtubule surfaces (Fig. II). At even lower concentrations of MAP2 on the surface, gliding takes over again, as in Fig. 1H. In the intermediate range where interlocking takes place the mean distance between MAP2 molecules is of the order of 25 nm or more, compared with only 7 nm for kinesin. In contrast to intact MAP2, neither the assembly nor the projection fragment of MAP2 (obtained by limited chymotryptic cleavage, as described by Vallee, 1980) has the repulsive or interlocking effect when used at comparable stoichiometric ratios (Fig. IF and G).

(2) Influence of MAP2c or tau on kinesin—microtubule interactionsCoating of the surface with kinesin plus MAP2c or tau does not show the same strong repulsive effect as with MAP2. In the case of MAP2c, the MAP2c:kinesin ratio has to be raised to 0.9:1 in order to observe repulsion; this is six times higher than the corresponding concentration of MAP2. For tau the minimum stoichiometry for repulsion is even higher, namely 8:1. In these conditions the calculated mean distances between MAP molecules would be about 7 and 2.5 nm for MAP2c and tau, respectively.

In contrast to MAP2, neither MAP2c nor tau produces an ‘interlocking’ effect, i.e. when the glass and micro­tubule surfaces are both coated with the MAPs one

122 S. Heins et al.

Fig. 2. Effect of MAP2 on the attachment of microtubules to the glass surface in the absence of kinesin. (A) Without pre­incubation with MAP2, microtubules (prepared from P C - tubulin and containing no MAPs) attach to the glass surface and can therefore be visualized by video microscopy. (B) When the glass surface is first covered with MAP2 and then microtubules are added, the microtubules are repelled from the surface and remain invisible in solution. For concentration ranges see text. Bar, 5 /on.

observes either repulsion (at high MAP concentrations, analogous to Fig. ID) or microtubule gliding (at low concentrations, analogous to Fig. 1G). In other words, whether or not a microtubule is coated with these MAPs has no obvious effect on the microtubule behavior. This is true even in hybrid situations, e.g. when the surface contains MAP2 and kinesin and the microtubules are coated with MAP2c or tau. In this case the strong repulsion of microtubules by MAP2 again dominates, and one observes no ‘interlocking’, i.e. stationary microtubules attached to the surface.

Discussion

These studies were originally prompted by the observation that crude kinesin preparations did not support micro-

Fig. 3. Effect of MAP2 on kinesin-induced microtubule motility across a boundary. The coverslip was first coated with kinesin (50,ugml-1 ), in addition a part of it was coated with MAP2 (50 (tigml-1 ). The approximate position of the boundary is indicated by the dashed line (upper left, only kinesin; lower right, kinesin plus MAP2). After adding microtubules and ATP they are visible and moving across the MAP2-free area (two of them are labeled 1 and 2; direction of movement indicated by white arrows). When they cross into the MAP2-containing area their leading part lifts off the surface and disappears from the image while the trailing part is still seen moving (compare (A) and (B), separated by a 22 s interval). The converse case can be observed as well (not shown). Microtubules that are initially invisible become attached in the MAP2-free area with their leading part; as they move further into it they gradually become visible along their whole length. Superficially they appear to be growing while moving, up to the point where they are fully visible. This effect is typical of MAP2 but not of MAP2c or tau. Bar, 5 um.

tubule gliding. A search for inhibitory factors led to the discovery of the effects of MAP2, which was present in these preparations and could be removed by gel filtration. One conclusion was that microtubule-bound MAP2 does not interfere with microtubule gliding while MAP2 preadsorbed to the glass does. Our working model (von Massow et al. 1989) is that MAP2 adsorbed to the glass surface forms a lawn which either repels microtubules

Effect o f MAPs on microtubule motility 123

from approaching the surface and thus prevents the interaction with kinesin, or, at lower concentrations, interacts somehow with MAP2 bound to microtubules, thereby keeping them close to the surface yet immobile. The effects occur at surprisingly low concentrations of glass-bound MAP2 (at least six times less than that of kinesin), where the mean separation of MAP2 molecules is expected to be of the order of 20 nm or more. In contrast, MAP2 bound to microtubules appears to have no effect on their motility per se, in spite of their high density on a microtubule. Saturated microtubules contain about 20 % MAP2 which translates into roughly one MAP2 molecule every 6 nm along the length. Thus far we have found only one condition where microtubule-bound MAPs affect gliding, that is, when the MAP2-MAP2 interlocking takes place (at low MAP2 densities on the surface).

We have now studied two other MAPs which are homologous to MAP2 in their C-terminal (microtubule- binding) region. Neither of them shows the pronounced repelling effect of MAP2, that is, in order to observe repulsion one requires six to fifty times higher molar concentrations than with MAP2 (for MAP2c and tau, respectively). Nevertheless, these effects appear to be specific, since controls with BSA show no effect at even much higher concentrations (100-fold and more). More­over, the interlocking effect is not observed with either MAP2c or tau.

One interpretation of these observations is suggested by the size of MAP2; it is four to five times larger than MAP2c or tau which makes the stronger repulsion of microtubules understandable. This view is probably oversimplified since the projection fragment of MAP2 lacks the repelling or interlocking capacity even though it is only 10-20% smaller than intact MAP2, Moreover, arguments based on size alone fail to explain the MAP2-MAP2 interlocking. It seems therefore likely that MAP2 contains specific sequences responsible for its effect. These sequences are unlikely to be in the C-terminal microtubule-binding domain where the three MAPs are largely homologous. However, the N-terminal projection domain of MAP2 contains about 1400 unique amino acid residues not found in MAP2c or tau which are therefore candidates for MAP2- specific functions. These considerations are derived from the published cDNA sequences of MAP2, MAP2c, and tau (see Lewis et al. 1988; Papandrikopoulou et al. 1989; Lee et al. 1988; Goedert et al. 1988), but they can only be approximate since we used proteins purified from brain tissue.

Finally, we note that one common feature of all MAPs tested is their capacity to repel microtubules and to overcome the attractive force of kinesin. There has been an ongoing debate as to whether MAPs act as crosslinkers or as spacers of microtubules. Recent reports have empha­sized the crosslinking function, since cells transfected with MAP2 or tau developed microtubule bundles (Kanai et al. 1989; Lewis et al. 1989). In contrast, biophysical exper­iments such as the ones discussed here tend to support the

view of MAPs as spacers, and X-ray experiments (to be described elsewhere) point in the same direction. This would mean that one function of MAPs (apart from microtubule stabilization) is to keep the space around microtubules free from structures (microtubules or other cytoplasmic components) that might interfere with kin- esin-based transport processes.

We thank Dr M. Suffness (US National Cancer Institute, Bethesda, MD) for providing taxol. This project was supported by the Bundesministerium für Forschung und Technologie.

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