* Adolf-Butenandt-Institut, Zellbiologie, 80336 München, Germany; Max-Planck-Unit for Structural Molecular Biology, 22607 Hamburg, Germany; and § Friedrich-Miescher Institute, CH-4002 Basel, Switzerland
Microtubules are flexible polymers whose mechanical properties are an important factor in the determination of cell architecture and function. It has been proposed that the two most prominent neuronal microtubule-associated proteins (MAPs), tau and MAP2, whose microtubule binding regions are largely homologous, make an important contribution to the formation and maintenance of neuronal processes, putatively by increasing the rigidity of microtubules. Using optical tweezers to manipulate single microtubules, we have measured their flexural rigidity in the presence of various constructs of tau and MAP2c. The results show a three- or fourfold increase of microtubule rigidity in the presence of wild-type tau or MAP2c, respectively. Unexpectedly, even low concentrations of MAPs promote a substantial increase in microtubule rigidity. Thus at ~20% saturation with full-length tau, a microtubule exhibits >80% of the rigidity observed at near saturating concentrations. Several different constructs of tau or MAP2 were used to determine the relative contribution of certain subdomains in the microtubule-binding region. All constructs tested increase microtubule rigidity, albeit to different extents. Thus, the repeat domains alone increase microtubule rigidity only marginally, whereas the domains flanking the repeats make a significant contribution. Overall, there is an excellent correlation between the strength of binding of a MAP construct to microtubules (as represented by its dissociation constant Kd) and the increase in microtubule rigidity. These findings demonstrate that neuronal MAPs as well as constructs derived from them increase microtubule rigidity, and that the changes in rigidity observed with different constructs correlate well with other biochemical and physiological parameters.
MICROTUBULES are important components of the
eukaryotic cytoskeleton that are involved in basic cellular activities such as cell shape determination, cell movement, mitosis, and organelle transport (for
comprehensive reviews see Dustin, 1984 Experiments in which MAP2 and tau have been expressed by transfection in nonneuronal cells have shown
that both MAPs significantly influence microtubule stability and arrangement. In their presence microtubules are
stabilized, form bundles, and become capable of supporting process formation in cells from which they are normally absent (Lewis et al., 1989 To serve a stabilizing and structuring function in the cell,
a microtubule must be able to resist tensile and compressive forces. This capacity is reflected in its flexural rigidity,
an important physical characteristic of these extended
polymers. The flexural rigidity of single microtubules has
recently been determined using a variety of approaches,
including the analysis of thermal fluctuations in shape
(Gittes et al., 1993 In a previous study addressing the mechanical properties of microtubules, we have used optical tweezers to deflect a single microtubule attached to an axoneme (Felgner et al., 1996 Optical Tweezers Setup and Image Processing
The setup used for the construction of the laser trap is described in detail
in Felgner et al. (1995 Preparation of Microtubules and Axonemes
Microtubules were prepared from phosphocellulose-purified pig brain tubulin according to the procedure of Shelanski et al. (1973) Adsorption to the Glass Surface and Levels of
Saturation of the Microtubules by MAPs
To determine eventual losses of tubulin and MAPs in our experimental
setup, we have measured their adsorption to the polylysine-covered glass
surface. 10 µg protein in 100 µl buffer was applied to an 18 × 18-mm coverslip as a thin film and incubated for 30-60 min in a wet chamber. The solution was removed and the protein concentration was measured, taking
into account that not all of the solution could be recovered from the coverslip. We find that 1.3 ± 0.4 µg of tubulin (n = 9) is adsorbed to the surface. This corresponds to one molecule per 42 nm2, a value that is in reasonable agreement with the surface area covered by one tubulin molecule
(16-32 nm2), assuming dimensions of tubulin of 4 × 4 × 8 nm. The adsorption of MAPs is 0.6 ± 0.2 µg (n = 12) for a construct of average size (40 kD). If a coverslip is incubated with tubulin first, followed by the addition
of MAPs, the adsorption of MAPs is reduced to <0.1 µg. This is important because in our experiments a coverslip is incubated with tubulin first
(to allow for polymerization onto the axonemes) before the addition of
MAPs in a 2-µl vol. The coverslip is then placed on a slide. On the slide
surface, we assume tubulin and MAPs adsorb with equal efficiency. The
total losses therefore are 1.3 µg (coverslip) plus half of 1.3 µg (slide) for
tubulin, and <0.1 µg (coverslip) plus half of 0.6 µg (slide) for MAPs. These losses of 2 µg total for tubulin and 0.4 µg total for MAPs were
taken into account in calculating the true molar ratio of MAPs to tubulin
and hence the level of saturation of microtubules by MAPs.
The molar ratios of MAPs to tubulin applied to the coverslip were 1:1
for full-length tau and MAP2c constructs, 30:1 for the low affinity tau constructs K10 and K18, 0.4:1 for the high affinity construct T8R2, and 1.5:1
for the others. The losses due to adsorption described above were subtracted subsequently. This leads to levels of saturation of microtubules by
the various tau constructs of between 79 and 93% (calculated according to
Gustke et al., 1994 Cloning and Expression of Tau Constructs
Recombinant full-length tau and other constructs were cloned in the
pNG2 expression vector and expressed in Escherichia coli as described by
Biernat et al. (1992)
Preparation of MAP2 and Constructs
MAP2c and deletion mutants were prepared as described by Ludin et al.
(1996) Statistical Analysis
A nonparametric Wilcoxon test was carried out according to Bronstein
(1985)
Measurement of Flexural Rigidity
The procedure used here is based on the calculation of
frictional hydrodynamic forces acting on a single moving
microtubule. A microtubule anchored at its minus end at
an axoneme is captured directly near its free end with the
laser tweezers and deflected in a direction perpendicular
to its long axis and parallel to the coverslip surface. The laser power is then switched off, allowing the microtubule to
relax back to its straight resting position. The flexural rigidity can be calculated from the relaxation movement, which is determined by the elastic force of the microtubule
that drives it back and the hydrodynamic force of the medium that resists the moving microtubule. An example is
shown in Fig. 2. The details of the theoretical considerations on which the analysis of this movement is based are
outlined in Felgner et al. (1996)
Various structural, biochemical, and physical factors
that affect the determination of the flexural rigidity in our
approach have already been discussed (Felgner et al.,
1996 Microtubule Rigidity in the Presence of Different
Tau Constructs
The COOH-terminal assembly domain of tau can be divided into a number of subdomains with different characteristics (Fig. 3). The most notable feature is the presence
of four repeats 31 or 32 amino acids in length (Lee et al.,
1988 To determine the relative contributions of tau domains
to the increase in microtubule stiffness, we tested a number of tau constructs. The data are summarized in Fig. 4.
As outlined in Materials and Methods, the molar ratio of a
given MAP construct to tubulin was adjusted so that the
level of saturation of microtubules by MAPs was ~80-90%
(except K23, where 65% saturation was reached). This is
higher than the presumptive level of saturation of tau in
vivo, which can be estimated to be 40-70% (Drubin and
Kirschner, 1986 The flexural rigidity of microtubules with bound full-length tau is almost three times higher than that of pure
microtubules. All tau constructs lacking one or more subdomains in the assembly portion caused microtubule stiffness to decrease relative to full-length tau. Apparently, all
subdomains of the assembly domain contribute to microtubule rigidity, albeit to different extents. Thus the four repeats alone (K18) increased microtubule rigidity only
slightly, but, when combined with either the P2 region
(K16) or the pseudorepeat (K11), microtubule rigidity
more than doubled. Constructs in which the repeat domains were deleted but the flanking domains were maintained yielded complementary results. The flanking domains alone already contributed substantially to microtubule
rigidity (K23); in combination with one or more repeat domains, it increased even more (K29, K30, and HT23). As a
rule, the differences in microtubule rigidity observed in
our measurements were statistically significant (Fig. 1) for
all pairs of constructs that differed by more than 2 U of EI
(10 An unusual observation was made with a construct in
which the four repeat domains are doubled up (T8R2). It
increased microtubule stiffness far beyond a value that
might be expected on the basis of the observations made
with the deletion constructs. Microtubule rigidity was almost three times higher than that with full-length tau and
almost eight times that of pure microtubules. Visual inspection of these microtubules clearly demonstrated that
their fluctuations in shape as a result of thermal motion
were greatly suppressed.
Several other constructs that we also wished to include
in this analysis unfortunately led to substantial microtubule bundling and therefore could not be tested.
Levels of Saturation of Microtubules by Tau
To determine how the level of saturation of a microtubule
with a given tau construct affects its flexural rigidity, experiments were made with full-length tau (HT40) at concentrations leading to ~2, 18, and 48% saturation, in addition to the experiments at 85% saturation listed in Fig. 4.
As shown in Table I, even a relatively low level of saturation (18%) already led to a substantial increase in microtubule rigidity. At 48 or 85% saturation, the additional increase in rigidity is comparatively small. These observations
were supported by findings with other constructs. For example, T8R2 was also used at a concentration that leads to
99% saturation of microtubules, but no difference was
found in microtubule rigidity (average 29.2 ± 4.8 in units
of 10 Table I.
Flexural Rigidity at Different Levels of Saturation by
Full-Length Tau (HT40)
; Hyams and Lloyd,
1993
). Two major classes of proteins interact with microtubule polymers: molecular motors and microtubule-associated proteins (MAPs).1 In contrast with motor molecules,
MAPs bind to microtubules in a nucleotide-independent
fashion. They copolymerize with microtubules through
cycles of assembly and disassembly, stimulate their nucleation, and stabilize the polymer (Olmsted, 1991
; Schoenfeld and Obar, 1994
; Mandelkow and Mandelkow, 1995
).
Two of the best-known families of MAPs are the tau proteins and MAP2. Both are expressed in neuronal cells,
with the high molecular weight form of MAP2 being confined to dendrites and tau being confined to axons (Bernhardt and Matus, 1984
; Binder et al., 1985
; Tucker et al.,
1988
). They are derived from different genes (Lee et al.,
1988
; Lewis et al., 1988
) and, in each case, alternative splicing gives rise to several isoforms (Himmler et al., 1989
;
Goedert et al., 1989
; Kindler et al., 1990
), the size of which
may vary considerably. Despite these size differences, MAP2 and tau are similar in overall domain composition,
including a set of three or four repeats of ~30 amino acids
in length. The repeats and the flanking proline-rich and
basic domain in the COOH-terminal half comprise the
"assembly domain" that is responsible for binding to microtubules (Butner and Kirschner 1991
; Brandt and Lee,
1993
; Coffey et al., 1994
; Gustke et al., 1994
; Ludin et al.,
1996
). The role of the NH2-terminal half that does not bind to microtubules and that extends into the surrounding medium ("projection domain") is less clear. Its size determines the separation between microtubules in a parallel
array (e.g., Black, 1987
; Chen et al., 1992
; Weisshaar et al.,
1992
). In addition, it may serve as an anchoring device for
cellular proteins, notably kinases and phosphatases (e.g.,
Mandelkow et al., 1992
; Sontag et al., 1995
). Whereas
MAP2 and tau are almost exclusively expressed in neurons, other cell types contain the more ubiquitous MAP4
that possesses many of the same features, including the repeated sequences (West et al., 1991
; Chapin and Bulinski,
1991
).
; Takemura et al., 1992
;
Knops et al., 1991
; Weisshaar et al., 1992
; Edson et al.,
1993
). These studies suggest that tau and MAP2 play a
crucial role in microtubule assembly and stability, and thus
contribute to the integrity and dynamics of the cells in
which they are expressed.
; Venier et al., 1994; Mickey and
Howard, 1995
), measurement of the deformation induced
by hydrodynamic flow (Dye et al., 1993
; Venier et al., 1994; Kurz and Williams, 1995
), and laser trap-induced
flexing with (Kurachi et al., 1995
) or without (Felgner et
al., 1996
) attached beads. These studies agree that microtubules are ~100 times more rigid than actin filaments, another major cytoskeletal filament of eukaryotic cells. Since
MAPs are important elements in determining the biochemical, and perhaps also the physical, properties of microtubules in the cell, some of these studies also addressed
the question of how MAPs affect microtubule rigidity. However, although the microtubule-supported processes
that MAP2 induces in fibroblastic cells are demonstrably
stiff (Ludin et al., 1996
), the question whether individual
microtubules are made more rigid by MAPs is still open.
). From the quantitative analysis of the
relaxation movement, the microtubule's flexural rigidity is
determined. It is a straightforward (though by no means
trivial) method to measure the stiffness of a biopolymer. In the present study we have determined the influence of
MAP2 and tau on the flexural rigidity of single microtubules in vitro. Unphosphorylated recombinant proteins
purified from bacterial extracts were used to avoid complications introduced by variations in phosphorylation that
affect MAP-microtubule interactions (Brugg and Matus,
1991
; Verde et al., 1991
; Drechsel et al., 1992
; Trinczek et
al., 1995
). To determine the contribution of different microtubule-binding domains of tau and MAP2 to microtubule rigidity, we have tested a number of constructs that
varied in the composition of subdomains in the microtubule-binding (assembly) domain. The results show that all
constructs tested increase microtubule rigidity and that the
contribution of certain subdomains correlates well with
their strength of binding to microtubules.
Materials and Methods
, 1996
). Briefly, the expanded beam of a continuous
wave Nd:YAG laser (Spectron, Rugby, UK) is coupled into an upright microscope using a 100×/1.3 NA objective (Zeiss, Oberkochen, Germany).
A laser output power of 1.5 W is coupled into the epifluorescence illumination path of the microscope, 40% of which reaches the sample. Whereas
the laser beam is fixed relative to the optical system, the specimen chamber is translated relative to the beam using a motorized microscope stage
(Maerzhaeuser, Wetzlar, Germany). A Newvicon camera (Hamamatsu, Herrsching, Germany) feeds the microscope image into a Macintosh IIfx
computer (Apple Computer Inc., Cupertino, CA). For image processing
details, see Felgner et al. (1996)
.
, with modifications, including MAP-depleting steps, according to Mandelkow et al.
(1985)
. Axonemes of Chlamydomonas reinhardtii were prepared according to Witman (1986)
and depleted of dynein and other endogenous proteins by salt extraction (King et al., 1986
). Polylysine coverslips were prepared by applying a drop of 1 mg/ml poly-L-lysine in water to a coverslip
for 30 s, followed by rinsing with water and air drying. Axonemes were attached to polylysine-coated coverslips, followed by the addition of tubulin (1 mg/ml) plus GTP (1 mM) in a buffer consisting of 100 mM Pipes, 2 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 10 µg/ml trypsin inhibitor, 10 µg/ml TAME, 10 µg/ml leupeptin, 10 µg/ml pepstatin,
and 10 µg/ml aprotinin, pH 6.9. The preparations were incubated at 30°C
for 10 min, which yielded axoneme-nucleated microtubules of 5-20 µm in
length. MAPs or tau constructs were added at the desired concentration
(see below), and the coverslip was placed on a slide and sealed with
VALAP (vaseline/lanoline/paraffin = 1:1:1). The temperature in the
chamber on the microscope stage during observation was 24-27°C.
); an exception is K23, where only 65% saturation is
reached for technical reasons. The errors due to pipetting are 1-3%.
Nearly complete saturation of microtubules by MAPs was not attempted because it would require unreasonably high concentrations of several of
the lower affinity constructs. In cells, the level of saturation by tau is estimated to be 40-70% (Drubin et al., 1985
; Drubin and Kirschner, 1986
).
and Gustke et al. (1994)
. The clones were derived
from isoforms of human tau (Goedert et al., 1989
). The numbering of residues follows that of human tau HT40, the largest of the six isoforms in the
central nervous system (441 residues). Variations included the following:
changes in the number of repeats, changes in the targeting domains flanking the repeats, omission of the projection domain, excision of the repeats,
and others (see Fig. 4).
Fig. 4.
Tau constructs and microtubule rigidity. For domain
organization see Fig. 3. The figure shows the flexural rigidity (EI)
of the different constructs and the number of experiments (n).
The values for MAP-free microtubules (Pure), taxol-stabilized
microtubules (Taxol), and microtubules in the presence of a mixture of brain MAPs (MAP Mix) are also included for comparison.
[View Larger Version of this Image (18K GIF file)]
Fig. 3.
Simplified diagrammatic representation of the domain
organization of tau (the largest isoform HT40) and MAP2c. The
31-amino acid repeats are numbered. In MAP2d a fourth repeat
is inserted between repeats 1 and 2. P1, P2, and P, proline-rich
domains. R, pseudorepeat.
[View Larger Version of this Image (8K GIF file)]
. Briefly, appropriate cDNAs were subcloned into the bacterial expression plasmid pET3d and grown in E. coli BL21(DE3). Protein expression was induced by the addition of 1 mM isopropylthio-
-D-galactoside for 3 h, and the protein was extracted and purified as described in
Ludin et al. (1996)
.
to compare the flexural rigidities of microtubules in the presence of
different constructs of tau. In most cases, the hypothesis that two populations of microtubules have the same flexural rigidity could be rejected
with a significance level of 1 or 5% (Fig. 1). No significant differences
were found for the MAP2 constructs.
Fig. 1.
Statistical analysis using the nonparametric Wilcoxon
test. H23 and H40, constructs HT23 and HT40, respectively; MM,
mixture of brain MAPs; 2c, 2d, and 2c3, MAP2 isoforms; Pure
and Taxol, microtubules prepared from phosphocellulose-purified tubulin and taxol-stabilized microtubules, respectively; 1%,
5%, and no, levels of significance.
[View Larger Version of this Image (25K GIF file)]
Results
. For a microtubule without MAPs, the flexural rigidity (EI; E = Young's modulus,
I = geometrical moment of inertia) is ~4 × 10
24 Nm2
(see Fig. 4). When microtubules are stabilized by taxol, we
find the value of EI to decrease by a factor of 2-3, suggesting that taxol, which makes microtubules less dynamic,
also makes them less rigid (Felgner et al., 1996
).
Fig. 2.
Representative experiment showing the time course of
the relaxation movement of a microtubule prepared from pure
tubulin (length 13 µm) relaxing from its deflected position (y(0)
~4 µm) to its resting position. The triangles show measured values with a time resolution of 50 Hz, and the line represents a fit to
the expected exponential decay from which EI is calculated.
[View Larger Version of this Image (11K GIF file)]
) and need not be reiterated here. Some of these factors introduce uncertainties that affect the determination
of EI, leading to either an underestimation or overestimation of its true value. These uncertainties have to be taken
into account when comparing our results with those obtained using other methods. However, even if this is done,
some discrepancies remain. For example, the rigidity of
untreated microtubules determined from thermal fluctuations by Mickey and Howard (1995)
, or from flow-induced
deformation by Kurz and Williams (1995)
, is about seven
to nine times higher than ours. On the other hand, Venier
et al. (1994) and Tran et al. (Tran, P.T., S.F. Parsons, R. Sterba, Z. Wang, M.P. Sheetz, and E.D. Salmon. 1995. Mol.
Biol. Cell. 6:260a) found a value of EI very similar to ours.
These discrepancies are difficult to explain at present, although it cannot be excluded that they arise from intrinsic
differences in the microtubules used or certain features of
the assay conditions. Regardless, the comparison between
MAP-containing and pure microtubules undertaken in
this study is not affected by these discrepancies because
identical procedures were used in all experiments.
; Himmler et al., 1989
; Goedert et al., 1989
). A fifth,
less conserved repeat-like domain (R
) is followed by a
COOH-terminal domain that apparently lacks distinctive
features. A proline-rich domain NH2-terminal to the repeats can be subdivided into portions located on either the projection (P1) or the assembly domain (P2). The domains
flanking the repeats can be considered targeting domains
that fix tau on the microtubule surface, while the repeats
enhance microtubule assembly ("jaws model"; Gustke et
al., 1994
).
). However, we reasoned that using a
higher level of saturation may help identify potentially
weak effects of these constructs on microtubule rigidity.
24 Nm2).
24 Nm2; n = 25) compared with experiments at 84%
saturation (see Fig. 4). Similarly, construct K10, at a concentration that saturated microtubules to only 40%, increased microtubule rigidity to a value (6.0 ± 1.3; n = 17)
almost identical to that measured at 93% saturation (Fig.
4). K18 seemed to be somewhat less effective as 34% saturation caused microtubule rigidity to increase to 4.9 ± 1.1 (n = 14). These findings suggest a nonlinear relationship between microtubule saturation by MAPs and flexural rigidity, where partial saturation may already lead to nearly
maximal microtubule stiffening.
Microtubule Rigidity in the Presence of MAP2
MAP2c is a low molecular weight isoform of MAP2 that is
expressed primarily in developing brain (Garner and Matus, 1988). Its COOH-terminal microtubule-binding domain begins at around amino acid 230 and encompasses a
proline-rich subdomain and three imperfect tandem repeats of 31 amino acids (Fig. 3) that are homologous to the
repeats found in tau. The entire assembly domain of
MAP2c is almost exactly as long as that of tau. Nevertheless, our measurements of the rigidity of microtubules in
the presence of MAP2c demonstrate an increase of ~50%
above that of tau (Table II). In comparison with MAP2c,
the recently discovered splice variant MAP2d, which contains a fourth conserved repeat inserted between the first and second (Doll et al., 1993
; Ferhat et al., 1994
), did not
significantly increase microtubule rigidity. Likewise, microtubules complexed with the deletion construct MAP2c3,
which contains only one of the 31-amino acid repeats from
the COOH-terminal binding domain (Ludin et al., 1996
),
fell within the same range. All three constructs resulted in
microtubules whose flexural rigidity was not significantly different from that of microtubules in the presence of a
mixture of brain MAPs, which presumably include various
isoforms of MAP2, tau, and other MAPs. This finding suggests that the effect of various MAPs on microtubule stiffness are not additive and dominated by the high molecular
weight MAPs.
Table II. Flexural Rigidity in the Presence of MAP2 Isoforms |
Relationship with Other Parameters of MAP-Microtubule Binding
We compared the ability of tau constructs and MAP2c to
alter microtubule rigidity with their binding to microtubules (dissociation constant, Kd) and their influence on microtubule dynamics (critical concentration at the plus end,
Cc) determined earlier (Gustke et al., 1994; Trinczek et al.,
1995
; Illenberger et al., 1996
). An excellent correlation
was found with the binding affinity for microtubules (Fig.
5 a), a high value of EI corresponding to a low dissociation
constant. A less perfect correlation also existed with the
critical concentration for tubulin assembly at the plus end
(Fig. 5 b), which is lower for constructs that induced a
higher rigidity. Only weak correlations or none at all were
found with other parameters of microtubule dynamics
such as the frequencies of catastrophe and rescue, the nucleation rate, or the initial rate of assembly in solution (not
shown).
Using optical tweezers as a micromanipulator to bend microtubules, we determined their flexural rigidity from the return movement to the resting position. We find that two types of MAPs, tau and MAP2c, both increase microtubule rigidity. Moreover, deletions of subdomains in the microtubule-binding region decrease microtubule rigidity in the case of tau but appear to have little effect in the case of MAP2c.
The subdomains of the microtubule-binding segment of
tau all contribute to the buildup of microtubule rigidity, although the potency of their contribution varies. Thus, unexpectedly, the four repeat domains, which are believed to
be important for microtubule binding (Lee et al., 1988;
Himmler et al., 1989
), and which make up half of the assembly domain, increase microtubule rigidity only marginally when used alone. However, in combination with either one of the flanking subdomains, rigidity is nearly as
high as with full-length tau, supporting the jaws model for
tau binding to microtubules (Gustke et al., 1994
). These
findings are also consistent with studies on MAP2c transfected into cultured cells that show that the repeat domain
itself binds poorly but, in combination with flanking sequences, binding as well as bundling and process formation are greatly improved (Ferralli et al., 1994
). Conversely, the potential of the flanking regions of tau alone
(K23) to increase microtubule rigidity is moderate, but it is
enhanced in a stepwise fashion by the addition of one or
more repeat domains, suggesting that each of the repeat
domains makes a significant contribution. In MAP2c, on
the other hand, the addition or deletion of repeat domains
has no significant effect on microtubule stiffness, although it is conceivable that a potentially small contribution could not be detected with the methods used in this study.
How tau and MAP2c interact with microtubules is not
known mainly because the molecular structure of these
molecules can not yet be determined. In solution, tau has
very little secondary structure and behaves as if it were a
random polymer (Schweers et al., 1994). Consequently, it
is unclear how much tau and MAP2c increase the hydrodynamically effective diameter of microtubules because
the shape and extension of the projection domain are unknown. It was not possible to test the influence of the projection domain on EI directly using constructs with identical assembly domains but with or without the projection
domain. However, even if the MAP projections were to
double the apparent diameter of the microtubules, which
probably is an overestimation, flexural rigidity would be underestimated by 20% at most, as calculated by Felgner
et al. (1996).
It is unlikely that some of the differences in microtubule
rigidity observed here are caused by uneven distribution
of MAPs along microtubules due to, e.g., cooperative
binding. An overwhelming body of evidence collected in
several laboratories suggests that MAPs are evenly distributed along a microtubule. Even though they may bind
tightly, MAPs can still redistribute quickly, owing to fast
on and off rates. MAP decoration of microtubules appears to be homogeneous even at low MAP/tubulin ratios (Murphy and Borisy, 1975; Sloboda and Rosenbaum, 1979
;
Stearns and Brown, 1979
; Olmsted et al., 1989
; Kowalski
and Williams, 1993). Evidence for cooperative binding of
MAP2 to microtubules in vitro is, at best, controversial (Wallis et al., 1993
; Coffey and Purich, 1995
), and with tau
it is rather unlikely (Biernat et al., 1993
; Gustke et al.,
1994
). Thus, we are reasonably confident that in the experiments reported here MAPs are distributed homogeneously along the length of a microtubule at all concentrations used.
All subregions of the assembly domain appear to contribute to microtubule binding, though to different extents. Binding is more efficient if the repeat domains plus
one or both of the flanking regions are involved (Gustke
et al., 1994). Remarkably, the same applies to the effect on
microtubule rigidity. A good correlation with microtubule
binding is revealed (Fig. 5 a), strongly supporting the conclusion that efficient binding also promotes efficient microtubule stiffening, with certain combinations of subdomains being more favorable than others.
Thus, the repeats alone (K18), which bind weakly to
microtubules also increase microtubule stiffness only marginally, suggesting that they cannot be considered a "microtubule-binding domain" by themselves. Conversely,
full-length tau or the series of constructs with both flanking domains and varying numbers of repeats (HT23, K23,
K29, and K30) binds strongly and also increases microtubule rigidity significantly. As tight binding of a tau construct usually implies a high potential for promoting microtubule growth (Trinczek et al., 1995), the correlation
between the potential to increase rigidity and the ability to
lower the critical concentration for assembly (Fig. 5 b) is
not surprising.
It may be premature to interpret the observed relationships in terms of molecular models for tau or MAP2c binding. The minimal conclusion is that the interaction of these
MAPs with microtubules leads to an increase in microtubule rigidity; how well a construct also promotes microtubule assembly may enter as a supplementary consideration. In agreement with this conclusion, only weak
correlations, or none at all, are found with parameters of
microtubule dynamics such as the rates of catastrophe and
rescue (Gustke et al., 1994; Trinczek et al., 1995
). Thus, it
seems that the influences of different MAPs on parameters of microtubule assembly and the mechanical properties of the polymer, respectively, are largely independent.
Interestingly, although the length and subdomain organization of the microtubule-binding segment of both tau
and MAP2c are remarkably similar, MAP2c is significantly more potent in promoting microtubule rigidity. This
may be related to the potency of the third repeat in MAP2.
In cell transfection assays, this single 31-amino acid domain is sufficient to confer microtubule binding on the rest
of the MAP2 sequence as well as the ability to support process outgrowth (Ludin et al., 1996). Processes formed
with single-repeat MAP2c3 show comparable stiffness to
those formed by cells expressing full-length MAP2c (Ludin et al., 1996
). This is confirmed by the physical measurements made on single microtubules in this study. Together these data indicate that the successive binding of
MAP repeats to neighboring tubulin subunits in the wall
of the microtubule lattice is neither a necessary nor major feature of microtubule stiffening by MAPs. Further experiments using different MAP2c constructs are required to
probe the mechanism by which a single MAP repeat, perhaps in association with other MAP2 sequence elements,
acts to induce stiffening.
Our findings on the influence of MAPs on microtubule
flexural rigidity are in partial agreement with studies from
other laboratories. The experiments of Kurachi et al.
(1995), though clouded by an unexpected dependence on
microtubule length, and those of Tran et al. (Tran, P.T.,
S.F. Parsons, R. Sterba, Z. Wang, M.P. Sheetz, and E.D.
Salmon. 1995. Mol. Biol. Cell. 6:260a.) both demonstrate, as we do, a large difference in the rigidity of microtubules
in the presence or absence of MAPs. On the other hand,
Mickey and Howard (1995)
and Kurz and Williams (1995)
conclude that the rigidity of single microtubules is only
marginally increased by the binding of tau or a mixture of
MAPs, respectively. While there is at present no satisfying
explanation for these discrepancies, it is evident from the
study reported here that microtubule rigidity does not change at random with different tau constructs but in a biologically consistent and interpretable manner. Moreover,
the changes we observe with different tau constructs and
MAP2c correlate well with other physiological parameters
such as their strength of binding to microtubules. These
factors lend strong support to the conclusion that MAPs
do indeed increase microtubule rigidity.
How do MAPs and MAP constructs modulate microtubule rigidity? Two mechanisms (that are not mutually exclusive) can be envisioned. In the first, the assembly domains of tau or MAP2 are proposed to associate with the
microtubule exterior in such a way that two or more tubulin subunits are interlinked. This would limit the freedom
of interdimer motions during the bending of a microtubule and thus increase microtubule stiffness (Matus, 1994). Alternatively, the binding of MAPs (or fragments thereof)
induces a conformational change in the tubulin dimer that
either affects interdimer binding or puts the dimer in a
strained conformation, analogous to that produced by
GTP hydrolysis (Caplow et al., 1994
; Mickey and Howard, 1995
); both these processes may help stiffen the polymer
(Bereiter-Hahn, 1978
; Mandelkow et al., 1991
). The magnitude of the conformational change promoted by the
binding of a given MAP construct to microtubules and,
hence, the resulting effect on microtubule stiffness would depend on the length and domain organization of the construct. At present it is not possible to distinguish between
these interpretations. As there is no strong evidence for
cooperative binding of tau to microtubules, the pronounced effect of a low concentration of tau (18% saturation causes 85% of the maximal stiffening effect; see Table
I) is unexpected and intriguing. It seems to suggest that the
actions of individial MAP molecules on microtubules are not merely additive, with each molecule contributing an
equal share until full saturation promotes maximal stiffening. Rather, subsaturation binding may induce conformational changes that affect more than just the tubulin subunits to which a MAP molecule binds. How this is achieved
remains to be determined.
What are the cell biological consequences of MAP-induced microtubule stiffening? In neurons, the length of
cell processes (axons and dendrites) may exceed the diameter of the cell body many hundredfold. This extreme cell
asymmetry requires mechanisms that confer mechanical
stability to these processes. Microtubules may well contribute to the morphological stability of these cell processes, and MAPs, in turn, may well contribute to the mechanical stability of microtubules. There are three properties
that MAPs could confer on microtubules to enhance their
ability to act as elements of structural support: reduction
of microtubule dynamic instability, increase in microtubule length, and increase in microtubule rigidity. Of these,
stabilization by reducing the growth and shrinkage of microtubules is less likely as MAP2 and tau do not significantly affect microtubule dynamic instability (Trinczek et
al., 1995; Kaech et al., 1996
). Increased microtubule length
and rigidity, on the other hand, are important factors, as
demonstrated by cell process formation in MAP-transfected nonneuronal cells (e.g., Kanai et al., 1989
; Edson et
al., 1993
). That neuronal MAPs do indeed alter microtubule rigidity in vivo is suggested by visual inspection of microtubule behavior in living cells: nonstabilized microtubules bend readily (Cassimeris et al., 1988
; Sammak and Borisy, 1988
), whereas MAP-stabilized microtubules appear much straighter and more resistant to bending even
when not bundled (Kaech et al., 1996
). In contrast with
MAP2 and tau, the microtubule-stabilizing drug taxol, which
also induces bundling of microtubules but has no stiffening
effect, is unable to induce cell process formation (Edson et
al., 1993
). Taken together, these observations indicate that
the increase in microtubule rigidity induced by tau and
MAP2 may be essential for the formation and maintenance
of neuronal processes. For this reason, the molecular mechanisms of the effects observed here are well worth studying.
Received for publication 23 January 1997 and in revised form 27 May 1997.
Please address all correspondence to Manfred Schliwa, Adolf-Butenandt-Institut, Zellbiologie, Schillerstr. 42, 80336 München, Germany. Tel.: (49) 89-5996-884. Fax: (49) 89-5996-882. e-mail: schliwa{at}bio.med.uni-muenchen.deWe thank Sabine Fuchs for expert technical assistance and Ulrike Henningsen for help with the preparation of axonemes.
This study was supported by the Deutsche Forschungsgemeinschaft priority program "Functional domains of cytoskeletal proteins" to E. Mandelkow, and SFB 266, the Friedrich Baur Stiftung, and a NATO travel grant to M. Schliwa.
MAP, microtubule-associated protein; EI, flexural rigidity.
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