From the Biochimie Cellulaire, CNRS FRE 2219, Université Pierre et Marie Curie, 9 quai Saint-Bernard, Case 265, 75252 Paris, Cedex 05, France, the § Department of Biology,
University of Milano, 20133 Milano, Italy, and ¶ Arpida AG,
Dammstrasse 36, Munchenstein 4142, BL, Switzerland
Received for publication, December 18, 2000, and in revised form, January 8, 2001
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ABSTRACT |
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The major neuronal post-translational
modification of tubulin, polyglutamylation, can act as a molecular
potentiometer to modulate microtubule-associated proteins (MAPs)
binding as a function of the polyglutamyl chain length. The relative
affinity of Tau, MAP2, and kinesin has been shown to be optimal for
tubulin modified by ~3 glutamyl units. Using blot overlay assays, we
have tested the ability of polyglutamylation to modulate the
interaction of two other structural MAPs, MAP1A and MAP1B, with
tubulin. MAP1A and MAP2 display distinct behavior in terms of tubulin
binding; they do not compete with each other, even when the
polyglutamyl chains of tubulin are removed, indicating that they have
distinct binding sites on tubulin. Binding of MAP1A and MAP1B to
tubulin is also controlled by polyglutamylation and, although the
modulation of MAP1B binding resembles that of MAP2, we found that
polyglutamylation can exert a different mode of regulation toward
MAP1A. Interestingly, although the affinity of the other MAPs tested so
far decreases sharply for tubulins carrying long polyglutamyl chains,
the affinity of MAP1A for these tubulins is maintained at a significant
level. This differential regulation exerted by polyglutamylation toward different MAPs might facilitate their selective recruitment into distinct microtubule populations, hence modulating their functional properties.
Microtubules (MTs)1 are
dynamic polymers, which are essential for a large variety of cellular
functions such as cell morphology and polarity, cell motility,
intracellular trafficking, and cell division. They are made up of Interactions between MTs and structural as well as motor MAPs are thus
of fundamental interest and must be tightly regulated by cells, both
locally and temporally, to ensure proper MT functions. MAPs generally
regulate their binding affinity for MTs by phosphorylation (see for
instance Refs. 5-8). However, observations that one motor binds to
only one MT in Chlamydomonas axoneme (9) or to only one MT
subset in lobster axon (10) or in the mitotic spindle (4) clearly imply
that MTs must also control their interactions with MAPs and somehow
deliver information to permit or restrict MAPs from binding. The
molecular basis for such control might be found in the tubulin
polymorphism. Indeed, tubulin diversity can generate MT diversity by
conferring to MTs heterogeneous interacting surfaces instead of a
monotonous succession of identical subunits all along the polymers. In
this context, post-translational modifications of tubulin represent
interesting potential means to play such a role: They can be easily
added to or removed from the MT surface through the intervention of
specific modifying enzymes that exhibit net substrate preferences for
either free or polymerized tubulin (for review see Refs. 1, 2). Dealing
with the genetic diversity of the diverse tubulin isotypes would
require active, time-consuming transcription and translation and would
not be as efficient as the rapid post-translational events, in
particular in the axonal compartment, where no protein synthesis
occurs, or in mitotic cells where transcription and translation are
turned off.
Among the multiple post-translational modifications of tubulin,
polyglutamylation was the first oligomeric modification discovered (11). Functionally, and in a close parallel with its oligomeric structure, polyglutamylation was further shown to behave as a molecular
potentiometer that modulates the binding of MAPs as a function of the
polyglutamyl chain length (12, 13). For instance, Tau, MAP2, and
kinesin motors have been shown to undergo the same mode of binding
regulation by polyglutamylation: The relative affinity of these
proteins first increases progressively for tubulin modified by 1 to 3 glutamyl units then progressively decreases when the chain lengthens
further, up to 6 units. This effect is likely to be achieved by
conformational changes of the C-terminal domain of tubulin, driven by
the growth of the polyglutamyl chain (12, 13).
Thus, polyglutamylation could control the targeted binding of a
particular MAP to MTs at a given level of glutamylation, without altering the binding of other MAPs. However, if polyglutamylation acts
as a general regulator of MAP binding, it is difficult to imagine the
manner in which it could distinguish between the different MAPs.
Consequently, we have sought to determine the binding behavior of other
MAPs, namely MAP1A and MAP1B, toward glutamylated tubulin and compared
it to that of another high molecular weight MAP, MAP2. We report that
polyglutamylation regulates the binding of MAP1A and MAP1B in a chain
length-dependent manner, MAP1B behaving like MAP2. On the
other hand, we found that the binding of MAP1A was differentially
regulated by polyglutamylation. In contrast to the other MAPs tested so
far, which all display an optimal affinity for tubulins modified by
around 3 glutamyl units, MAP1A has the selective property to maintain a
high affinity for highly glutamylated Antibodies--
The general anti- Purification of Microtubule Proteins--
Tubulin was prepared
from a 150,000 × g supernatant of adult mouse brain by
one cycle of assembly-disassembly (15) in MEM buffer (50 mM
MES, pH 6.7, 2 mM EGTA, 1 mM MgCl2)
containing a mixture of protease inhibitors (10 µg.ml
MAP1A was purified from twice-cycled MTs prepared from whole
bovine brain according to Pedrotti and Islam (16). The protocol is
based on a differential binding of MAPs to MTs depending on the
sulfonate buffer used (17). Briefly, twice-cycled MT proteins were
assembled in MEM buffer in the presence of 20 µM taxol.
After centrifugation, the pellet was resuspended in warm PEM buffer containing 20 µM taxol and incubated 20 min at
37 °C prior to centrifugation. Most of MAP1A was released into
the supernatant, with some other components, and the purification was
achieved by a Mono-Q anion-exchange chromatography.
MAP1B was prepared from whole calf brain by two ion-exchange
chromatographies (18). After pelleting assembled MTs, the supernatant was submitted to a Mono-Q anion-exchange chromatography in MEM buffer
containing 0.2 M NaCl. Fractions eluted at 0.4 M NaCl were pooled, dialyzed against MEM buffer, and loaded
on a Mono-S anion-exchange chromatographic column. MAP1B, eluted at
0.33 M NaCl, was finally dialyzed against MEM buffer,
rapidly frozen in liquid nitrogen, and stored at
MAP2 was purified by the heat treatment method according to Herzog and
Weber (19). Briefly, MAP2 was prepared from two-cycled calf brain MTs,
which were depolymerized at 4 °C, cleared by centrifugation, brought
to 0.75 M NaCl and boiled for 5 min. The supernatant was collected and submitted to a 50% (w/v) ammonium sulfate precipitation. MAP2 and Tau were then separated by phosphocellulose cation-exchange chromatography followed by a Sepharose 4B exclusion chromatography.
Protein concentrations were determined using the bicinchoninic acid
method (Micro BCA protein assay reagent kit, Pierce), with bovine serum
albumin being used as standard.
Limited Proteolysis of Tubulin by
Subtilisin--
Phosphocellulose-purified tubulin from mouse brain was
digested by subtilisin (Sigma) in a molar ratio of 240/1 at room
temperature for increasing times (0-150 min) in 10 mM MES,
pH 6.7, 0.1 mM MgCl2, 0.1 mM EGTA,
2 mM GTP. 10 µM EDTA and 50 µM
dithiothreitol were added to the digestion buffer to inhibit
contaminating proteases. Aliquots were drawn at increasing times and
analyzed by one-dimensional PAGE to follow the kinetics of proteolysis.
One- and Two-dimensional PAGE--
Protein separation by
one-dimensional (20) or two-dimensional PAGE (21) was carried out as
described previously (13). Transfer of proteins onto nitrocellulose
membranes (Hybond C, Amersham Pharmacia Biotech) was performed
essentially as described (22), and the blots were saturated in TBS-T
(20 mM Tris, pH 7.5, 136.8 mM NaCl, 0.1% v/v
Tween 20) containing 2% (w/v) low fat milk. Antibodies were incubated
in TBS-T overnight at room temperature, and their binding was revealed
by the chemiluminescence system ECL (Amersham Pharmacia Biotech).
Signals were quantified by densitometric scanning using an integrating
densitometer (Vernon) and the MultiAnalyst System (Bio-Rad).
Blot Overlay Assay--
Binding of MAPs to high speed
supernatant proteins or tubulin, separated by one-dimensional or
two-dimensional PAGE and transferred onto nitrocellulose membranes
(Hybond C, Amersham Pharmacia Biotech) was performed essentially as
described previously (12). Briefly, precise locations of blotted
tubulin subunits after one-dimensional PAGE or blotted tubulin isoforms
after two-dimensional PAGE were registered with pencil marks on the
membranes after Ponceau red staining following electrotransfer.
Strips corresponding to lanes of one-dimensional gels, or rectangles
corresponding to tubulin regions of the two-dimensional gels, were cut
from the blots and placed into the grooves of a hand-made, Plexiglas
incubation device adjusted to the size of the nitrocellulose pieces.
Membranes were blocked overnight in overlay (OV) buffer (MEM buffer
containing 1 mM dithiothreitol, 0.1% v/v Tween 20, and
0.1% w/v gelatin), incubated 1 h at room temperature with the
overlaying protein fraction (1 ml) then washed 5 × 5 min with OV
buffer. Protein interactions were stabilized with 0.5% (v/v)
formaldehyde in OV buffer. Blots were then equilibrated in TBS-T buffer
and processed for MAP immunodetection as described. Signals were
scanned and processed by the MultiAnalyst System (Bio-Rad).
Statistical Processing of Two-dimensional Overlay
Data--
Two-dimensional overlay experiments with the different MAPs
were done independently several times (6 with MAP1A, 2 with MAP1B, and
5 with MAP2). After MAP overlays, each blot was reincubated with
anti- Specificity of MAP Binding by Blot Overlay Experiments--
The
specificity of the interactions between purified MAP1A, MAP1B, or MAP2
and tubulin was tested by blot overlay experiments. In a first step,
proteins from whole mouse brain, high speed supernatant fraction
(So) were separated by one-dimensional PAGE and transferred onto
nitrocellulose. Increasing concentrations of each MAP were separately
overlaid onto identical duplicate membranes, and MAPs bound to their
protein targets were detected using specific antibodies. Fig.
1A shows that, under the
experimental conditions used and among the large number of soluble
brain proteins present on the membrane,
In a second step, increasing concentrations of each MAP were overlaid
onto a constant amount of phosphocellulose-purified tubulin separated
by one-dimensional PAGE and immobilized onto nitrocellulose. Fig. 1
(B-D) shows that the saturation curves of the Physicochemical Parameters of MAP1A and MAP2 Binding to
Tubulin--
The protein-protein interactions between
nitrocellulose-bound tubulin and soluble MAP1A or MAP2 in the presence
of increasing concentrations of salt or urea were also tested by blot
overlay. Binding of MAP1A and MAP2 to tubulin was differentially
affected by these treatments (Fig. 2).
Interaction of MAP1A appeared to be significantly more sensitive to
salt when compared with MAP2 (Fig. 2, A and B).
For example, addition of 50 mM NaCl resulted in a 50%
decrease in MAP1A binding (Fig. 2A), whereas the presence of
up to 75 mM NaCl did not affect the binding of MAP2 (Fig.
2B). However, neither MAP exhibited a detectable difference
in its interaction with the
In the presence of increasing concentrations of urea, the binding of
MAP2 appeared to be highly sensitive to the presence of this chaotropic
agent and decreased linearly as a function of urea concentration (Fig.
2D). A concentration of 1 M urea was sufficient
to reduce MAP2 binding by 50%, and no interaction could be detected
above 2 M urea. Once again, no difference could be observed
between the two tubulin subunits. By contrast, MAP1A in the presence of
urea exhibited important differences in binding to the Tubulin Polyglutamylation Modulates Differentially the Binding of
the Various MAPs--
We previously reported that polyglutamylation of
tubulin can regulate the binding of Tau, MAP2, and different kinesin
motors as a function of the polyglutamyl chain length through
progressive conformational changes of the C-terminal domain of tubulin,
the post-translational polyglutamyl chain acting as a molecular
potentiometer (12, 13). Because Tau and MAP2, on one hand, and kinesin
motors, on the other hand, are thought to interact at separate but
close sites within the C-terminal domain of tubulin (13), we observed logically a similar effect of polyglutamylation on the modulation of
binding of these three different proteins. In view of the observations that the putative tubulin binding sites present on MAP1A and MAP1B are
quite different from those described for Tau or MAP2 (see "Discussion"), we asked whether MAP1A and MAP1B binding could also
be affected by tubulin polyglutamylation. Consequently, we tested their
binding to tubulins carrying glutamyl chains of various lengths (from 0 to 6 or 7 glutamyl units). Because polyglutamylation is, by far, the
major post-translational modification of both
A closer examination of the positions of the MAPs bound onto tubulin in
Fig. 3 (B-D) shows that each MAP displays a distinct tubulin pattern of preferential binding. Compared with MAP2 (Fig. 3D), MAP1B appears to bind roughly to the same moderately
glutamylated tubulin isoforms (Fig. 3C). MAP1A, however,
seems to bind to additional more acidic
To analyze more precisely the binding preference of the diverse MAPs,
signals obtained from control immunoblots and from overlays (such as
those shown in Fig. 3, A-D) were integrated by
densitometric scanning. Several independent experiments with different
MAPs were processed and submitted to statistical analysis to confirm or
invalidate the differences observed in the binding of distinct MAPs to
a given subset of tubulin isoforms. To compare the relative affinities
of the three MAPs for tubulins at different levels of glutamylation,
scans were divided into 12 regions of identical size along the pI
dimension from the basic to the acidic side of the tubulin
two-dimensional spots, that is, from unglutamylated to highly
glutamylated tubulin isoforms, respectively. Results for both tubulin
subunits are shown in Fig. 4. In the
upper panels are represented the mean distribution of the
different
From data shown in Fig. 4, it can be calculated that tubulins with
short (<3 units) polyglutamyl chains can bind MAP1B or MAP2 ~2 times
more efficiently than MAP1A. This ratio is reversed for tubulins
carrying long polyglutamyl chains (>4 units). Although the relative
affinity of MAP1B or MAP2 for tubulins comprising more than four
glutamates shows a drastic 70% decrease, MAP1A shows a unique
capacity to significantly maintain a high affinity for these highly
glutamylated tubulins (80-90% of the optimal value for MAP1A and MAP2 Do Not Compete Each Other for Binding to
Tubulin--
As shown above, MAP1A and MAP2 display distinct
affinities toward the different polyglutamylated tubulins. However, it
is important to mention that mid-glutamylated tubulin isoforms are efficient targets for both MAPs. In this context, MAP1A and MAP2 could
compete for binding to the same site on tubulin. In the hypothesis of a
single common site, a high level of polyglutamylation could well alter
the binding of MAP2 but not that of MAP1A, suggesting erroneously the
existence of two distinct sites. For a mid level of glutamylation,
however, competition could be more apparent. To answer this question,
we performed competition binding experiments by overlay assay onto
tubulin separated by two-dimensional PAGE (Fig.
5) and one-dimensional PAGE (Fig.
6).
MAP1A and MAP2 were mixed and overlaid together onto tubulin separated
by two-dimensional PAGE and immobilized onto nitrocellulose (Fig. 5).
The experiments were performed in duplicate, and the binding of each
MAP was detected using specific anti-MAP antibodies on separate
membranes (Fig. 5, upper panels). A control membrane with no
MAP added was used to precisely locate the different tubulin isoforms
(Fig. 5, lower panels). The results confirm the data from
Figs. 3 and 4 and show that, besides the moderately glutamylated
To test the possible competition between MAP1A and MAP2 for the same
tubulin binding sites, we then performed two complementary competition
overlay protocols with tubulin separated by one-dimensional PAGE (Fig.
6). In a first approach, MAP1A and MAP2 were mixed and overlaid
simultaneously onto immobilized tubulin, MAP2 at a constant
concentration and MAP1A at increasing concentrations (Fig.
6A) and, conversely, MAP1A at a constant concentration and MAP2 at increasing concentrations (Fig. 6B). In the second
approach, increasing concentrations of one MAP were added first to
progressively saturate the binding sites on the tubulin molecules, then
the other MAP was added at a constant concentration (Fig. 6,
C and D). In this latter series of experiments,
interactions established between tubulin and the first MAP were
stabilized with formaldehyde prior to the addition of the other one to
avoid any displacement of the initially bound MAP by the other one. The
binding of each MAP to tubulin was then detected using specific
anti-MAP antibodies. The binding curves in Fig. 6 show that,
independently of the protocol used, when the two MAPs are added
simultaneously or successively the binding of a given MAP is always
representative of its respective concentration and is not affected at
all by that of the other one. These data strongly argue that
MAP1A and MAP2 bind to distinct and independent sites on the tubulin molecules.
Finally, an additional experiment was performed to rule out the
possibility of a competition between MAP1A and MAP2 for a single
binding site on tubulin that would not have been detectable in the
experimental conditions of Figs. 5 and 6. Because polyglutamylation is
thought to play a central role by differentially modulating MAP
binding, we tested the binding of MAP1A and MAP2 to tubulin cleaved
with subtilisin (Fig. 7). After protease
treatment, the distal C-terminal sequences of
In summary, polyglutamylation of tubulin, in addition to Tau, MAP2, and
kinesin motors, can also regulate the binding of the high molecular
weight MAP1A and MAP1B. Interestingly, we observed that
polyglutamylation can exert a differential control on the binding of
these MAPs. Changes in the relative affinity of MAPs for tubulins as a
function of the length of the polyglutamyl chains do not follow a
unique pattern for all of the MAPs. On the contrary, polyglutamylation
appears to act as a differential rather than a general regulator of
tubulin-MAP interactions. Subtilisin experiments indicate that the
polyglutamyl chains do not represent the direct physical target for
these MAPs (Refs. 12 and 13 and Fig. 7 in this paper). But as the
binding sites for most of these MAPs are located within the C-terminal
domain of tubulin, these sites likely undergo conformational changes
driven and modulated by the length of the polyglutamyl chain, the
optimal binding of a given MAP to tubulin, or the optimal accessibility
of this MAP for tubulin, being achieved for a defined polyglutamyl
chain length. In the case of MAP1A and MAP2, the establishment and/or
the stability of MAP1A-tubulin complexes tends to be retained for
tubulin carrying long polyglutamyl chains, lengths for which
MAP2-tubulin interactions are largely destabilized.
Blot Overlay Approach to Study Tubulin-MAP Interactions--
Blot
overlay assay represents a rapid and useful means to study
protein-protein interactions. This is particularly useful in the case
of tubulin that comprises a very large number of isoforms arising from
different gene products and multiple post-translational modifications.
These isoforms cannot be readily purified for use in classical
interaction experiments. Separation of such isoforms by two-dimensional
PAGE and their subsequent immobilization onto the surface of a
nitrocellulose membrane, however, provides the advantageous opportunity
for analyzing their respective binding capacities toward a given MAP
added in solution as a potential interaction partner.
Accurate positioning of tubulin and MAP to engage in their mutual
interaction depends on an active protein conformation involving the
establishment of critical bonds. In overlay experiments, in which
tubulin is first submitted to SDS-PAGE and transferred onto nitrocellulose, the recovery of a functional conformation is therefore essential. First, SDS is released from the protein during its transfer
onto the membrane, the transfer buffer containing 20% methanol (22).
Second, renaturation in an appropriate sulfonate buffer (or at least
the remodeling of an active local conformations) is largely
time-dependent. After transfer, nitrocellulose membranes need to be incubated at least overnight in OV buffer to observe a
subsequent efficient protein binding capacity of the membrane-bound tubulin. The same is also true for reverse overlay, where soluble tubulin interacts with membrane-bound MAPs (12).
By this technique, the specificity of the interactions can also be
easily tested. When soluble proteins from a whole mouse brain
supernatant are presented on the membrane to the overlaying MAP,
When the physicochemical conditions were altered by the addition of
salt or urea, differences in the binding of MAP1A and MAP2 were
observed. Binding of MAP1A to tubulin is much more sensitive to salt
than that of MAP2, indicating that ionic bonds are involved to a
greater extent in tubulin-MAP1A than in tubulin-MAP2 interactions. Moreover, this differential sensitivity would suggest that the two MAPs
bind to different sites on the tubulin molecule, and this is further
confirmed by the competition binding experiments shown in Figs. 5-7.
Differences were also observed between Differential Regulation of MAP Binding by
Polyglutamylation--
Polyglutamylation of tubulin (11) is an
oligomeric post-translational modification comprising a secondary chain
added close to the C terminus of
Polyglutamyl chains can be removed together with the extreme C-terminal
peptides of
The modulation of MAP1B binding to tubulin isoforms at different states
of glutamylation resembles roughly that of MAP2. MAP1B preferentially
interacts with moderately glutamylated A Model for MAP Transition Driven by Polyglutamylation--
The
original property of polyglutamylation to act as a differential
regulator of MAP binding could fulfill an interesting function in
neurons concerning MAP transitions during development or along axonal
and dendritic processes. During development, polyglutamylation is
regulated differently on
In adult neurons, several MAPs are expressed simultaneously and could
compete for binding to MTs. On the other hand, for noncompetitive MAPs,
the question of their specific recruitment to different MTs, to give
them distinct structural and/or functional properties, is of importance
and should require specific mechanisms. Tau and MAP2 share nearly
identical tubulin binding motifs and are under the same mode of
regulation by polyglutamylation. The problem of competition between
them is topologically resolved, because Tau is predominantly found in
axons (60) and MAP2 in dendrites (61, 62). Because MAP1A is present in
both types of processes (14), the problem of its differential
recruitment by MTs arises with MAP2 in dendrites and with Tau in
axons. Polyglutamylation of tubulin could potentially solve this
problem. In our previous studies on the axonal transport of tubulin and
Tau, we observed that Tau proteins were found associated with axonal
MTs in the very proximal segments of the sciatic motor axons but
apparently detach from them rapidly and become undetectable in the
following, more distal axonal
segments.3 Similar evidences
of asymmetrical distribution have been reported for different MAPs,
including Tau, MAP1B, and MT motors (63-68). In light of our results
on the differential regulation of MAP binding by polyglutamylation, we
propose that this modification could control a transition between Tau
and MAP1A on the axonal MTs along neuronal processes (Fig.
8). Indeed, when one considers tubulins
with polyglutamyl chains beyond 3 units, MAP1A can maintain a strong
binding to MTs, whereas at this level of modification both Tau and MAP2
tend to leave or are destabilized from MTs. Fig. 8 shows that, in the
neuronal cell body, tubulins are synthesized and assembled with Tau
into MTs that are then sent into the axons (for a review, see Ref. 69).
Tubulin polyglutamylase proceeds on these newly assembled MTs (70) and
lengthens the glutamyl chains as MTs move into the axons (26). When the
length of polyglutamyl chains exceeds 3 units, and given the relative
affinities of Tau and MAP1A, Tau would be destabilized and detached
from the MTs and would be progressively replaced by MAP1A, which can
interact with a broader range of polyglutamylated tubulins. This MAP
replacement could consequently confer new structural and functional
properties to the MTs (e.g. the ability to interact with
another set of axonal proteins, change the spacing between MTs or the
parameters of their movement down the axons, etc.).
This model could be generalized to other biological situations;
polyglutamylation of MTs or of specific MT subsets could offer the
capacity to sort the different MAPs to be recruited on these MTs to
modulate their properties. In non-neuronal cells, if centrioles are
constantly and strongly polyglutamylated, the polyglutamylation level
of cytoplasmic MTs is highly variable from one cell type to an other
and generally lower than that found in neurons or in axonemal
structures. However, even a low level of glutamylation could give a
substantial advantage to the modified MTs to recruit a given MAP or a
specific motor needed for a given function. Such a situation is
typically illustrated by the mitotic spindle. The different
subpopulations of astral, polar, and kinetochore MTs are each
characterized by specific dynamic and functional properties and
particularly by selective interactions with motor proteins. Moreover,
glutamylation of tubulin increases during mitosis, and polar/kinetochore but not astral MTs appear more glutamylated than
interphase MTs (71, 72), in good agreement with the proposed role for
glutamylation as a differential regulator of MAP interactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
and
-tubulin heterodimers, the two related subunits displaying a
large isoform polymorphism due to the expression of multiple genes
whose products are substrates for several post-translational
modifications (for review, see Refs. 1 and 2). MTs are polymerized
under the control of MT-associated proteins (MAPs), which also shape
the MT networks and confer on them distinct functional properties. For
instance, changes in MAP expression and/or activity when cells enter
mitosis is accompanied by drastic changes in dynamic, structural, and functional properties of spindle MTs, as compared with interphasic MTs.
Additional differences can also be observed in the binding of
structural or motor MAPs to the various MT subpopulations
astral, polar, and kinetochore MTs
of the mitotic spindle (for review, see
Refs. 3 and 4).
- and
-tubulins. A model
for a transition between MAPs along axonal processes is presented.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin and
anti-
-tubulin monoclonal antibodies DM1A (working dilution, 1:1000)
and DM1B (1:500) were purchased from Amersham Pharmacia Biotech, and
monoclonal anti-MAP1B (anti-MAP5, 1:3000) and monoclonal anti-MAP2
(1:2000) were purchased from Sigma Chemical Co. The anti-MAP1A (1A-1,
14) monoclonal antibody (1:20,000) was a generous gift of Dr. R. B. Vallee (University of Massachusetts Medical School, Worcester, MA).
Peroxidase-conjugated anti-mouse IgG serum (1:10,000) was from Byosis (France).
1
aprotinin, 10 µg.ml
1 leupeptin, 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride). Tubulin was further purified
by phosphocellulose cation-exchange chromatography (P11, Whatman) in
PEM buffer (75 mM PIPES, pH 6.8, 2 mM EGTA, 1 mM MgCl2). Tubulin aliquots were stored in PEM
buffer containing 1 mM Mg-GTP in liquid nitrogen.
80 °C.
- and anti-
-tubulin antibodies (post-control immunoblots) to
ensure a perfect positioning of the MAP signals onto the different tubulins immobilized on the membrane. For each overlay experiment and
for each
- and
-tubulin subunit, signals from overlays and from
control immunoblots were scanned (MultiAnalyst System, Bio-Rad), giving
one value for each 40 µm. Data were exported and processed using
Kaleidagraph (Abelbeck Software). Using marks prepositioned on the
membranes and on the autoradiographic films, overlay signals were
carefully aligned with those from the corresponding control immunoblots. Control immunoblots were then aligned together. Background was subtracted, and data were expressed as fractions of the maximal value taken as 1.0. Aligned scans were then divided into 12 sections, each one encompassing 75- to 90-point values and representing 3-3.6 mm
of a tubulin two-dimensional spot in the pI dimension. For each
section, the 75-90 values were added and the sums were exported into
Excel files. Files corresponding to repeated experiments with a given
MAP were used to calculate mean values and standard deviations (per
tubulin subunit and per overlaid MAP for each of the 12 sections).
These final values were used to create the graphics in Fig. 4
(curves and histograms). The mean values were also used for a statistical comparative test (Student's t
test) between MAP1A and MAP1B, MAP1A and MAP2, and MAP1B and MAP2. From this test, p values were calculated for each section.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
-tubulin represent
by far the main protein targets for each of the three MAP tested. Few
other unidentified proteins that bound MAPs were also detected albeit
at much lower level.
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Fig. 1.
MAP1A, MAP1B, and MAP2 binding to
- and
-tubulin
subunits. A, increasing concentrations (µg/ml) of
MAP1A, MAP1B, and MAP2 were overlaid onto whole supernatant proteins
from mouse brain separated by one-dimensional PAGE and transferred onto
nitrocellulose. Binding of MAPs, detected by specific anti-MAP
antibodies, is mostly restricted to
- and
-tubulin. Endogenous
MAPs present in brain samples are also revealed (top of
autoradiographs, indicated only for MAP2 immunodetection, right
panel). In the MAP1B experiment, the primary antibody recognizes
in the tubulin region a protein (noted by an asterisk) that
does not interact with MAP1B. B, C, and
D, phosphocellulose-purified tubulin (2 µg) was separated
by one-dimensional PAGE, blotted onto nitrocellulose, and overlaid with
increasing concentrations of MAP1A (B), MAP1B
(C), or MAP2 (D). MAPs bound to separated
-
and
-tubulin subunits were detected with specific anti-MAP
antibodies, and the resulting signals (upper panels) were
quantified by densitometric scanning and expressed in arbitrary units
(a.u.). Lower panels:
-tubulin, open
circles and solid lines;
-tubulin, filled
circles and dashed lines.
- and the
-tubulin subunits with the different MAPs are completely
superimposed, indicating that MAP1A, MAP1B, and MAP2 each have the same
intrinsic capacity to bind to either tubulin subunit. In these
experimental overlay conditions, the half-saturation values were found
to be similar (20-30 nM) for the different MAPs.
- or
-tubulin subunit.
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[in a new window]
Fig. 2.
MAP1A and MAP2 binding to
- and
-tubulin subunits in
the presence of NaCl or urea. For each experiment, 2 µg of
tubulin separated by one-dimensional PAGE and blotted onto
nitrocellulose were overlaid with 7 µg of MAP1A (A and
C) or MAP2 (B and D) in 1 ml of OV
buffer containing 0-250 mM NaCl (A and
B) or 0-5 M urea (C and
D). MAPs bound to separated
-tubulin
(
-T, solid lines) and
-tubulin
(
-T, broken lines) subunits were
detected with specific anti-MAP antibodies. The resulting signals were
quantified by densitometric scanning and expressed in arbitrary units
(a.u.).
- and
-tubulin subunit (Fig. 2C). As noted above with MAP2, a
50% decrease in the binding of MAP1A to tubulin was observed in the
presence of 1 M urea. However, as the urea concentration was raised further, binding of MAP1A to
-tubulin decreased rapidly but the binding to
-tubulin appeared to remain largely and
reproducibly unaffected. These results suggest that interaction of
MAP1A with tubulin mainly depends on ionic bonds, to a larger extent
than for MAP2, and that binding of MAP2 depends, to a larger extent than for MAP1A, on the protein conformation through the implication of
critical hydrogen bonds. Additionally, MAP1A appears to interact differently with the two tubulin subunits implying that, although MAP1A
binds to both subunits, the binding occurs at nonhomologous domains.
Taken together, these results suggest that different amino acid
residues of tubulin are involved in the interaction with MAP1A and
MAP2. A possible effect of calcium ions (from 1 to 10 mM)
on the binding of MAP1A was also investigated, but no qualitative or
quantitative modification in the binding of MAP was observed (data not shown).
- and
-tubulin in
adult brain (11, 23, 24), an electrophoretic separation of brain
tubulin by two-dimensional PAGE allowed us to differentiate
between the various glutamylated forms of both subunits toward
the acidic portion of the gel, proportionally to the number of the
additional acidic charges of the glutamyl units, in other words,
proportionally to the polyglutamyl chain length. After two-dimensional
PAGE, the separated glutamylated isoforms of tubulin were immobilized
by electrotransfer onto a nitrocellulose membrane where they could be
easily located and probed for their capacity to interact with a given
MAP. Fig. 3A shows a typical
two-dimensional pattern of adult brain tubulin after immunodetection
with anti-
- and anti-
-tubulin antibodies. The unmodified primary
translation products are present at the extreme basic side of the
different tubulin spots (25, 26), and the glutamylated derivatives are
spread toward the acidic sides of the spots (24, 27), as a function of
the number of glutamyl units oligomerized into the post-translational
chains. The extreme acidic sides of the spots correspond to
- or
-tubulins carrying polyglutamylated chains 6-7 units in length.
Nitrocellulose membranes, identical to those shown in Fig.
3A, were overlaid with purified MAP1A, MAP1B, or MAP2. Fig.
3 (B-D) shows the immunodetection of MAP1A, MAP1B, and
MAP2, respectively, that bound to
- and
-tubulin isoforms.
Compared with Fig. 3A, which displays the whole repertoire
of tubulin isoforms present on the membrane and available for MAP
interaction, MAP1A, MAP1B, and MAP2 did not obviously bind to all of
the tubulins with the same intensity but rather bound to a restricted
subpopulation of mid-glutamylated tubulin isoforms. This observation
strongly suggests that, as already reported for Tau, MAP2, or kinesin
(12, 13), the length of the polyglutamyl chains linked to
- and
-tubulin can also regulate the binding of MAP1A and MAP1B.
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Fig. 3.
MAP1A, MAP1B, and MAP2 binding to
- and
-isotubulins
separated by two-dimensional PAGE. Tubulin (20 µg) was separated
by high resolution two-dimensional PAGE, transferred onto
nitrocellulose, and stained with Ponceau red. Positions of tubulin
isoforms were carefully registered with pencil marks on the
nitrocellulose membranes for accurate comparisons between the different
membranes (brackets). Tubulin was either immunodetected with
general anti-tubulin antibodies (A) or overlaid with 16 µg/ml MAP1A (B), 12 µg/ml MAP1B (C), and 10 µg/ml MAP2 (D). A, control: the different
isotubulins were immunodetected first with DM1A (anti-
-tubulin) then
with DM1B (anti-
-tubulin). B, C, and
D, immunodetection of tubulin-bound MAP1A, MAP1B, and MAP2,
respectively, after overlay with the corresponding purified proteins.
Brackets are localized precisely in the same coordinates on
the four panels and indicate the positions of the whole range of
-
and
-tubulin isoforms.
- and
-tubulin
isoforms, i.e. those carrying longer polyglutamyl chains
(Fig. 3B).
- and
-tubulin isoforms present on the membrane and the
mean relative amounts of MAP1A, MAP1B, and MAP2 bound to the tubulin
isoforms as a function of pI. These mean scans from control immunoblots and overlay experiments were accurately aligned one to the other relative to the pI on the x axis (see "Experimental
Procedures" for details). The lower panels show the
comparison of the binding capacities of MAP1A versus MAP1B,
MAP1A versus MAP2, and MAP1B versus MAP2 toward
the different tubulin isoforms as a function of their degree of
glutamylation. Asterisks positioned in sections indicate
that the differences observed in the binding behavior of two MAPs
toward a given isotubulin subset can be considered as significant
according to Student t test calculations (see
"Experimental Procedures"). Fig. 4 shows that the relative affinity
of MAP2 for
- and
-tubulin increases with the polyglutamyl chain
length, as reported previously (12), reaching an optimum for a length of about 3 units then gradually decreasing for longer chains. Similarly, the tubulin isoform binding range of MAP1B is also centered
around triglutamylated tubulins and does not differ significantly from
that of MAP2. By contrast, the tubulin isoform binding range of MAP1A
is much broader and clearly comprises more acidic isoforms from both
tubulin subunits. Compared with MAP2 or MAP1B, the optimal preference
of MAP1A is shifted toward tubulins with longer polyglutamyl chains.
All of these differences were confirmed to be statistically significant.
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Fig. 4.
Preferential binding of MAP1A, MAP1B, and
MAP2 to the different - and
-tubulin isoforms. Signals detected over the
- and the
-tubulin two-dimensional spots from control tubulin
immunoblots and from MAP immunodetections after overlay (such as those
shown in Fig. 3) were scanned and integrated. Several independent
experiments were carried out (6 for MAP1A, 3 for MAP1B, and 5 for
MAP2). Data were processed as indicated under "Experimental
Procedures" after dividing the tubulin spots into 12 equal sections
in the pI dimension. Curves shown in the upper
panels represent the preferential binding of MAP1A, MAP1B, and
MAP2 to
- (left panel) and
-tubulin (right
panel) isoforms as a function of their pI (in the different
sections, from basic, left, to acidic, right),
that is, of the length of their polyglutamyl chains (from 0 to 6 or 7 units). Solid lines represent the distribution of total
-
and
-tubulins present on the membranes and detected by general
anti-tubulin antibodies (control immunoblots). Histograms of the
lower panels represent the mean values (± S.D.) of the
amounts of MAP1A, MAP1B, and MAP2 (expressed in arbitrary units) bound
to the different subsets of isotubulins present in the 12 sections. For
each section, data from couples of MAPs were submitted to Student's
t test. When mean values representing the binding of two
different MAPs to a given subset of tubulins within a given section
were assessed as "statistically different values" (confidence
test), the corresponding sections were labeled with one, two, three, or
four asterisks, representing p values < 0.05, 0.02, 0.01, or 0.001, respectively.
-tubulins;
75-120% for
-tubulins). In conclusion, it appears that
polyglutamylation of tubulin has the property to regulate
differentially the binding of distinct structural MAPs.
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Fig. 5.
Simultaneous overlay of MAP1A and MAP2 on
tubulin separated by high resolution two-dimensional PAGE. Tubulin
(20 µg) was separated by high resolution two-dimensional PAGE and
transferred onto nitrocellulose membrane. Positions of - and
-tubulin isoforms were carefully registered with pencil
marks after Ponceau red staining (brackets above the
upper panels). This experiment was done in triplicate. Two
blots were overlaid simultaneously with MAP1A (8 µg/ml) and MAP2 (4 µg/ml). MAP binding was detected using specific anti-MAP antibodies
(MAP1A on the first blot, upper panels; MAP2 on the second
blot, middle panels). Tubulin isoforms were immunodetected
on the third blot (lower panels) with DM1A (general
anti-
-tubulin) then with DM1B (general anti-
-tubulin). To
facilitate the comparison between the resulting signals, the
- and
-tubulin regions of the blots were cut and precisely aligned
vertically.
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Fig. 6.
Competition binding experiments with MAP1A
and MAP2 on tubulin separated by one-dimensional PAGE. Tubulin (2 µg per lane) was separated by one-dimensional PAGE, transferred onto
nitrocellulose, and overlaid simultaneously (A,
B) or successively (C, D) with MAP1A
and MAP2 at increasing or constant concentration (symbolized by
triangles or rectangles, respectively, at the
top of the figure). A, co-overlay with increasing
concentrations of MAP1A (0-16 µg/ml) and a constant concentration of
MAP2 (12 µg/ml); B, co-overlay with increasing
concentrations of MAP2 (0-12 µg/ml) and a constant concentration of
MAP1A (16 µg/ml); C, first overlay with increasing
concentrations of MAP1A (0-16 µg/ml) then second overlay with a
constant concentration of MAP2 (12 µg/ml); D, first
overlay with increasing concentrations of MAP2 (0-12 µg/ml) then
second overlay with a constant concentration of MAP1A (16 µg/ml).
Tubulin-bound MAP1A (solid lines) and MAP2 (broken
lines) were detected with specific anti-MAP antibodies and signals
were quantified by densitometric scanning (expressed in arbitrary
units, a.u.). When MAPs were overlaid one after the other,
interactions between tubulin and the first MAP were stabilized with
formaldehyde prior to the second overlay to avoid any displacement of
the first MAP by the second one.
-
and
-tubulins that share the property to bind both MAP1A and MAP2,
the
- and
-tubulin species carrying longer polyglutamyl chains
are efficient and specific targets for MAP1A.
- and
-tubulins
carrying the polyglutamyl chains are cut off and hence
polyglutamylation cannot interfere anymore. Purified tubulin was
submitted to limited proteolysis with subtilisin, and aliquots were
drawn at various times of digestion. After one-dimensional PAGE
analysis, we chose a time point corresponding to a partially digested
sample containing both subtilisin-cleaved tubulins (named
S and
S) and uncleaved tubulin subunits to serve as internal binding
controls (Fig. 7, lanes 1 and 2). Binding of
MAP1A (lanes 3-5) and MAP2 (lanes 6-8) was then
probed on a duplicate series of nitrocellulose strips where both MAPs
had been overlaid in the following orders: MAP1A then MAP2 (lanes 4 and 7) and MAP2 then MAP1A (lanes 5 and
8). Removal of the C-terminal sequences of tubulin by
subtilisin does not prevent MAP1A and MAP2 from binding to
S- and
S-tubulins, although the binding of both MAPs appears significantly
reduced on
S-tubulin. When compared with the overlay controls (MAP1A
alone, lane 3; MAP2 alone, lane 6), it is clear
that the prior binding of one MAP does not affect at all the subsequent
binding of the other, which strongly suggests, again, that MAP1A and
MAP2 do bind tubulin at different sites.
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Fig. 7.
Binding of MAP1A and MAP2 to
subtilisin-cleaved tubulin. Purified mouse brain tubulin was
submitted to limited proteolysis with subtilisin. Time of digestion was
chosen to obtain both cleaved and uncleaved tubulin for each subunit.
Digested samples were subjected to one-dimensional PAGE (4 µg per
lane), transferred onto nitrocellulose, and either immunoprobed with
DM1A (lane 1) and DM1B (lane 2) to localize the
cleaved ( S and
S) and
uncleaved (
and
) tubulin subunits or
overlaid with MAP1A or/and MAP2 (lanes 3-8). Competition
overlays with MAP1A and MAP2 were performed in duplicate as indicated:
first overlay with MAP1A then second overlay with MAP2 (lanes
4, 7), first overlay with MAP2 then second overlay with
MAP1A (lanes 5, 8), followed by the
immunodetection of MAP1A (lanes 4, 5) or MAP2
(lanes 7, 8). Lanes 3 and 6 show the binding of MAP1A and MAP2, respectively, in the absence of the
other MAP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
and
-tubulin subunits represent the main protein targets (see Fig.
1A), although other minor MAP-interacting proteins are also
detected. For instance, in experiments performed with higher MAP
concentrations, two other proteins were found to specifically bind
MAP1A under these conditions (data not shown): Actin, according to the
previously reported actin-binding activity of MAP1A (28), and an 80-kDa
protein, which remains unidentified.
- and
-tubulin subunits in
the binding of MAP1A. In the presence of urea, MAP1A can still
efficiently engage in an interaction with the
- but not with the
-tubulin subunit. The establishment of the complex between MAP1A and
-tubulin, as those between MAP2 and
- or
-tubulin, appears to
be more dependent on precise hydrogen bonding between the two protein
partners. These results suggest that, contrary to the other MAPs tested
so far, MAP1A contacts
- and
-tubulin subunits at asymmetrical,
nonrelated sequences but apparently with a similar efficiency. An
alternative possibility, in view of the fact that multiple tubulin
binding motifs differing in their physicochemical properties have been
reported for MAP1A (29-31), is that the different binding conditions
selectively affect one motif without affecting the functional state of
others. In particular, the latter hypothesis may be more applicable to
the
-tubulin subunit.
- and
-tubulin to the
-carboxylic group of the lateral chain of a glutamate residue. This
chain is made of 1 to 6 or 7 glutamyl units linked one to the other by
peptidic and/or isopeptidic bonds (11, 27, 32-37). This oligomeric
structure works as a molecular potentiometer to regulate in a
length-dependent manner the affinity of the C-terminal
domains of tubulins for structural and motor MAPs such as Tau, MAP2
(12), or kinesin motors (13). Masking the polyglutamyl chains with the
specific monoclonal antibody GT335 (27) leads to a strong inhibition of
MAP binding in vitro (12, 13) as well as a strong inhibition of motility of axonemes of flagella or cilia (38-40). Microinjection or electroporation of GT335 into proliferating HeLa cells has been
shown to be followed by a transient and reversible disappearance of
centrioles (41). Microinjection of GT335 into Atlantic cod melanophores
has also been shown to interfere with kinesin-based pigment granule
dispersion but not with its cytoplasmic dynein-based aggregation,
suggesting that polyglutamylation could also differentially regulate
both motor proteins (42). A common feature of these results obtained
in vivo might be that the antibody blocks the function of
the polyglutamyl chains, thus preventing or drastically altering the
interaction of structural and motor MAPs with tubulins, just as
observed in the in vitro experiments cited above.
- and
-tubulin by enzymatic cleavage with subtilisin
(33, 43). In this case, subtilisin-cleaved tubulin was shown to retain
the ability, albeit to a lesser extent, to bind Tau, MAP2, or kinesin
(12, 13). Similar results were obtained with MAP1A (see Fig. 7). These
results indicate that the polyglutamyl chains are not the direct
targets for MAP binding but, rather, could play an indirect role in
modulating the overall conformation of the C-terminal domain of
tubulin. Such conformational changes may fine-tune the opening or the
closure of the MAP binding sites. Tau and kinesin are thought to bind
tubulin at close but independent sites inside of a predicted
two-
-helix motif closed by ionic bonds (12, 13) and located close to
the C terminus, exposed at the outer surface of the MT, facing the
incoming MAPs. This motif is now clearly visible in the electron
crystallography structure of the tubulin heterodimer (
-helices H11
and H12 in Ref. 44). It is therefore possible that this two-
-helix
motif may gradually open as a consequence of the incoming of the
negative charges from the growing polyglutamyl chain when it lengthens up to 3 units and consequently increase the accessibility to the MAP
binding sites (12, 13). Because Tau, MAP2, and kinesin all bind to this
same structural motif, it is not surprising that they exhibit a similar
mode of regulation by polyglutamylation. The location of the MAP
binding site could then correlate with the mode of regulation driven by
polyglutamylation. For instance, we observed that the binding of STOP
proteins (55) was insensitive to
polyglutamylation,2
suggesting that the corresponding binding site should be out of the
structural influence of polyglutamylation. On the other hand, MAP1A and
MAP1B have tubulin-binding motifs (29-31, 45, 46) unrelated to those
shared by Tau, MAP2, or MAP4 (47-49) and are thought to bind to
tubulin at different sites. Although related, MAP1A and MAP1B display
different affinities for tubulin and induce distinct MT shapes (17,
50). Furthermore, MAP2 can compete with MAP1B for binding to tubulin
(51, 52) but not with MAP1A (16, 53, 54; see also this paper, Figs.
5-7). MAP1A and MAP1B therefore offer the possibility to test whether
their binding sites could be under a different mode of regulation than
that of MAP2.
- and
-tubulin isoforms
(see Figs. 3-5). This observation is in agreement with previous
reports that MAP1B and MAP2 bind to similar sites on MTs (51, 52).
Consequently, their binding is modulated in a similar manner.
Interestingly, MAP1A displays a significantly distinct binding behavior
toward the various glutamylated tubulin isoforms (see Figs. 3-5). In
contrast to all of the other MAPs tested so far, MAP1A can maintain a
selective and high relative affinity for
- and
-tubulin isoforms
carrying long polyglutamyl chains, from 3 to 5 or 6 units in length.
MAP1A thus appears to be the only adult MAP capable of an efficient
binding to highly glutamylated
- and
-tubulins; such isoforms are
present in large amounts in neuronal (24, 27, 56) and axonemal (40, 57)
MTs. The presence of MAP1A could then be particularly useful for
stabilizing MTs made of these highly glutamylated tubulins for which
other MAPs like Tau, MAP2, or MAP1B have but a weak affinity.
- and
-tubulin, both qualitatively and
quantitatively. At birth, most of neuronal
-tubulin is already glutamylated, with chain lengths covering the whole range from 1 to 6 or 7 units. By contrast,
-tubulin is quantitatively far less
modified and in particular the glutamyl chains progressively increase
in size throughout postnatal development (56). During the same period,
in developing neurons, the juvenile MAP1B is progressively replaced by
MAP1A (58, 59). It is striking to note that MAP1B is expressed in these
neurons up to a time when tubulin is glutamylated at a maximal level of
2 or 3 units (at least in the case of the
-subunit), which
corresponds to the optimal affinity for this MAP. As development
proceeds, the length of glutamyl chains continues to increase while
MAP1A progressively replaces MAP1B. In other words, MAP1B is
substituted by MAP1A, which is much better adapted to interact with the
-tubulin isoforms equipped with longer polyglutamyl chains.
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Fig. 8.
Model for a Tau-MAP1A transition on axonal
microtubules driven by tubulin polyglutamylation. Schematic
representation of a neuronal cell showing part of the cell body and the
beginning of the axon. After synthesis of tubulin heterodimers
(step 1) and their assembly with Tau proteins (step
2), MTs are conveyed into the axon. Because MTs are formed and as
they move down the axon, assembled tubulins are substrates to the
enzyme tubulin-polyglutamylase that gradually lengthens the glutamyl
chains (En) (steps 3 and 4).
When the length of the glutamyl chains increases beyond 3 units, and up
to 6 or 7 units (step 4), the relative affinity of Tau for
these MTs decreases drastically and Tau can but detach from them. At
the same time Tau is destabilized, and given the affinity of MAP1A for
highly glutamylated tubulins, MAP1A can bind and/or stay efficiently
bound to these axonal MTs to maintain their stability and confer new
structural, functional, and interacting properties. According to the
role of polyglutamylation as a differential regulator of MAP
interactions, this post-translational modification could drive
automatic MAP transitions (Tau-MAP1A in axons, MAP2-MAP1A in dendrites,
MAP1B-MAP1A during development, etc.) and, more generally, could
possibly control the recruitment of specific MAPs or motor proteins
onto specialized subsets of MTs such as axonemes or mitotic spindles in
non-neuronal cells.
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ACKNOWLEDGEMENTS |
---|
We are very grateful to Dr. Richard B. Vallee (University of Massachusetts Medical School, Worcester, MA) for the generously (and repeatedly) providing the anti-MAP1A monoclonal antibodies.
![]() |
FOOTNOTES |
---|
* This work was supported by CNRS FRE 2219 and by the Association pour la Recherche sur le Cancer (Grant ARC 9241).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Tel.:
33-1-44-27-22-94; Fax: 33-1-44-27-22-15; E-mail:
jclarche@snv.jussieu.fr.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M011380200
2 C. Bonnet, C. Bosc, E. Denarier, and J.-C. Larcher, personal communication.
3 J. C. Larcher, D. Boucher, and P. Denoulet, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MT(s), microtubule(s); PAGE, polyacrylamide gel electrophoresis; MAP(s), microtubule-associated protein(s); MES, 2-(N-morpholino)ethanesulfonic acid; OV buffer, overlay buffer; PIPES, 1,4-piperazinediethanesulfonic acid; Tris, tris(hydroxymethyl)aminomethane.
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