1 Département de pathologie et biologie cellulaire, Université de
Montréal, Montréal, Québec, Canada, H3T 1J8
2 Montreal General Hospital Research Institute in Neuroscience, McGill
University, Montréal, Canada, H3G 1A4
* These authors contributed equally to the experimental work in this study
Author for correspondence (e-mail:
nicole.leclerc{at}umontreal.ca
)
Accepted 19 December 2001
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Summary |
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Key words: MAP2, Microtubules, Process formation, Sf9 cells, Projection domain
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Introduction |
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Diverse MAP2 isoforms generated by alternative splicing contribute to
neuronal differentiation (Tucker,
1990). In the COOH-terminus, these isoforms present a
microtubule-binding domain similar to that found in MAP4 and in the low
molecular weight MAP, tau (Lewis et al.,
1989
; Lewis et al.,
1988
). It contains 3 to 4 imperfect repeats of 18 amino acids
(a.a.) responsible for the binding to microtubules. The NH2-
terminus is called the projection domain since it projects at the surface of
microtubules. There exists at least four isoforms of MAP2: MAP2a, MAP2b, MAP2c
and MAP2d. MAP2a and MAP2b, the high molecular weight isoforms, are the
predominant isoforms found in the neurons of the adult CNS
(Chung et al., 1996
). They are
only expressed in dendrites. MAP2b is expressed before MAP2a during neuronal
differentiation (Tucker,
1990
). Recently, new variants of the high molecular weight MAP2
have been identified that contain additional sequences in the N- and
C-terminus (Couchie et al.,
1996
; Forleo et al.,
1996
; Kalcheva et al.,
1997
). MAP2c and MAP2d, the low molecular weight isoforms of MAP2
differ by the fact that MAP2d contains four repeats in the C-terminus for
binding to microtubules whereas MAP2c has three repeats
(Ferhat et al., 1994
). MAP2c
is found in growing dendrites and, in certain populations of neurons, in
growing axons (Tucker, 1990
).
Interestingly, it remains expressed in neurons that have the capacity to
regenerate in the adult CNS (Tucker,
1990
).
MAP2b and MAP2c are the two best characterized isoforms of MAP2. MAP2c is a
467 amino acid (a.a.) protein whereas MAP2b is a 1828 a.a. protein that
contains an additional sequence of 1372 a.a. in the N-terminus whose function
is unknown (Neve et al., 1986;
Papandrikopoulou et al.,
1989
). This additional sequence shifts the apparent molecular
weight of MAP2b from 70 kDa to 280 kDa. The most studied functions of these
isoforms are their ability to promote microtubule assembly and to stabilize
microtubules (Ludin and Matus,
1993
). They also induce microtubule bundling but the spacing
between microtubules is less in MAP2c-induced microtubules than in
MAP2b-induced microtubules (Chen et al.,
1992
; Leclerc et al.,
1996
). Thus, it has been concluded that the length of the
projection domain of MAP2 is one primary determinant of the spacing between
microtubules.
When overexpressed in cultured non-neuronal cells, MAP2b and MAP2c induce
microtubule protrusion from cell surface thus causing the formation of
cytoplasmic processes (Boucher et al.,
1999; Edson et al.,
1993
; Langkopf et al.,
1995
; Leclerc et al.,
1996
; Leclerc et al.,
1993
). In some systems, depolymerisation of the actin cortical
network is required to allow microtubule protrusion from the cell surface
(Edson et al., 1993
). In other
systems such as Sf9 cells, spontaneous protrusion of microtubules and process
formation occurs upon overexpressing MAP2b or MAP2c
(Leclerc et al., 1996
;
Leclerc et al., 1993
). In this
study, we used the baculoviral expression system in Sf9 cells to characterize
the role of the projection domain of MAP2 in the protrusion of microtubules
from the cell surface, and the subsequent process formation in these cells. In
Sf9 cells, MAP2c has a higher capacity than MAP2b to induce process formation
(Leclerc et al., 1996
;
Leclerc et al., 1993
). Thus,
it appears that the additional sequence of 1372 a.a. contained in the
projection domain of MAP2b influences the protrusion of microtubules from the
cell surface in these cells. In the present study, we generated truncated
forms of MAP2b and MAP2c that had partial or complete deletion of the
projection domain. Our results indicate that the formation of processes is
induced by the microtubule-binding domain and is regulated by the projection
domain of these MAP2 proteins. Furthermore, our results indicate that the
structural properties of the microtubule bundles induced by MAP2c and MAP2b
truncated forms are not the sole factors contributing to microtubule
protrusion from Sf9 cells. The regulatory effects of the projection domain on
microtubule protrusion would involve intramolecular interactions between this
domain and the microtubule-binding domain.
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Materials and Methods |
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Five MAP2b constructs having a deletion of nucleotides 656-1921 (MAP2b-1), 1921-3320 (MAP2b-2), 3329-4772 (MAP2b-3), 1-4772 (Mt) and 4772-5881 (Prob) were produced. To delete the nucleotides 656-1921 (MAP2b-1), a ScaI site was inserted in 3' of nucleotide 656 by PCR. A PCR product containing the MAP2b sequence from nucleotide 378-656-ScaI (PCR-C1) was cloned into the blunt end Pstblue vector. The insertion of PCR-C1 into the MAP2b-BacPAK His2 vector was done in three steps. First, the sequence from 1-5471 of MAP2b-His sequence was subcloned into the BamHI and XhoI sites of the Bluescript vector (MAP2b1-5471-BS). The nucleotides from 378-1921 of the MAP2b sequence were deleted by digestion of MAP2b1-5471-BS with KasI and ScaI. Second, the PCR-C1 cloned into the Pstblue vector was digested with KasI and ScaI and subcloned into the sites KasI and ScaI of the MAP2b1-5471-BS vector (C1-MAP2b1-5471-BS). Third, the sequence of MAP2b containing the deletion from 656-1921 was cut from the MAP2b1-5471-BS by digestion with BamHI and XhoI and re-inserted in the MAP2b-BacPAK His2 vector.
To delete the nucleotides 1921-3329 (MAP2b-2), an Xba1 site was inserted in 3' of nucleotide 1921 by PCR. A PCR product containing the MAP2b sequence from nucleotides 378-1921-Xba1 (PCR-C2) was cloned into the vector Pstblue. The insertion of PCR-C2 into MAP2b-BacPAK His2 vector was done in three steps. First the nucleotides 1-3121 of MAP2b-His sequence were subcloned into the Bluescript vector (MAP2b1-3121-BS). Second, the PCR-C2 cloned into the PSTblue vector was digested with Kas1 and Xba1 and subcloned into the corresponding sites of MAP2b1-3121-BS (C2-MAP2b1-3121-BS). Third, the sequence of MAP2b containing the deletion from 1921 to 3329 was cut from the C2-MAP2b1-3121-BS by digestion with BamHI and XbaI and re-inserted in the MAP2b-BacPAK His2 vector.
To delete the nucleotides 3329 to 4472 (MAP2b-3), an XbaI site was inserted in 3' of nucleotide 4472 by PCR. A PCR product containing the MAP2b sequence from nucleotides 3329-4472-XbaI (PCR-C3) was cloned into the blunt end Pstblue vector. The insertion of PCR-C3 into MAP2b-BacPAK His2 vector was done in three steps. First, the nucleotides 3329-5513 of MAP2b-His sequence was subcloned into the Pstblue vector (MAP2b-3329-5513-Pb) at the SphI and XbaI sites. Second, the PCR-C3 cloned into the Pstblue vector was digested with SmaI and XbaI and subcloned into the corresponding sites of MAP2b-3329-5513-Pb (C3-MAP2b-3329-5513-Pb). Third, the sequence of MAP2b containing the deletion from 3329 to 4772 was cut from the C3-MAP2b-3329-5513-Pb by digestion with XbaI and SphI and re-inserted in the MAP2b-BacPAK His2 vector.
A truncated form of MAP2b corresponding to the microtubule-binding domain (Mt) was generated by deleting the nucleotides 1-4772. To do so, a BamHI site was inserted at the 4765 nucleotide by PCR. MAP2b-pBacPAK His2 was digested with BamHI and SphI. The deleted sequence was replaced by the PCR produce digested with the same restriction enzymes.
A truncated form of MAP2b corresponding to the projection domain of MAP2b (Prob) was produced by deleting the nucleotides 4772-5881. First, a NotI site was inserted at the 4772 nucleotide of the MAP2b sequence by PCR. MAP2b-pBacPAK His2 was digested with NotI and XbaI. The deleted sequence was replaced by the PCR product digested with the same restriction enzymes.
We also engineered a truncated form that corresponds to the projection domain of MAP2c (Proc) (nucleotides 1-444). To do so, the MAP2c sequence was inserted into the pBacPAK vector. MAP2c was removed from the pCMV vector by digestion with NotI and inserted into the pBacPAK His2 at the NotI site. The start codon of MAP2c and the 5' untranslated region was removed. To do so, a BamHI was inserted at the start codon of MAP2c by directed mutagenesis. MAP2c-pBacPAK was digested with BamHI and PstI. The deleted part was replaced by the PCR product digested with the same restriction enzymes. To generate a truncated form of MAP2c corresponding to its projection domain, a NotI site was inserted by directed mutagenesis at the nucleotide 444 of the MAP2c sequence. MAP2c-pBacPAK His2 was digested with NotI and NcoI. The deleted sequence was replaced by the PCR product digested with the same restriction enzymes.
The transfer vector containing the different mutated forms of MAP2b and MAP2c were co-transfected with the Bsu361-digested BacPAK6 viral DNA onto the Spodoptera frugigerpa (Sf9) cells using bacfectin (Clontech, Palo Alto, CA).
Cell culture
The Sf9 cells were obtained from the American Type Culture Collection (ATCC
# CRL 1711; Rockville, MD). Sf9 cells were grown in Grace's medium (Gibco BRL,
Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum
(Immunocorp, Montreal, Quebec, Canada) as a monolayer at 27°C. For
infection, cells were plated on glass coverslips at a density of
1x106 cells/ 60 mm dish. Cells were infected for 24 or 72
hours with viral stock at various multiplicities of infection (m.o.i.).
Immunofluorescence
For immunochemistry, the cells were fixed in 4% paraformaldehyde in PBS for
20 minutes. Then the cells were permeabilized with 0.2% Triton X-100 in PBS
for 5 minutes. The expression of the truncated forms of MAP2b and MAP2c except
MT was revealed by using a monoclonal antibody directed against MAP2 (clone
HM2, dilution 1:200) purchased from Sigma (Mississauga, Ontario, Canada). The
Mt mutant was revealed by the monoclonal antibody 46.1 directed against the
microtubule-binding domain of MAP2 (kindly provided by V. Lee, University of
Pennsylvania). To visualize the microtubule reorganization, a monoclonal
antibody directed against -tubulin (Sigma, dilution 1:500) was used.
The actin reorganization was visualized with Rhodamin-phalloidin (Molecular
Probes, Eugene, OR), at a dilution of 1:200. We used the following secondary
antibodies (Jackson Immunoresearch Laboratories, Bio/Cam, Mississauga,
Ontario, Canada): the anti-mouse Fab fragment conjugated to rhodamine
(dilution 1:500) and a donkey anti-mouse conjugated to FITC (dilution 1:500).
All these antibodies were diluted in PBS plus 5% BSA. Incubation was carried
out at room temperature for 1 hour. After three washes in PBS, the coverslips
were mounted in moviol and visualized by fluorescence microscopy.
Preparation of microtubules
Microtubules were purified from Sf9 cells as previously described, with
slight modifications (Vallee and Collins,
1986). Briefly, cells were collected by centrifugation at 250
g for 3 minutes. The cells were then suspended in the PEM
buffer (0.1 M Pipes-NaOH, pH 6.6, 1 mM EGTA and 1 mM MgSO4) to
which a cocktail of protease inhibitors had been added before use. Sf9 cells
were then homogenized using a Dounce homogenizer and centrifuged for 30
minutes at 39,000 g at 2°C. The supernatant was recovered
and centrifuged at 23,000 g for 90 minutes at 2°C. The
supernatant was collected again and taxol added to 20 µM and GTP to 1 mM.
The solution was warmed to 37°C for 5 minutes and then chilled on ice for
15 minutes before being transferred to a centrifuge tube. Ice cold sucrose
underlayer solution (to which 20 µM taxol and 1 mM GTP were added just
before use) was introduced at the bottom of the tube with a Pasteur pipette,
and the sample centrifuged at 2°C at 39,000 g for 30
minutes. The microtubule pellet was then resuspended in ice-cold PEM buffer
(containing taxol and GTP) and centrifuged again in at 2°C.
Extraction of the cytoskeleton
At 48 hours post-infection, Sf9 cells were washed in PBS and then suspended
in the extraction buffer (80 mM Pipes pH 6.8, 0.05% IGEPAL, 1 mM
MgCl2, 5 mM EGTA), to which protease inhibitors were added prior to
use. Extraction was allowed to proceed for 2 minutes. Cells were then
centrifuged at 250 g for 2 minutes. The pellet was resuspended
in 80 mM Pipes pH 6.8, 1 mM MgCl2 and 5 mM EGTA and cells lysed
using PBS buffer containing 1% IGEPAL and 0.1% SDS. For drug treatment, 10
µM colchicine was added to the cultures 2 hours before extraction. For cold
treatment, cultures were incubated in an ice-bath for 30 minutes before the
extraction was performed.
Immunoblotting and dot blotting
The expression of the truncated forms of MAP2 in the Sf9 cells was
confirmed by western blot. To do so, the transfected cells were centrifugated
at 250 g for 3 minutes. The pellets were resuspended into PBS
containing protease inhibitors (5 µg/ml of antipain, aprotinin and
leupeptin, 1 mM EDTA, 100 µg/ml PMSF and 7 mM DFP). An equal volume of
sample buffer was added to the cell suspension before it was boiled for 5
minutes. The protein were separated on a 7.5% polyacrylamide gel and
transferred on nitrocellulose membrane. The antibodies used to visualized the
proteins were HM2 (Sigma) 46.1 (kindly provided by V. Lee, University of
Pennsylvania) or an anti-His antibody (Santa Cruz, Santa Cruz, CA). The
secondary antibodies were conjugated to HRP (Jackson Immunoresearch
Laboratories, Bio/Cam, Mississauga, Ontario, Canada) and revealed by
chemiluminescence (Roche, Laval, Québec, Canada). For dot blotting, 30
µg of total protein extract prepared from infected cells were applied to a
nitrocellulose membrane using a dot blot Manifold apparatus. The membrane was
air dried for 30 minutes and incubated in the primary antibody against MAP2
for 60 minutes at room temperature. The membrane was then washed and incubated
in the secondary antibody conjugated to HRP (Jackson Immunoresearch
Laboratories) and revealed by chemiluminescence (Roche). To quantify the
protein level, the autoradiographic dots were scanned and the digitized data
were quantified using the program ImageQuant (Molecular Dynamics).
Co-immunoprecipitation
The cells were washed twice in PBS and lysed in RIPA buffer (50 mM Tris,
150 mM NaCl, 0.02% sodium azide, 1% Nonidet P-40, 0.5% sodium dexycholate and
0.1% SDS) containing protease inhibitors for 30 minutes at 4°C. The cell
lysates were centrifuged for 10 minutes at 12,000 g at 4°C. The
primary antibodies directed against MAP2, AP18 (1:100) or AP20 (1:400), were
added to the cell lysates and incubated for 1 hour at 4°C on a rocking
platform. Then 50 µl of protein A-agarose was added to the cell lysates and
incubated overnight at 4°C on a rocking platform. The complexes were
collected by centrifugation for 20 seconds at 12,000 g at 4°C.
The supernatant was carefully removed and the beads were resuspended in 1 ml
of RIPA and incubated for 20 minutes at 4°C on a rocking platform. This
step was repeated four times. Finally, the beads were resuspended in 40 µl
of loading buffer and boiled for 5 minutes. The protein A-agarose was removed
by centrifugation at 12,000 g for 20 seconds at room temperature; the
remaining sample was analyzed by SDS-PAGE.
Electron microscopy
For transmission electron microscopy, Sf9 cells were grown on glass
coverslips at a density of 2.0x106 cells/60 mm dish. Cells
were infected for 48 or 72 hours with viral stock at a multiplicity of
infection (m.o.i.) of 5. The cultures were fixed in a solution containing 2%
glutaraldehyde and 2 mg/ml tannic acid for 15 minutes, rinsed in a solution of
5% sucrose in 0.1 M cacodylate, postfixed for 10 minutes with 1% osmium
tetroxide, dehydrated with increasing concentrations of ethanol, and embedded
using EPON resin (Cedarlane Laboratories, Hornby, Ontario, Canada). After
curing the resin, cells were sectioned parallel to the long axis of the
processes. The spacing between microtubules was measured using either the NIH
Image 1.62 program or the Pro AnalySIS program. Measurements were made in the
proximal region of the processes. We performed 50 random measurements per
process.
Quantitative morphological analysis
The morphological analysis was performed by two observers. Three sets of
experiments were analyzed. The morphological phenotypes observed with the
different truncated forms of MAP2 were highly reproducible from one set of
experiments to another. To evaluate the number of processes per cell, 150
cells were measured for each truncated form of MAP2 in each set of experiment.
To analyze the process length, 50 cells were used for each truncated form of
MAP2 in each set of experiments.
Statistical analysis
The distribution of the percentage of cells with one, two or more than two
processes was analyzed for each truncated form and full-length MAP2c and MAP2b
in three sets of experiments. To analyze the reproducibility of the data from
one set of experiments to another, a chi-square test followed by the Fisher's
Exact test was performed. Since the distribution was not statistically
different from one experiment to another, the three experiments were combined
for the statistical analysis. The differences among truncated and full-length
MAP2c and MAP2b in the distribution of the number of processes per cell were
analyzed by a chi-square test followed by the Fisher's Exact test. The length
of process was analyzed by one-way ANOVA followed by the Sheffe test. For
electron microscopy analysis, the statistical significance of the spacing
between microtubules was determined using one-way ANOVA followed by Fisher's
PLSD test. Statistical significance was accepted if P<0.05.
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Results |
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The pattern of process formation of Mt in Sf9 cells was analyzed according
to two parameters: number of cells with processes and number of processes per
cell. To evaluate the percentage of cells having processes, at 24 and 72 hours
post-infection the cells were fixed and stained with the anti-MAP2 antibody,
HM2, which recognizes MAP2b and MAP2c, and the antibody 46.1 directed against
an epitope located in Mt (Kosik et al.,
1988). The percentage of cells positive to HM2 or 46.1 and having
cell processes was determined. 44% of the Mt-expressing cells presented
processes compared with 7% and 80% of MAP2b- and MAP2c-expressing cells,
respectively (Fig. 1A). This
indicates that the projection domain of MAP2b negatively regulates the
capacity of the microtubule-binding domain to induce process formation,
whereas the projection domain of MAP2c appears to increase its capacity to
initiate process formation. For the cells expressing Mt and presenting
processes, the number of processes per cell was examined and compared with
that of MAP2b and MAP2c. As reported before, MAP2b induced the formation of
one process, whereas MAP2c had the tendency to induce the formation of
multiple processes (Figs 2,
3). Interestingly, in the cells
expressing Mt, the number of processes per cell were significantly different
from that of MAP2c- and MAP2b-expressing cells. Indeed, 39% of Mt-expressing
cells had multiple processes compared with 57% and 9% of MAP2c- and
MAP2b-expressing cells (Fig.
3). However, Mt-expressing cells (37%) had a slightly higher
tendency than MAP2c-expressing cells (30%) to develop one process but,
importantly, a lower tendency than MAP2b-expressing cells (74%). Thus, the
projection domain of MAP2c seems to contribute to the production of multiple
processes by Sf9 cells whereas that of MAP2b impairs it. To eliminate the
possibility that the difference in the Mt pattern of process formation,
compared with that of full-length MAP2b and MAP2c, was attributable to
different levels of protein expression, their protein level was analyzed by
dot blotting. The antibody, 46.1, which recognized an epitope located in the
C-terminus of MAP2, was used to compare the protein level of MAP2b, MAP2c and
Mt (Kosik et al., 1988
). At 72
hours post-infection, the expression of Mt was similar to that of MAP2c and
MAP2b (Table 1). This indicates
that the molar expression of Mt was approximately two times higher than that
of MAP2c and approximately seven times higher than that of MAP2b at this time
of infection. While the molar expression of Mt was higher than that of MAP2c,
it induced a lower percentage of cells with processes and a lower number of
proceses per cell than MAP2c. Thus, the protein level does not influence the
pattern of process formation of MAP2 proteins in Sf9 cells as reported before
(Leclerc et al., 1996
). At 24
hours post-infection, Mt and MAP2c had a similar percentage of cells with
processes (
20%) despite the fact that the molar expression of Mt is
higher than that of MAP2c (Table
1). By contrast, at 48 hours post-infection, 44% of Mt-expressing
cells had processes compared with 80% of cells expressing MAP2c. These results
indicate that Mt is less efficient than MAP2c at inducing process outgrowth in
parallel to the increase of its protein level.
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Regions of Prob that regulate the process formation by the
microtubule-binding domain
To identify which region(s) of the 1372 a.a. domain were involved in
regulating the process formation by Mt, we subdivided this sequence into three
portions of equal size corresponding to the region adjacent to the N-terminus
common to MAP2c and MAP2b (MAP2b-1), the median region (MAP2b-2) and the
region adjacent to the microtubule-binding domain (MAP2b-3)
(Fig. 1A). MAP2b-1 is deleted
of amino acids 147-569-, 147 a.a. is the amino acid located at the splicing
site of MAP2c. MAP2b-2 has a deletion from amino acids 569-1035. This deleted
sequence includes a phylogenic conserved sequence in the MAP2b projection
domain extending from 650-940 (Kindler et
al., 1990). MAP2b-3 is deleted from amino acid 1035 to 1519; 1519
corresponds to the splicing site of MAP2c. These truncated forms were
expressed in Sf9 cells. Their expression was analyzed by western blotting and
each truncated form migrated at the expected apparent molecular weight on
SDS-PAGE (Fig. 1B). Their
protein level was evaluated by dot blotting
(Table 1). At 24 and 72 hours
post-infection, MAP2b-3 presented the highest protein level. The protein level
of MAP2b-1 was similar to that of MAP2b, whereas that of MAP2b-2 was slightly
lower than that of MAP2b at 72 hours post-infection.
We performed a quantitative morphological analysis as described above to
verify whether these truncated forms of MAP2b induced different patterns of
process formation in Sf9 cells. As noted for MAP2b, they had the tendency to
induce one process per cell (Figs
2,
3). The percentage of cells
with processes induced by MAP2b-3 (21%) was significantly higher than that
induced by MAP2b (7%), MAP2b-1 (12%) and MAP2b-2 (11%)
(Fig. 1A). The percentage of
cells with processes induced by MAP2b-1 and MAP2b-2 was not significantly
different from that of MAP2b. For the cells that expressed MAP2b-1, MAP2b-2 or
MAP2b-3 and presented processes, the number of processes per cell was
examined. The number of processes per cell induced by MAP2b-1, MAP2b-2 and
MAP2b-3 was reminiscent of that of MAP2b. Indeed, 74% of the
MAP2b-expressing cells had one process compared with 75%, 69% and 71% of the
MAP2b-1-, MAP2b-2- and MAP2b-3-expressing cells, respectively. Furthermore,
the percentage of MAP2b-1-, MAP2b-2- and MAP2b-3-expressing cells having two
or more than two processes was similar to that of MAP2b. Thus, in the present
expression system, it seems that the deletion of the amino acids 1035 to 1519
deleted in MAP2b-3 had the most important positive effect on the formation of
processes by MAP2b in Sf9 cells. This could be related to the fact that
MAP2b-3 presented the highest protein level at 72 hours post-infection.
Indeed, there is an increase in the number of cells with processes in parallel
to the increase of MAP2 protein expression in Sf9 cells. However, the level of
MAP2 proteins does not seem to be the sole determinant involved in the
production of processes by these cells. For instance, at 24 hours
post-infection, MAP2b-3 protein level was ten times lower than that of MAP2b
at 72 hours post-infection, but the percentage of cells presenting processes
(
10%) was identical to that of MAP2b at 72 hours post-infection
(Table 1). This suggests that
MAP2b-3 has a higher capacity than MAP2b, MAP2b-1 and MAP2b-2 to initiate
process formation.
Process length induced by MAP2 truncated forms
The process length was analyzed for each truncated form of MAP2b and MAP2c
(Fig. 4). We first compare the
process length of cells bearing one process. MAP2c-expressing cells presented
the longest process (72.7±2.2 µm) followed by MAP2b-3
(62.4±2.5 µm), MAP2b-1 (52.4±1.9 µm), Mt (47.9±2.0
µm), MAP2b-2 (41.9±1.6 µm) and MAP2b (35.8±1.4 µm).
These differences in process length were statistically significant except for
Mt and MAP2b-1. Thus, a partial deletion in the 1372 a.a. domain had a
positive effect on process length, whereas a deletion of Proc had a negative
one. However, the process length of cells expressing MAP2b-1, MAP2b-2 and
MAP2b-3 seems to correlate with their level of protein expression
(Table 1). Indeed,
MAP2b-2-expressing cells present the lowest protein expression and the
shortest processes, whereas MAP2b-3-expressing cells present the highest
protein expression and the longest processes. However, the level of protein
expression does not seem to be the only parameter that determines process
length since MAP2b, which has an expression level that is lower than that of
MAP2b, induces the formation of processes longer than those generated by
MAP2b. Moreover, the process length induced by Mt, whose level of expression
is much higher than that of MAP2b-1 and MAP2b-2, is similar or slightly longer
than the process length induced by these MAP2b constructs. From the above
observations, it appears that the additional domain of 1372 a.a. in the
projection domain of MAP2b not only is involved in determining the number of
processes per cell but also exerts an effect on process length.
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Finally, we examined the process length of cells bearing multiple processes. Since MAP2c and Mt induced the highest percentage of cells with multiple processes, the process length was analyzed only for these two constructs. The process length of MAP2c- and Mt-expressing cells bearing multiple processes was 31.5±1.4 µm and 29.6±1.4 µm, respectively. These lengths were not statistically different but were significantly shorter than the process length of MAP2c- and Mt-expressing cells that have one process.
Distribution of the microtubules and actin microfilaments in Sf9
cells expressing MAP2c and MAP2b truncated forms
According to our previous work in Sf9 cells, microtubule formation is
required for process outgrowth in Sf9 cells. Thus, we examined the
distribution of microtubules in cells expressing the full-length and the
truncated forms of MAP2b and MAP2c. As shown previously, in MAP2c-expressing
cells that have multiple processes, thin microtubule bundles radiate
tangentially from the cell surface to form processes
(Boucher et al., 1999). In
MAP2b-expressing cells that develop one process, a thick bundle of
microtubules originating in the cell body extends into the process. In
MAP2b-expressing cells that have no processes, microtubules are organized in a
thick bundle that forms a ring under the plasma membrane
(Fig. 5).
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We analyzed the distribution of microtubules in cells expressing the
different truncated forms of MAP2b and MAP2c using confocal microscopy
(Fig. 5). In cells expressing
Mt and having one process, several thin bundles of microtubules were found in
the cell body that formed a thick bundle at the hillock region of the process
and extended into the process. In cells having multiple processes, the
distribution of the microtubules was reminiscent of that found in
MAP2c-expressing cells presenting multiple processes. In MAP2b-1, MAP2b-2 and
MAP2b-3-expressing cells, a thick bundle of microtubules was found in the cell
body that extended into a process at one pole of the cell body as previously
described for MAP2b (Leclerc et al.,
1996). In cells expressing either of these MAP2b truncated forms
that did not have processes, microtubules formed a ring under the plasma
membrane as reported for full-length MAP2b
(Boucher et al., 1999
). From
these observations, it appears that the projection domain is not necessary to
induce microtubule bundling since the construct Mt promoted microtubule
bundling. However, the 1372 a.a. domain in the MAP2b projection domain seems
to favor the formation of a thick bundle of microtubules rather than the
formation of multiple thin bundles. Furthermore, the packing density of
microtubules does not seem to influence their capacity to protrude from cell
surface. For instance, Mt can induce the formation of one thick bundle of
microtubules (Fig. 5) or of
multiple thin bundles. The thick bundles seem to have a capacity to protrude
from the cell surface similar to that of the thin ones. Indeed, an equal
number of Mt-expressing cells develop multiple processes (37%) and one process
(39%).
In cells expressing Prob, there was no formation of microtubules
(Fig. 6). Interestingly, Prob
expression was concentrated in the cell periphery. In these cells, F-actin
formed a ring under the plasma membrane and seemed to co-localize with Prob
(Fig. 6). In addition, Proc
expression had the tendency to be concentrated in the cell periphery and to
co-localize with F-actin (Fig.
6). F-actin nuclear staining was often noted in cells infected
with any truncated form of MAP2b and MAP2c as noted in wild-type baculovirus
infection (Charlton and Volkman,
1991). Finally, in Sf9 cells expressing either full-length or
truncated forms of MAP2b and MAP2c and presenting processes, F-actin was found
in the cell body and in the processes (Fig.
5).
|
Bundling of microtubules in Sf9 cells expressing MAP2c and MAP2b
truncated forms
As revealed by light microscopy, MAP2c and Mt have a higher tendency to
induce multiple thin microtubule bundles than MAP2b, MAP2b-1, MAP2b-2 and
MAP2b-3. This suggests that the 1372 a.a. domain favors the formation of a
unique thick bundle of microtubules. To better understand its role in
microtubule bundling, we examined the effect of partial or complete deletion
of this domain on the spacing between microtubules along the processes in Sf9
cells. This was examined in cells expressing MAP2b-1, MAP2b-2, MAP2b-3 or Mt
as well as full-length MAP2b and MAP2c 72 hours after infection. Longitudinal
sections were used (Fig. 7). We
measured wall to wall spacing between neighboring microtubules at 50 randomly
selected locations in the proximal region of the process
(Table 2). Because of the very
low capacity of MAP2b to induce process formation in Sf9 cells, measurements
were made in the cell bodies of the round-infected cells expressing MAP2b. In
these cells, microtubules form a ring under the plasma membrane
(Fig. 7). To confirm that the
spacing between microtubules is not different between the cell body of the
round infected cells and the processes, we performed measurements in the cell
bodies of the round cells expressing MAP2b-2 or MAP2b-3. The microtubules are
also organized as bundles under the plasma membrane in these cells
(Fig. 7). There was no
statistically significant difference between the cell bodies and the processes
for MAP2b-2 and MAP2b-3 expressing cells. Thus, the measurements in these two
compartments were combined for those constructs
(Table 2). However, differences
were observed in the case of MAP2c-expressing cells where the average spacing
between microtubules was 13.05±0.23 nm in the cell bodies compared with
16.40±0.51 nm in the processes.
|
|
The average spacing between microtubules in MAP2b and MAP2c induced bundles
was 53±1.90 and 16.40±0.51 nm, respectively. These values
confirm previous findings (Chen et al.,
1992; Leclerc et al.,
1996
). As for MAP2b-1, MAP2b-2 and MAP2b-3, the average spacing
between microtubules is 33.40±1.11, 39.70±1.10 and
41.00±1.00 nm, respectively. Thus, the spacing between microtubules is
significantly different between MAP2b-1 and MAP2b-2 and between MAP2b-1 and
MAP2b-3. These results indicate that equal size deletions in the 1372 a.a.
domain of MAP2b give different microtubule spacing. Moreover, MAP2b-1, which
deleted sequence was slightly shorter than that of MAP2b-2 and MAP2b-3, gave a
narrower microtubule spacing than these constructs. This suggests that the
length of the projection domain of MAP2, although important, is not the sole
determinant of the spacing between microtubules. As for Mt-induced processes,
the microtubules were so tightly packed that it was impossible to make
measurements (Fig. 7).
To verify whether the expression level of the protein affects the spacing between microtubules, measurements were made 48 hours post-infection and no differences were observed (data not shown).
The above observations suggest that the structural properties of microtubule bundling are not the sole parameters that regulate microtubule protrusion from Sf9 cells. Indeed, MAP2b-1, which gives rise to a narrower spacing than MAP2b, MAP2b-2 and MAP2b-3, does not induce higher microtubule protrusion than that induced by these proteins. Furthermore, MAP2b-3 induces a microtubule spacing similar to that of MAP2b-2 but has a higher capacity to promote microtubule protrusion than MAP2b-2.
Microtubule-binding properties of MAP2c and MAP2b truncated
forms
The microtubule-binding affinity of MAP2b and MAP2c truncated forms was
evaluated by quantifying the amount of each truncated form in a preparation of
microtubules by dot-blot. The microtubules were prepared as described
previously (Vallee and Collins,
1986). As illustrated in Fig.
8, similar amounts of tubulin were found in the microtubule
preparations from Sf9 cells expressing either the truncated forms of MAP2 or
fulllength MAP2b and MAP2c. The amount of MAP2b, MAP2c and MAP2 truncated
forms in the microtubule preparation is presented as a percentage of the total
amount of protein in 4x106 Sf9 cells. All the deletion in the
1372 a.a. domain had a positive effect on the microtubule-binding affinity of
MAP2b (Fig. 8). 5% of MAP2b was
found in the microtubule preparation, whereas 9%, 50% and 40% of MAP2b-1,
MAP2b-2 and MAP2b-3 was bound to microtubules, respectively. Interestingly,
MAP2b-2, which has the lowest protein level, presented the highest
microtubule-binding affinity. Moreover, the microtubule-binding affinity does
not seem to influence the capacity of microtubules to protrude from Sf9 cells
since MAP2b-2 induced a lower percentage of cells with microtubule protrusion
than MAP2b-3. Moreover, MAP2c and Mt, which showed a significantly higher
percentage of cells with microtubule protrusion than MAP2b-2 and MAP2b-3,
presented an equal or lower percentage of protein bound to microtubules than
these MAP2b truncated forms. Indeed, 34% and 39% of MAP2c and Mt was bound to
microtubules compared with 50% and 40% for MAP2b-2 and MAP2b-3.
|
The amount of polymerized tubulin induced by the expression of MAP2c and MAP2b truncated forms was also evaluated by extracting the cytoskeleton 48 hours following infection, as described in Materials and Methods. Similar amounts of tubulin were found in the extracted cytoskeletal pellets indicating that differences in the binding affinities of MAP2c and MAP2b truncated forms to microtubules did not affect the amount of polymerized tubulin. Thus, the amount of polymerized tubulin is not a limiting factor in microtubule protrusion from the cell surface in Sf9 cells. For instance, MAP2b-3, which has a higher protrusion activity than MAP2b, MAP2b-1 and MAP2b-2, induces a similar amount of polymerized tubulin to these proteins. Furthermore, we examined the effect of cold treatment (30 minutes) on the stability of the polymerized microtubules in the cells expressing MAP2c and MAP2b truncated forms. The amounts of tubulin remaining in the extracted cytoskeletal pellet following cold treatment, compared with control levels, are the following: MAP2b, 30%; MAP2c, 30%; MAP2b-1, 35%; MAP2b-2, 28%; MAP2b-3, 25%; and Mt, 26%. These results indicate that cold treatment destabilized the microtubule polymers induced by MAP2c and MAP2b truncated forms as well as those induced by full-length MAP2b and MAP2c, to the same extent. Finally, we examined the effect of treatment with the microtubule depolymerizing agent colchicine (2 hours) on the stability of the microtubule polymers induced by MAP2c and MAP2b truncated forms. The amounts of tubulin remaining in the extracted cytoskeletal pellet following treatment with colchicine, compared with control levels, are the following: MAP2b, 34%; MAP2c, 12%; MAP2b-1, 17%; MAP2b-2, 33%; MAP2b-3, 28%; and Mt, 25%. Thus, treatment with colchicine destabilized the microtubule polymers induced by MAP2c and MAP2b truncated forms as well as those induced by full-length MAP2c and MAP2b, but to different extents. After 2 hours of treatment with colchicine, the amount of polymerized tubulin in the extracted cytoskeletal pellet was higher for MAP2b than for MAP2c indicating that MAP2b might confer more resistance to drug treatment. Furthermore, partial deletion in the 1372 a.a. domain of MAP2b seems to render microtubule polymers less resistant to drug treatment. This is particularly evident for MAP2b-1. Therefore, the 1372 a.a. domain might play a role in conferring drug resistance to polymerized microtubules. Moreover, MAP2c seems to induce the formation of microtubule polymers more sensitive to colchicine treatment than Mt. This indicates that the projection domain of MAP2c confers drug sensitivity to polymerized microtubules. Thus, the microtubule resistance to colchicine seems to be induced mainly by Mt, but Proc decreases the resistance whereas the 1372 a.a. domain enhances it.
As reported above for the structural properties of microtubules, one can conclude from the present observations that the microtubule-binding affinity and the polymerizing activity of MAP2c and MAP2b truncated forms are not the only factors that control microtubule protrusion and process formation in Sf9 cells.
Interactions between the projection domain and the
microtubule-binding domain
To explore the mechanism by which Prob regulates microtubule protrusion and
process formation in Sf9 cells, we first verified whether Prob has to be
attached to Mt to impair the protrusion of microtubule bundles. Thus, Sf9
cells were co-infected with Prob and Mt recombinant baculovirus. A
quantitative morphological analysis was performed as described in the previous
section. To identify the cells that co-expressed Prob and Mt, the cells were
double stained with a polyclonal anti-MAP2 antibody (kindly provided by
Richard Vallee, University of Massachusetts Medical School, Worcester, MA),
which recognizes an epitope contained in the projection domain of MAP2b, and
the antibody 46.1, which recognizes an epitope found in Mt as described above.
For the quantitative morphological analysis, only the cells presenting a high
protein level of Prob and Mt were selected
(Fig. 9). The percentage of
Prob/Mt co-infected cells presenting processes was significantly lower (30%)
than that of Mt-infected cells (44%) (Fig.
10). Moreover, the number of processes per cell was significantly
lower in Prob/Mt-expressing cells than in Mt-expressing cells
(Fig. 10). In Mt-infected
cells, 37% and 39% of the cells had one and multiple processes, respectively,
compared with 51% and 28% of co-infected cells. From this set of experiments,
it appears that Prob does not have to be attached to Mt to impair its capacity
to induce process formation in Sf9 cells. Interestingly, co-expression of Proc
with Mt did not result in the pattern of process formation of full-length
MAP2c but rather gave rise to a pattern resembling that of Mt: 38% of cells
co-expressing Proc and Mt had processes compared with 44% of Mt-expressing
cells (Fig. 10).
|
|
The co-infection experiments demonstrated that the projection domain of
MAP2b impairs process formation activity of Mt. This could be done by
intramolecular interactions between these two domains, which would result in
masking functional domain(s) involved in process formation. Another
possibility is that Prob could compete with Mt for a common element that when
bound to Mt allows microtubule protrusion and process formation and, when
bound to Prob, blocks them. However, previous studies on MAP2 structure have
suggested that MAP2b could adopt different conformations, including the
formation of hairpin structures (Wille et
al., 1992a; Wille et al.,
1992b
). This indicates that Prob and Mt would interact to give
rise to such a conformation. Moreover, these electron microscopic studies
demonstrated that MAP2b can form anti-parallel dimers reinforcing the
possibility that Prob and Mt can interact. To verify this possibility we
performed a co-immunoprecipitation experiment
(Fig. 11). We used the
anti-MAP2 antibody, AP20, which recognizes an epitope located in the
additional sequence of 1372 a.a. in the MAP2b projection domain, or the
antibody HM2 to immunoprecipitate Prob. The membrane was revealed with AP20 or
HM2 to show that Prob was immunoprecipitated. Then, the same membrane was
probed with the antibody, 46.1, which recognizes the microtubule-binding
domain of MAP2 isoforms to reveal the presence of Mt. As shown in
Fig. 11, a band corresponding
to the apparent molecular weight of Mt co-immunoprecipitated with Prob (lanes
2,3). A similar band having a higher intensity was found when Mt was
immunoprecipitated with the antibody 46.1 in cells expressing only Mt (lane
4). However, this band was not detected when Prob was immunoprecipitated from
cells only expressing Prob (lanes 6,7). These results indicate that Prob
interacts with Mt in Sf9 cells. We also verified whether Proc interacts with
Mt in Sf9 cells. In the present conditions, no interaction was detected
between Proc and Mt.
|
The present study indicates that the additional domain of 1372 a.a. in Prob increases the spacing between microtubules and favors the formation of a single thick bundle of microtubules. Most notably, it regulates the capacity of this microtubule bundle to protrude from cells. This could be through its interaction with Mt.
![]() |
Discussion |
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The microtubule-binding domain and the proline-rich region of MAP2
proteins are involved in microtubule protrusion and process formation
Our present results show that the expression of the construct Mt, which
contains the microtubule-binding domain and the adjacent proline-rich region,
promotes microtubule bundling, microtubule protrusion and process formation in
Sf9 cells. Indeed, it was previously shown that the proline-rich region and
the microtubule-binding domain were sufficient to induce microtubule bundling
in non-neuronal cells (Ferralli et al.,
1994; Umeyama et al.,
1993
). The microtubule-binding domain contains the three repeated
sequences of 18 a.a. responsible for microtubule assembly that share sequence
homology with MAP4 and tau (Lewis et al.,
1988
). Furthermore, MAP2, MAP4 and tau present sequence homology
in the proline-rich region adjacent to the microtubule-binding domain
(Chapin and Bulinski, 1991
;
Ferralli et al., 1994
;
West et al., 1991
). The
homology is found in the 25-30 a.a. adjacent to the first repeat. Two
residues, Lys215 and Arg221, are highly conserved. These
amino acids are known to enhance the microtubule-binding activity of the
microtubule-binding domain of tau (Goode
et al., 1997
). The sequence homology indicates that this function
is conserved between MAP2, MAP4 and tau. Furthermore, previous studies
demonstrated that the proline-rich region adjacent to the microtubule-binding
domain in tau interacts with the src-family of non-receptor tyrosine kinases
such as fyn through the SH3 domains of these kinases
(Lee et al., 1998
).
Interestingly, the binding of tau to fyn alters cell morphology, which is
associated with a reorganization of the microtubules. A similar binding
sequence to SH3 domains is found in MAP2 isoforms from amino acids 286 to 294.
However, this region does not seem to be involved in the binding of the SH3
domains of Src and Grb2 to MAP2c (Lim and
Halpain, 2000
). This interaction is rather mediated by the 300-400
a.a. region located in the microtubule-binding domain. Moreover, Src and Grb2
interact preferentially with non-microtubule-associated MAP2c
(Lim and Halpain, 2000
).
Nonetheless, these data indicate that, as noted for tau, the proline-rich
region in MAP2 proteins could influence the microtubule bundling and
protrusion from cells by its binding to signaling proteins.
Effects of the additional domain of 1372 a.a. in the projection
domain of MAP2b on microtubule protrusion and process formation
Here, we show that a partial or complete deletion of the 1372 a.a. domain
has a strong positive effect on microtubule protrusion and process formation
in Sf9 cells. However, Proc, the projection domain of MAP2c, seems to enhance
these events since MAP2c presents the highest percentage of cells with
processes. This enhancing effect of Proc on MAP2c ability to induce process
formation was also reported in human hepatoma cell line PLC
(Ferralli et al., 1994).
Previous studies have suggested that the capacity of MAP2c constructs to
support process formation was related to their strength of binding to
microtubules (Ferralli et al.,
1994
). However, in Sf9 cells this correlation does not seem to
exist. Indeed, Mt has a slightly higher binding affinity than MAP2c but
presents a lower capacity to induce process formation in Sf9 cells. This
difference might be explained by the fact that, in previous studies, cortical
actin had to be depolymerized by cytochalasin D to induce process formation,
whereas in Sf9 cells process formation occurs spontaneously
(Ferralli et al., 1994
;
Leclerc et al., 1996
;
Leclerc et al., 1993
).
Therefore, in Sf9 cells microtubule protrusion and process formation might
require additional cellular elements that influence MAP2 protein activity.
Moreover, Proc seems to decrease microubule stability as revealed by the lower
percentage of microtubules resistant to colchicine treatment in
MAP2c-expressing cells compared with Mt-expressing cells. Our results
correlate with previous studies showing that MAP2c does not confer colchicine
resistance to microtubules (Caceres et al.,
1992
; Olmsted et al.,
1989
; Takemura et al.,
1995
). However, some studies reported that MAP2c can induce drug
resistance to microtubules (Ferhat et al.,
1996
; Takemura et al.,
1992
). These studies used immunocytochemistry to evaluate the
amount of polymerized tubulin whereas, in our study, we used a biochemical
approach. Furthermore, a different cellular system was used in these studies.
These experimental differences might explain the discrepancy between the
results. In neurons, the suppression of MAP2 protein expression blocks the
induction of labile or tyrosinated microtubules but does not affect the
population of stable or acetylated microtubules resistant to colchicine
(Caceres et al., 1992
).
Moreover, it was shown that, in the presence of MAP2c, microtubules display
dynamic instability (Kaech et al.,
1996
). The high capacity of MAP2c to induce microtubule protrusion
and process formation might be due to the induction of labile microtubules
that are mainly located in the growth region in neurons.
Our present results indicate that the additional domain of 1372 a.a. in the
MAP2b projection domain decreases the positive effects of Proc on process
formation in Sf9 cells. This could be explained by the fact that it
significantly decreases the microtubule-binding affinity of MAP2b. However,
MAP2b-2 and MAP2b-3, which have a higher or similar binding affinity to
microtubules than MAP2c, present a lower percentage of cells with processes
than MAP2c. Thus, the microtubule-binding activity does not seem to be the
sole factor involved in process formation by MAP2b in Sf9 cells.
Interestingly, the 1372 a.a. domain seems to favor the formation of a unique
thick bundle of microtubules resulting in the formation of a unique process,
whereas MAP2c induces the formation of multiple thin bundles that give rise to
the formation of multiple processes. The induction of multiple thin bundles of
microtubules was reported in other non-neuronal cell lines
(Ferhat et al., 1996). In
cells expressing full-length or MAP2b truncated forms that do not have
processes, the thick bundle of microtubules forms a ring under the plasma
membrane. Since microtubules have to penetrate the actin network to protrude
from cell surface, the thickness of the microtubule bundles might be a
limiting factor (Tanaka and Sabry,
1995
). However, this does not seem to be the case in Sf9 cells
since MAP2b-3, which induces a thick bundle of microtubules, can induce
three-times as much process formation as MAP2b. Futhermore, the 1372 a.a.
domain seems to enhance the resistance of microtubules to colchicine,
resistance that is mainly induced by Mt expression. This could contribute to
lower microtubule dynamics and thereby their capacity to protrude from cells.
The distinct organization of microtubule bundles by MAP2c and MAP2b might
reflect their distinct role in the elaboration of the dendritic arborization.
MAP2c, which leads to the formation of thin and labile microtubule bundles
that could easily penetrate the actin network of the growth cone, would be
involved in the initial stage of dendritic outgrowth
(Tanaka and Sabry, 1995
). By
contrast, MAP2b would be involved in the production of thicker bundles to
increase the diameter of dendrites and stabilize microtubules to consolidate
the newly formed dendritic branches
(Hillman, 1988
).
Mechanisms regulating the effects of the additional domain of 1372
a.a in the projection domain of MAP2b on microtubule protrusion
Microtubule protrusion and process formation induced by MAP2 proteins
depend on the actin cytoskeleton, as previously shown
(Boucher et al., 1999;
Edson et al., 1993
). Thus, the
distinct effect of MAP2b and MAP2c on microtubule protrusion might be related
to their distinct effect on actin cytoskeleton. Indeed, MAP2b and MAP2c
organize differentially F-actin in vitro. MAP2c is able to induce the
formation of an isotropic gel of F-actin, whereas MAP2b induces the formation
of F-actin bundles (Cunningham et al.,
1997
). To bundle or crosslink F-actin, a protein has to contain
two actin-binding domains or to have the capacity to dimerize
(Puius et al., 1998
). In
either case, the 1372 a.a. domain, by allowing different conformational states
of MAP2b, might modify the structural relation between the actin-binding
domains and thereby the organization of F-actin by MAP2b. An actin-binding
domain was identified in one of the repeats of the microtubule-binding domain
(Correas et al., 1990
).
Futhermore, as revealed in the present study, Proc and Prob co-localize with
F-actin in Sf9 cells indicating that an F-actin-binding site could be located
in the region common to MAP2c and MAP2b. This needs to be confirmed by in
vitro studies. Finally, since simultaneous changes in microtubule and actin
organization are observed in process outgrowth, the 1372 a.a. domain might
compromise the molecular link between these cytoskeletal elements and thereby
impair process formation (Tanaka and
Sabry, 1995
). The 1372 a.a. domain also influences the length of
the processes since a partial deletion in this domain increases process length
in Sf9 cells. This could occur through its effect on actin organization.
Indeed, depolymerization of F-actin by cytochalasin increased the rate of
process outgrowth in Sf9 cells (Knowles et
al., 1994
). Thus, the organization of F-actin induced by these
MAP2b truncated forms could favor a higher rate of process elongation.
The effect of the domain of 1372 a.a. on process formation can be also
mediated through its binding to signaling proteins. This domain contains a
binding site for calmodulin, which is known to decrease the actin-binding
activity of MAP2b (Kindler et al.,
1990; Kotani et al.,
1985
). This calmodulin-binding domain was deleted in MAP2b-3, the
MAP2b truncated form that gave rise to the highest number of cells with
processes. Moreover, the domain of 1372 a.a. also contains a high affinity
phosphatidylinositol-binding site (Burns
and Surridge, 1995
; Surridge
and Burns, 1994
). A recent study demonstrated that MAP2c process
outgrowth activity can be inhibited by the co-expression of a subtype of
metabotropic glutamate receptors, mGluR1, in Sf9 cells
(Huang and Hampson, 2000
).
mGluR1 stimulates phosphoinositide (PI) hydrolysis
(Pickering et al., 1993
).
Treatment of the cells with a phospholipase C inhibitor reversed the
inhibitory effect of mGluR1 suggesting that the PI pathway was involved in the
suppression of MAP2c-mediated process formation in Sf9 cells
(Huang and Hampson, 2000
). It
was proposed that binding of PI to MAP2c reduced its binding to tubulin and
consequently its microtubule assembly activity
(Yamauchi and Purich, 1987
).
In Sf9 cells, MAP2b promotes microtubule assembly
(Leclerc et al., 1996
).
Therefore, if binding of PI to MAP2b is responsible for its low capacity to
induce process formation, it regulates a function of MAP2b other than that of
microtubule assembly. PI is also known to decrease the binding of MAP2 to
actin (Yamauchi and Purich,
1993
). Since MAP2b contains a high affinity PI-binding site, its
actin-binding activity might be lower than that of MAP2c.
By co-immunoprecipitation, we showed that intermolecular interactions occur
between Prob and Mt in Sf9 cells. Previous studies highlighted the possibility
of such interactions by demonstrating that MAP2b can form antiparallel dimers
that are nearly in complete overlap (Wille
et al., 1992a; Wille et al.,
1992b
). However, another study reported that autonomous
dimerization of MAP2c did not occur in human hepatoma cell line PLC or Hela
cells (Burgin et al., 1994
). It
was reported that the ERM protein, ezrin, forms oligomers and that the
formation of oligomers depends on its state of phosphorylation
(Gautreau et al., 2000
).
Similarly, the interaction between MAP2 proteins might depend on their state
of phosphorylation, which could vary from one cell type to another. Our
present data indicate that, in Sf9 cells, interactions seem to exist between
the projection domain and the microtubule-binding domain of MAP2b, suggesting
that MAP2 proteins could form antiparallel dimers in Sf9 cells. Moreover,
these interactions might be responsible for the negative effect that Prob
exerts on process formation by Mt. The inhibitory effect of Prob is most
likely mediated by the additional domain of 1372 a.a. since we could not
co-immunoprecipitate Proc and Mt in Sf9 cells. However, this does not exclude
the possibility that Proc interacts with Mt. Indeed, the effect of Prob on
process outgrowth could occur through its interaction with Mt and/or by
modulating the interaction of Proc with Mt. Furthermore, our data does not
indicate whether the interactions between Prob and Mt are direct or indirect.
For example, the interactions between these two domains could be mediated by
signaling proteins involved in neurite outgrowth and neuronal plasticity.
The length of the projection domain of MAP2b is not the sole
determinant of the spacing between microtubules
One known function of the projection domain is to set the spacing between
microtubules. Previous studies suggested that the primary sequence of this
domain is one primary determinant of the spacing between microtubules
(Chen et al., 1992;
Leclerc et al., 1996
).
Furthermore, the loss of MAP2 and MAP1B in MAP2/MAP1B knock-out mice results
in a decreased microtubule spacing in axons and dendrites
(Teng et al., 2001
). In this
study, we show that the deletion of equal portions of the projection domain of
MAP2b gives different microtubule spacing. Moreover, MAP2b-1, which was
deleted of a slightly shorter sequence than MAP2b-2 and MAP2b-3, induces a
narrower spacing between microtubules than these proteins. This indicates that
the primary sequence of the projection domain of MAP2, although important, is
not the sole determinant of the spacing between microtubules. One possibility
is that the deleted portions in the 1372 a.a. domain contain different
phosphorylation sites. Previous studies suggested that the phosphorylation of
the projection domain of MAP2 causes it to expand due to an increase in
intramolecular repulsion. This in turn could cause the distance between
adjacent microtubules to increase
(Mukhopadhyay and Hoh, 2001
).
Thus, the phosphorylation state of MAP2b might also be a key determinant of
the spacing between microtubules. This has previously been shown for
neurofilament proteins whose phosphorylation state regulates the spacing
between them by regulating their structural features
(Glicksman et al., 1987
;
Myers et al., 1987
). It is
also well known that the phosphorylation of tau protein increases its rigidity
(Hagestedt et al., 1989
).
Thus, the phosphorylation state of MAP2 might regulate its flexibility,
determining the spacing between microtubules.
Another possibility is that those deleted portions are involved in
different structural configuration regulating the length of the protein.
Previous atomic force microscopy studies suggested that the projection domain
of MAP2b could arbor different structural conformations due to the existence
of repulsive intramolecular forces
(Mukhopadhyay and Hoh, 2001).
In this study, we demonstrate by co-immunoprecipitation the interaction of the
projection domain of MAP2b with its microtubule-binding domain. Thus, the
deletion of different portions in the 1372 a.a. domain, although of equal
length, might have affected differently the conformation of the projection
domain.
Structural conformation of operative and inoperative MAP2b
Several studies, using different approaches, highlighted the possibility
that Prob can exist in different structural configurations. First, this domain
was shown to be flexible (Woody et al.,
1983). Computer-generated secondary structure predictions suggest
that the projection domain of MAP2b has a very important stretch of helices
separated by short turns (Kindler et al.,
1990
). This secondary structure could contribute to its
flexibility. Interestingly, tau's flexibility decreases considerably when it
binds to microtubules but not that of MAP2b
(Woody et al., 1983
).
Phosphorylation also diminishes tau's flexibility
(Hagestedt et al., 1989
). Such
data do not exist for MAP2b. Given that the projection domain of MAP2b
contains several sites of phosphorylation, it is possible that its
phosphorylation has also some effects on MAP2's flexibility. Variation of
phosphorylation of MAP2b could allow a higher or lower degree of extension,
which could result in masking or unmasking sites involved in process
formation. Second, several studies point out that the length of the projection
domain can vary. Voter and Erickson demonstrated by rotary shadowing that the
length of MAP2b varies by folding back
(Voter and Erickson, 1982
).
Moreover, it was shown that the length of the microtubule-binding domain is
half of the length of the total protein, despite the fact that it contains
only one-sixth of the mass (Wille et al.,
1992a
). Third, there is evidence from electron microscopy studies
that MAP2b can form hairpin structures
(Wille et al., 1992a
). This
indicates that Prob could fold back on the microtubule-binding domain in
full-length MAP2b and thus interactions between these two domains could
contribute to the low capacity of MAP2b to induce process formation in Sf9
cells. The detection of an interaction between Prob and Mt in cells that
co-express these two domains indicates that such a situation might exist in
Sf9 cells. However, the fact co-expression of Prob and Mt does not completely
reconstitute the low capacity of MAP2b to induce process formation might
indicate that in these conditions the interactions between these two domains
do not fully match.
The folding back of the projection domain could mask binding sites to the
cytoskeletal or signaling proteins located in the microtubule-binding domain
and in the proline-rich region. This situation was reported for ezrin, a
member of the ERM family of proteins that links the actin cytoskeleton to the
membrane. It was shown that ezrin exists in a dormant form in which its
actin-binding site located in the C-terminus is masked by the N-terminus that
is folded back (Gary and Bretscher,
1995). Moreover, the folding back of the N-terminus of ezrin masks
its binding site for EBP50, the ezrin-radixin-moesin-binding phosphoprotein 50
(Reezek and Bretscher, 1998
).
These conformational changes of ezrin are controlled by intramolecular
interactions. Similarly, MAP2b function could be regulated by intramolecular
interactions as suggested by our data.
The truncated form, MAP2b-3, which has a deletion from 1035 to 1519, had
the highest capacity to induce process formation in Sf9 cells. This region
comprises a proline-rich region extending from 1370 to 1650 a.a., which
includes the splicing site of MAP2c (1519 a.a.)
(Kindler et al., 1990). The
computer programs predict that there are several secondary structures, 19
helices separated by very short turns, in the last two-thirds of the
projection domain adjacent to the MAP2c splicing site. Therefore, this region
could serve as an hinge that would determine the position of projection
domain. In the truncated form, MAP2b-3, one part of the hinge (1370 to 1519
a.a.) was removed. This could compromise the folding back of the projection
domain on the microtubule-binding domain. Consequently, it would have reduced
the possibility of intramolecular interactions between the projection domain
and the microtubule-binding domain and thereby increase process formation by
this MAP2b truncated forms.
None of the deletions performed in the 1372 a.a. could induce the formation
of multiple processes such as MAP2c. In a previous study, we reported that the
expression of a truncated form of MAP2b, that has a deletion from amino acid
228 to 1621 induces half as many cells with multiple processes than MAP2c
(Leclerc et al., 1993).
Therefore, the formation of multiple processes would not depend solely on the
unfolding of the projection domain but it appears that the junctional sequence
at the splicing site of MAP2c plays a role in this event.
Our data demonstrate that the projection domain of MAP2b regulates the capacity of the microtubule-binding domain to induce microtubule protrusion and process formation. As suggested by our present data, this regulation could happen through intramolecular interactions between the projection domain and the microtubule-binding domain, most probably involving the 1372 a.a. domain present in MAP2b. Our data suggest that the projection domain would allow MAP2b to exist in an operative form that is able to induce microtubule protrusion and process formation and in an inoperative form that would not induce microtubule protrusion and process formation. In the latter form, the projection domain would be folded back on the microtubule-binding domain, reducing the capacity of the microtubule-binding domain to induce process formation. In the inoperative form, MAP2b would stabilize the cytoskeleton to maintain the dendritic shape whereas, in the operative form, it would promote process formation and remodeling of dendrites.
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