1 Department of Molecular Biology, University of Umea, S-901 87 Umea,
Sweden
2 Department of Medical Biosciences, University of Umea, S-901 87 Umea,
Sweden
* Author for correspondence (e-mail: martin.gullberg{at}molbiol.umu.se)
Accepted 27 May 2003
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Summary |
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Key words: Microtubules, Tubulin, Microtubule-associated proteins, Recombinant fusion proteins, MAP4
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Introduction |
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It is widely recognized that a balance between MT-stabilizing and
MT-destabilizing factors regulates the dynamics of MT polymerization, and that
phosphorylation of these factors is important for cell-cycle-specific
alterations of MT dynamics (reviewed by
Andersen and Wittmann, 2002).
Studies in egg extracts and using purified components have shown the
importance of the XKCM1 catastrophe promotor and the MT-associated XMAP215
protein (Kinoshita et al.,
2001
; Tournebize et al.,
2000
). It has been proposed that much of the observed MT dynamics
in intact cells can be attributed to the antagonizing activities of these two
proteins (reviewed by Kinoshita et al.,
2002
).
In addition to the XMAP215 protein (termed TOGp in humans)
(Charrasse et al., 1998), the
structurally unrelated and ubiquitously expressed MAP4 protein has been
implicated as being important for MT stability in vertebrates (reviewed by
Olmsted, 1999
). It has been
reported that MAP4 promotes a large increase in transitions from shrinking to
growing MTs (i.e. rescues) during in vitro MT assembly without altering other
parameters that define dynamic instability (namely, the rate of growth and
shrinkage, and the frequency of catastrophes)
(Ookata et al., 1995
).
Interestingly, it was shown in the same study that phosphorylation of MAP4
results in loss of in vitro rescue-promoting activity without an apparent
effect on MT binding. Studies in intact cells have revealed
hyperphosphorylation during mitosis, which might suggest functional
inactivation. However, given the complexity of MAP4 phosphorylation, the
consequences of phosphorylation at specific sites remain largely unknown
(Chang et al., 2001
;
Ookata et al., 1995
;
Ookata et al., 1997
). MAP4
appears to be associated with MTs to the same extent throughout the cell cycle
and has been proposed to localize the mitotically active cyclin-dependent
kinase/cyclin B complex to the mitotic spindle by serving as a cyclin B
binding site (Ookata et al.,
1995
). Determination of net polymer levels in HeLa cells
expressing antisense RNA constructs has indicated that MAP4 has a role in
maintenance of normal MT polymer levels during interphase
(Nguyen et al., 1999
).
However, an antibody injection study has suggested that MAP4 ablation does not
have an immediate detrimental effect on interphase or mitotic MTs
(Wang et al., 1996
). Thus, the
physiological role of MAP4 in the cell remains unclear.
Previous reports on the outcome of overexpressed MAP4 in various cell types
have been somewhat inconsistent. Whereas some have reported increased cellular
MT content (Yoshida et al.,
1996; Zhang et al.,
1998
), others have reported that the MT content is unaltered
(Barlow et al., 1994
;
Nguyen et al., 1997
). We have
recently analyzed the effect of ectopic MAP4 in human leukemic cells with
microtubules partially destabilized by either ectopic tubulin-sequestering
proteins or proteins that promote catastrophe
(Holmfeldt et al., 2002
). It
was found that coexpression of tubulin-sequestration-specific truncation
derivatives of Op18/stathmin family members abolish microtubule stabilization
by MAP4, whereas MAP4 successfully counteracts MT destabilization by two
distinct and specific catastrophe promoters, namely XKCM1 and a
low-sequestering truncation derivative of Op18/stathmin.
MT destabilization by catastrophe promotion and tubulin sequestering have opposite effects on the concentration of free tubulin heterodimers. Hence, a possible explanation for our previous data is that MAP4 is active at the increased free tubulin concentrations that result from catastrophe promotion, whereas its activity is reduced under tubulin-sequestering conditions. Here, we have evaluated this model by coexpression of MAP4 with truncated and/or chimeric Op18/stathmin family member derivatives with either catastrophe-promoting or tubulin-sequestering properties and distinct cellular localizations. The results support a model in which MAP4 activity is decreased in direct response to the lowered free tubulin concentrations that are still within a range that permits spindle formation.
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Materials and Methods |
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Immunoblotting and immunofluorescence
Immunoblotting and subsequent detection using the electrochemiluminescence
(ECL) detection system (Amersham Pharmacia Biotech) were performed using
anti-MAP4 (M75820; Transduction Laboratories), anti--tubulin (B-5-1-2,
Sigma) and anti-rat CD2 (MRC OX-34) (He et
al., 1988
). To allow simultaneous and equivalent detection of all
Op18/stathmin family members, rabbit antibodies were raised against a peptide
corresponding to a completely conserved region of Op18 (SLEEIQKKLEAAE)
corresponding to residues 46 to 58. The resulting antibodies were affinity
purified by adsorption to native Op18 coupled to Sepharose. Analysis of
cellular MT content by flow cytometry (>95% of all cells were included in
the acquisition gate and >200,000 cells were collected) was performed using
a FACS Calibur instrument (Becton and Dickinson) as described previously
(Holmfeldt et al., 2001
), but
with the following modifications: first, soluble tubulin was pre-extracted in
a saponin-containing MT-stabilizing buffer modified by increased pH (pH 7.4),
omission of glycerol, and reduced paclitaxel concentration (from 4 µM to 20
nM). These modifications minimize nonspecific MT polymerization during the
fixation step. In experiments including cells transfected with
plasma-membrane-associated CD2-RB3, saponin in the MT-stabilizing buffer was
replaced with 0.2% Triton X-100 to ensure that tubulin-associated CD2-RB3
complexes were solubilized. This protocol ensures that MT-specific
fluorescence is not obscured by retention of tubulin heterodimers by CD2-RB3.
The final modification involved addition of an equal volume of 4%
paraformaldehyde dissolved in PEM buffer (80 mM
piperazine-N,N'-bis[2-ethanesulfonic acid], 1 mM EGTA, 4 mM
Mg2+, pH 7.4) added directly to cells resuspended in MT-stabilizing
buffer. After gentle mixing and 15 minutes incubation at 37°C, the cells
were washed and stained for
-tubulin and DNA as described
(Holmfeldt et al., 2001
). In
some experiments, these cells were also stained in parallel with
anti-phospho-histone H3 (Ser28; Upstate Biotechnology). Quantification of MAP4
expression by flow cytometry was performed on cells chilled on ice to
depolymerize MTs, followed by paraformaldehyde fixation (4%) and staining with
anti-MAP4 (5 µg/ml). Fluorescein-conjugated rabbit anti-mouse
immunoglobulin was used as secondary antibody. For immunolocalization of
centrosomes, cells were fixed with methanol at -20°C and stained with
rabbit anti-pericentrin (PRB-432;
http://www.babco.com).
For characterization of spindles by immunofluorescence analysis, cells were
permeabilized with saponin (0.2%) in MT-stabilizing buffer and subsequently
fixed in 4% paraformaldehyde/0.5% glutaraldehyde, followed by quenching with
NaBH4. MTs and DNA were co-stained using Alexa Fluor488-conjugated
anti-
-tubulin and propidium iodide, and analyzed by epifluorescence. To
evaluate tubulin sequestering in cells expressing CD2 chimeras, cells were
fixed directly in 4% paraformaldehyde at 37°C. CD2 chimeras and tubulin
were co-stained with biotinylated anti-CD2/R-phycoerythrin-conjugated
streptavidin and Alexa Fluor488-conjugated anti-
-tubulin, respectively,
and analyzed using a Leica SP2 confocal imager system
(Marklund et al., 1996
). To
estimate cytosolic levels of nonpolymerized tubulin in mitotic cells,
fluorescence intensities of Alexa Fluor488 within ten randomly chosen circular
areas (radius approx. 0.25 µm) in the cytosol, which were free from MTs,
were evaluated in a confocal section. To ascertain that only tubulin-specific
fluorescence was quantified, the analysis was performed on cells stained with
Alexa Fluor488-conjugated anti-
-tubulin alone. Using the same general
approach, plasma-membrane-associated tubulin was analyzed from the same
confocal section within ten randomly chosen rectangular areas of the plasma
membrane. The cytosolic and plasma membrane fluorescence intensities were
averaged for each of a total of 50 cells analyzed. Special care was taken not
to bleach the fluorescence signal prior to confocal scanning.
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Results |
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The penetrance of the monoastral mitotic overexpression phenotype of MAP4 in K562 cells is very high and it is evident from Table 1 that the same fraction of mitotic cells is generated as with treatment using the MT-stabilizing drug paclitaxel (Table 1), which indicates that ectopic MAP4 imposes a complete M-block. However, paclitaxel does not cause monoastral spindles in K562 cells. Finally, it is evident from the data shown in Fig. 1D that the mitotic overexpression phenotype of MAP4 is not unique for K562 cells, since high frequencies of monoastral spindles are also observed in Burkitt's B-cell lymphoma (DG75) and acute T-cell leukemia (Jurkat) cell lines.
|
|
The distribution of MT content was determined within the G2- and M-phase
gates validated above. The histograms derived from control and MAP4-expressing
cells reveal relatively homogeneous distributions of MT content and,
consistent with a previous study on normal cycling cells
(Zhai et al., 1996), that the
average MT content becomes somewhat reduced during mitosis
(Fig. 2C; nonspecific
fluorescence was <1% and is not shown). A comparison between an
unsynchronous mitotic control population and cells blocked at mitosis by
overexpressed MAP4 reveals that the latter results in a more symmetrical and
narrow distribution of MT content. It is also noteworthy that this
distribution is even narrower than the distribution of MT content among cells
blocked in mitosis by the MT-stabilizing drug paclitaxel, again indicating the
homogeneity of the cell population blocked by ectopic MAP4. Given that the
majority of cells within each cell-cycle-stage-specific gate show a relatively
tight distribution (Fig. 2C;
data for G1 cells are not shown), the mean MT-specific fluorescence intensity
provides a good estimate of the relative MT content at different stages of the
cell cycle. A plot of these data reveals that ectopic MAP4, similar to
paclitaxel, causes an increased average level of MT polymers in all phases of
the cell cycle (Fig. 2D), which
in turn shows that MAP4 stabilizes MTs all through the cell cycle. It should
be noted that the observed cell-cycle fluctuations of MT content shown in
Fig. 2D faithfully reproduce
data of others (Zhai and Borisy,
1994
; Zhai et al.,
1996
), with G2 cells having double the MT content of G1 cells,
which is consistent with a doubling of cell mass, and with the MT content
dropping somewhat during mitosis. Given that immunoblotting reveals no
increase in the total cellular tubulin level in K562 cells within the time
period analyzed (Fig. 1A)
(Holmfeldt et al., 2002
), it
appears that ectopic MAP4 increases MT polymers in both interphase and mitotic
K562 cells at the expense of the available pool of free tubulin. This
stabilization seems likely to interfere with spindle formation on many
different levels, including the process of centrosome separation.
Coexpression of a tubulin-sequesteration-specific Op18 truncation
derivative suppresses the mitotic phenotype of ectopic MAP4
We have shown previously that tubulin sequestering, but not catastrophe
promotion, is functionally dominant over MAP4-mediated MT stabilization during
the interphase of the cell cycle
(Holmfeldt et al., 2002). To
compare counteraction of MAP4 activity during interphase and mitosis, MAP4 was
coexpressed with either the tubulin-sequestering Op18(25-149)-triA or
catastrophe-promoting Op18(1-99)-tetraA derivatives outlined in
Fig. 3A. Both of these Op18
truncation derivatives are non-phosphorylatable (i.e. they posses Ser to Ala
substitutions at phosphorylation sites) and are consequently not inactivated
by phosphorylation during mitosis, which is the fate of endogenous Op18
(Larsson et al., 1997
;
Marklund et al., 1996
). For
conditional coexpression, a pMEP-vector-based cotransfection approach was used
that would allow stringently regulated expression of two gene products from
the hMTIIa promoter (Gradin et al.,
1998
). As compared with our previous report
(Holmfeldt et al., 2002
), the
ratio of pMEP DNAs was altered such that (1) the levels of coexpressed Op18
derivatives were increased at the expense of MAP4, and (2) the MT
destabilization by the two derivatives when expressed alone is equivalent
(Fig. 3B). These coexpression
conditions caused a modest increase in MT content by ectopic MAP4 and an
approximately 50% decrease in MT content by either of the two truncated Op18
derivatives (Fig. 3B). The data
depict MT content determined specifically in G2 cells after 20 hours of
induced expression, and it is clear that our previous findings on a mixed
cell-cycle-stage population induced for 8 hours are faithfully reproduced
under these modified conditions. Thus, coexpressed MAP4 did not counteract MT
destabilization caused by the coexpressed tubulin-sequestering
Op18(25-149)-triA derivative, whereas the action of the catastrophe-promoting
Op18(1-99)-tetraA was counteracted (Fig.
3B).
|
Analysis of DNA profiles, shown in Fig. 4, confirms that the two Op18 truncation derivatives interfere with spindle formation to different extents, and only Op18(1-99)-tetraA causes a prominent mitotic block. To uncover both suppressive and enhancing effects on monoastral-mitotic phenotypes, the cotransfection conditions were optimized so that the MAP4 levels would not cause a complete mitotic block. Interestingly, the cell-cycle profiles of cotransfected cells show that the mitotic block mediated by MAP4 is somewhat suppressed by coexpressed Op18(25-149)-triA, whereas coexpressed Op18(1-99)-tetraA has the opposite effect (Fig. 4, compare - and + MAP4). This apparent suppression by coexpressed Op18(25-149)-triA was not due to a general cell-cycle arrest or potential interference with a metaphase checkpoint, as evidenced by accumulation of G2/M cells in the presence of paclitaxel (see insert in Fig. 4, top panels). Thus, MT destabilization by two distinct mechanisms has opposite effects on the potency shown by the MT-stabilizing MAP4 protein in blocking cell division.
|
Since the monoastral spindle is a hallmark of the mitotic phenotype of
ectopic MAP4, we evaluated cotransfected mitotic cells with respect to
monoastral or bipolar spindles. For simplicity, data on bipolar spindles are
presented in Fig. 4 without
consideration either of MT density or whether the spindles appeared aberrant
or not. Consistent with our previous report
(Holmfeldt et al., 2001) and
the DNA profiles, the results show that expression of Op18(25-149)-triA alone
only causes a modest increase in the mitotic index. Moreover, as previously
reported (Holmfeldt et al.,
2001
), Op18(1-99)-tetraA causes a much more pronounced mitotic
block, and a major fraction of all spindles were bipolar. These spindles were
clearly abnormal, with MTs appearing as two small star-like asters (data not
shown) (Holmfeldt et al.,
2001
). Analysis of cells coexpressing MAP4 with either of the Op18
truncation derivatives revealed a large increase in the frequency of mitotic
cells. The appearance of the mitotic figures was heterogeneous, some of which
were difficult to categorize as normal or aberrant. Nevertheless, a major
proportion of the abundant bipolar spindles found in
MAP4/Op18(1-99)-tetraA-coexpressing cells appeared similar to the abnormal
mitotic figures of cells expressing the catastrophe-promoting Op18 derivative
alone (data not shown) (Holmfeldt et al.,
2001
).
Monoastral spindles were scored as being either intermediate-to-large or small (see Fig. 4, lower panel). In the latter category, MTs appeared as a single, centrally located bright dot without or with very short, individually distinguishable MTs. Importantly, and in agreement with a partial relief of a mitotic block, the data in Fig. 4 demonstrate that coexpression of the tubulin-sequesterer Op18(25-149)triA with MAP4 causes a 50% reduction in the total frequency of monoastral spindles. The data also show that the frequency of intermediate-to-large monoastral spindles in MAP4-expressing cells is reduced by a factor of 4 to 5 by coexpressed Op18(25-149)-triA, which reveals a general decrease in the MT content of monoastral spindles.
As shown in Fig. 4, the fraction of small monoastral spindles in MAP4-overexpressing cells is increased by coexpression of Op18(1-99)-tetraA. However, despite this apparent MAP4-antagonizing destabilizing activity, the total frequency of monoastral spindles was unaltered and the potency of the mitotic block was further increased by the catastrophe-promoting Op18(1-99)-tetraA derivative. It follows that the observed suppression of MAP4-mediated monoastral-mitotic phenotype by the tubulin-sequesterer Op18(25-149)-triA cannot be explained by a general mechanism involving antagonistic activities. This suggests that suppression is caused by downregulation of MAP4 activity, which would be consistent with the observed inability of MAP4 to antagonize MT destabilization by Op18(25-149)-triA in interphase cells (Fig. 3B). To address whether the mechanism involves direct interactions with Op18(25-149)-triA, we lysed MAP4/Op18(25-149)-triA-Flag-coexpressing cells and used antibodies directed against the Flag-epitope tag to precipitate the Op18 derivative. These experiments revealed the expected co-precipitation of tubulin but not MAP4 (data not shown). Thus, it appears that MAP4 activity is not downregulated by direct interactions with Op18(25-149)-triA.
A decrease in free tubulin concentration, caused by a
plasma-membrane-located high-affinity tubulin-binding chimera, suppresses MAP4
activity throughout the cell cycle
Mechanistically distinct types of MT destabilization have opposite effects
on the concentration of free tubulin heterodimers. Thus, while catastrophe
promotion increases the free tubulin concentration, tubulin sequestering has
the reverse effect. It follows that suppression of MAP4 activity by the
tubulin-sequestering Op18(25-149)-triA derivative might be explained if MAP4
is less active under conditions of reduced free tubulin concentrations. To
assess directly the effect of decreased free tubulin concentrations on MAP4
activity, we took advantage of the RB3 neural member of the Op18/stathmin
family, which binds tubulin with affinity that is orders of magnitude higher
than that of Op18, and consequently forms much more stable
tubulin-sequestering complexes (Charbaut et
al., 2001). To localize sequestering complexes to a defined
cellular location as far as possible from the mitotic spindle, RB3 was located
specifically to the plasma membrane by creating a chimera between RB3 and the
extracellular and transmembrane region of the T-cell-specific cell-surface
protein CD2 (Fig. 5A). As a
control, a CD2 chimera was prepared in which the RB3 part was replaced by a
95-residue C-terminal part of Op18 lacking significant tubulin affinity
(CD2-Co). A confocal section through a mitotic cell shows that CD2-Co and
CD2-RB3 were expressed to similar degrees at the plasma membrane, as detected
by an anti-CD2 antibody (Fig.
5B). It is also evident that the CD2-RB3, but not CD2-Co, chimera
causes membrane localization of a significant fraction of cytosolic tubulin
(Fig. 5B).
|
|
|
Immunoblot analysis, using antibodies against a peptide corresponding to a
completely conserved region among members of the Op18/stathmin family
(anti-SLEEIQ), showed that the CD2-RB3 chimera is expressed at higher levels
than endogenous Op18 (Fig. 6).
Since the CD2 cell-surface receptor is extensively glycosylated, CD2 chimeras
migrate as a broad heterogeneous band, as detected by either anti-CD2 or by
anti-SLEEIQ. Arbitrary quantification of CD2-RB3 relative to endogenous Op18
was achieved from serial dilutions of cell lysates (data not shown).
Integration of the heterogeneous band signal indicated that CD2-RB3 is
expressed at a fourfold higher level than endogenous Op18. On the basis of
previous estimates of cytosolic concentration of Op18 in K562 cells (i.e. 10
µM) (Larsson et al., 1999),
it can be predicted that the CD2-RB3 chimera is expressed at approximately 40
µM, which should be compared with an estimated total tubulin heterodimer
concentration of 23 µM in K562 cells
(Larsson et al., 1999
). These
approximations suggest that CD2-RB3 is expressed at an approximately twofold
molar excess over total tubulin. Under these conditions of decreased free
tubulin concentrations, determination of MT content in interphase (G2) cells
revealed a 65% reduction in polymerized tubulin and, most significantly, that
this drop is not counteracted by coexpressed MAP4
(Fig. 6).
The RB3 protein lacks the cyclin-dependent kinase phosphorylation sites of native Op18 and is not inactivated by phosphorylation during mitosis. Analysis of cell-cycle profiles revealed that expression of CD2-RB3 causes a 2.5-fold increase in the mitotic index as compared with Vector-Co cells (Fig. 7, percentage of mitotic cells is shown below the cell-cycle profiles). This result is not surprising given the high tubulin-sequestering potential of this derivative, as revealed by its potent destabilization of interphase MTs. The DNA ratio used for coexpression was adjusted such that MAP4 expression caused a major accumulation of G2/M cells. Significantly, coexpression of the CD2-RB3 chimera potently and specifically suppressed this accumulation of G2/M cells. This was not due to a general cell-cycle arrest, as shown by accumulation of G2/M cells in the presence of paclitaxel (see insert in Fig. 7, top panels).
Cotransfected cells were evaluated with respect to monoastral and bipolar spindles according to the criteria described in Fig. 4. It is shown in Fig. 7, lower panel, that essentially all mitotic CD2-RB3-expressing cells contained bipolar spindles. Under these conditions, expression of MAP4 alone or coexpressed with CD2-Co resulted in an essentially homogeneous population of mitotic cells with monoastral spindles of intermediate-to-large size. Consistent with the cell-cycle profile, this phenotype of MAP4 was efficiently suppressed by the tubulin sequestration-specific CD2-RB3 derivative.
It seems reasonable to assume that suppression of monoastral spindles and consequent formation of functional spindles is a stringent criterion for downregulation of MAP4 activity by the coexpressed CD2-RB3 chimera. This chimera has a defined plasma membrane location, which precludes direct contact with the mitotic machinery. As expected, analysis of MAP4-specific fluorescence revealed that MAP4 was present both in the cytosol and in association with MTs, and this pattern of localization appeared to be unaltered by CD2-RB3 expression (data not shown). Thus, since MAP4 and CD2-RB3 did not colocalize, the data indicate that the decline in free tubulin concentration was a direct cause of downregulating MAP4 activity.
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Discussion |
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The net balance of opposing MT-regulatory factors seems likely to determine
the length of MTs, and thus the total MT content
(Andersen and Wittmann, 2002;
Kinoshita et al., 2001
;
Tournebize et al., 2000
). We
have previously shown that MAP4 counteracts MT destabilization by XKCM1 and a
C-terminally truncated Op18 derivative (two catastrophe promotors that both
lack significant tubulin-sequestering activity), but not MT destabilization
caused by tubulin-sequestering competent regulators
(Holmfeldt et al., 2002
). This
suggested that MAP4 differentiates between mechanistically distinct types of
MT destabilization. However, from these analyses of the MT content of
interphase cells, it could not be excluded that MAP4 coexpressed with
tubulin-sequestering regulators modulated MT dynamics without altering
cellular MT content to a significant extent. However, in mitotic cells,
interference with dynamic parameters of MTs by coexpressed
stabilizing/destabilizing proteins will cause a mitotic block. Thus, if the
combined phenotype of coexpressed ectopic MT regulators is manifested as a
suppression of MAP4-mediated spindle defects to allow subsequent cell
division, it seems reasonable to assume that essential dynamic parameters of
spindle MTs in coexpressing cells are close to the normal range.
In the present study, counteraction of ectopic MAP4 activity was evaluated in mitotic cells by determining (1) suppression of the monoastral phenotype, (2) size of monoastral spindles, and (3) formation of functional dipolar spindles. Our result demonstrated that both catastrophe-promoting and tubulinsequestering Op18 derivatives antagonize MT stabilization by MAP4 in mitotic cells, since coexpression in both cases altered the ratio of monoastral spindles with intermediate-to-large and small MT content (Figs 4, 7). Given that only the tubulinsequestration-competent derivatives suppressed MAP4-mediated formation of monoastral spindles and facilitated cell division in coexpressing cells, it seems clear that an antagonizing activity is not sufficient to suppress the deleterious MT-stabilizing effect of excess MAP4 activity during mitosis. This does not seem surprising, considering that spindle formation and subsequent cell division involves a complex series of events requiring finely tuned regulation of MT dynamics. Thus, the improbability that the spindle-disrupting effect of ectopic MAP4 can be partially reversed by an antagonizing MT-destabilizing activity provided an indication that the cytosolic tubulin-sequestering Op18(25-149)-tetraA derivative might suppress the mitotic phenotype by downregulation of MAP4 activity.
To link downregulation of MAP4 activity directly to a decrease in free
tubulin concentrations requires a high-affinity tubulin-sequestering molecule
that is spatially separated from both the MT system and the
cytosolic/MT-associated MAP4 protein. In interphase cells, the MT system is in
extensive contact with the entire cytoplasmic space, whereas mitotic MTs are
mainly located in the vicinity of chromosomes and have minimal contact with
the plasma membrane. Thus, the transmembrane CD2-RB3 chimera provides an ideal
tool for studying how decreased free tubulin concentrations modulate ectopic
MAP4 activity in a mitotic cell. Expression of CD2-RB3 alone causes only a
modest (2.5-fold) increased frequency of mitotic cells
(Fig. 7). This weak mitotic
phenotype may appear surprising, but is in line with the previously observed
resistance of spindle formation to tubulinsequestering conditions
(Holmfeldt et al., 2001). The
relative robustness of spindle formation under tubulin-sequestering conditions
greatly simplified interpretation of data from coexpression experiments that,
taken together, provide compelling evidence that the activity of ectopic MAP4
is downregulated by the coexpressed CD3-RB3 derivative. Since CD2-RB3 and MAP4
are spatially separated in mitotic cells, it seems reasonable to conclude that
downregulation of MAP4 activity is directly coupled to decreased free tubulin
concentrations. Moreover, since a functional spindle can still be formed at
these lowered concentrations of free tubulin, albeit at a decreased rate, our
results indicate that the activity of MAP4 is modulated within the
physiological range of free tubulin concentrations.
The free concentration of tubulin heterodimers modulates the rate of
tubulin gene expression in many, but not all, cellular systems
(Cleveland et al., 1981).
Paclitaxel or MAP4-mediated over-polymerization of MTs or tubulin
sequestration does not result in a detectable increase in tubulin synthesis
within a 24-hour time period in the leukemic K562 cell line
(Holmfeldt et al., 2002
). This
property of K562 greatly simplifies interpretation of data in the present
study. However, the specific mechanism behind downregulated MAP4 activity by
lowered free tubulin concentration is still unclear. Analysis of MAP4-specific
fluorescence reveals no obvious redistribution of cytosolic and MT-associated
MAP4 in cells expressing tubulin-sequestering regulators (data not shown).
Moreover, downregulated MAP4 activity could in principle be explained by
either (1) direct physical association with MAP4, (2) phosphorylations by a
putative tubulin-sequestration-regulated protein kinase, or (3) reduction of
the MT-stabilizing properties of MAP4 at lowered free tubulin concentrations.
Given that CD2-RB3 and MAP4 are spatially separated during mitosis, it is
highly improbable that direct association suppresses MAP4 activity. However,
we are presently unable to distinguish between the two remaining alternatives.
For example, lowered tubulin concentrations may activate members of the
MAP/microtubule affinity regulating kinase (MARK) family, which have been
shown to phosphorylate and thereby downregulate the activity of several MAPs
including MAP4 (Drewes et al.,
1997
; Ebneth et al.,
1999
). Alternatively, it is also possible that low free tubulin
concentration might directly limit MAP4 activity. Thus, increased knowledge on
the mechanism by which MAP4 stabilizes MTs, combined with functional
dissection of MAP4 phosphorylation sites, is required to resolve these
issues.
Downregulation of MAP4 activity at lowered free tubulin concentrations in K562 cells has general implications for regulation of MT dynamics in intact cells. For example, excessive polymerization will lead to deceased MAP4 activity, whereas depolymerization by catastrophe promotion will increase MAP4 activity. Such a negative autoregulatory loop might contribute to the homeostasis of the MT system. Given that the free tubulin concentration could vary in different cell types (e.g. by differences in tubulin partitioning between monomer-polymer pools), the present results provide a rationale for explaining the lack of consensus among previous reports describing the consequences of ectopic MAP4-overexpression phenotypes.
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Acknowledgments |
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