European Molecular Biology Laboratory, Cell Biology Programme, D-69117 Heidelberg, Germany
To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Domínguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289-1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5-10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985-993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation.
THE function of the mitotic spindle is to ensure even
segregation of the duplicated chromosomes to the
two daughter cells (Kirschner and Mitchison, 1986 To proceed with the identification and characterization
of non-neuronal MAPs, we have raised mAbs against MT
proteins bound to taxol stabilized MTs isolated from interphase Xenopus laevis egg extracts (Andersen et al., 1994 Extracts, Antibodies, and Immunofluorescence
Xenopus egg extracts were prepared as described by Murray (1991) Purification of XMAP310 from Xenopus Egg Extracts
Interphase egg extracts were prepared by activation of dejellied eggs with
calcium ionophore A23187 (Sigma Chem. Co., St. Louis, MO) at a 1/10,000
dilution (stock 2 mg/ml in DMSO) in MMR plus 200 µg/ml cycloheximide
for 5 min (see also Murray, 1991
Video Microscopy and MT Cross-linking Assay
Chambers with a volume of ~4 µl were used. Two strips of double-sided
tape were fixed on a microscope slide (76 x 22mm, 0.8 mm thick, Select
Micro Slide model BS3836-1975; Chance Propper Ltd., UK) ~3 mm apart
and perpendicular to the long axis of the slide. A 22 x 22mm coverslip
(gold seal cover glass model 3306; Clay Adams, UK) was then placed on
top of the two tape strips and firmly pressed to attach it well. Slide and
coverslip were ethanol and water washed before use. The channel formed
by the tape strips, the slide and the coverslip made up the chamber. Before use, centrosomes (Bornens et al., 1987 Tubulin at 40 µM plus 2 µM rhodamine-labeled tubulin (Hyman et al.,
1991 Miscellaneous
For microsequencing XMAP230 and XMAP310 were prepared in preparative amounts by immunoprecipitation. Dynabeads (model 110.12; Dynal
GmbH, Hamburg, Germany) with the Q4 or L7 mAb precoupled were incubated in extracts for 30 min at 4°C. Beads were washed 10 times for 1 min, each time with 1 ml PBS, 150 mM NaCl, 0.5% TX-100, 1 mM PMSF
at 4°C before analysis on 4% SDS-PAGE. The bands corresponding to
XMAP230 and XMAP310 were excised and digested with trypsin. Peptides were separated as described by Tetaz et al. (1993) Identification and Tissue Distribution of XMAP310
Three major MAPs larger than 200 kD cosediment with
taxol stabilized MTs from interphasic Xenopus egg extracts:
XMAP215, XMAP230, and a 310-kD MAP (XMAP310)
that was not previously characterized (see Fig. 1 A in
Andersen et al., 1994
Since XMAP310 had been identified biochemically due
to its affinity for MTs, we wanted to determine whether it
localized to MTs in vivo. The Q4 mAb was used to probe
the localization of XMAP310 at different stages during the
cell cycle of Xenopus XL177 tissue culture cells (Fig. 1). In
interphase cells XMAP310 was present only in the nucleus
(Fig. 1, a-c). The analysis of confocal sections showed that
XMAP310 was homogeneously localized throughout the
nucleus (data not shown). This localization was maintained through prophase, but XMAP310 relocalized to
spindle MTs at the exclusion of astral MTs during metaphase (data not shown and Fig. 1, d-f). The metaphase localization was resistant to preextraction of the cells with
detergents (Fig. 1, d-f). During anaphase, XMAP310 was
concentrated to areas of high MT density (Fig. 1, g-i), but this staining could be removed by preextraction. During
cytokinesis XMAP310 began to reaccumulate in the nucleus (Fig. 1, j and k). Some of the protein that remained
diffusely localized in the cytoplasm, was completely removed by preextraction (Fig. 1, l and m, note the faint
staining of the nucleus after preextraction). The localization of XMAP310 in the nucleus during interphase and to
the spindle during mitosis was confirmed by immunostaining of Xenopus embryos at the mid blastulae stage (data
not shown). Double immunostaining of XL177 cells with
XMAP230 and XMAP310 mAbs and observation by confocal microscopy revealed that the two proteins colocalize in the mitotic spindle (Fig. 2). However, immunoprecipitation and native molecular weight estimation showed that
XMAP230 and XMAP310 do not form a complex in the
extract, suggesting that the co-localization in the spindle
MTs is due to independent regulation of binding for these
two proteins (data not shown; hydrodynamic data for
XMAP230 and XMAP310: stokes radius (ao), 13.3 nm and 16.5 nm, respectively; sedimentation coefficients, (S20w) of
5.2 x 10
Purification of XMAP310 from Xenopus Egg Extracts
To determine the effect of XMAP310 on MTs it was purified from interphase Xenopus egg extracts. The first purification step consisted in the preparation of MAPs using
taxol stabilized MTs, as shown in Fig. 3 A and B, lane 1.
XMAP310 could be eluted from MTs by 400 mM salt together with XMAP230 and XMAP215 (Fig. 3, lane 1).
However, instead of eluting the MAPs with salt, we used Ca2+ ions to depolymerize taxol stabilized MTs on ice
(Collins, 1991 We checked that XMAP310 is a bona fide MAP by assaying the capacity of the pure protein to bind back to taxol
stabilized MTs. The experiment showed that XMAP310
bound back quantitatively to MTs (Fig. 3 D, arrow). These
experiments also showed that EF1- XMAP310 Causes Microtubule Cross-linking
As a first step in the characterization of the effect of pure
XMAP310 on MTs, we polymerized rhodamine labeled
tubulin, either in the presence or absence of XMAP310.
The morphology of the MTs obtained suggested that they
formed bundles in the presence of XMAP310 (data not
shown). This was confirmed by electron microscopy, which showed long bundles of 2-5 MTs, in the presence of
XMAP310 (Fig. 4). The short distance between the individual MTs resembles the cross-links obtained with 280 kD Syncolin (Feick et al., 1991
XMAP310 Primarily Promotes Rescues
To determine how XMAP310 affected the parameters of
MT dynamic instability, we examined MT growth in the
presence and absence of XMAP310 by time lapse video
microscopy (Fig. 5). Centrosomes and tubulin were preabsorbed to the glass of a microscope chamber on ice, followed by further blocking of the glass with casein. The
sample containing soluble tubulin with or without XMAP310 was then injected into the chamber and observed under a
microscope at 35°C. The polymerization of individual MTs
was then followed in real time (Fig. 5). The average
growth rate of MTs (vg) was not significantly influenced by
XMAP310, whereas the shrinkage rate (vs) was reduced
twofold compared with the control (Table I). Moreover,
XMAP310 promoted rescues 5-10-fold (fres) without having any effect on the catastrophe frequency (fcat, Table I). The dynamicity of MTs in the presence of XMAP310 relative to the control was calculated to 0.6, showing that
XMAP310 dampens MT dynamics (Toso et al., 1993
Table I.
Effects on MT Dynamics of XMAP310
;
McIntosh, 1994
; Inoué and Salmon, 1995
; Hyman and
Karsenti, 1996
; Waters and Salmon, 1997
). Assembly of
the mitotic spindle involves a dramatic reorganization of
the radial interphase microtubule (MT)1 network into an
elliptical, bipolar shape. This reorganization results from a
significant increase in MT turnover at the onset of mitosis
(Olmsted et al., 1989
; Belmont et al., 1990
; Verde et al.,
1990
, 1992
; Vale, 1991
; Shiina et al., 1992a
; Sawin and Mitchison, 1994
; Hyman and Karsenti, 1996
; McNally et
al., 1996
; Zhai et al., 1996
; Tournebize et al., 1997
). MT
turnover is largely due to the dynamic instability of MTs
(Kirschner and Mitchison, 1986
), meaning that MTs are
not stable rods but instead alternate between phases of
growth and shrinkage. Therefore a given turnover state for
MTs will be determined by the respective values of four parameters: the growth rate (vg), the shrinkage rate (vs),
and the transition between growth and shrinkage, a catastrophe which occurs with a certain frequency (fcat). The
opposite event, when a MT transit from shrinkage to
growth, a rescue also occurs with a certain frequency (fres)
(Walker et al., 1988
). Spindle MTs are stabilized and have
reduced dynamicity compared with their cytoplasmic
counterparts (Karsenti et al., 1984
; Saxton et al., 1984
;
Toso et al., 1993
; Vasquez et al., 1994
; Dogterom et al., 1996
; Heald et al., 1996
; Zhai et al., 1996
). Microtubule-
associated proteins (MAPs) may have an important function in the stabilization of spindle MTs, since they in general
dampen MT dynamics and since their activity can be regulated by phosphorylation. However, little is known about
the properties of non-neuronal MAPs in general (for reviews see Olmsted, 1986
; Matus, 1990
; Wiche et al., 1991
; Bulinski, 1994
; Hirokawa, 1994
; Mandelkow and Mandelkow, 1995
; Hyman and Karsenti, 1996
). Most probably,
several different MAPs are involved in regulation of MT
turnover during the cell cycle and in the spindle. However,
only XMAP230, MAP4, and recently XMAP215 have
been localized to the spindle and functionally characterized in vitro (Gard and Kirschner, 1987
; Andersen et al.,
1994
; Vasquez et al., 1994
; Ookata et al., 1995
; Charrasse,
S., M. Schroeder, C. Gauthier-Rouviere, L. Cassimeris,
D.L. Gard and C. Larroque. 1996. Mol. Biol. Cell. 7:222a).
).
Several studies indicate that the MT binding activity of
MAPs is finely tuned during the cell cycle, rather than
modulated by a strict on- off- switch (Zieve and Solomon,
1982
; Olmsted et al., 1989
; Shiina et al., 1992b
; Andersen
et al., 1994
; Masson and Kreis, 1995
). Therefore, from the
interphase MAP fraction, it should be possible to identify MAPs associated with the interphasic MT network, the
mitotic spindle MTs, or both. Here we report on the structural and functional in vitro characterization of a new 310-kD
Xenopus MAP (XMAP310) that localizes to the mitotic
spindle. Interestingly, this MAP turns out to be a strong
rescue factor.
Materials and Methods
. Interphase and mitotic extracts (CSF-arrested) had H1 kinase activities of 2-5
and 15-25 pmol/µl/min (Félix et al., 1993
), respectively. Total MAPs for
mouse mAb production were produced as described by Andersen et al.
(1994)
. The screening procedure to obtain the Q4 mAb consisted of immunofluorescence and western blotting in parallel; the mouse polyclonal
antiserum gave the same immunofluorescence pattern as the final anti-XMAP310 Q4 mAb (subtyped as IgG1; Mouse-Hybridoma-Subtyping
Kit, Boehringer Mannheim GmbH, Mannheim, Germany). Treatment of
Western blots with alkaline phosphatase did not result in loss of reactivity
with the Q4 mAb, indicating that Q4 does not recognize a phosphorylated
epitope (data not shown). For the experiment described here either ascites or culture supernatants of Q4 were used at a 1/500 and 1/3 dilution, respectively, for Westerns. The L7 anti-XMAP230 mAb was as described by Andersen et al. (1994)
. Immunofluorescence was performed as described by (Andersen et al., 1994
). The Q4 ascites was used at a 1/200 dilution. Preextraction of cells was performed twice for 3 s in BRB80, 5 mM
EGTA, 0.5% Tx-100, pH 6.8. Confocal fluorescence microscopy was performed as described by Reinsch and Karsenti (1994)
.
). Concentrated extracts were frozen in
liquid nitrogen in 200-µl aliquots and stored at
80°C. Concentrated extracts (5-7 ml/purification) were rapidly thawed at 37°C, transferred to ice,
and diluted with 1 vol 50 mM sucrose, 10 mM K-Hepes (pH 7.7), 1 mM
EGTA, 1 mM MgCl2, 0.1%
-mercaptoethanol, protease inhibitors. This
mix was centrifuged at 40,000 rpm (192,000 g) for 40 min at 4°C in a SW50
rotor. 20 µM (final) taxol (10 mM stock in DMSO; Molecular Probes) was
added to this clarified extract on ice, together with 1/10 vol prepolymerized MTs: 400 µl cycled tubulin at 9 mg/ml was mixed with 400 µl 66% glycerol, BRB80 (80 mM K-Pipes, 1 mM EGTA, 1 mM MgCl2, pH 6.8), 10 mM MgCl2, and 1 mM GTP final and incubated for 30 min at 37°C; Taxol
was then added to 20 µM (final) and incubation continued for 5 min before addition to the extract. The extract with the prepolymerized MTs was
incubated on ice for 10 min, and then loaded on 40% sucrose cushions,
BRB80, 20 µM taxol, and protease inhibitors at a 1:1 volume ratio. The
MTs were spun through the cushion at 40,000 rpm (70,000 g) for 20 min at
4°C in a TLA100.2 rotor. The supernatant was discarded and the MT pellets resuspended in 1/4 the volume of the clarified extract (2 ml) using
buffer A (20 mM K-Pipes (pH 7), 50 mM NaCl) plus 5 mM CaCl2, protease inhibitors and 0.1%
-mercaptoethanol. This mix was incubated for
10 min on ice to allow depolymerization of the taxol stabilized MTs and then centrifuged twice for 10 min, 50,000 rpm (110,000 g), 4°C. The supernatant (see Fig. 3, lane 3), enriched in XMAP310 and tubulin, was applied
onto a PC 1.6/5 MonoS column using the SMART system (Pharmacia
LKB Biotechnology, Uppsala, Sweden) at 4°C and 100 µl/min; After loading the column was washed with 1 ml 5% B buffer (20 mM K-Pipes (pH
7), 800 mM NaCl), a step to 20% B resulted in elution of XMAP310 along
with other proteins; XMAP310 was enriched in the early fractions after
shift to 20% B; pooled fractions (100-150 µl) from the MonoS column
(see Fig. 3, lane 5) were loaded on a PC 3.2/30 Superose 6 column equilibrated in 100 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10% glycerol, 10 mM
K-Hepes (pH 7.7) using the SMART system at 4°C and 40 µl/min. Fractions containing pure XMAP310 (see Fig. 3, lane 6) were stored separately
or pooled with later fractions (see Fig. 3, lanes 7-8), aliquoted by 20 µl
and stored in liquid nitrogen. The concentration of XMAP310 in the extract is ~200 nM.
Fig. 3.
Purification of XMAP310. A and B show the different
steps of the purification by Coomassie-stained 4% (A) and 10%
(B) SDS-PAGE: lane 1, total MAPs eluted with salt (Andersen
et al., 1994) from taxol stabilized MTs; lane 2, MT pellet of Ca2+
undepolymerized MTs; lane 3, Supernatant of Ca2+ depolymerized MTs/Sample loaded on the MonoS column; lane 4, Flow-through from the MonoS column; lane 5, pooled fractions from the MonoS column (the major 49-kD protein was determined as
EF1-
); lanes 6-8, fractions from the Superose 6 column. (C)
Steps of the purification monitored with the Q4 mAb: lane I, start
extract; lane II, extract after depletion with MTs; lane III, total
MTs (V = 2 ml); lane IV, Ca2+-stable MT fraction (V = 500 µl);
lane V, pooled fractions from the MonoS column (compare with
A and B, lanes 5). (D) Purified XMAP310 rebinds to and pellets
(P) with taxol stabilized MTs. Binding to MTs was for 10 min on
ice. In lane 1 the MT concentration (in tubulin equivalents) was
2.5 µM and in 2 0.6 µM. However, the total amount of MTs used
was the same in lanes 1 and 2 (S, supernatant after pelleting MTs).
[View Larger Version of this Image (89K GIF file)]
) were diluted to 3 x 103/µl
with working concentration of tubulin, and injected into the chamber and
left for 5 min on ice. 10 µl 5 mg/ml Casein in BRB80 was then by capillary
forces flowed through the chamber and left for 5 min on ice. The chamber
was then washed with 15 µl BRB80 and the sample was injected. The volume of the sample was always 30 µl and mixed immediately before use.
For most of the preparations the sample consisted of: 10.0 µl Superose 6 column buffer with XMAP310 or without XMAP310 (control), 15.1 µl
BRB80, 4.83 µl cycled tubulin at 87 µM (final tubulin concentration 14 µM [using BSA as Standard]), 0.3 µl 0.1 M GTP. After application of the
sample the chamber was sealed at both ends with grease and placed on the
stage of a Zeiss Axiovert 10 photomicroscope (Zeiss GmbH, Oberkochen,
Germany) equipped with a Zeiss 100x achrostigmat 100x/1.25 oil objective and a Hamamatsu Phototonics (Japan) C3077 CCD camera. Objective,
condenser, and stage were heated to 37°C. The temperature in the chamber was 35°C. Measurements of MT dynamics were performed as described
(Andersen et al., 1994
) using 4-s time interval between individual video
frames and NIH 1.55 and Microsoft Excel 5 programs. No samples were
observed for more than 40 min.
) in BRB80 and 1 mM GTP was mixed on ice with 1/2 vol MAP or
control buffer (same dilution of MAP as used in the video experiments)
and incubated for 15 min at 37°C. The sample was then processed for negative stain electron microscopy. Alternatively, the sample was diluted 200-fold into BRB80 plus 20 µM taxol at room temperature (without fixation),
loaded on top of a 3 ml 15% glycerol cushion, BRB80, 20 µM taxol in
modified Corex tubes (Evans et al., 1985
) and then centrifuged at 12,000 rpm for 20 min at 20°C using a HB4 rotor. Coverslips were fixed for 5 min
in
20°C methanol and mounted in 90% glycerol, 50 mM Tris-HCl (pH = 8), and analyzed. For negative stain EM, 3 µl MT-MAP mix was absorbed onto a glow-discharged 300 mesh carbon grid for 1 min at room temperature, followed by three 5-µl washes and staining with 1% uranyl acetate
for 1 min and analysis with a Zeiss 10C electron microscope.
. Edman degradation was performed using an Applied Biosystems Machine (model 477A;
Foster City, San Francisco, CA). These microsequences as well as the
identity of the Q4 immunoprecipitations, the purified XMAP310 protein
(see Fig. 3, lane 6), and EF1-
(see Fig. 3, lane 5) were confirmed by Matrix-assisted laser desorption ionization mass spectroscopy (MALDI MS).
MALDI MS consists of tryptic cleavage of the protein followed by mass
spectroscopy of the peptides (Jensen et al., 1996
). Therefore, using
MALDI MS one gets a fingerprint spectrum of the tryptic cleavage sites of
the protein. The spectrum can then be compared with hypothetical tryptic
cleavage spectra of proteins in the data bases. Very conserved proteins
will show homology by this method because trypsin sites will be conserved in a conserved sequence. Calf brain tubulin was prepared as described by
Ashford et al. (1998)
. The relative molecular mass for XMAP310 of 310 kD was estimated on the basis of the migration on 4% SDS-PAGE of bovine brain MAP1B (Mr 325 kD). The hydrodynamic data for XMAP230 and XMAP310 were determined as described by Wilhelm et al. (1997)
. Gel electrophoresis and Western blotting were performed as described by
Andersen et al. (1994)
. Western blots were developed using alkaline phosphatase conjugated secondary antibodies or ECL (Amersham Corp.).
Protein concentrations were measured with Bradford (Bio-Rad Labs.,
Hercules, CA) or BCA (Pierce, BA oud-Beijerland, Holland) reagents.
Tissue extracts were prepared as described in Andersen et al., (1994). Unless otherwise indicated, chemicals were from Sigma or Boehringer Mannheim GmbH.
Results
and Fig. 3 A, lane 1 in this study:
XMAP215 (
), XMAP230 (o), XMAP310 (*). We raised
mAbs against XMAP310, and obtained one clone (Q4)
that recognized XMAP310 in extracts of the Xenopus tissue culture cell line XL177 (Miller and Daniel, 1977
), in
total Xenopus egg extracts, and in the MAP fraction (data
not shown). When Xenopus tissue extracts were probed with the Q4 mAb, XMAP310 was very abundant in brain
and testis tissues but could not be detected in heart, lung,
stomach, or muscle tissues (data not shown).
Fig. 1.
XMAP310 localization in XL177 cells (Miller and
Daniel, 1977) during the cell cycle. XL177 cells were stained with:
Hoechst (a, d, g), a rabbit anti-tubulin pAb (b, e, h, j, l), and the Q4 mAb (c, f, i, k, m). Interphase (a-c), metaphase/early
anaphase after pr-extraction with 0.5% TX-100 (d-f), late
anaphase (g-i), late telophase without preextraction (j and k),
late telophase with preextraction (l and m). Bar: (a-k) 10 µm; (l
and m) 12.5 µm.
[View Larger Version of this Image (95K GIF file)]
13 s
1 and 10.1 x 10
13 s
1, respectively; native
molecular masses, 285 and 685 kD, respectively).
Fig. 2.
(Top) XMAP310 localization in the mitotic spindle:
MTs stained with a rabbit anti-tublin pAb in the rhodamine channel (Tubulin), XMAP310 in the fluorescein channel (XMAP310)
and in overlay (Overlay). (Bottom) XMAP230 and XMAP310
colocalize (Overlay) to the central region of the mitotic spindle in
XL177 cells. Images were obtained by confocal microscopy. Bar,
2.5 µm.
[View Larger Version of this Image (77K GIF file)]
; Bulinski and Bossler, 1994
). This results in
the depolymerization of ~50% of the taxol MTs (Fig. 3 B,
lanes 2 and 3; the large band at ~50 kD is tubulin), and
most of XMAP310 was released from the MT pellet by
this treatment, although a heterogeneous fraction remained bound to MTs (Fig. 3 A, *, lanes 3 and 2, note the
doublet in lane 2). XMAP230 was also eluted after Ca2+
depolymerization (Fig. 3 A o, lane 3). However, XMAP215
was highly enriched in the Ca2+-stable MT fraction (Fig. 3
A,
, lane 2). The Ca2+-eluted XMAP310 was then loaded
on a MonoS column, and eluted with a salt gradient (Fig. 3
A, lanes 4 and 5). The MonoS fractions enriched in
XMAP310 (Fig. 3 A, lane 5) contained one major contaminant of ~49 kD (Fig. 3 B, lane 5), that was identified as
EF1-
by MALDI MS (data not shown, Cormier et al.,
1991
; Hershey, 1991
; Jensen et al., 1996
). EF1-
, was eliminated from the preparation by gel filtration through a Superose 6 column (Fig. 3 A and B, lane 6 and later fractions,
lanes 7 and 8). The purification was monitored by western
blotting with the Q4 mAb. A fraction appeared to remain
bound to MTs after Ca2+-depolymerization (Fig. 3 C, lane
IV). However, analysis of the same fractions by Coomassie
staining (Fig. 3 A, lane 3) revealed that most of the protein
was eluted from the MTs. This could be due to the presence of different proteins of the same molecular weight,
the presence of isoforms (see Fig. 3 A, lane 2; occasionally three bands were resolved) or the loss of the epitope during the purification. To resolve this issue we checked by
MALDI MS whether the protein recognized by the Q4
mAb and the purified protein were the same. The MALDI
MS analysis showed that the protein immunoprecipitated by the Q4 mAb and the protein purified on the Superose 6 column were the same (data not shown). We conclude that
the poor recognition of pure XMAP310 by the Q4 mAb
reflects the presence of isoforms (as previously reported
for XMAP230) and perhaps reduced recognition of the
epitope on the pure XMAP310.
does not rebind to
MTs (data not shown), and that the apparent dissociation
constant of XMAP310 for MTs was ~200 nM (data not shown).
), but is different from the
larger spacing reported for brain MAPs (Hirokawa et al.,
1988
; Sato-Yoshitake et al., 1989
; Chen et al., 1992
).
Fig. 4.
Electron micrographs showing cross-linked
MTs in bundles formed in
the presence of XMAP310.
After spontaneous assembly
in the presence XMAP310,
MTs were spotted onto carbon grids and visualized by
negative stain and electron
microscopy. (a) A long bundle consisting of three cross-linked MTs (b) magnification
of the arrow-outlined area in
a, note the uniform short
spacing of the MTs. Bars: (a) 200 nm; (b) 60 nm.
[View Larger Version of this Image (50K GIF file)]
;
Vasquez et al., 1994
). In these experiments the concentration of XMAP310 in the chamber varied between 50 and
150 nM depending on the batch used, and the total tubulin concentration was 14 µM. Assuming a stoichiometry <1:5,
we calculated (Andersen et al., 1994
) that MTs should be
saturated with XMAP310, even at a concentration of 50 nM (data not shown). At higher concentrations (>150
nM) catastrophes were rare (data not shown). We believe that this is caused by a stronger promotion of rescues
which would cause a MT that undergoes a catastrophe to
be rescued immediately. This idea is supported by the observation of pauses, that only occurred in the presence of
XMAP310. We verified that the activity of pure XMAP310
from Superose 6 fractions without (Fig. 3, lane 6) and with
(Fig. 3, lane 8) EF1-
was the same, indicating that EF1-
does not influence MT polymerization (data not shown).
These data show that XMAP310 stabilizes MTs by moderately decreasing the shrinkage rate and increasing the rescue frequency 5-10-fold.
Fig. 5.
MT dynamics visualized by video microscopy. MTs are
nucleated by a centrosome (center of the MT asters). (a) MTs polymerized in the absence of XMAP310. (b-d) MTs polymerized
with XMAP310. The MT indicated with an arrow experienced a
catastrophe shortly after the frame shown in b, and shrank to the
length shown in c where it was rescued and continued to grow, as
shown in d. Time since transfer to 37°C is indicated by xx:xx:xx,
which represents hours:minutes:seconds, respectively. Bar, 10 µm.
[View Larger Version of this Image (133K GIF file)]
XMAP310, A Novel Mitotic Spindle MAP
Like XMAP310, other MAPs localized to the nucleus in
interphase and to spindle MTs during mitosis have been
described previously (i.e., Izant et al., 1982; Pepper et al.,
1984
; Bonifacino, 1985
; and NuMA: Compton et al., 1991
;
Cleveland, 1995
; Merdes et al., 1996
). This could suggest
that XMAP310 is homologous to known proteins. However, by Edman degradation we obtained the following amino acid sequences for XMAP310: IVQPFGGK,
LETAY(P or V)MI and VLPINAA, and these sequences
did not show any significant sequence homology with
other known proteins. Moreover, MALDI MS (Jensen et
al., 1996
; Materials and Methods) analysis also did not reveal any homology between XMAP310 or other proteins
in the data bases. Moreover, Edman degradation of
XMAP230 gave the following sequences that did not show
homology with any proteins in the data bases: APLAKPSTALAL, LPTA(S or T)NVTNAA, T(Q or K)EAKRTNQGE, QSIVVEE, NGEPAAAPTEGDNQR, (S or A)-
VKIVE, and ATVPAE. MALDI MS of XMAP230 also
did not reveal any homologous proteins in the data bases. However, a MALDI MS analysis of XMAP215 revealed
that it has strong homology with the human protein TOGp,
as previously reported (Charrasse, S., M. Schroeder, C. Gauthier-Rouviere, L. Cassimeris, D.L. Gard and C. Larroque. 1996. Mol. Biol. Cell. 7:222a; Podtelejnikov, A., and
M. Mann, unpublished observation). Thus, XMAP310 and
XMAP230 appear to be new MAPs whereas XMAP215 is
a TOGp homologue (Charasse et al., 1995
).
Interestingly, XMAP230 and E-MAP-115 (Masson and
Kreis, 1995) are localized to spindle MTs although they do
not have maximal binding affinity for MTs during mitosis.
Preliminary results indicate that XMAP310 is a phosphoprotein and that its binding to MTs, like XMAP230 and
E-MAP-115, is reduced by phosphorylation during mitosis
(Andersen, S.S.L., unpublished observation). We previously hypothesized that MAPs may be locally less phosphorylated around chromosomes during mitosis, allowing
their specific binding to spindle MTs (Andersen et al.,
1994
). It has been shown that mitotic chromatin can regulate the phosphorylation level of the MT polymerization inhibitor Stathmin/Op18, and it seems likely that a similar
mechanism could locally regulate the binding activity/
phosphorylation of MAPs like XMAP310, XMAP230, and
E-MAP-115 to spindle MTs (Sobel, 1991
; Belmont and
Mitchison, 1996
; Andersen et al., 1997
). However, more
work will be required to determine how preferential binding of MAPs to spindle MTs is achieved during metaphase.
Effects of XMAP310 and other MAPs on MT Dynamics In Vitro
Brain MAPs and XMAP230 strongly reduce fcat and increase vg whereas XMAP310 primarily increases rescues
(Drechsel et al., 1992; Pryer et al., 1992
; Andersen et al.,
1994
; Itoh and Hotani, 1994
; Trinczek et al., 1995
). MAP4,
a nonneuronal MAP resembling MAP2 and tau at the molecular level, strongly promotes rescues without having
any effect on any of the other dynamic instability parameters (Ookata et al., 1995
). However, the experimental conditions between our study and the former are so different
that a direct comparison is difficult. Moreover, an important function of MAP4 seems to be the targeting of cdc2
kinase to MTs (Ookata et al., 1995
). XMAP215 promotes
MT turnover by strongly increasing vg at the MT plus end,
increasing vs, and decreasing fres without affecting fcat
(Gard and Kirschner, 1987
; Vasquez et al., 1994
). Comparatively, the brain MAPs together with XMAP230 are the
most potent MAP MT stablizers, followed by XMAP310,
MAP4, and then XMAP215.
The effect of XMAP310 on fres is unusual and raises interesting questions about the mechanism of MT polymerization. How could XMAP310 increase rescues and not
growth? Protofilaments tend to change from a straight
configuration during the assembly phase to a curved one
during disassembly. This change in curvature is thought to
drive MT depolymerization (Simon and Salmon, 1990; Mandelkow et al., 1991
; Chrétien et al., 1995
). We propose
that XMAP310 does not increase vg because it does not
bind along the length of tubulin dimers as brain MAPs are
thought to do (Drechsel et al., 1992
; Pryer et al., 1992
).
Based on our observations, XMAP310 rather cross-links
MTs and protofilaments. As shown in the model (Fig. 6),
XMAP310 could, by cross-linking protofilaments, reduce
the tendency of protofilaments to change to a curved conformation during MT depolymerization. Together with a
reduction in vs, this may increase the rescue frequency by
allowing new tubulin-GTP to recap depolymerizing MTs
(Fig. 6). This model fits also with the unusually high frequency of pauses observed in MTs assembled in the presence of XMAP310. The plus end of MTs with laterally cross-linked protofilaments could stay in a metastable
state, some subunits being added to the end of some
protofilaments while other subunits would be removed
from other protofilaments.
Role of MAPs in Regulation of Spindle MT Dynamics
At present the role of MAPs in spindle assembly is unclear. Some reports suggest that MAPs are redundant
(Pereira et al., 1992; Wang et al., 1996
), whereas others report that they are involved in spindle assembly, cell
growth, and differentiation (Mangan and Olmsted, 1996
;
Nguyen et al., 1997
; Saunders et al., 1997
, and discussion of
the MAP4 literature therein). On the basis of what is
known about the subcellular localization and the effect of
Xenopus MAPs on MT dynamics in vitro, it seems that
XMAP215 may produce the rapid polymer growth and
turnover observed for spindle MTs. Such a MAP would
significantly increase the search efficiency of MTs searching for a kinetochore during spindle assembly (Kirschner
and Mitchison, 1986
; Holy and Leibler, 1994
). Since XMAP310 is localized to the nucleus during interphase
and does not stably localize to spindle MTs until metaphase,
this MAP may, like XMAP215, be involved in enhancing
the searching efficiency of future kinetochore MTs. XMAP230
and XMAP310 may be involved in generating stable kinetochore MTs (XMAP230; Andersen et al., 1994
), and
other types of stable spindle MTs, such as cross-linked interpolar MTs (XMAP310; Mastronarde et al., 1993
). Thus,
the abundance of XMAP310 in brain tissue, together with
the functional in vitro data, suggest that an important in
vivo function of this protein is to cross-link and stabilize MTs.
Our immunofluorescence results show that XMAP310 is
tightly associated to metaphase MTs but released from
anaphase MTs, indicating that XMAP310 could specifically stabilize metaphase MTs and may not be required
during anaphase. The release of XMAP310 from anaphase
MTs may indicate that MT rescues are undesirable events
during anaphase, or perhaps other MAPs, like XMAP230,
become reactivated, suppressing catastrophes and therefore the occurrence of rescues.
In mitotic Xenopus egg extracts lacking chromosomes,
MTs display a high growth rate, a high catastrophe frequency and rescues and pauses are very infrequent (Belmont et al., 1990; Verde et al., 1992
; Tournebize et al., 1997
).
One could wonder why, when a protein like XMAP310 is
present in such extracts. The absence of rescues in mitotic
extracts could be explained if during mitosis XMAP310 is
inactivated globally by phosphorylation in the cytoplasm. In contrast, due to the presence of XMAP310 on spindle
MTs, the rescue frequency of spindle MTs could be high.
Therefore, it seems likely that XMAP310 and XMAP230
could be part of the machinery that ensures preferential
growth of MTs towards chromosomes during spindle assembly (Andersen et al., 1997
), XMAP310 by locally increasing fres and XMAP230 by locally reducing fcat (Andersen et al., 1994
). In contrast, XMAP215 seems to be
globally active during mitosis, since the MT growth rate
remains high and XMAP215 probably is the main regulator of vg in the extract.
It is interesting to see that different MAPs affect different parameters of MT dynamic instability in a complex system like Xenopus. When dynamic instability was discovered, one of the immediate predictions was that different molecules could affect each parameter specifically, and now this is becoming reality. In the next phase, it will be very exciting to determine precisely the function of XMAP215, XMAP230, and XMAP310 in the global and local regulation of MT dynamics during mitosis and in spindle assembly. Before this becomes feasible, these molecules have to be cloned and efficient tools developed.
Address correspondence to Dr. Søren S.L. Andersen, Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014. Tel.: (609) 258-2828. FAX: (609) 258-6175. e-mail: Andersen@EMBL-Heidelberg.DE
Received for publication 26 June 1997 and in revised form 18 August 1997.
1. Abbreviations used in this paper: fcat, catastrophe frequency; fres, rescue frequency; mAb, monoclonal antibody; MALDI MS, Matrix Assisted Laser Desorption Ionization Mass Spectroscopy; MAP, microtubule-associated protein; MT, microtubule; pAb, polyclonal antibody; vg, growth rate; vs, shrinkage rate.We are grateful to Byeong Cha, Emily Harwood, and David L. Gard for the gifts of the XMAP215 and XMAP230 pAbs and IF of Xenopus embryos. We are indebted to Thomas Kreis for the antitubulin pAb. Special thanks to Alan Sawyer who was of great help in the preparation of the mAbs. Thanks to Alexandre Podtelejnikov, Ole Jensen, and Juri Rappsilber for the MALDI MS analysis, to Keith Ashman for Edman data, and to Anja Habermann for assistance with the EM. Thanks to Rebecca Heald, and Torsten Wittmann for comments on the manuscript.
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