Localization of the Kinesin-like Protein Xklp2 to Spindle
Poles Requires a Leucine Zipper, a Microtubule-associated
Protein, and Dynein
Torsten Wittmann,* Haralabia Boleti,‡ Claude Antony,§ Eric Karsenti,* and Isabelle Vernos*
*European Molecular Biology Laboratory, Cell Biology and Cell Biophysics Programs, D-69117 Heidelberg, Germany;
‡
Institut Pasteur, 75724 Paris Cedex 15, France; and §Institut Curie, 75248 Paris Cedex 05, France
sin-like protein localized at spindle poles and required
for centrosome separation during spindle assembly in
Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of
Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49–59). Now, we have examined the
mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that
Xklp2 and GST-Xklp2-Tail localize specifically to the
minus ends of spindle pole and aster microtubules in
mitotic, but not in interphase, Xenopus egg extracts.
We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single
D
uring mitosis, chromosomes are segregated by a
complex microtubule-based structure, the mitotic
spindle. Current models of spindle assembly invoke the action of a variety of microtubule motors exerting forces on microtubules (reviewed in Barton and Goldstein, 1996; Hyman and Karsenti, 1996; Merdes and
Cleveland, 1997; Vernos and Karsenti, 1996; Walczak and
Mitchison, 1996). To understand how motors participate
in spindle morphogenesis, we need to examine their function at different levels. One level concerns the mechanisms
of motor localization through specific interactions between the variable nonmotor domains and their cargoes.
For example, Nod, a kinesin-like protein (KLP)1 associated with chromatin in Drosophila meiotic spindles, binds
directly to DNA through a sequence homologous to the
point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two
additional factors in mitotic egg extracts are required
for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated
protein that we named TPX2 (Targeting protein for
Xklp2) that mediates the binding of GST-Xklp2-Tail to
microtubules and (b) the dynein–dynactin complex that
is required for the accumulation of GST-Xklp2-Tail at
microtubule minus ends. We propose two molecular
mechanisms that could account for the localization of
Xklp2 to microtubule minus ends.
Key words: kinesin-like protein • microtubule-associated protein • spindle • microtubule • dynein
1. Abbreviations used in this paper: GST, glutathione-S-transferase; KLP,
kinesin-like protein; MAP, microtubule-associated protein; Tm, melting
temperature; TPX2, Targeting protein for Xklp2.
DNA-binding domain of HMG 14/17 (Afshar et al., 1995a,
b). A second level deals with the actual motor function in
relation to the activity of other plus and minus end–
directed motors. For example, the BimC family KLPs,
Kip1 and Cin8, counteract the activity of the minus end–
directed COOH-terminal motor Kar3 in yeast (Hoyt et al.,
1992; Saunders and Hoyt, 1992) and opposing motor activities have been implicated in the formation of mammalian
spindle poles (Gaglio et al., 1996). A third level concerns
the regulation of the activity and localization of motors in
time and space by posttranslational modifications. For example, phosphorylation at specific cdc2 sites is required
for the localization of human and Xenopus Eg5, a member
of the BimC family, to spindle microtubules (Blangy et al.,
1995; Sawin and Mitchison, 1995) and for the redistribution of the human KLP, CENP-E, from kinetochores to
the spindle midzone during anaphase (Liao et al., 1994).
We recently reported that a Xenopus KLP, Xklp2, localizes to centrosomes and participates in their separation
during mitosis (Boleti et al., 1996). A similar function has
been proposed for motors of the BimC family (reviewed in
Karsenti et al., 1996; Walczak and Mitchison, 1996; Kashina et al., 1997). Motors of the BimC family form bipolar
The Rockefeller University Press, 0021-9525/98/11/673/13 $2.00
The Journal of Cell Biology, Volume 143, Number 3, November 2, 1998 673–685
http://www.jcb.org
673
Address all correspondence to Isabelle Vernos, EMBL Heidelberg, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Tel.: 49 6221 387 324. Fax:
49 6221 387 306. E-mail: vernos@embl-heidelberg.de
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
Abstract. Xklp2 is a plus end–directed Xenopus kine-
Materials and Methods
Xenopus Egg Extracts
CSF-arrested extracts (mitotic extracts) were prepared according to Murray (1991). They were released to interphase by addition of 0.5 mM CaCl2
and 200 mg/ml cycloheximide and subsequent incubation at 208C for 45–60
min. High speed extracts were centrifuged for 60 min at 150,000 g at 48C.
Recombinant Proteins
egg extract containing 0.2 mg/ml rhodamine-labeled tubulin (Hyman et al.,
1991). Asters were assembled either by addition of human centrosomes
purified from KE37 lymphoid cells as described (Bornens et al., 1987;
Domínguez et al., 1994), 5% DMSO or 1 mM taxol (paclitaxel; Molecular
Probes, Eugene, OR). The reactions were incubated for 30–60 min at
208C, diluted with 1 ml BRB80 (80 mM K-PIPES, pH 6.8, 1 mM EGTA,
and 1 mM MgCl2) containing 10% glycerol, 0.25% glutaraldehyde, 1 mM
GTP, and 0.1% Triton X-100 and subsequently centrifuged (HB4 rotor,
12,000 rpm, 12 min, 168C) through a 25% glycerol cushion in BRB80 onto
coverslips as described (Sawin and Mitchison, 1991). The coverslips were
fixed in 2208C methanol, incubated twice for 10 min in 0.1% NaBH4 in
PBS and processed for immunofluorescence with the anti-GST-antibody.
For immunolocalization with the anti-Xklp2-Tail antibody the dilution
buffer did not contain glutaraldehyde but 5 mM taxol were included. All
pictures were taken with Zeiss or Leica confocal microscopes. For biochemical analysis, asters were assembled in 150,000 g mitotic egg extracts
with 1 mM taxol for 30 min at 208C. The reactions were diluted 1:10 in
BRB80 containing 5 mM EGTA, 0.1% Triton X-100, 1 mM DTT, protease inhibitors and 10 mg/ml cytochalasin D and spun through a 25%
glycerol cushion in BRB80 (TLS-55 rotor, 20,000 rpm, 15 min, 208C). The
supernatant and pellet fractions were solubilized in SDS-PAGE sample
buffer and analyzed by Western blotting.
Electron Microscopy
Asters were assembled around human centrosomes, fixed with 1 ml
BRB80 containing 0.25% glutaraldehyde, 1 mM GTP, and 0.1% Triton
X-100, and spun onto polylysine-coated coverslips as for immunofluorescence. The coverslips were fixed for 10 min in the same buffer, rinsed in
PBS, quenched for 30 min in 0.12% glycine in PBS and blocked for 15 min
in 5% FCS in PBS. The coverslips were then incubated with the anti-GST
antibody (2.5 mg/ml) in PBS, 5% FCS, 0.1% Triton X-100 for 45 min,
washed with PBS, 1% FCS and stained with 10 nm protein A–gold (from
J.W. Slot, Utrecht University, Utrecht, The Netherlands). After washing
extensively with PBS, 1% FCS and once with PBS the samples were postfixed in 2% glutaraldehyde in PBS for 15 min, washed with 100 mM phosphate buffer, pH 7.2, and incubated for 15 min in 1.5% glutaraldehyde,
0.4% tannic acid in phosphate buffer. The samples were rinsed in phosphate buffer, treated with 0.5% OsO4 in phosphate buffer for 10 min on
ice, washed with water, stained with 1% uranyl acetate for 2 h at 48C, dehydrated and flat embedded in Epon. After sectioning, the samples were
contrasted with uranyl acetate and lead citrate and observed in a Philips
CM120 electron microscope at 80 kV.
Circular Dichroism Spectroscopy
The truncated Xklp2-Tail fragments were produced by PCR introducing
BamHI and EcoRI restriction sites at their 59- and 39-ends, respectively,
and cloned into a modified pGEX-2T vector (Pharmacia Biotech Sverige,
Uppsala, Sweden). The construct GST-LtoK carrying a point mutation at
amino acid 1370 was produced by overlap extension PCR with primers
changing the codon CTG to AAG. All constructs were sequenced and did
not contain mutations altering the amino acid sequence. The GST-fusion
proteins were overexpressed in E. coli and purified by glutathione affinity
chromatography using standard protocols. Subsequently the proteins were
dialyzed against CSF-XB (10 mM K-Hepes, pH 7.7, 50 mM sucrose, 100
mM KCl, 2 mM MgCl2, 0.1 mM CaCl2, and 5 mM EGTA), frozen in liquid
nitrogen and stored at 2808C.
GST-fusion proteins (z2 mg) were dialyzed against 150 mM Tris-HCl, pH
7.8, 150 mM NaCl, 5 mM MgCl2, 2.5 mM CaCl2, and 1 mM DTT and incubated with 10 units (3.2 mg) of thrombin (Sigma Chemical Co.) overnight
at 48C. The thrombin was removed by addition of 100 ml p-aminobenzamidine agarose (Sigma Chemical Co.) for 30 min on ice. Uncleaved protein
and GST were removed by two subsequent incubations with 250 ml glutathione agarose for 30 min on ice. The proteins were dialyzed against 20 mM
potassium phosphate pH 7.5, 20 mM KCl, 0.1 mM DTT, frozen in liquid
nitrogen and stored at 2808C. The concentration of the cleaved proteins
was determined with the BCA assay (Pierce Chemical Co., Rockford, IL).
CD-spectra were recorded on a Jasco J-710 spectropolarimeter using cuvettes with 0.2- and 1-cm path length. Thermal unfolding was performed
at a heating rate of 50 K/h and recorded at 0.2 K steps.
Antibodies
Glutaraldehyde Cross-linking
The anti-GST antibody was affinity purified against GST from a rabbit serum immunized with an unrelated GST-fusion protein. The anti-Xklp2Tail antibody was an affinity-purified rabbit serum (Boleti et al., 1996)
raised either against MBP- or GST-Xklp2-Tail fusion proteins. The anticentrosome antibody was a human autoimmune serum strongly recognizing centrosomes in mammalian cells (Domínguez et al., 1994). The monoclonal m70.1 anti-dynein intermediate chain antibody was from Sigma
Chemical Co. (St. Louis, MO). Fluorescent- and horseradish peroxidase–
labeled antibodies were from Jackson ImmunoResearch Laboratories,
Inc. (West Grove, PA).
2 mM Xklp2-Tail were incubated with varying concentrations of glutaraldehyde (Grade I; Sigma Chemical Co.) in 20 mM potassium phosphate
pH 7.5, 20 mM KCl, 1 mM DTT for 30 min at 208C. The reactions were
stopped by the addition of one-half volume of 2 M glycine, mixed with
SDS-PAGE sample buffer, and run on a 10% SDS-PAGE.
Hydrodynamic Analysis
Recombinant GST-Xklp2-Tail fusion proteins were added to 20 ml mitotic
Mitotic or interphase egg extracts were centrifuged at 280,000 g for 20 min
at 48C. The Stokes radius of Xklp2 was determined by gel filtration chromatography of a 50-ml sample on a SMART Superose 6 column (Pharmacia Biotech Sverige) in CSF-XB. The sedimentation coefficient was estimated by sucrose gradient centrifugation. 100–200-ml samples were
loaded on 4 ml 5–27% sucrose gradients in CSF-XB and spun for 16 h at
The Journal of Cell Biology, Volume 143, 1998
674
Localization Assay
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
tetramers suggesting that they may act by sliding antiparallel microtubules against each other (Kashina et al.,
1996). Xklp2 was proposed to function in a different way.
Motors tethered to one centrosome could move towards
the plus end of microtubules emanating from the other,
leading to their separation (Boleti et al., 1996; Karsenti et al.,
1996). To better understand the role of Xklp2 in spindle
pole separation we have examined in more detail the
structural organization of Xklp2 and its mechanism of localization. We had previously reported (Boleti et al., 1996)
that a GST-fusion protein containing the COOH-terminal
domain of Xklp2 (amino acids 1137–1387; GST-Xklp2Tail) was sufficient for its localization to spindle poles.
Longer fragments including the tail showed the same localization, whereas the stalk domain alone (amino acids
363–1137) did not localize. Furthermore, only fusion proteins containing the tail and thus localizing to spindle poles
had a dominant negative effect on spindle assembly pointing to the importance of this localization in Xklp2 function. Therefore, to understand how Xklp2 functions in
centrosome separation, we have used GST-Xklp2-Tail to
examine how Xklp2 is localized to spindle poles.
We now report that Xklp2 is a homodimer that localizes
to the minus ends of microtubules rather than directly to
centrosomes. This localization is cell cycle dependent, requires a COOH-terminal leucine zipper found in Xklp2, a
novel microtubule-associated protein (MAP), and the activity of the dynein–dynactin complex.
48C at 27,000 rpm in a SW60 rotor. The elution profile of Xklp2 in both
cases was determined by Western blotting of the fractions and probing
with the anti-Xklp2-Tail antibody and compared with a variety of standard proteins of known Stokes radius and sedimentation coefficient, respectively. The standard proteins included rabbit muscle myosin (17.6 nm,
6.4 S), thyroglobulin (tetramer: 10.7 nm, 15 S, dimer: 6.7 nm, 12 S), g-globulin (5.4 nm, 7.0 S), b-amylase (4.1 nm, 8.9 S), ovalbumin (2.8 nm, 3.5 S),
catalase (11.3 S), aldolase (7.35 S), and BSA (4.6 S). The calculations were
performed according to (Wilhelm et al., 1997).
Purification of TPX2
Results
Cell Cycle–dependent Targeting of the
COOH-terminal Domain of Xklp2 to Microtubule
Minus Ends in Xenopus Egg Extracts
Figure 1. Localization of GST-Xklp2-Tail is cell cycle dependent.
(A) Asters were assembled around human centrosomes in 10,000 g
mitotic egg extract (a–c), the same extract released into interphase by the addition of calcium and cycloheximide (d–f), and cycled back to mitosis by addition of cyclin D90 (g–i). The asters
were assembled in the presence of rhodaminated tubulin (a, d,
and g), and stained with a human anti-centrosome antibody (b, e,
and h), and an anti-GST antibody (c, f, and i) that were revealed
with FITC- and Cy5-conjugated secondary antibodies, respectively. GST-Xklp2-Tail accumulates at the center of mitotic asters (a–c). This localization is abolished during interphase (d–f)
and reappears after reentry into mitosis (g–i). (B) Asters assembled in the presence of 5% DMSO in 150,000 g mitotic egg extract (a and b), and microtubules in the same extract released to
interphase before addition of DMSO (c and d). (a and c) Microtubules, (b and d) anti-GST-staining. GST-Xklp2-Tail is strongly
concentrated at the center of mitotic DMSO asters (b) and
stained diffusely along microtubules in interphase (d). All reactions contained 2 mM GST-Xklp2-Tail. Bars, 10 mm.
We previously reported that Xklp2 is localized to centrosomes with a faint staining in interphase and a fairly
strong staining at spindle poles. This suggested that there
was a recruitment of Xklp2 at centrosomes (or spindle
poles) during mitosis. To determine more precisely the localization of Xklp2 and its cell cycle dependence, we studied the targeting properties of a GST-fusion protein containing the COOH-terminal 250 amino acids of Xklp2
(GST-Xklp2-Tail) that localized to the poles of spindles
formed around sperm nuclei in Xenopus egg extracts (Boleti et al., 1996). A simple localization assay was designed
using microtubule asters formed in egg extracts by the addition of purified centrosomes. In extracts prepared from
eggs naturally arrested in second metaphase of meiosis,
the GST-Xklp2-Tail protein accumulated in the center of
the asters around the centrosomes (Figs. 1 A, a–c and 2, A
and G). When the extract was released into interphase by
addition of calcium, the microtubules formed an extended
network and we did not observe any accumulation of the
fusion protein anywhere in the sample and in particular,
there was no enrichment of GST-Xklp2-Tail around centrosomes (Fig. 1 A, d–f). However, we observed a reproducible but very faint granular staining along a subset of
Wittmann et al. Targeting of Xklp2 to Microtubule Minus Ends
675
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
CSF-arrested egg extract was diluted with two volumes of motor buffer
(100 mM K-PIPES, pH 7.0, 0.5 mM EGTA, 2.5 mM magnesium acetate,
and 1 mM DTT) containing 10 mg/ml pepstatin, leupeptin, aprotinin, and
1 mM PMSF, and then centrifuged for 90 min at 180,000 g at 48C. The cytoplasmic layer was collected and supplemented with 0.6 mg/ml bovine
brain tubulin (Ashford et al., 1998) and 20 mM taxol and microtubules
were polymerized at room temperature for 30 min. The microtubules were
then centrifuged through a 15% sucrose cushion in motor buffer (30,000 g,
20 min, 228C). The supernatant was discarded, the cushion once washed
with water, removed and the microtubule pellet resuspended in 1/3 of the
original volume in motor buffer, and then centrifuged through a sucrose
cushion again. MAPs were eluted in 100 mM steps of NaCl in motor
buffer for 15 min at room temperature. Between the elution steps microtubules were recovered by centrifugation (30,000 g, 15 min, 228C). TPX2
was enriched in the fraction eluted with 300 mM NaCl. This fraction was
diluted to reduce the salt concentration and applied onto a PC 1.6/5 Mono
S column (SMART system; Pharmacia Biotech Sverige). The column was
eluted with a 1-ml linear gradient of 100–500 mM KCl in 20 mM K-PIPES,
pH 7.0, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.01% Tween-20 at 48C
at 25 ml/min and 50-ml fractions were collected. TPX2 eluted at z350 mM
KCl. The peak fraction from the Mono S chromatography was applied to
a PC 3.2/30 Superdex 200 gel filtration column (SMART system; Pharmacia Biotech Sverige) equilibrated with 20 mM K-Hepes, pH 7.0, 300 mM
KCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, and 0.01% Tween-20 at
48C. The column was eluted with the same buffer at a flow rate of 20 ml/
min and 40-ml fractions were collected. The column fractions were assayed
in the following way: 4 mg/ml cycled bovine brain tubulin was polymerized in BRB80 containing 5 mM MgCl2, 33% glycerol and 1 mM GTP at
378C for 30 min. The microtubules were stabilized by the addition of 20 mM
taxol. 5 ml MAPs or column fractions were mixed with 10 ml BRB80 containing 1 mM DTT, 5 mM taxol, 0.1% Triton X-100 and 1 mM GST-Xklp2Tail. 5 ml of the prepolymerized microtubules were added and the reactions incubated at 208C for 30 min. The reactions were then diluted 1:5
with BRB80 containing 1 mM DTT, 5 mM taxol, and 0.1% Triton X-100,
and then centrifuged through a 10% sucrose cushion in BRB80, 5 mM
taxol at 200,000 g at 208C for 15 min. The cushion was washed once with
water and the microtubule pellet solubilized in SDS-PAGE sample buffer
and analyzed by Western blotting with the anti-GST antibody.
The Journal of Cell Biology, Volume 143, 1998
Figure 2. Both endogenous Xklp2 and GST-Xklp2-Tail localize
to microtubule minus ends in mitotic asters in the absence of centrosomes. (A–C) Localization of GST-Xklp2-Tail. Microtubules
are red, anti-GST-staining is green. (A) Aster nucleated by human centrosomes, (B) Aster assembled in the presence of 5%
DMSO, (C) Spindle assembled around chromatin beads (Heald
et al., 1996). Although there are no centrosomes present in B and
C, GST-Xklp2-Tail accumulates at the center of the asters or the
spindle poles, respectively. (D–F) The COOH-terminal domain
of Xklp2 is necessary and sufficient for localization. (D) Coomassie-stained SDS-PAGE of GST-Xklp2-Stalk (amino acids
363–1137, S) and GST-Xklp2-Tail (amino acids 1137–1387, T).
GST-Xklp2-Stalk does not localize to the center of taxol-induced
mitotic asters (E) whereas GST-Xklp2-Tail does in a parallel experiment (F). (G and H) Depolymerization of microtubules eliminates GST-Xklp2-Tail staining around centrosomes. Centrosomes are red, anti-GST-staining is green, microtubules are
not shown. (G) Aster nucleated by human centrosomes in the
presence of 2 mM GST-Xklp2-Tail and stained with the human
anti-centrosome antibody, (H) Asters assembled as in G, but
treated with 5 mM nocodazole and incubated for 5 min on ice before fixation. (J) Western blot of 0.5 ml egg extract probed with
an affinity-purified anti-Xklp2-Tail antibody. The antibody
strongly recognizes a band of 160 kD. (K) Localization of the endogenous Xklp2. Microtubules are red, anti-Xklp2-staining is
green. Asters were assembled with 5% DMSO in the presence of
FITC-labeled tubulin and stained with the anti-Xklp2-Tail antibody (z5 mg/ml) and a Cy3-conjugated secondary antibody. The
anti-Xklp2-Tail antibody stained the center of the DMSO-asters.
Bars, 10 mm.
676
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
microtubules. This binding to microtubules was more obvious in high speed interphase extracts where microtubule
polymerization was induced by addition of 5% DMSO.
Under these conditions microtubules formed a disorganized network but did not form asters and the GST-Xklp2Tail appeared to be spread along microtubules (Fig. 1 B,
d). When the interphase extract was driven back into mitosis by addition of cyclin D90 (Glotzer et al., 1991), the
GST-Xklp2-Tail was again observed strongly localized at
the center of centrosome-nucleated asters (Fig. 1 A, g–i).
These results indicated that the COOH-terminal domain
of Xklp2 was sufficient to target Xklp2 to the center of microtubule asters specifically during mitosis. However, the
apparent binding along microtubules in interphase extracts and the lack of association of GST-Xklp2-Tail with
centrosomes in interphase raised the question of whether
it was really interacting with centrosomes as indicated by
our previous immunofluorescence results (Boleti et al.,
1996).
To determine whether centrosomes were required for
localization of the GST-Xklp2-Tail, asters and spindles were
assembled in mitotic extracts in the absence of centrosomes (Stearns and Kirschner, 1994; Verde et al., 1991).
The fusion protein localized efficiently to the center of asters assembled in the presence of 5% DMSO or 1 mM
taxol in low speed (Fig. 2 B, F) or high speed mitotic extracts (Fig. 1 B, b) and to the poles of spindles assembled
around DNA coated beads (Fig. 2 C; Heald et al., 1996). A
fusion protein containing amino acids 363–1137, GSTXklp2-Stalk, did not localize to the center of taxol-induced
mitotic asters (Fig. 2 E) confirming that all the targeting
information is contained in the GST-Xklp2-Tail construct.
When the microtubules were depolymerized with nocodazole in centrosome-nucleated asters, the anti-GST-staining
around the centrosomes disappeared completely (Fig. 2
G, H).
To test whether the endogenous Xklp2 was also recruited to the minus ends of microtubules in the absence
of centrosomes, we performed immunofluorescence with
an affinity-purified antibody raised against GST-Xklp2Tail. This antibody strongly recognized Xklp2 on a Western blot of total egg extract (Fig. 2 J) and stained the center of asters assembled in the presence of 5% DMSO (Fig.
2 K). This result confirmed that Xklp2 is localized to the
minus ends of microtubules rather than to the centrosomes
themselves.
Immunogold electron microscopy of centrosomal asters
assembled in mitotic egg extracts in the presence of the
GST-Xklp2-Tail fusion protein also supported this idea.
Staining with the anti-GST-antibody and protein A–gold
revealed a strong accumulation of the protein on electron
dense material along the minus ends of microtubules (Fig.
3 A), extending up to 1–2 mm away from the center of the
aster. Gold particles were observed at a distance of up to
20–30 nm around the microtubules (note cross-sectioned
microtubules, arrowheads in Fig. 3 B). Very few gold particles were found along microtubules further away from
the center of the aster and the transition between the two
areas was very sharp. In agreement with the immunofluorescence data presented above, no gold particles were
found on the centrioles nor on the pericentriolar material
itself (Fig. 3 B). No gold particles were found along mi-
crotubules in negative control samples incubated with
protein A–gold alone or with a mutant fusion protein
that does not accumulate in the center of the asters
(GST-Xklp2-CDel2, see below). These results indicate
that Xklp2 is targeted to the minus ends of microtubules
in a mitotic extract via its COOH-terminal domain. In interphase, the COOH-terminal domain of Xklp2 seems to
bind weakly along the whole length of microtubules.
To confirm these results we compared the behavior of
the COOH-terminal domain with that of the full-length
endogenous Xklp2 protein using a biochemical analysis.
Microtubules were assembled in the presence of 1 mM
taxol in high speed mitotic and interphase egg extracts
and sedimented by centrifugation through a glycerol
cushion. A Coomassie-stained gel of the microtubule
pellets showed that they contained mainly tubulin and a
range of associated proteins (Fig. 4). Western blot analysis showed that z5% of the total amount of Xklp2 (Fig.
4 A, lane 2) was recovered in the mitotic microtubule
pellet whereas a minor amount was detected in the interphase microtubule pellet and none at all in the absence
of taxol (Fig. 4 A). Our previous biochemical analysis
(Boleti et al., 1996) indicated that Xklp2 was released
from microtubules by ATP. Since the assay was done in
the presence of ATP, it reflected binding of Xklp2 to microtubules that was not due to its motor domain. The
relatively low amount of Xklp2 that associates with microtubules under these conditions might be due to a low
affinity of the ATP-independent binding site of Xklp2
for microtubules. This may lead to the dissociation of a
significant amount of Xklp2 from microtubules during
dilution of the extract and centrifugation of the microtubules through the cushion. When the GST-Xklp2-Tail
fusion protein was present in the reactions at a concentration of 0.3 mM it behaved similarly to Xklp2 although
it appeared to associate somewhat better with microtubules since z10% of the fusion protein pelleted with mitotic microtubules (Fig. 4 B). Interestingly, under these
conditions the amount of endogenous Xklp2 associated
with the microtubule pellet was reduced indicating that
the GST-Xklp2-Tail competed with the binding of the
endogenous protein (Fig. 4 B, lane 2). The GST-Xklp2Tail also copelleted more efficiently than Xklp2 with interphase microtubules. The increased binding of GSTXklp2-Tail to microtubules might be due to the fact that
the construct was added in a slight molar excess (three- to
Wittmann et al. Targeting of Xklp2 to Microtubule Minus Ends
677
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
Figure 3. Localization of GST-Xklp2-Tail by
immunoelectron microscopy. (A) Aster nucleated by purified human centrosomes in mitotic 10,000 g egg extract in the presence of 2
mM GST-Xklp2-Tail, stained with an antiGST-antibody. The centrosomes are not visible in this section. (B) Centrosomes in the
center of mitotic asters from the same sample.
Gold particles were observed along microtubule minus ends but not on centrosomes. Arrowheads point to microtubules in cross-section, revealing the distance between gold
particles and the microtubules. We did not
observe any labeling in negative controls
when the primary antibody was omitted or
when the mutant fusion protein missing the
leucine zipper (GST-Xklp2-CDel2) was
added to the reaction instead of the tail (data
not shown). Bars, 200 nm.
The Journal of Cell Biology, Volume 143, 1998
678
Figure 4. Biochemical analysis of Xklp2 cosedimentation with
microtubules. Taxol (1 mM) and the m70.1 antibody (1–2 mg/ml)
were added as indicated to 150,000 g egg extracts and the reactions incubated for 30 min at 208C. M, mitotic extract, I, interphase extract. Microtubule pellets (corresponding to 5 ml of extract) and soluble fractions (corresponding to 0.25 ml of extract)
were analyzed on Coomassie-stained gels and Western blots
probed for the proteins indicated (DIC, dynein intermediate
chain). (A) Sedimentation of endogenous Xklp2. (B) Sedimentation when either GST-Xklp2-Tail, T, or GST-Xklp2-LtoK, K,
were added to the extract at 0.3 mM. Xklp2 as well as the GSTXklp2-Tail sediment with microtubules in a mitotic cytoplasm.
GST-Xklp2-Tail (52 kD) runs very close to the tubulin doublet.
This is why the band on the Western blot of the microtubule pellet appears broadened. Also the dynein intermediate chain is enriched in mitotic microtubule pellets. When the m70.1 antibody is
added the dynein intermediate chain is not associated with microtubules anymore and the binding of Xklp2 and GST-Xklp2-Tail
to microtubules is reduced. The molecular mass of marker proteins is indicated on the right.
fivefold) over the endogenous protein. We conclude from
these results that the GST-Xklp2-Tail has a similar behavior as the endogenous Xklp2 and is actively targeted to microtubule minus ends specifically during mitosis.
Targeting of Xklp2-Tail Requires Dimerization and a
Leucine Zipper Domain
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
To understand how the biophysical properties of Xklp2
could contribute to its localization and function, we first
examined its quaternary structure. We determined its native molecular mass and hydrodynamic properties using
gel filtration and sucrose gradient centrifugation. In both
cases, Xklp2 behaved as a homogenous species. The results were identical for interphase and mitotic extracts
within the experimental error. Therefore, we combined
them to determine average values for the Stokes radius
(10.4 6 0.5 nm) and the sedimentation coefficient (8.1 6
0.6 S). From these numbers we calculated a native molecu-
lar mass of 330 6 40 kD, which is consistent with Xklp2
being an homodimer (the calculated molecular mass for an
Xklp2 homodimer is 320 kD). In addition, these data indicated that Xklp2 is a highly asymmetric molecule with an
approximate axial ratio of 20, as one would expect for a
rod-shaped protein containing a fairly long (z1,000 amino
acids) coiled-coil domain.
To examine whether a leucine zipper that we had previously identified at the COOH-terminal end of Xklp2 (Boleti et al., 1996) was important for localization, we prepared a series of GST-fusion proteins which truncated the
tail domain either at the NH2- or at the COOH-terminal
ends (Fig. 5, A and B). Asters were assembled in mitotic
egg extracts in the presence of 2 mM of the corresponding
fusion protein and their localization was determined by
immunofluorescence with an anti-GST antibody. The
same results were obtained in the three conditions tested:
on asters nucleated by human centrosomes in low speed
extracts, on DMSO-asters generated in high speed extracts
(Fig. 5 C) and on half spindles assembled around Xenopus
sperm nuclei (data not shown). With the NH2-terminal deletion series we observed a gradual decrease in localization efficiency as estimated from the fluorescence intensity
of the anti-GST staining: GST-NDel1 was localized with
z40% efficiency compared with GST-Xklp2-Tail whereas
GST-NDel2 was not visible on centrosomal asters and
weakly on DMSO-asters (probably reflecting the higher
microtubule density in this later case). GST-NDel3, which
contains only the leucine zipper, did not show any localization at all. The first deletion from the COOH-terminal
end, GST-CDel1, missing the last five amino acids (not involved in coiled-coil interactions) localized as efficiently as
GST-Xklp2-Tail. GST-CDel2 that lacks the entire leucine
zipper (33 amino acids) was not targeted at all. To determine whether the leucine zipper structure was required for
the localization, we introduced a charged amino acid into
its hydrophobic core. In this mutant, GST-LtoK, the leucine 1370 was changed to a lysine. This protein was not localized in any of the three conditions tested. In addition,
GST-LtoK did not cosediment significantly with mitotic
microtubules and did not compete with the binding of the
endogenous Xklp2 (Fig. 4 B, lane 4). These results showed
that the leucine zipper is essential but not sufficient for localization to the center of mitotic asters and that some regions NH2-terminal to it were also required.
Since the NH2-terminal region of the Xklp2-Tail was
predicted to have the highest coiled-coil forming potential
(Fig. 5 A) apart from the leucine zipper itself, we examined whether dimerization was important for localization.
We first determined the oligomeric state of the Xklp2-Tail
by chemical cross-linking. This technique had been used
previously to characterize coiled-coil interactions (Frère et
al., 1995; Harborth et al., 1995). Xklp2-Tail from which the
GST had been removed by thrombin treatment was incubated with increasing concentrations of glutaraldehyde
and analyzed by SDS–gel electrophoresis (Fig. 6 A). At
the highest glutaraldehyde concentration the 32-kD band
corresponding to the monomer disappeared completely
and was replaced by a single other band of z60 kD. This
strongly suggests that Xklp2-Tail mainly exists as a dimer
in solution. The lack of reaction products of higher apparent molecular mass suggested that the cross-linking was
Targeting of Xklp2-Tail to
Microtubule Minus Ends Requires a Functional
Dynein–Dynactin Complex
specific. Cross-linking with formaldehyde was less efficient
but yielded similar results (data not shown).
We then determined the respective role of the NH2-terminal region and the leucine zipper in stable dimerization.
Far UV circular dichroism spectroscopy showed that
Xklp2-Tail as well as NDel2 and CDel2 are mainly a-helical at a concentration of 2 mM (Fig. 6 B). The helical content was estimated to be z80% according to the method
described by Zhong and Johnson (1992). Furthermore, the
ratio [u]222/[u]208 was close to 1.0 for all three fragments
suggesting a coiled-coil conformation (Lau et al., 1984).
To examine the relative stability of the different fragments, we monitored the thermal unfolding at 222 nm
(Fig. 6 C). At a concentration of 10 mM, Xklp2-Tail had a
Next, we wanted to understand how Xklp2 could be localized to microtubule minus ends. This was not due to its
own motor activity since the GST-Xklp2-Tail has no motor domain and, furthermore, Xklp2 is a plus end–directed
motor. NuMA, a protein associated with mitotic spindle
poles, has been shown to form a complex with cytoplasmic
dynein and dynactin and to be recruited to the spindle
poles in a dynein dependent way (Gaglio et al., 1996; Merdes et al., 1996). We therefore tested whether dynein could
also transport Xklp2 towards microtubule minus ends. We
used the monoclonal antibody m70.1 directed against the
dynein intermediate chain to inhibit dynein function in egg
extracts (Steuer et al., 1990; Heald et al., 1996; Gaglio et al.,
1997).
Using the microtubule aster sedimentation assay described above, we determined by Western blot analysis
that the dynein intermediate chain was enriched in the microtubule pellets from mitotic extract (Fig. 4 A, lane 2, and
B, lanes 2 and 4). Addition of the m70.1 antibody to the
extract reduced the level of dynein intermediate chain in
the microtubule pellet to a nondetectable level (Fig. 4 A,
lane 4, and B, lane 5), supporting the idea that this antibody disrupts the interaction of the dynein intermediate
and heavy chains with spindle microtubules (Heald et al.,
1997). Under these conditions, almost no endogenous
Xklp2 was found in the microtubule pellet (Fig. 4 A, lane
4) whereas the binding of the GST-Xklp2-Tail was only
slightly reduced (Fig. 4 B, lane 5). Again, this difference
might be due to a difference in affinity for microtubules
between the full-length protein and the tail construct or to
the slight molar excess of GST-Xklp2-Tail added to the
extract. In any case, these results indicated that inhibition
of the interaction between dynein and microtubules af-
Wittmann et al. Targeting of Xklp2 to Microtubule Minus Ends
679
Figure 5. Localization of the truncated GST-Xklp2-Tail fusion
proteins to mitotic asters. (A) Coiled-coil prediction for the
Xklp2-Tail obtained with the Coils- (solid line; Lupas et al., 1991)
and the Paircoil-algorithm (dashed line; Berger et al., 1995), and
schematic representation of the different constructs. Vertical bars
represent the four leucines present in the leucine zipper. (B)
Coomassie Brilliant blue–stained SDS-gel of the purified GSTfusion proteins and the thrombin-cleaved proteins used for circular dichroism spectroscopy. The molecular mass of marker proteins is indicated on the left. (C) Localization of the GST-fusion
proteins to asters nucleated by human centrosomes in 10,000 g
egg extract and to DMSO-asters in 150,000 g egg extract. The
fusion proteins were added at a concentration of 2 mM and are indicated on the left. Both the NH2-terminal region and the
COOH-terminal leucine zipper are required for localization of
the GST-Xklp2-Tail to the center of mitotic asters. Microtubules
are red, anti-GST-staining is green. Bar, 10 mm.
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
melting temperature (Tm) of z408C. Deletion of the leucine zipper, CDel2, did not affect the stability of the protein severely, whereas deletions of the NH2-terminal
coiled-coil domain, NDel1 and NDel2, clearly lowered Tm.
Therefore, the NH2-terminal sequences are essential for
dimer stability. This was further confirmed by looking at
the concentration dependence of Tm for the different constructs. A 10-fold dilution of Xklp2-Tail or of CDel2 did
not affect their Tm significantly, indicating a very strong
dimerization, whereas a 10-fold dilution of NDel2 significantly lowered its Tm indicating a weakened dimerization
(Fig. 6 D). The presence of GST had no effect on the stability of the dimeric Xklp2-Tail moiety (Fig. 6 E). The
melting curves of GST-Xklp2-Tail and Xklp2-Tail were
overlapping in the region where the melting of Xklp2Tail occurred. The GST-moiety denatured irreversibly at
higher temperature (above 508C). The same was observed
for other fusion proteins tested (data not shown) indicating that the results obtained by circular dichroism reflected the differential localization of the fusion proteins
in the extract.
Taken together these data show that both dimerization
of Xklp2 and the COOH-terminal leucine zipper are essential for its targeting to microtubule minus ends.
fected the binding of Xklp2 to microtubules in some way
but did not address whether it affected its targeting to microtubule minus ends.
To examine the effect of dynein inhibition on the localization of GST-Xklp2-Tail on microtubule asters, centrosome-nucleated asters were assembled in the presence
of the m70.1 antibody. Although some microtubules appeared to be bundled and detached from the centrosomes,
asters could still be detected reasonably well. Under these
conditions, the localization of GST-Xklp2-Tail to the center of the asters was severely reduced, but a very weak
staining was observed along microtubule bundles (Fig. 7
A, b). The m70.1 antibody blocked more dramatically the
formation of asters in mitotic extracts treated with 5%
DMSO or taxol as predicted by previous studies on dynein
dependence of aster formation in the absence of centrosomes (Verde et al., 1991). Again, the GST-Xklp2-Tail
was found spread along microtubules (Fig. 7 A, d) indicating that dynein function was required for the accumulation
of Xklp2 to microtubule minus ends in mitotic asters. It
also showed that when dynein function was blocked in the
Figure 7. Cytoplasmic dynein and the dynactin complex are required for localization of GST-Xklp2-Tail. (A) Human centrosomes were added to 10,000 g mitotic egg extract and incubated at 208C. After 10 min, 1–2 mg/ml m70.1 antibody was
added to b, after another 5 min 2 mM GST-Xklp2-Tail was added
and the reaction incubated for 45 min. (d) 1–2 mg/ml m70.1 was
added to 150,000 g egg extract and incubated for 5 min on ice. Microtubule polymerization was then induced by addition of 5%
DMSO and incubation for 30 min at 208C. In both cases addition
of m70.1 disrupts the localization of GST-Xklp2-Tail to the center of mitotic asters. (B) Disruption of the dynactin complex inhibits localization of GST-Xklp2-Tail. (b) Asters nucleated by
human centrosomes in the presence of partially purified bacterially expressed p50/dynamitin (z1 mg/ml) in 10,000 g mitotic egg
extract for 60 min at 208C. (d) Asters assembled by 1 mM taxol in
the presence of 1 mg/ml p50/dynamitin in 10,000 g mitotic egg extract for 30 min at 208C. Microtubules are red, anti-GST-staining
is green. Bars, 10 mm.
The Journal of Cell Biology, Volume 143, 1998
680
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
Figure 6. Dimerization of the Xklp2-Tail. (A) Silver-stained
SDS-PAGE of thrombin-cleaved Xklp2-Tail incubated with increasing amounts of glutaraldehyde (from left to right: markers,
0, 0.0003, 0.001, 0.003, and 0.01%). With rising glutaraldehyde
concentrations, a new band appears at 60 kD corresponding to
the dimeric Xklp2-Tail. The molecular mass of marker proteins is
indicated on the left. (B) Far UV circular dichroism spectra of
Xklp2-Tail (solid line), CDel2 (long dash), and NDel2 (short
dash) at 2 mM concentration at 48C. (C) Thermal unfolding of
Xklp2-Tail (filled circle), CDel2 (open circle), NDel1 (triangle),
NDel2 (upsidedown triangle) at 10 mM. (D) Thermal unfolding of
Xklp2-NDel2 at a concentration of 10 mM (filled circle) and 1 mM
(open circle). (E) Thermal unfolding of GST-Xklp2-Tail (filled
circle) and Xklp2-Tail (open circle) at 10 mM concentration. See
text for details.
Binding of Xklp2-Tail to Microtubules Requires TPX2,
a Novel 100-kD MAP
The above set of experiments suggested that the targeting
of Xklp2-Tail to microtubule minus ends occurred in two
steps: (a) binding to microtubules and (b) localization to
microtubule minus ends through the action of the dynein–
dynactin complex. Although dynein could mediate the interaction between endogenous Xklp2 and microtubules in
some way it could not be responsible for microtubule
binding since even in the presence of dynein inhibitors we
still detected binding of GST-Xklp2-Tail to microtubules
both biochemically and by immunofluorescence (see Fig. 4
B, lane 5 and Fig. 7). Moreover, we could not detect any
interaction between dynein or dynactin and Xklp2 or GSTXklp2-Tail in immunoprecipitation experiments (data not
shown).
We then wanted to test whether the GST-Xklp2-Tail itself contained a microtubule-binding activity using a biochemical assay. Pure taxol-stabilized microtubules were
mixed with the GST-Xklp2-Tail and the microtubules
were then sedimented by centrifugation through a sucrose
cushion and analyzed by Western blot. Under these conditions, no GST-Xklp2-Tail was detected in the microtubule
pellet (Fig. 8 A, lane 1). To determine whether an additional protein was required for GST-Xklp2-Tail binding to
microtubules, we prepared MAPs from CSF-arrested egg
Figure 8. Purification of TPX2, a MAP that mediates the binding
of Xklp2 to microtubules. (A) GST-Xklp2-Tail alone does not
bind to pure taxol-stabilized microtubules (lane 1). However, a
fraction of MAPs contains an activity that mediates the binding
of GST-Xklp2-Tail to microtubules (lane 3). This activity was assayed in the following way: lane 1, prepolymerized microtubules
were mixed with either GST-Xklp2-Tail and control buffer; lane
2, GST-Xklp2-Tail and soluble proteins (0.6 mg/ml) from CSFarrested egg extract; lane 3, GST-Xklp2-Tail and MAPs (0.6 mg/
ml); lane 4, or GST-Xklp2-CDel2 and MAPs. The microtubules
were sedimented by centrifugation through a sucrose cushion and
the soluble fractions and pellets (four times more loaded than of
the soluble fraction) were analyzed by Western blotting, probed
with an anti-GST antibody. (B) Fractionation of MAPs by sequential salt elution from microtubules. Microtubules polymerized in CSF-arrested egg extract were purified by centrifugation
through a sucrose cushion and eluted in one step with 500 mM
NaCl (MAPs) or sequentially with the NaCl concentration indicated. The fractions were assayed as described above and the pellets were analyzed by Western blotting and probed with an antiGST antibody. (C) Mono S chromatography of the 300 mM NaCl
fraction eluted from microtubules. (a) Silver-stained SDS-PAGE
of 5-ml aliquots of the fractions indicated. The molecular mass of
marker proteins is indicated on the left. (b) Assay for the activity
mediating the binding of GST-Xklp2-Tail to microtubules of fractions from the Mono S chromatography as indicated. (D) The
peak fraction of the Mono S chromatography was applied to a
Superdex 200 gel filtration column. (a) Silver-stained gel of 5-ml
aliquots of the fractions indicated. (b) Assay for the activity mediating the binding of GST-Xklp2-Tail to microtubules of frac-
tions from the Superdex 200 gel filtration chromatography as indicated. In both chromatographic steps the activity copurified
with a 100-kD protein.
Wittmann et al. Targeting of Xklp2 to Microtubule Minus Ends
681
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
presence of the m70.1 antibody the GST-Xklp2-Tail could
still bind all along microtubules.
We then examined if the dynactin complex was also involved in Xklp2-Tail localization to microtubule minus
ends. Overexpression of the p50/dynamitin protein in tissue culture cells has been shown to disrupt the dynactin
complex (Echeverri et al., 1996). Addition of bacterially
expressed and partially purified p50/dynamitin to Xenopus
egg extracts resulted in the reduction of the sedimentation
velocity of the p150Glued subunit of dynactin indicating that
the complex had dissociated (Wittmann and Hyman, 1999)
as previously reported for tissue culture cells (Echeverri
et al., 1996). In the presence of p50/dynamitin, centrosomes still nucleated asters that looked similar to those
generated in the presence of the m70.1 antibody. Again,
this abolished the localization of the GST-Xklp2-Tail to
the center of the asters (Fig. 7 B, b). When microtubule
polymerization was induced by the addition of taxol in the
presence of p50/dynamitin the asters were highly disorganized and GST-Xklp2-Tail failed to localize (Fig. 7 B, d).
Also in the presence of p50/dynamitin we could observe a
significant binding of GST-Xklp2-Tail along microtubules.
Altogether these results show that cytoplasmic dynein as
well as the dynactin complex are required for the localization of GST-Xklp2-Tail to microtubule minus ends during
mitosis. Interestingly in the absence of dynein function,
GST-Xklp2-Tail could still bind all along microtubules although with a lower efficiency as determined biochemically.
careful examination of this localization revealed that Eg5
is actually distributed throughout the spindle (Sawin and
Mitchison, 1995). On the basis of initial immunofluorescence results we also reported that Xklp2 was on centrosomes. Here, we have clearly established that at least a
fraction of Xklp2 and the recombinant GST-Xklp2-Tail do
not bind to centrosomes directly. They are rather localized
to the minus ends of microtubules in mitosis and at least a
fraction appears to bind all over the microtubule length in
interphase. Our earlier conclusion was based on the fact
that some staining remained associated with centrosomes
after depolymerization of microtubules by nocodazole in
XL177 tissue culture cells (Boleti et al., 1996). However,
there are alternative explanations for this observation,
e.g., incomplete disassembly of microtubules close to the
centrosome. In any case, the important result as far as
Xklp2 function in centrosome separation is concerned is
that it is localized close to microtubule minus ends during
mitosis. Interestingly, a similar discrepancy has been observed for the localization of the microtubule-severing
ATPase katanin. Katanin is localized to the centrosomal
area and its localization has been reported to be microtubule dependent in sea urchin embryos (McNally et al.,
1996) whereas the centrosomal localization in tissue culture cells was not affected by microtubule depolymerization (Hartman et al., 1998).
The structural organization and localization of Xklp2 indicates that its mechanism of action is different from Eg5
and probably complements it. Eg5 belongs to the BimC
family that form bipolar tetramers with motor domains on
both ends (reviewed by Kashina et al., 1997). Tetrameric
Eg5 moves towards the plus ends of microtubules while simultaneously bundling and sorting them into antiparallel
arrays. Xklp2 is proposed to function as a dimer concentrated at microtubule minus ends which separates the
poles by exerting a plus end–directed force on microtubules emanating from the opposite pole (Boleti et al.,
1996). The difference between the two motors is mainly
the site where the force is produced and it is likely that
both functions are essential to establish spindle bipolarity
and to stabilize the bipolar shape. This is why the inactivation of any of these plus end–directed motors gives an apparently similar phenotype whereas their specific function
in defining spindle shape is entirely different. The function
of Xklp2 may be mostly to help the initiation of centrosome separation in the initial phases of spindle assembly. This is consistent with a recent study showing that inhibition of Xklp2 has little effect on spindles assembled in
the absence of centrosomes around chromatin beads
(Walczak et al., 1998).
Discussion
Mechanism of Xklp2 Localization to Microtubule
Minus Ends
Xklp2 Accumulates at Microtubule Minus Ends
during Mitosis
We have previously shown that Xklp2 is required for centrosome separation. This function is shared with other motors like those of the BimC family. At first sight, both
Xklp2 and the Xenopus member of the BimC family, Eg5,
had a similar localization at spindle poles and even on centrosomes (Sawin et al., 1992; Boleti et al., 1996). A more
Although we still do not understand very well how KLPs
are localized to their different sites of action within the
cell, it is generally accepted that regions in the nonconserved nonmotor domains of KLPs confer targeting specificity (reviewed by Goldstein, 1993). KLPs could be attached to their target in two different ways: through a
direct interaction as it has been determined for Nod, a
chromatin bound KLP, that has been shown to interact di-
The Journal of Cell Biology, Volume 143, 1998
682
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
extract. Identical amounts of either the proteins that remained soluble or MAPs were included in the reaction
(Fig. 8 A, lanes 2 and 3, respectively). Only in the presence
of the MAP-fraction a substantial amount of GST-Xklp2Tail was recovered in the microtubule pellet. We observed
by immunofluorescence that in this case GST-Xklp2-Tail
bound all along the prepolymerized microtubules showing
no preference for either end (data not shown). The fusion
protein lacking the leucine zipper, GST-Xklp2-CDel2, did
not cosediment with MAPs and microtubules demonstrating the specificity of the assay (Fig. 8 A, lane 4). These results indicated the presence of a factor in mitotic egg extracts required for the binding of the COOH-terminal
domain of Xklp2 to microtubules that was enriched in a
MAP-fraction prepared from CSF-arrested egg extract.
Affinity chromatography of total extracts or MAPs
passed over a GST-Xklp2-Tail protein column did not
yield any interacting proteins (data not shown). Therefore,
we attempted the purification of the factor mediating the
binding of GST-Xklp2-Tail to microtubules using classical
biochemistry. As a first purification step, mitotic MAPs
were eluted from microtubules with steps of 100 mM NaCl
and the different fractions were tested whether they could
promote GST-Xklp2-Tail binding to microtubules (Fig. 8 B).
Most of the targeting factor remained bound to microtubules up to a salt concentration of 200 mM but was eluted
by 300 mM salt. This protein fraction was then applied to a
Mono S column and eluted with a linear salt gradient. The
activity that mediated the binding of GST-Xklp2-Tail to
microtubules eluted from the Mono S column in a single
peak at around 350 mM KCl and corresponded to a doublet of polypeptides with molecular masses of z100 kD
(Fig. 8 C). The strong affinity of these proteins for the
Mono S suggested that they were highly basic. Since the
Mono S peak fraction still contained some minor contaminants it was further purified on a Superdex 200 gel filtration column (Fig. 8 D). Also on this column the activity
mediating the binding of GST-Xklp2-Tail copurified with
the 100-kD band. The peaks of minor contaminating
bands left after the Mono S did not coelute with the activity. These results showed that a MAP that we named
TPX2 (Targeting Protein for Xklp2), mediated the binding
of the COOH-terminal domain of Xklp2 to microtubules.
Purified TPX2 itself was able to rebind to pure microtubules (data not shown). Interestingly, the Xklp2-Tail carrying a single point mutation in the leucine zipper (GSTLtoK) did not bind at all to microtubules in the presence
of this MAP. This strongly suggests that TPX2 is the receptor for the leucine zipper found at the COOH terminus
of Xklp2.
Figure 9. Two alternative
models for how Xklp2 and
TPX2 could be localized to
microtubule minus ends in a
dynein dependent way. (A)
Direct mechanism: Xklp2
and TPX2 interact with a
dynein containing complex
that moves towards the microtubule minus end or they
bind directly to a component
that has been transported
there by dynein before. (B)
Indirect mechanism: a protein that is transported to the minus
ends by dynein creates a gradient along the microtubule (possibly
by some enzymatic activity) that changes the affinity of TPX2 to
Xklp2 and/or to microtubules.
Wittmann et al. Targeting of Xklp2 to Microtubule Minus Ends
683
think that this dimerization is important to stabilize the
ternary complex on the microtubules. The weak interaction between Xklp2 and TPX2/microtubules may also account for the apparent differences in binding properties of
the GST-Xklp2-Tail and the endogenous protein. We usually add an excess of GST-Xklp2-Tail to our assays and
this seems to be sufficient to increase the binding efficiency of the tail relative to the endogenous protein to microtubules.
Localization of Xklp2 to the Minus Ends by Dynein. Our
data indicate that the targeting of Xklp2 to microtubule
minus ends involves the dynein–dynactin complex. Cytoplasmic dynein, a minus end–directed motor, and the dynactin complex, a modulator of cytoplasmic dynein function, are localized to spindle poles and required for the
generation and maintenance of focused poles (Steuer et
al., 1990; Paschal et al., 1993; Echeverri et al., 1996; Heald
et al., 1996, 1997). Moreover, cytoplasmic dynein is required for the movement of exogenously added microtubule seeds to the minus end of microtubules, suggesting
that it is involved in the transport of microtubules towards
the spindle poles. In addition, other proteins are localized
to spindle poles in a dynein dependent manner. NuMA, a
coiled-coil protein that cross-links microtubules at the
spindle poles, has been shown to interact directly with dynactin and cytoplasmic dynein (Merdes et al., 1996) and
appears to be displaced at least partially when dynein
function is inhibited (Gaglio et al., 1996; Heald et al.,
1997).
We found that the localization of GST-Xklp2-Tail to microtubule minus ends was disrupted by interfering with either dynein using the m70.1 antibody or dynactin by addition of p50/dynamitin to the extract. Since the m70.1
antibody dissociates both the dynein intermediate and
heavy chains from spindle microtubules (Gaglio et al.,
1997; Heald et al., 1997) the present findings demonstrate
that dynein is required for the localization of Xklp2 to microtubule minus ends.
Therefore, we are left with two findings: a MAP, TPX2,
mediates the binding of Xklp2 to microtubules and the dynein–dynactin complex is required to bring Xklp2 to the
minus end of microtubules. We can think of two different
mechanisms leading to Xklp2 localization at microtubule
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
rectly with DNA via an 82–amino acid region in its
COOH-terminal domain (Afshar et al., 1995b); or through
a complex with other proteins such as the binding of kinesin to vesicles, which involves kinectin, an integral membrane protein that anchors kinesin to the surface of membrane vesicles (Kumar et al., 1995; reviewed in Vallee and
Sheetz, 1996).
The targeting of Xklp2 to microtubule minus ends is
complex and seems to involve several steps. To dissect this
mechanism, we have used the COOH-terminal domain of
Xklp2 that was shown to be necessary and sufficient to target the protein to spindle poles.
Targeting of Xklp2 to Microtubules by TPX2. We have
identified TPX2, a 100-kD MAP, that is required to mediate the binding of Xklp2 to microtubules. However,
TPX2 is not sufficient to localize Xklp2 to microtubule
minus ends. Most MAPs have been identified by their
ability to bind microtubules and for some of them the effect on microtubule dynamics has been studied (e.g.,
Andersen and Karsenti, 1997). Only few examples are
known of MAPs that link other cellular components to
microtubules. It has been suggested that p34cdc2 kinase
associates with microtubules in the mitotic spindle because of its interaction with MAP4 (Ookata et al., 1995).
CLIP-170 has been characterized as a cytoplasmic linker
protein that mediates the binding of endocytic vesicles to
microtubules (Pierre et al., 1992). A similar brain-specific protein, CLIP-115, links dendritic lamellar bodies to
microtubules (De Zeeuw et al., 1997). Both CLIPs share a
homologous microtubule binding motif with p150Glued, a
component of the dynactin complex, that has also been
shown to bind directly to microtubules (WatermanStorer et al., 1995). NuMA has been shown to bind both
microtubules and the dynein–dynactin complex (Merdes
et al., 1996). Cytoplasmic dynein binds through its intermediate chain to p150Glued (Karki and Holzbaur, 1995).
Interestingly, human Eg5 seems to interact also with
p150Glued (Blangy et al., 1997). To our knowledge TPX2
represents the only other example of a MAP that binds
directly to a KLP. The microtubule localization of the
yeast KLP Kar3 is dependent on its interaction with
Cik1 (Page et al., 1994), but since Cik1 does not show a microtubule binding activity on its own it is more similar to a
kinesin light chain than to a MAP.
Our data show that a very COOH-terminal leucine zipper is required for the interaction between TPX2 and
Xklp2, whereas the more NH2-terminal regions of the
Xklp2 COOH-terminal domain are required for the stability of the dimeric structure. This dimerization is also required for Xklp2 targeting. Therefore, the COOH-terminal domain of Xklp2 interacts through its leucine zipper
with TPX2 that mediates the binding of Xklp2 to microtubules. It is interesting to note that Xklp2 does not seem to
bind to TPX2 in the absence of microtubules. Since Xklp2
does not bind either to microtubules in the absence of
TPX2, it seems that the stable binding of Xklp2 to microtubules involves a ternary complex between microtubules,
TPX2 and the tail of Xklp2. Why dimerization of the tail is
required is not yet understood. Because of the apparent
weak interaction between Xklp2 and microtubules in the
extract (GST-Xklp2-Tail can inhibit the endogenous protein in equimolar amounts, see Boleti et al., 1996), we
Afshar, K., N.R. Barton, R.S. Hawley, and L.S. Goldstein. 1995a. DNA binding
and meiotic chromosomal localization of the Drosophila nod kinesin-like
protein. Cell. 81:129–138.
Afshar, K., J. Scholey, and R.S. Hawley. 1995b. Identification of the chromosome localization domain of the Drosophila Nod kinesin-like protein. J. Cell
Biol. 131:833–843.
Andersen, S.S.L., and E. Karsenti. 1997. XMAP310: A Xenopus rescue-promoting factor localized to the mitotic spindle. J. Cell Biol. 139:975–983.
Ashford, A.J., S.S.L. Andersen, and A.A. Hyman. 1998. Preparation of tubulin
from bovine brain. In Cell Biology: A Laboratory Handbook. Vol. 2. J.E.
Celis, editor. Academic Press, San Diego, CA. 205–212.
Barton, N.R., and L.S.B. Goldstein. 1996. Going mobile: microtubule motors
and chromosome segregation. Proc. Natl. Acad. Sci. USA. 93:1735–1742.
Berger, B., D.B. Wilson, E. Wolf, T. Tonchev, M. Milla, and P.S. Kim. 1995.
Predicting coiled coils by use of pairwise residue correlations. Proc. Natl.
Acad. Sci. USA. 92:8259–8263.
Blangy, A., H.A. Lane, P. d’Herin, M. Harper, M. Kress, and E.A. Nigg. 1995.
Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell. 83:
1159–1169.
Blangy, A., L. Arnaud, and E.A. Nigg. 1997. Phosphorylation by p34cdc2 protein
kinase regulates binding of the kinesin-related motor HsEg5 to the dynactin
subunit p150Glued. J. Biol. Chem. 272:19418–19424.
Boleti, H., E. Karsenti, and I. Vernos. 1996. Xklp2, a novel Xenopus centroso-
mal kinesin-like protein required for centrosome separation during mitosis.
Cell. 84:49–59.
Bornens, M., M. Paintrand, J. Berges, M.C. Marty, and E. Karsenti. 1987. Structural and chemical characterization of isolated centrosomes. Cell Motil. Cytoskel. 8:238–249.
De Zeeuw, C.I., C.C. Hoogenraad, E. Goedknegt, E. Hertzberg, A. Neubauer,
F. Grosveld, and N. Galjart. 1997. CLIP-115, a novel brain-specific cytoplasmic linker protein, mediates the localization of dendritic lamellar bodies.
Neuron. 19:1187–1199.
Domínguez, J.E., B. Buendia, C. Lopez-Otin, C. Antony, E. Karsenti, and J.
Avila. 1994. A protein related to brain microtubule-associated protein
MAP1B is a component of the mammalian centrosome. J. Cell Sci. 107:601–611.
Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. Molecular
characterization of the 50-kD subunit of dynactin reveals function for the
complex in chromosome alignment and spindle organization during mitosis.
J. Cell Biol. 132:617–633.
Frère, V., F. Sourgen, M. Monnot, F. Troalen, and S. Fermandjian. 1995. A peptide fragment of human DNA topoisomerase II alpha forms a stable coiledcoil structure in solution. J. Biol. Chem. 270:17502–17507.
Gaglio, T., A. Saredi, J.B. Bingham, M.J. Hasbani, S.R. Gill, T.A. Schroer, and
D.A. Compton. 1996. Opposing motor activities are required for the organization of the mammalian mitotic spindle pole. J. Cell Biol. 135:399–414.
Gaglio, T., M.A. Dionne, and D.A. Compton. 1997. Mitotic spindle poles are
organized by structural and motor proteins in addition to centrosom J. Cell
Biol. 138:1055–1066.
Glotzer, M., A.W. Murray, and M.W. Kirschner. 1991. Cyclin is degraded by
the ubiquitin pathway. Nature. 349:132–138.
Goldstein, L.S.B. 1993. With apologies to Scheherazade: tails of 1001 kinesin
motors. Annu. Rev. Genet. 27:319–351.
Harborth, J., K. Weber, and M. Osborn. 1995. Epitope mapping and direct visualization of the parallel, in-register arrangement of the double-stranded
coiled-coil in the NuMA protein. EMBO J. 14:2447–2460.
Hartman, J.J., J. Mahr, K. McNally, K. Okawa, A. Iwamatsu, S. Thomas, S.
Cheesman, J. Heuser, R.D. Vale, and F.J. McNally. 1998. Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit. Cell. 93:277–287.
Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman,
and E. Karsenti. 1996. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature. 382:
420–425.
Heald, R., R. Tournebize, A. Habermann, E. Karsenti, and A. Hyman. 1997.
Spindle assembly in Xenopus egg extracts: Respective roles of centrosomes
and microtubule self-organization. J. Cell Biol. 138:615–628.
Hoyt, M.A., L. He, K.K. Loo, and W.S. Saunders. 1992. Two Saccharomyces
cerevisiae kinesin-related gene products required for mitotic spindle assembly. J. Cell Biol. 118:109–120.
Hyman, A.A., and E. Karsenti. 1996. Morphogenetic properties of microtubules and mitotic spindle assembly. Cell. 84:401–410.
Hyman, A.A., D. Drechsel, D. Kellogg, S. Salser, K. Sawin, P. Steffen, L. Wordeman, and T.J. Mitchison. 1991. Preparation of modified tubulins. In Methods in Enzymology. Vol. 196. R.B. Vallee, editor. Academic Press, San Diego, CA. 478–485.
Karki, S., and E.L.F. Holzbaur. 1995. Affinity chromatography demonstrates a
direct binding between cytoplasmic dynein and the dynactin complex. J.
Biol. Chem. 270:28806–28811.
Karsenti, E., H. Boleti, and I. Vernos. 1996. The role of microtubule dependent
motors in centrosome movements and spindle pole organization during mitosis. Sem. Cell Biol. 7:367–378.
Kashina, A.S., R.J. Baskin, D.G. Cole, K.P. Wedaman, W.M. Saxton, and J.M.
Scholey. 1996. A bipolar kinesin. Nature. 379:270–272.
Kashina, A.S., G.C. Rogers, and J.M. Scholey. 1997. The bimC family of kinesins: essential bipolar mitotic motors driving centrosome separation. Biochim. Biophys. Acta. 1357:257–271.
Kumar, J., H. Yu, and M.P. Sheetz. 1995. Kinectin, an essential anchor for kinesin-driven vesicle motility. Science. 267:1834–1837.
Lau, S.Y.M., A.K. Taneja, and S.H. Hodges. 1984. Synthesis of a model protein
of defined secondary and quaternary structure. J. Biol. Chem. 259:13253–
13261.
Liao, H., G. Li, and T.J. Yen. 1994. Mitotic regulation of microtubule crosslinking activity of CENP-E kinetochore protein. Science. 265:394–398.
Lupas, A., M. van Dyke, and J. Stock. 1991. Predicting coiled coils from protein
sequences. Science. 252:1162–1164.
McNally, F.J., K. Okawa, A. Iwamatsu, and R.D. Vale. 1996. Katanin, the microtubule-severing ATPase, is concentrated at centrosomes. J. Cell. Sci. 109:
561–567.
Merdes, A., and D.W. Cleveland. 1997. Pathways of spindle pole formation: different mechanisms; conserved components. J. Cell Biol. 138:953–956.
Merdes, A., K. Ramyar, J.D. Vechio, and D.W. Cleveland. 1996. A complex of
NuMA and cytoplasmic dynein is essential for spindle assembly. Cell. 87:
447–458.
Murray, A.W. 1991. Cell cycle extracts. In Methods in Cell Biology. Vol. 36.
B.K. Kay and H.B. Peng, editors. Academic Press, San Diego, CA. 581–605.
Ookata, K., S. Hisanaga, J.C. Bulinski, H. Murofushi, H. Aizawa, T.J. Itoh, H.
Hotani, E. Okumura, K. Tachibana, and T. Kishimoto. 1995. Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase
The Journal of Cell Biology, Volume 143, 1998
684
We would like to thank L. Serrano for his invaluable help with the circular
dichroism spectroscopy and S. Reinsch, R. Heald, J. Domínguez, T. Hyman, and M. Glotzer for advice and gifts of reagents. We also thank N. Le
Bot, A. Westerholm, R. Tournebize, and S. Andersen for critical reading
of the manuscript.
Received for publication 23 February 1998 and in revised form 9 September 1998.
References
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
minus ends: (a) a direct mechanism in which Xklp2 and
TPX2 may be part of a larger complex containing dynein/
dynactin, NuMA and maybe other proteins that are
moved towards the spindle poles by the motor activity of
dynein (Fig. 9 A). Xklp2 and TPX2 could also be recruited
to the pole by binding to components that have been
transported there by dynein already, but this would still involve a series of direct interactions between Xklp2 and
TPX2 on one side and dynein–dynactin on the other; (b)
an indirect mechanism in which an enzymatic activity (e.g.,
a protein kinase or phosphatase) is transported towards
microtubule minus ends by dynein. Because in an aster,
dynein accumulates at microtubule minus ends, this could
create a gradient of enzymatic activity along the microtubules of the aster. This could, in turn, increase the affinity
of Xklp2 and TPX2 for each other and/or for microtubules
close to the microtubule minus end (Fig. 9 B).
From our present data we can not distinguish between
these two possibilities and further work will be necessary
to clarify how certain proteins are enriched at a specific
end of a microtubule. It is interesting to note that in interphase or in mitotic extracts in which dynein has been inactivated Xklp2 binds along microtubules. This suggests that
the cell cycle regulation of Xklp2 localization to microtubule minus ends may involve a specific function of dynein
during mitosis.
In any case, this work has unraveled a very interesting
mechanism where a minus end–directed motor, dynein,
can organize spindle poles and at the same time participates in the localization of a plus end–directed motor to
the poles, an event required for their separation: this is another example of how several levels of self-organizing processes can lead to the assembly of a machine as complex as
a mitotic spindle.
Vallee, R.B., and M.P. Sheetz. 1996. Targeting of motor proteins. Science. 271:
1539–1544.
Verde, F., J.M. Berrez, C. Antony, and E. Karsenti. 1991. Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: requirement for phosphorylated factors and cytoplasmic dynein. J. Cell Biol. 112:1177–1187.
Vernos, I., and E. Karsenti. 1996. Motors involved in spindle assembly and
chromosome segregation. Curr. Opin. Cell Biol. 8:4–9.
Walczak, C.E., and T.J. Mitchison. 1996. Kinesin-related proteins at mitotic
spindle poles: function and regulation. Cell. 85:943–946.
Walczak, C.E., I. Vernos, T.J. Mitchison, E. Karsenti, and R. Heald. 1998. A
model for the proposed roles of different microtubule-based motor proteins
in establishing spindle bipolarity. Curr. Biol. 8:903–913.
Waterman-Storer, C.M., S. Karki, and E.L.F. Holzbaur. 1995. The p150Glued
component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1). Proc. Natl. Acad. Sci. USA. 92:1634–
1638.
Wilhelm, H., S.S.L. Andersen, and E. Karsenti. 1997. Purification of recombinant cyclin B1/cdc2 kinase from Xenopus egg extracts. In Methods in Enzymology. Vol. 283. W.G. Dunphy, editor. Academic Press, San Diego, CA.
12–28.
Wittmann, T., and T. Hyman. 1999. Recombinant p50/dynamitin as a tool to examine the role of dynactin in intracellular processes. In Methods in Cell Biology. Vol. 61. C. Rieder, editor. Academic Press, San Diego, CA. 137–143.
Zhong, L., and W.C. Johnson. 1992. Environment affects amino acid preference
for secondary structure. Proc. Natl. Acad. Sci. USA. 89:4462–4465.
Wittmann et al. Targeting of Xklp2 to Microtubule Minus Ends
685
Downloaded from http://rupress.org/jcb/article-pdf/143/3/673/1281103/9802126.pdf by guest on 18 March 2023
to microtubules and is a potential regulator of M-phase microtubule dynamics. J. Cell Biol. 128:849–862.
Page, B.D., L.L. Satterwhite, M.D. Rose, and M. Snyder. 1994. Localization of
the Kar3 kinesin heavy chain-related protein requires the Cik1 interacting
protein. J. Cell Biol. 124:507–519.
Paschal, B.M., E.L.F. Holzbaur, K.K. Pfister, S. Clark, D.I. Meyer, and R.B.
Vallee. 1993. Characterization of a 50-kDa polypeptide in cytoplasmic dynein preparations reveals a complex with p150Glued and a novel actin. J. Biol.
Chem. 268:15318–15323.
Pierre, P., J. Scheel, J.E. Rickard, and T.E. Kreis. 1992. CLIP-170 links endocytic vesicles to microtubules. Cell. 70:887–900.
Saunders, W.S., and M.A. Hoyt. 1992. Kinesin-related proteins required for
structural integrity of the mitotic spindle. Cell. 70:451–458.
Sawin, K.E., and T.J. Mitchison. 1991. Mitotic spindle assembly by two different
pathways in vitro. J. Cell Biol. 112:925–940.
Sawin, K.E., and T.J. Mitchison. 1995. Mutations in the kinesin-like protein Eg5
disrupting localization to the mitotic spindle. Proc. Natl. Acad. Sci. USA. 92:
4289–4293.
Sawin, K.E., K. LeGuellec, M. Philippe, and T.J. Mitchison. 1992. Mitotic spindle organization by a plus-end-directed microtubule motor. Nature. 359:
540–543.
Stearns, T., and M. Kirschner. 1994. In vitro reconstitution of centrosome assembly and function: the central role of g-tubulin. Cell. 76:623–637.
Steuer, E.R., L. Wordeman, T.A. Schroer, and M.P. Sheetz. 1990. Localization
of cytoplasmic dynein to mitotic spindles and kinetochores. Nature. 345:266–268.