First publ. in : Current biology ; 17 (2007), 6. - S. 488-498
DOI : 10.1016/j.cub.2007.02.036
The Human Kinesin Kif18A
Is a Motile Microtubule Depolymerase
Essential for Chromosome Congression
Monika I. Mayr,1 Stefan HOmmer,1 Jenny Bormann,1
Tamara GrOner,1,2 Sarah Adio,3 Guenther Woehlke,3
and Thomas U. Mayer1,.
1 Chemical Genetics
Independent Research Group
Department of Cell Biology
Max-Planck-Institute of Biochemistry
Am Klopferspitz 18
82152 Martinsried
Germany
2London Research Institute
Lincoln's Inn Fields Laboratories
44 Lincoln's Inn Fields
London WC2A 3PX
United Kingdom
31nstitute for Cell Biology
University of Munich
Schillerstr. 42
80336 Munich
Germany
Summary
Background: The accurate alignment of chromosomes
at the spindle equator is fundamental for the equal distribution of the genome in mitosis and thus for the genetic
integrity of eukaryotes. Although it is well established
that chromosome movements are coupled to microtubule dynamics, the underlying mechanism is not well
understood.
Results: By combining RNAi-depletion experiments
with in vitro biochemical assays, we demonstrate that
the human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression in
mammalian tissue culture cells. We show that in vitro
Kif18A is a slow plus-end-directed kinesin that possesses microtubule depolymerizing activity. Notably,
Kif18A like its yeast ortholog Kip3p depolymerizes
longer microtubules more quickly than shorter ones.
In vivo, Kif18A accumulates in mitosis where it localizes
close to the plus ends of kinetochore microtubules. The
depletion of Kif18A induces aberrantly long mitotic spindles and loss of tension across sister kinetochores,
resulting in the activation of the Mad2-dependent spindle-assembly checkpoint. Live-cell microscopy studies
revealed that in Kif18A-depleted cells, chromosomes
move at reduced speed and completely fail to align at
the spindle equator.
Conclusions: These studies identify Kif18A as a dualfunctional kinesin and a key component of chromosome
congression in mammalian cells.
'Correspondence: [email protected]
Introduction
The function of mitosis is to equally divide the duplicated parental genome into two newly forming daughter
cells. A key component of this process is the mitotic
spindle, a microtubule-based bipolar structure whose
dynamic properties are vital for both chromosome alignment and separation before and upon anaphase onset,
respectively [1]. The assembly and function of the mitotic spindle depends on a complex network regulating-in a temporally and spatially controlled mannermicrotubule dynamics and activities of motor proteins
of the dynein and kinesin superfamilies, microtubulebased ATPases that convert chemical energy into
mechanical force [2-4].
Prerequisite for the equal partitioning of replicated
chromosomes is their proper bipolar attachment on
the mitotic spindle; i.e., sister kinetochores of chromatid
pairs are attached to microtubules emanating from opposite spindle poles. In higher eukaryotes, the capture
of kinetochores by spindle microtubules starts with
nuclear-envelope breakdown resulting, commonly, in
mono-oriented chromosomes with only one kinetochore
attached to microtubules. Subsequently, monooriented chromosomes alternate between movements
away from and toward the spindle pole until the unattached kinetochore is encountered by microtubules
from the opposite spindle pole. Bivalent-oriented chromosomes congress toward the spindle equator where
they oscillate until the sudden loss of cohesion at anaphase onset allows the separation of sister chromatids
to opposite spindle poles.
Although the basic concepts of chromosome congression have been recognized for a long time (see [5]
for review), the discovery of the underlying molecular
mechanisms has been started only recently. Yet, given
that microtubule plus ends remain attached to kinetochores of congressing and oscillating chromosomes, it
is apparent that chromosome movements have to be
tightly coupled to microtubule dynamics. The identification of kinetochores as the major sites of microtubule
assembly and disassembly associated with chromosome movements [6, 7] suggested that the kinetochore-associated motor proteins CENP-E and MCAKI
XKCM1 play a key role in chromosome congression.
However, inactivation of neither CENP-E, a plus-enddirected kinesin [8], nor MCAKlXKCM1, a nonmotile
microtubule depolymerase [9, 10], impaired the congression of bivalently oriented chromosomes [11, 12].
Members of the Kinesin-8 family of kinesins (Kif18A,
H. sapiens; Klp67A, D. me/anogaster; KipB, A. nidulans;
Kip3p, S. cerevisiae; klp5/6+, S. pombe) [13] have been
classified as microtubule depolymerases based on the
observation that loss of their activity results in aberrantly
long spindles with hyperstable microtubules [14-23]. In
the current study, we show that the human kinesin
Kif18A, which is enriched at the plus ends of kinetochore
microtubules, possesses both plus-end-directed
Konstanzer Online-Publikations-System (KOPS)
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-1401416
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Figure 1. The Human Kinesin Kif18A Peaks in
Mitosis
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(A) Schematic representation of Kif18A
indicating the regions used for antibody
production.
(6) HeLa cell extracts; in vitro-translated
Kif18AFL and un programmed wheat-germ extract were probed by western blotting with
Kif18A antibody (Kif18AAbN). Anti-a.-tubulin
immunoblot serves as a loading control.
(C) HeLa cells synchronized in S phase were
released and analyzed at the indicated time
points by western blotting with Kif18A
(Kif18AAbN) and cyclin 61 antibodies. Anti-a;tubulin immunoblot serves as a loading control. The total cell extract loaded was 15 I-'g.
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motility and microtubule depolymerizing activity. Because recently a dual functionality has been demonstrated for the yeast ortholog Kip3p [24, 25], it is suggested that this remarkable characteristic applies to all
members of the Kinesin-8 family. Our cellular studies revealed that chromosome congress ion relies on the presence of Kif18A, suggesting that this motile microtubule
depolymerase is key for the dynamics of kinetochore
microtubules driving chromosome alignment in the preanaphase state of the cell cycle in human cells.
Results and Discussion
Kif18A Is a Cell-Cycle-Regulated Kinesin
For exploring the function and localization of endogenous human Kif18A, polyclonal Kif18A antibodies were
raised against an N-terminal GST-tagged fragment (residues 1-367) and against a peptide composed of amino
acids 879-894 (Figure 1A). Western-blot analyses of
Hela extracts revealed that the affinity-purified antibody raised against the N terminus of Kif18A (Kif18 AbN)
detected a protein of the expected size of approximately
100 kDa (Figure 1 B). The antibody also recognized
in vitro-translated (IVT) Kif18A but did not detect a protein in unprogrammed wheat-germ extract (Figure 1B).
The depletion of endogenous Kif18A with short-interfering RNAs (siRNAs) caused the almost complete loss
of the immunoreactive signal in Hela extracts,
demonstrating that the detected protein was indeed
Kif18A (Figure 1B).ldentical results were obtained with
the immunopurified peptide antibody (Kif18Abc) (data
not shown).
Immunoblot analyses indicated that Kif18A was more
abundant in mitotic than in interphase cells (Figure 1B).
For investigating regulation of abundance in more detail,
extracts of synchronized Hela cells released from a thymidine arrest were immunoblotted for Kif18A. Figure 1 C
shows that the endogenous levels of Kif18A were low in
S phase, increased approximately 9 hr after release, and
decreased abruptly after approximately 10.5 hr (Figure 1 C). For defining the timing of entry into and exit
from mitosis, identical samples were immunoblotted
for cyclin B1. As shown in Figure 1C, endogenous levels
of cyclin B1 increased and declined with similar kinetics
as Kif18A. Immunoblots against (I.-tubulin confirmed
that for each time point equal amounts of proteins
were loaded (Figure 1C). Taken together, these data
show that Kif18A accumulates during mitosiS, suggesting a mitotic function for this kinesin.
Kif18A localizes Close to Plus Ends
of Kinetochore Microtubules
Next, we determined Kif18A's intracellular localization
by indirect immunofluorescence with affinity-purified
Kif18 Abc antibodies. In interphase cells, Kif18A was
detected in the nucleus (Figure 2A). As a bipolar spindle
490
B
Figure 2. Kif18A Localizes near the Plus Ends of Kinetochore Microtubules
(A) Immunofluorescence images of HeLa cells at different cell-cycle stages stained with DAPI (blue) and antibodies directed against Kif18A (red)
and anti ,,-tubulin (green). The scale bar represents 10 IIm.
(8) HeLa cells stained for Kif18A (red), the kinetochore protein Hec1 (green), and chromatin (blue). The scale bar represents 10 ~lm.
(C) Localization of transiently expressed GFP-tagged full-length Kif18A (GFP- Kif18A FL , 1-898) during metaphase. HeLa cells were stained for the
kinetochore-microtubule-binding protein HURP (red), and Kif18A was visualized by GFP-tag. The scale bars represent 10 ~lm.
491
formed, Kif18A localized along spindle microtubules
before it accumulated in metaphase at the tips of microtubules pointing toward chromosomes or being connected to kinetochores (Figure 2A). Upon anaphase onset, the Kif18A signal diminished and finally disappeared
almost completely as cells left mitosis (Figure 2A), and
this is in line with the immunoblot analyses (see
Figure 1C). No signal was detected in HeLa cells
depleted of Kif18A by siRNA approaches and thus confirmed that the observed immunofluorescence signal
was specific for Kif18A (Figure 5A). Identical localization
patterns were observed with the affinity-purified
Kif18AbN antibody (data not shown). To determine the localization of Kif18A on the metaphase spindle in greater
detail, we costained cells with the outer-kinetochore
protein Hec1 [26, 27]. As shown in Figure 2B, Kif18A
was enriched at the kinetochore microtubules' tips,
distal to the outer-kinetochore protein Hec1. Next, we
stained HeLa cells transiently expressing greenfluorescent-protein (GFP)-tagged full-length Kif18A
(GFP-Kif18AFL) for Hec1 and HURP, a mitotic spindle
protein that localizes to kinetochore microtubules in
the vicinity of chromosomes [28, 29]. As shown by
triple-immunofluorescence images, Kif18A was enriched at kinetochore microtubules flanked by Hec1
and HURP on the chromosome-proximal and -distal
site, respectively (Figure 2C). Quantification of Kif18Aimmunoreactive punctae and the number of kinetochores per cell-with Hec1 as a marker-revealed that
both numbers closely match (average Hec1 and Kif18A
punctae per HeLa cell: 62.6 and 58.3 respectively [n =
10]), further supporting the observation that under these
experimental conditions, Kif18A binds exclusively to kinetochore microtubules. In nocodazole-release experiments, we observed that GFP-Kif18A FL was enriched
in the proximity of centrosomes in the presence of nocodazole before it accumulated upon nocodazole washout
at the tips of regrown microtubules in the vicinity of
chromosomes (Figure S1 in the Supplemental Data
available online). Taken together, these data demonstrate that Kif18A is enriched near the plus ends of kinetochore microtubules.
Loss of Kif18A Activates the Mad2-Dependent
Spindle-Assembly Checkpoint
Previously performed organism-wide kinesin-siRNA
studies have shown that Kif18A seems to be required
for chromosome congression in mitotic cells [23]. However, the underlying mechanism leading to chromosome
alignment defects in the absence of Kif18A has not been
addressed in this study. To gain detailed insights into
the mitotic function of Kif18A, we first depleted Kif18A
in asynchronous HeLa cells by using two different siRNA
oligos. Immunoblot analyses confirmed that both siRNA
oligos decreased the endogenous levels of Kif18A to
less than 25% compared to cells treated with a luciferase
control (GL2) siRNA oligo (Figure 3A). Cell-cycle analyses with laser scanning microscopy (LSM) (see Supplemental Experimental Procedures) revealed that cells depleted of Kif18A accumulated in a G2IM state with a 4N
DNA content (Figure 3B). This indicates that Kif18A is required for timed progression through mitosis. The observed delay in mitosis was mediated by the spindle-assembly checkpoint (SAC) because cells depleted of
both Kif18A and Mad2 did not show an accumulation
of mitotic cells but a dramatic increase in the number
of multinucleated cells (Figure 3C). In line with these results, we observed that Mad2 was enriched at kinetochores of mal oriented chromosomes in Kif18A-depleted
cells (Figure 3D). Notably, in the rare cases when chromosomes were properly aligned at the metaphase plate
in Kif18A-RNAi cells, Mad2 did not accumulate at these
kinetochores (Figure 3D) and thus suggests that
Kif18A-in contrast to dynein-dynactin [30]-does not
play a role in checkpoint inactivation, i.e., in the depletion of checkpoint proteins from correctly attached kinetochores.
Cells Depleted of Kif18A Fail to Establish a Stable
Metaphase Plate
To gain further insight into the defects caused by Kif18A
depletion, we performed live-cell analyses of control
and Kif18A-depleted HeLa cells expressing GFP-tagged
histone-H2B. Whereas control-treated cells efficiently
aligned chromosomes at the metaphase plate, Kif18ARNAi cells had severe problems in chromosome congression and displayed a prolonged prometaphase
characterized by oscillating chromosomes that failed
to form a stable metaphase plate (Figure 4A; Movies
S1 and S2). Within 60 min (median: 52 min, n 31) after
the first signs of chromosome condensation, 65% of
control RNAi cells aligned all chromosomes correctly
(Figure 4B). In contrast, only approximately 6% of
Kif18A-depleted cells managed to do so within the
same period of time (median: 170 min, n = 35) (Figure 4B
and Movie S2). Next, we examined whether alterations in
the velocity of chromosomes in Kif18A-depleted cells
could account for the observed chromosome alignment
defects. To this end, we quantified the rate of chromosome movements in GL2- and Kif18A-RNAi cells by using a yellow fluorescent-protein (yFP)-tagged form of
the centromeric protein CENP-A to visualize individual
chromosomes. These analyses revealed that in the period between nuclear-envelope breakdown and the
end of metaphase (PM-M, prometa-, metaphase), chromosomes moved away from and toward the spindle
equator in GL2-RNAi cells at an average rate of approximately 2l-tm/min (Figure 4C, left, and Movie S3), a velocity consistent with previous reports [31, 32]. Upon
anaphase onset, chromosomes started to move polewards at 2.5 I-tm/min (EA, early anaphase) before they
slowed down to 1.1 I-tm/min during late anaphase (LA in
the left panel of Figure 4C). In contrast, chromosomes
in Kif18A-depleted cells displayed sustained but slow
movements (betwe.en 0.8 and 1.3 I-tm/min and Movie
S4) while trying to align properly at the metaphase spindle (Figure 4C, right). Because ofthe prolonged prometaphase arrest of Kif18A-depleted cells (Figure 3B), no anaphase movements of chromosomes could be recorded
during the course of the experiments. Taken together,
these data indicate that Kif18A is essential for dynamic
movements of chromosomes during prometaphase
and their proper alignment at the metaphase plate.
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Depletion of Kif18A Induces Elongated Spindles and
Decreases Tension across Sister Kinetochores
The observed defects in chromosome congression
upon Kif18A depletion prompted us to examine more
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Figure 3. Loss of Kif18A Activates the Mad2-Dependent Spindle-Assembly Checkpoint
(A) HeLa cells treated with GL2 control or Kif18A-RNAi oligos were probed by western blotting with Kif18A (Kif18AAbN) antibody. For Kif18A-RNAi
cells, 10 j.lg of total protein was loaded. An anti-a-tubulin blot serves as a loading control.
(B) Cell-cycle analyses of control (GL2) or Kif18A-depleted cells.
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Figure 4. Kif18A Is Required for Timed Progression through Mitosis and Chromosome Congression
(A) Selected live-cell images of HeLa cells expressing GFP-tagged Histon-H2B transfected with control or Kif18A siRNA oligos. The scale bar
represents 10 !!m.
(B) Quantitative analyses of the live-cell images shown in (A) documenting the time required for anaphase onset in control or Kif18A-depleted
HeLa cells (n = 30). The first signs of chromosome condensation is defined at t = 0 min.
(C) Quantification of chromosome movements in GL2-treated (left panel) and Kif18A-depleted (right panel) HeLa cells stably expressing
YFP-CENP-A during different cell-cycle stages (I, interphase; PM-M, prometa-, metaphase; EA, early anaphase; LA, late anaphase; C, cytokinesis). (Regarding GL2-RNAi cells, each bar represents average chromosome velocity at the indicated cell-cycle stages calculated by the analyses of a total of 116 individual CENP-A dots in five different cells; for Kif18A-RNAi cells, each bar represents average chromosome velocity in
one Kif18A-RNAi cell. In total, 108 individual CENP-A dots in five different cells were analyzed.) Values represent the average ± SE.
closely the spindle morphology in these cells. As shown
in Figure 5A, HeLa cells depleted of Kif18A below the
detection limit frequently formed elongated "bananashaped" spindles with wavy and bent microtubules, indicating that these microtubules are abnormally long.
Quantitative analyses with pericentrin as a marker for
spindle poles demonstrated that the average distance
between the two spindle poles in Kif18A-RNAi cells
(n = 10) was approximately 18.1 I1m (±1.5) compared
to 9.3 I1m (±1.1) in GL2-RNAi cells, (n = 10, Figure 58
and Figure 84). To rule out the possibility that the increase in spindle length in Kif18A-RNAi cells was
caused by the prolonged arrest in prometaphase, we
determined spindle length in cells depleted of both
Mad2 and Kif18A. Although the codepletion of Mad2
eliminated the mitotic delay caused by Kif18A depletion
(Figure 3e), spindle length in Kif18-/Mad2-RNAi cells
(n = 10) was still significantly increased to 16.0 I1m
(±1.0) compared to GL2-/Mad2-RNAi control cells
(Figure 58 and Figure 84). Thus, abnormally long spindle
(C) Quantitative cell-cycle analyses of HeLa cells 48 hr after transfection with GL2IGL2, GL2IMad2, GL2IKif18A, or Kif18A1Mad2 siRNA-oligos.
For each combination of double siRNA, 100 cells were counted.
(D) The checkpoint protein Mad2 is enriched at kinetochores of maloriented chromosomes but not at those of properly aligned chromosomes in
GL2- and Kif18A-siRNA HeLa cells. Cells were transfected with GL2 control or Kif18A-RNAi oligos for 48 hr and then stained for Mad2 (red), Hec1
(blue), and a-tubulin (green). The scale bar represents 10 ~lm.
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Figure 5. Kif18A Depletion Affects the Integrity of the Mitotic Spindle
(A) Hela cells transfected with GL2- or Kif18A-RNAi oligos were stained with Kif18A (Kif18AAbc , red) and et-tubulin (green) antibodies and DAPI
(blue). The scale bar represents 1 Ilm.
(B) Quantification of pole-to-pole distance in Gl2lMad2, Gl2lKif18A, or Kif18A1Mad2-depleted mitotic cells (n =10 for each condition). Values
represent the average ± SE.
(C) Spindle microtubules are hyperstable in Kif18A-depleted cells. Hela cells were treated for 48 hr with control (GL2-) or Kif18A-siRNA, and this
was followed by a 35 min incubation on ice. Cells were stained for et-tubulin. The scale bar represents 1 I'm.
(D) Depletion of Kif18A decreases tension on sister kinetochore pairs. Representative immunofluorescence images of Hela cells 48 hr after
transfection with Gl2- or Kif18A-siRNA are shown. Tubulin and DNA are shown in green and blue, respectively. Kinetochore pairs on sister chromatids were observed by CREST signal (red) at either side of the sister chromatid. The scale bar represents 15 l'ffi.
(E) Quantification of interkinetochore distances. In total, the interkinetochore distance of 113 (Gl2-RNAi cells, n = 10), 78 (Kif18A-RNAi cells,
n =10), and 73 (GL2-RNAi cells + 5 I'M nocodazole for 6 hr, n =10) bioriented chromatid pairs were measured.
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Figure 6. Kif18A Is a Plus-End-Directed Microtubule Depolymerase
(A) Immunofluorescence images of polarity-marked microtubules moving in the presence of His-Kif18AFL and ATP with the brightly labeled plusend trailing. The scale bar represents 10 flm.
(B) GMP-CPP microtubules (final tubulin concentration: 0.35 flM) in BRB80 buffer supplemented with KCI (final CI- concentration: 110 mM) were
incubated with buffer or two different concentrations of insect-derived full-length Kif18A protein in the presence of ATP (at a final concentration
of 1 mM). Where indicated, the reactions were supplemented with various concentrations of AMP-PNP, a nonhydrolyzable ATP analog. After
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496
microtubules and elongated spindle shapes are direct
consequences of Kif18A depletion, suggesting that
Kif18A negatively affects length of spindle microtubules.
Consistently, in the absence of Kif18A, spindle microtubules were more resistant to cold-induced depolymerization than those in GL2-RNAi control cells (Figure 5C).
These data suggest that Kif18A is required for the dynamic behavior of kinetochore microtubules. Changes
in the dynamics of kinetochore microtubules affect
tension acting on sister kinetochores [33], and this can
be analyzed by measurement of the distance between
sister kinetochores. As shown in Figures 5D and 5E,
loss of Kif18A decreased the interkinetochore distance
of bioriented chromosomes from 1.55 Ilm (±0.17) (113
kinetochore pairs from ten different GL2-RNAi cells
were analyzed) to 1.31 Ilm (±0.17) (78 kinetochore pairs
from ten different Kif18A-RNAi cells were analyzed). In
the absence of microtubules, the distance between
kinetochore pairs was reduced to 0.86 Ilm (±0.12) (73
kinetochore pairs from ten different nocodazole-treated
GL2-RNAi cells were analyzed), indicating that in the absence of Kif18A, kinetochores are still attached to microtubules albeit under reduced tension. Together, all data
obtained so far indicate that Kif18A has a destabilizing
effect on kinetochore microtubules, and this function
seems to be vital for tension acting on sister kinetochores (Figure 5E) and thus for proper chromosome
congression during prometaphase (Figure 4A).
Kif18A Is a Plus-End-Directed Microtubule
Depolymerase
The phenotype of Kif18A-depleted cells prompted us to
investigate the enzymatic properties of Kif18A in vitro.
First, we tested whether Kif18A possesses motility activity by using in vitro microtubule-gliding assays. In
the presence of recombinant full-length Kif18A (HisKif18A FL) and ATP, we observed motility offluorescently
labeled microtubules at an average speed of approximately 0.02 (±0.007) Ilm/s (Movie 85). To determine
the directionality of Kif18A, we analyzed the movement
of polarity-marked microtubules. As shown in Figure 6A
and Movie 85, microtubules moved with the brightly labeled plus-end trailing (n = 20) indicative of a plus-enddirected motor activity. Thus, these studies identify
Kif18A similarly to its orthologs Klp67A and Kip3p from
Drosophila and budding yeast [25, 34J, respectively, as
a slow plus-end-directed kinesin. Next, it was investigated whether Kif18A possesses microtubule-destabilizing activity in vitro. For these experiments, full-length
His-tagged Kif18A (His-Kif18AFL) expressed in insect
cells and purified to homogeneity was incubated with
fluorescently labeled microtubule polymer that were
stabilized by polymerization in the presence of the
slowly hydrolyzable GTP analog GMPCPP (guanylyl(rx,13)-methylene-diphosphonate) [35]. In the presence
of ATP and recombinant His-Kif18AFL , rhodaminelabeled GMP-CPP-microtubules (resuspended to a
0.35 IlM tubulin-dimer concentration) de polymerized
within 25 min at RT (Figure 6B). This effect was dose dependent on His-Kif18AFL and specific as indicated by
the fact that identical buffer conditions did not affect
microtubule polymers (Figure 6B). Previously, it was
shown that the nonmotile Kinesin-13 kinesins are capable of de polymerizing microtubules in an ATP-dependent manner [10, 36, 37]. To address whether Kif18Alike the Kinesin-13 proteins-requires ATP hydrolysis
for its microtubule depolymerization activity, the effect
of Kif18A on microtubule polymers was studied in the
presence of the nonhydrolyzable ATP analog AMPPNP (13,y-imidoadenosine 5' -triphosphate). As shown
in Figure 6B, 2.5 mM AMP-PNP did not significantly
affect His-Kif18AFL-dependent microtubule destabilization, whereas 5 mM attenuated and 7.5 mM completely
blocked the depolymerization of microtubules. In these
assays, ATP was present at a concentration of 1 mM
because His-Kif18AFL was purified and stored in ATPcontaining buffer for ensuring protein stability. Thus,
these data suggest that Kif18A's destabilizing activity
is sensitive to high concentrations ofthe ATP competitor
AMP-PNP. The fact that identical results were previously reported for Kip3p, whose microtubule-destabilizing activity was attenuated but not blocked in the presence of 2 mM AMP-PNP [24], suggests that Kinesin-8
proteins share common mechanochemical properties.
To corroborate our findings, we incubated GMP-CPP
microtubules (resuspended to a 0.35 IlM tubulin-dimer
concentration) with His-Kif18AFL and 1 mM ATP and
then ultracentrifugated them to separate soluble tubulin
dimer from pelletable microtubule polymer. When GMPCPP microtubule polymer was incubated with 100 nM
His-Kif18AFL, almost all tubulin was recovered from the
supernatant fraction, whereas the majority of tubulin
was recovered from the pellet fraction in the absence
of Kif18A (Figure 6C). In line with the microscopic analyses, AMP-PNP suppressed in a dose-dependent manner the destabilizing activity of 100 nM His-Kif18AFL in
the presence of 1 mM ATP (Figure 6C). Under lowersalt condition, 100 nM His-Kif18FL did not efficiently
depolymerize GMPCPP microtubules in the presence
of 1 mM ATP (Figure 6D, 60mM final CI- concentration
compared to 110 mM in Figure 6C). Thus, the microtubule depolymerization activity of Kif18A is highly
sensitive to lower-salt conditions.
Recently, Kip3p was identified as a length-dependent
depolymerase that de polymerizes long microtubules
more efficiently than short ones [25]. As further noted
(C) Microtubule pelleting assay. Samples treated as in (A) (control buffer or 100 nM His-Kif18A FL, 0.35 JlM tubulin as polymer, 1 mM ATP,
110 mM CI-, and the indicated concentrations of AMP-PNP) were not fixed but pelleted by ultracentrifugation. Supernatant and pellet fractions
were analyzed by SDS-PAGE. Asterisks mark His-Kif18AFL•
(D) Kif18A does not depolymerize GMP-CPP microtubules under low-salt conditions. GMP-CPP microtubules (final tubulin concentration of
0.35 I'M) in BRB80 buffer were incubated with 100 nM HiS-Kif18AFL (final CI- concentration: 60 mM), and 1 mM ATP were incubated for
25 min at RT, pelleted by ultracentrifugation and analyzed by SDS-PAGE.
(E) Kif18A depolymerizes longer GMP-CPP microtubules more efficiently than shorter ones. Short or long fluorescently labeled GMP-CPP
microtubules in the amount of 0.35 I'M were incubated with 100 nM His-Kif18AFL and 1 mM ATP for the indicated time points at RT. Microtubule
length was determined by fluorescence microscopy. For each bar, the lengths of 20 microtubules were measured. Values represent the average ± SE.
497
in this study, longer microtubules accumulated more
Kip3p at their plus ends than shorter ones, a finding
that seems to be critical for the rate of microtubule
depolymerization. In agreement with these results, we
observed that in the presence of 1 mM ATP, 100 nM
His-Kif1SAFL depolymerized long (14.9 ± 1.44 llm)
GMPCPP microtubules at a rate of 1.25 ± 0.14 llm/min,
whereas short ones (5.7 ± 0.4S11m) were depolymerized
at a rate of 0.21 ± O.OS llm/min. As expected, this effect
was specific because in the absence of Kif1SA, long and
short microtubules did not significantly change length
(Figure 60). Thus, under conditions where Kif1SA cannot
accumulate to sufficient amounts at the plus ends (50
nM His-Kif1SAFL or 100 nM His-Kif1SAFL + high concentrations of AMP-PNP), microtubules are not efficiently
depolymerized. Because AMP-PNP inhibits Kif1SA's
motility (data not shown), we were not able to elucidate
whether Kif1SA also requires its ATPase activity to depolymerize microtubules. Taken together, these data
demonstrate that Kif1SA is a motile microtubule depolymerase that depolymerizes longer microtubules more
quickly than shorter ones.
The ATPase Activity of Kif1SA Is Stimulated Not Only
by Microtubule Polymer but Also by Tubulin Oimer
The nonmotile microtubule depolymerases of the Kinesin-13 family share the characteristic that their ATPase
activity-unlike that of conventional kinesins-is stimulated not only by microtubule polymer but also by tubulin dimers [36]. In vitro assays revealed that Kif1SA's
ATPase activity like that of Kip3p [24] was stimulated
in a concentration-dependent manner by microtubule
polymer and albeit to a lesser extent by tubulin dimer
(Figure S6 in the Supplemental Data). Thus, the microtubule depolymerases of the Kinesin-S and the Kinesin-13
families distinguish themselves from the Kar3p-a motile kinesin that utilizes its minus-end-directed motility
to depolymerize microtubules from their plus ends
[3S]-in their tubulin-stimulated ATPase activity.
In conclusion, we have identified the human kinesin
Kif1SA as a motile plus-end-directed microtubule depolymerase. Thus, Kif1SA integrates both motility and
microtubule depolymerization. Most recent reports
demonstrated that this dual functionality also applies
for Kip3p, the yeast Kinesin-S member [24, 25]. Previously, it has been demonstrated that microtubule depoIymerization that occurs predominantly at kinetochore
sites [6] can create forces sufficient for moving chromosomes [39]. For the congression of chromosomes in
prometaphase, the depolymerase activities of the nonmotile Kinesin-13 proteins Kif2a and Kif2c (MCAK)
seem to be dispensable because cells depleted of
both kinesins have no defect in chromosome movement
and alignment and mitotic progression [40]. In contrast,
in Kif1SA-depleted cells, chromosomes oscillate at
reduced velocity and completely fail to align at the metaphase plate. Furthermore, a lack of Kif1SA causes hyperstable spindle microtubules and the loss of tension
across sister kinetochores; the latter results in the activation of the Mad2-dependent spindle-assembly checkpoint. This loss of tension could also accounts for sustained chromosomes oscillations in Kif1S-RNAi cells
because, as previously shown, tension on kinetochores
controls chromosome movements [41]. Thus, in view of
our data we postulate that Kif1SA is an essential component for chromosome congression in prometaphase by
regulating microtubule dynamics at the plus ends of kinetochore microtubules. Clearly, the activity of Kif1SA
has to be tightly controlled. It is tempting to speculate
that the length dependency of the Kif1SA-mediated depolymerization might play a role in the alignment of chromosomes at the mitotic spindle's center, where microtubules on both sites of kinetochore pairs are equally long.
In the future, it will be important to elucidate the molecular mechanism and regulation of Kinesin-S proteins to
understand precisely how this family of motile depolymerases contribute to the function of the mitotic spindle
in eukaryotes.
Acknowledgments
We are exceptionally grateful to D.R. Foltz and D.W. Cleveland for
providing HeLa stably expressing YFP-CENPA and Vladimir Varga
for providing helpful comments on experiments. We also thank
members of the Mayer lab and Nigg department for helpful comments on the manuscript. TUM and SH were supported by the Deutschen Forschungsgemeinschaft (MA 1559/4-4) and the Fonds der
Chemischen Industrie, respectively.
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