From the Department of Biology, Johns Hopkins
University, Baltimore, Maryland 21218 and the ¶ Department of
Biomedical Engineering, Johns Hopkins School of Medicine,
Baltimore, Maryland 21205
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ABSTRACT |
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We have developed microtubule binding and
motility assays for Cin8p, a kinesin-related mitotic spindle motor
protein from Saccharomyces cerevisiae. The methods examine
Cin8p rapidly purified from crude yeast cell extracts. We created a
recombinant form of CIN8 that fused the biotin carrying
polypeptide from yeast pyruvate carboxylase to the carboxyl terminus of
Cin8p. This form was biotinated in yeast cells and provided Cin8p
activity in vivo. Avidin-coated glass surfaces were used to
specifically bind biotinated Cin8p from crude extracts. Microtubules
bound to the Cin8p-coated surfaces and moved at 3.4 ± 0.5 µm/min in the presence of ATP. Force production by Cin8p was directed
toward the plus ends of microtubules. A mutation affecting the
microtubule-binding site within the motor domain
(cin8-F467A) decreased Cin8p's ability to bind
microtubules to the glass surface by >10-fold, but reduced gliding
velocity by only 35%. The cin8-3 mutant form, affecting the Eukaryotic chromosome segregation is mediated by the mitotic
spindle, a microtubule-based motile structure that undergoes a distinct
program of morphological changes. It is now clear that many spindle
movements are accomplished by microtubule-based motor proteins. Perhaps
the best characterized type of spindle motor is that of the BimC
subfamily of kinesin-related proteins. Members of the BimC family,
first discovered in Aspergillus nidulans (1), have been
found in numerous eukaryotic species (2-7). These proteins are
conserved in amino acid sequence of the motor (force-producing) domain
and apparently perform similar roles in many different cell types
(8-10). BimC motors are required for bipolar spindle assembly;
elimination of their function blocked this essential early mitotic step
in fungal, insect, and mammalian cells. The yeast Saccharomyces
cerevisiae expresses two BimC-related motors that overlap in
function, Cin8p and Kip1p. Although neither is individually essential,
one of the pair is required for viability (3, 4, 11). Loss of
KIP1 causes less severe phenotypes than loss of
CIN8, suggesting that Cin8p is more important for successful
yeast spindle function (3). Besides essential roles in spindle
assembly, Cin8p and Kip1p are also required for the maintenance of
spindle bipolarity following assembly and are responsible for producing
most of the spindle-elongating force during anaphase (11, 12).
In addition to genetic experiments, several lines of evidence suggest
that BimC motors act by cross-linking and sliding antiparallel microtubules found in the spindle midzone. BimC motors have been localized exclusively to the microtubules that lie between the spindle
poles (3, 4, 13-15). In vitro, BimC motors from
Drosophila melanogaster and Xenopus laevis have
been found to move exclusively toward microtubule plus ends, albeit at
a rate much slower than that achieved by kinesin (~2 µm/min
versus 20-40 µm/min for kinesin) (16). In particular, the
"bipolar" molecular structure determined for the
Drosophila BimC motor Klp61F suggested a mechanism by which
these motors may contribute to spindle structure and elongation. Klp61F
is an elongated bipolar homotetramer, with two motor domains positioned
at each end of a rodlike structure (17). Such a bipolar molecule would
have the ability to cross-link and slide antiparallel microtubules
(10). Despite these suggestive observations, the actual molecular role
of BimC motors during spindle dynamics has yet to be established.
In this study, we developed in vitro assays for the analysis
of the S. cerevisiae Cin8p motor. Our purpose was 2-fold.
First, the in vitro properties of Cin8p have not been
described. Mere analogies to other BimC members are clearly indirect
and unsatisfactory. Second, the ease of genetic and cell cycle
manipulations in yeast demands a similarly direct method to examine and
dissect Cin8p functions in vitro. Although exogenous
expression of motors allows initial reconstituted studies, only
endogenous expression in yeast allows direct study of Cin8p cell cycle
regulation and different mitotic roles. With these goals in mind, we
developed a rapid assay for the activity of Cin8p expressed in yeast
cells. For a one-step purification and adsorption to avidin-coated
surface, the Cin8 protein was fused to a peptide (biotin carrier
peptide or BCP) that is endogenously biotinated in yeast. Subsequent
analysis could distinguish microtubule binding from translocation
activities, and photoactivation of caged-ATP further increased the
sensitivity of our assays. Using these assays, the study of two Cin8p
mutants revealed the relative importance of binding and motility for
Cin8p in vivo functions.
Yeast Strains and Media--
The S. cerevisiae
strains used in these experiments are derivatives of S288C and are
listed in Table I. The
cin8::HIS3 and kip1::HIS3
alleles were described previously (12). Rich (YPD) and minimal (SD)
media were as described by Sherman et al. (18). To derepress
galactose-inducible genes, cells were grown in 2% raffinose minimal
media at 26 °C for 24 h prior to induction by the addition of
galactose to 2%. Cycloheximide (Sigma) was added to a final
concentration of 5 µg/ml. All media for expression of biotinated
Cin8p were supplemented with 24 mg/liter biotin (Sigma) (19). Cells
were synchronized in G1 phase by the addition of 4-6
µg/ml of DNA Manipulations--
To create a biotinated version of Cin8p,
we used the polymerase chain reaction to amplify the sequence of yeast
PYC1, encoding the last 90 amino acids of pyruvate
carboxylase (20, 21), and add NotI sites at each end. The 5'
side primer used was AATAAAAGCGGCCGCACTGTTACTAAATCCAAAGCA, and the 3'
primer was AATAAATGCGGCCGCTGCCTTAGTTTCAACAGG. The BCP fragment was inserted into a NotI site that had previously
been created at the 3'-end of
CIN81 in the CEN
vector pRS317, (22), creating pLG3. Standard DNA manipulations were
used to place CIN8-BCP under the control of the
galactose-inducible promoter on a CEN vector (pLG8). The cin8-3 (cin8-R196K) mutation was introduced into CIN8-BCP
constructs using an MscI to PstI fragment
containing the mutant allele. The presence of cin8-3 was
confirmed by sequencing. The cin8-F467A mutation was created
by the unique site elimination method (23). The primer for mutagenesis
was GTCAATTTCGATTCACGGGCAGGTATATGGCCGCTTTTATC, which also
introduced a BspMI site with the amino acid change. The
presence of the cin8-F467A mutation was confirmed by
digestion with BspMI.
Yeast Extracts and Protein Analysis--
Yeast protein extracts
for SDS gel electrophoresis were prepared by vortexing cells with glass
beads (diameter of 425-600 µm) in 50 mM Tris-HCl (pH
7.4), 250 mM NaCl, 50 mM NaF, 5 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride. Cells were
vortexed eight times at the maximum speed for 15 s, with 15 s
on ice in between. Lysates was separated from cell debris and glass
beads via centrifugation for 5 min in the cold. An aliquot was reserved
for protein concentration determination using the BCA assay (Pierce) to
ensure equal protein loading in gel lanes. Samples were run on 7%
acrylamide gels, and the proteins were transferred to polyvinylidene
difluoride Immobilon-P (Millipore Corp.) membranes using standard
techniques (24). Reagents for immunoblot analysis, including
streptavidin-conjugated alkaline phosphatase, were obtained from
Tropix. Band intensities were quantified by scanning film images of
blots and analyzing with commercial software, IDL (Research Systems).
For the in vitro assays, 20-50-ml cultures were grown to
midlog phase. Cells were pelleted, washed, and ground with a mortar and
pestle under liquid nitrogen in 1 ml of motility buffer 1 (MB1): 30 mM Tris, 35 mM PIPES, final pH 7.2, 175 mM NaCl, 2 mM EDTA, 1 mM EGTA, 10%
glycerol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol. Extracts were clarified by
centrifugation at 13,000 rpm in a microcentrifuge at 4 °C. Protein
concentration in these extracts was typically 1-3 mg/ml.
In Vitro Microtubule Binding and Motility Assays--
To assay
Cin8p-BCP-induced microtubule gliding and binding on avidin- or
streptavidin-coated surfaces, we modified the protocol of Berliner
et al. (25). Acid-treated number 1 borosilicate glass
coverslips were prepared by overnight incubation in Chromerge (Thomas
Scientific), followed by washing under running filtered deionized water
for 20-30 min and spin-dried on a custom coverslip spinner. Flow
chambers (~10 µl) were formed between microscope slide and the
coverslip mounted with double stick tape. For side-by-side comparison
of different conditions, up to four chambers separated by silicone
grease lines were prepared on a single slide and coverslip. All
incubations below were for 10 min at room temperature.
Biotinamidocaproyl bovine albumin (Sigma), 10 µl of a 1.7 mg/ml
solution in H2O, was introduced to the chambers, incubated,
and washed twice with H2O. The chambers were treated with
10 µl of 5 mg/ml UltraAvidinTM (deglycosylated and
charge-neutralized avidin; from Leinco Technologies) in 10 mM Tris, pH 8, 1 mM EDTA, incubated, and rinsed
twice with H2O, incubated with casein (up to 5 mg/ml) in
H2O, rinsed twice with H2O, and then rinsed
twice with MB1. Crude yeast extracts, 10 µl of 0.2-1 mg/ml total
protein, were introduced, incubated for 4-6 min, and washed twice with
MB1. In experiments in which the ability of biotin to block binding was
assessed, the H2O rinse after UltraAvidin incubation was
replaced with biotin-saturated H2O, and the consecutive
rinses and incubations contained a 1% dilution of biotin-saturated
H2O. The final rinse in MB1 and motility and binding assays
were performed without the addition of biotin. For motility, taxol (40 µM)-stabilized microtubules (0.05 mg/ml) in MB1 were
added to the flow chamber along with 1-5 mM ATP. Average microtubule length was 5 µm. When tested, other nucleotides were added to a final concentration of 1 mM. For motility
assays, MB1 was supplemented with 5 mM MgSO4.
To visualize microtubules, video-enhanced DIC microscopy was used as
described previously (26). Briefly, an inverted Zeiss microscope
(Axiovert 100TV, 100× Plan Neofluar 1.3 NA objective) was modified by
installing high transmission polarizers to project images onto a
Newvicon camera (Dage VE1000), and images were enhanced using an image
processor (Hamamatsu Argus 100). Video sequences were recorded on sVHS
tape (Panasonic AG7650 recorder), and microtubule gliding velocities
were measured using custom software (Imaging Technologies AFG image
processor) from these videos.
ATP uncaging experiments were done in the presence of 1 mM
of NPE-ATP2 (Molecular
Probes, Inc., Eugene, OR), a "caged" nucleotide from which ATP is
liberated by exposure to UV irradiation. The assay was performed
similarly to experiments with myosin (27, 28). Photoactivation of caged
ATP required a custom dichroic (400DCLPL from Chroma Technology Corp.)
to reflect light from a 75-watt xenon arc lamp. To prevent uncaging
during video-enhanced differential interference contrast microscopy,
the mercury arc illumination passed through an OG515 Schott Glass
optical filter as well as through a 546-nm interference filter. UV
illumination of the sample was between 1 and 2 min, followed by
observation of the sample for an additional minute. Under these
conditions, no effect on the length of microtubules was observed.
Polarity-marked microtubules were prepared according to Refs. 29 and 30
by using a 1:1 ratio of NEM-treated to NEM-untreated tubulin with a
total tubulin concentration of 2 µg/ml. Sea urchin (Strongylocentrotus purpuratus) axonemes (a gift from Herb
Miller and Jennifer Gregory) were used as seeds and were prepared by the method of Bell et al. (31) as modified by Walker
et al. (32). The correct orientation of microtubules
labeling was confirmed by a motility assay using bovine brain kinesin (Cytoskeleton).
The microtubule bundling assay was performed using flow chambers
constructed as described above. Yeast cell extracts (4 µl of 2 mg/ml)
were mixed with taxol-stabilized microtubules in MB1 (16 µl of 3.3 µg/ml). Bundling was observed in solution and on the glass surface by
video-enhanced differential interference contrast optics.
A Biotinated Form of Cin8p Provides Activity in Vivo--
For this
study, we developed an in vitro assay to examine the
microtubule binding and motile properties of the Cin8p kinesin-related motor from S. cerevisiae. We constructed a version of
CIN8 that encodes a fusion between Cin8p and a peptide from
S. cerevisiae pyruvate carboxylase (product of
PYC1; Refs. 20 and 21) attached to the Cin8p carboxyl
terminus. This region of pyruvate carboxylase, derived from its
carboxyl terminus, acts as a covalent acceptor of biotin from a
specific ligase (20, 25). We refer to this region of pyruvate
carboxylase as BCP for biotin-carrying polypeptide. Fusion proteins
that contained the homologous region of E. coli pyruvate
carboxylase become endogenously biotinated both in E. coli
and S. cerevisiae (19). The CIN8-BCP fusion gene
was cloned into low copy centromere-containing (CEN) and high copy
(2-µm origin) vectors and was expressed from either the endogenous
CIN8 promoter or a high level galactose-inducible promoter.
Relative to the level of expression provided by the endogenous
promoter/CEN plasmid construct, the endogenous promoter/2-µm version
and the galactose promoter/CEN version (induced for 4 h) produced
approximately 35- and 500-fold more Cin8p-BCP, respectively.
On account of overlapping functions (3, 4), the ability of
CIN8-BCP to complement a CIN8 deletion allele was
tested in a strain that was deleted for both CIN8 and
KIP1. Since cin8
The Cin8p-BCP fusion protein was endogenously biotinated in S. cerevisiae (Fig. 1B). Biotination was detected by gel
electrophoresis of total cell protein extract followed by blotting and
probing with streptavidin-conjugated alkaline phosphatase. In cells
expressing CIN8-BCP from the galactose-inducible promoter
(on a CEN plasmid), the addition of galactose resulted in the
appearance of an ~128-kDa protein, consistent with the predicted
molecular weight of the fusion protein (Fig. 1B). This
protein band increased in intensity with the time of galactose
induction and was absent from cells that are deleted for
CIN8 (Fig. 1B). In cells expressing
CIN8-BCP from its native promoter, either on low copy (CEN)
or high copy (2-µm) plasmids, the amount of Cin8p-BCP produced was
correspondingly lower (Fig. 1B). The 2-µm expression level
was intermediate between that of CEN and PGAL expression levels
(Fig. 1B). The noninducible biotinated protein band of
~120 kDa is probably S. cerevisiae pyruvate carboxylase,
one of the five endogenously biotinated proteins in this organism
(33).
Bundling of Microtubules in Extracts Containing High Levels of
Cin8p--
We observed that extracts from yeast cells overproducing
Cin8p exhibited a microtubule bundling activity (Fig.
2 and Table II). This activity may be related to the
ability of BimC motors to cross-link spindle microtubules (10). When
extracts from cells overproducing Cin8p or Cin8p-BCP under the control
of the galactose promoter were mixed with taxol-stabilized microtubule, bundles were observed. In the presence of 1 mM AMP-PNP, a
nonhydrolyzable ATP analogue, 90% of the observable microtubule
structures were bundles. Bundles were found in 70-80% of scored
microscope fields (n = 100 fields of 400 µm (2).
Without the addition of AMP-PNP, the degree of bundling was slightly
reduced. At lower overexpression levels (Cin8p-BCP from its normal
promoter but on a high copy 2-µm plasmid) bundling was observed, but
at a reduced level (Table II). No bundling was observed using extracts
from yeast producing wild-type levels of Cin8p or deleted for
CIN8 (n > 100 fields for each). We found
that the addition of avidin, which interacts with the biotin moiety of
BCP, did not alter the extent of Cin8p-BCP-specific bundling. This
indicates that the ability of Cin8p-BCP to interact with microtubules
is independent of its interaction with avidin. When 1 mM
ATP was added to bundles formed in the absence of added nucleotides,
dissolution of bundles occurred within 5-10 min. In some cases, we
were able to observe the sliding apart of bundles following the
addition of ATP. The relative polarity of the sliding microtubules
(parallel versus antiparallel) was not determined, however.
The bundling in crude cell extracts was specific for overproduced
Cin8p. Under the conditions described above, extracts from cells that
overproduced either a Cin8p motor domain mutant (Cin8p-R394A, H396A) or
kinesin-related Kip2p or Kip3p never bundled microtubules. Interestingly, Cin8p-871, a tail deletion mutant that forms a dimer
instead of the wild-type
tetramer,3 was unable to form
bundles. This suggests that bundling reflects the ability of BimC
motors to cross-link microtubules via their bipolar arrangement of
motor domains.
Cin8p-BCP Specifically Binds Microtubules to Avidin-coated
Surfaces--
Avidin-coated glass coverslips were used to specifically
bind Cin8p-BCP from crude protein extracts. Two criteria were used to
establish that any observed microtubule binding to these surfaces was
specific for Cin8p: 1) extracts that lack biotinated Cin8p should
exhibit no microtubule binding, and 2) added biotin should block
microtubule binding. Fig. 3 summarizes
six experiments in which microtubule binding was assessed in the
absence of added nucleotide. The microtubule binding activity was high
from extracts in which Cin8p-BCP was overproduced but was greatly
reduced by the addition of biotin or from extracts of
cin8 In Vitro Motile Activity of Cin8p--
The spindle pole-separating
activity of Cin8p is most easily explained by a plus end-directed
motile activity (3, 12, 34). To observe Cin8p motility in
vitro, 1 mM ATP was added to microtubules preadsorbed
to Cin8p-BCP on avidin-coated surfaces. The Cin8p-BCP source for these
initial experiments was an extract from cells in which it had been
overproduced from the galactose promoter. Under these conditions,
efficient microtubule gliding was observed (Fig.
4A). Of the 3-10 microtubules
visible per 400-µm (2) field, 70-80% moved in the presence of ATP.
The nonmotile microtubules divided approximately equally between two
categories: immobile microtubules and those that detached. For about
10-20% of the moving microtubules, pivoting around a single point on the surface was observed during movement. Such movement probably reflects the activity of a single surface motor (35). For about 10% of
the moving microtubules, the movement was episodic, where changes in
velocity or stops and starts were observed. Buckling of microtubules,
which would indicate the presence of binding but nonmotile motors on
the surface (36), was not observed.
To determine the directionality of Cin8p-induced motility, we used
minus end-marked microtubules nucleated from sea urchin sperm axonemes
(see "Materials and Methods"). The direction of all gliding
microtubules was minus end leading (n = 16), indicating that Cin8p exhibits plus end-directed activity (Fig.
4B).
The velocity distribution of Cin8p-induced microtubule gliding is shown
in Fig. 5A. This distribution
represents 85 moving microtubules in tests of four different Cin8p-BCP
overproducing extracts. The average velocity of Cin8p-BCP induced
gliding was 3.4 ± 0.5 µm/min (S.E. shown in parentheses). A
small but significant number of microtubules moved at rates as much as
3 times this average, causing the velocity distribution to be quite
broad. The distribution of the overall distance moved by microtubules (running length) is shown in Fig. 5B. About 75% of
microtubules moved relatively short distances, smaller than 5 µm,
before the microtubule either paused or detached from the glass
surface. High microtubule densities did not change the episodic nature of movement. The short running length may suggest that Cin8p is less
processive than kinesin (37, 38), which was also suggested in a kinetic
study on the vertebrate BimC motor Eg5 (39).
The effects of added nucleotide on microtubule binding and gliding are
shown in Table III. The number of
microtubules found on the surface in the presence of AMP-PNP was more
than 10-fold larger than that with ATP. However, unlike experiments
that lacked added nucleotide (Table III and Fig. 3), a high fraction of
the AMP-PNP-bound microtubules could not be blocked by preincubation with biotin. Nonetheless, this residual microtubule binding with AMP-PNP appeared specific to Cin8p; extracts from cin8
The above microtubule binding and motility experiments were performed
using extracts in which Cin8p-BCP had been overexpressed. We also used
this assay to examine Cin8p activity at closer to normal expression
levels. While we have not been able to detect microtubule binding to
the glass surface using Cin8p-BCP from cells producing it from its
native promoter on a low copy (CEN) plasmid, we have been able to assay
Cin8p-BCP activity when produced from its native promoter on a high
copy (2-µm) plasmid. Under these conditions, Cin8p levels are
~35-fold higher than wild type but lower than the ~500-fold
amplification produced by PGAL. PGAL overexpressed
CIN8 is toxic to cells, but the 2-µm plasmid is tolerated
and complements the growth defect of cin8
Although Cin8p-BCP from 2-µm extracts could immobilize microtubules
in the presence of AMP-PNP, introducing ATP released all the bound
microtubules before motility could be analyzed (Table III). To overcome
this problem, we used NPE-ATP, a "caged" nucleotide from which ATP
is liberated by exposure to UV irradiation. Prior to uncaging with UV,
microtubules were bound to the surface, similar to the
absence-of-nucleotide rigor state. Upon activation with UV light, the
pulse of released ATP caused gliding of the preadsorbed microtubules.
The motility characteristics in caged ATP protocol were slightly
different from the simple ATP addition protocol, described above. In
direct comparison with the PGAL extracts, fewer microtubules
moved, and running lengths were shorter in the caged ATP experiment.
The average running length was 3.3 ± 0.3 and 1.1 ± 0.1 µm
for steady ATP and caged ATP, respectively. However, microtubule
gliding velocities were similar for PGAL caged ATP,
PGAL steady ATP, and 2-µm caged ATP assays (Table III),
validating the use of caged ATP to examine Cin8p activity in situations
where increased sensitivity is required.
Analysis of Cin8p Motor Domain Mutants in Vivo and in
Vitro--
An advantage of our experimental system is that it allows
us to analyze both the phenotypic and biochemical consequences of motor
gene mutations. To demonstrate this potential, we examined two Cin8p
forms mutant within their motor domains. One mutant is encoded by
cin8-3, a form that has been subjected to significant in vivo analyses (3, 11, 12). cin8-3 causes a
change from arginine to lysine at amino acid 196, located in the region
corresponding to the
Fig. 6 compares the ability of the Cin8p
mutants to bind microtubule in vitro. Extracts producing the
Cin8p-BCP forms were passed over avidin-coated glass surfaces to adsorb
the motor. Microtubules were added in the presence of AMP-PNP, and the
number bound per 400-µm (2) field was determined by microscopy. We also determined, by immunoblotting, that all three forms were expressed
at comparable levels (Fig. 6B) and were bound to the glass
surface equally well (data not shown). Cin8p-F467A displayed severely
reduced microtubule binding. Even higher expression levels from longer
induction times (Fig. 6; 4.5 h of induction) did not rescue the
reduction in microtubule binding. Cin8p-3 also exhibited a reduction in
microtubule binding, but this was less pronounced (typically ~50%
reduced). By optically focusing on the glass surface, we could estimate
the rates of microtubule capture and release. For wild-type Cin8p
surfaces, almost all microtubules that landed adhered for at least 5 min (the length of the observation time). For Cin8p-F467A and Cin8p-3
surfaces, 30-70% of the landing microtubules rapidly detached (within
1 s). However, of the microtubules that were captured to the
mutant surfaces, most remained attached for at least 5 min. This
suggests that the reduced microtubule binding affinity of the mutant
proteins reflects a diminished capture rate rather than an increased
detachment rate.
Because of their reduced ability to capture microtubules, experiments
for in vitro motility of mutants required the more sensitive caged ATP method (see above). In this set of experiments (Table IV), the average gliding velocity of
wild-type Cin8p-BCP measured after uncaging ATP was 2.8 ± 0.5 µm/min, within the margin of error of that measured using ATP
(3.4 ± 0.5 µm/min). Although Cin8p-F467A was greatly reduced in
its ability to bind microtubules, the gliding velocity achieved was
reduced by only 35% (Table IV). In addition, the fraction of
surface-bound microtubules that responded to ATP uncaging by either
moving or detaching was similar to that of the wild type. This finding,
combined with the results in Fig. 6, leads us to conclude that the
effect of this mutation is mainly on the ability of Cin8p to bind
microtubules rather than on motility. In contrast, for Cin8p-3, ATP
uncaging resulted in a lower fraction of microtubules moving or
detaching from the surface. However, the few motile microtubules moved
at a velocity close to the wild type.
To summarize mutant reconstitution studies, the main defect of
Cin8p-F467A mutant was its ability to bind microtubules in vitro. In comparison, Cin8p-3 mutant was less impaired for
microtubule binding but was able to move fewer microtubules after ATP uncaging.
A major mitotic role for BimC motors is the assembly of the bipolar
spindle. By monitoring the extent of spindle bipolarity and
pole-to-pole distances, we were able to detect differential effects of
the two Cin8p motor mutants on the spindle assembly process (Fig.
7). After release from The phenotypic analyses of S. cerevisiae cells mutant
for BimC-related Cin8p have revealed many aspects of the in
vivo function of this family of mitotic spindle motors (8-10).
The observed requirement for Cin8p during spindle assembly and
elongation and its localization to microtubules between the spindle
poles have led to the suggestion that Cin8p acts to cross-link and
slide spindle midzone microtubules. The in vitro analysis
presented here demonstrates that Cin8p indeed exhibits the properties
expected for a kinesin-related microtubule-based motor protein. Cin8p
can bind and bundle microtubules in the absence of nucleotide or in the
presence of AMP-PNP. In the presence of ATP, Cin8p induced microtubule
gliding in a plus end-directed fashion. If Cin8p acts to cross-link
antiparallel midzone microtubules, plus end-directed sliding would lead
to spindle elongation.
The assay we developed examines Cin8p isolated from crude extracts of
S. cerevisiae cells. An active biotinated form was expressed in S. cerevisiae and rapidly isolated by adsorption to an
avidin-coated glass surface. In principle, this assay has several
advantages over studies of motors produced in exogenous host cells or
assays in which extensive purification steps are required. Since Cin8p acts in a cell cycle-specific fashion, it seems likely that its activity will be subject to cell cycle regulation. We have determined that Cin8p levels fluctuate during the cell cycle, peaking in mitosis
and rapidly decreasing during the G1
stage.5 It is possible that
other forms of post-translational regulation are also imposed upon
Cin8p. The rapid purification used in this assay, requiring minimal
manipulation of the protein extracts, may permit (hypothetical)
regulatory proteins to maintain their association with Cin8p, allowing
their influence to be monitored. On account of the low intracellular
abundance of Cin8p, we were unable to detect Cin8p activity in extracts
of cells in which it was produced at normal levels. We were successful,
however, with assays using cells harboring the gene (expressed from its normal promoter) in extra copies on a 2 µm-based plasmid. Our crude
extract assay is currently being used to examine differences in
microtubule binding and translocation proficiency of Cin8p during cell
cycle progression.
With our biotin-based assay, we characterized the microtubule binding
and motility properties of Cin8p. The nucleotide specificity for
microtubule binding was similar to that reported for other kinesins and
exhibits strong binding in the presence of AMP-PNP and weak binding in
the presence of ADP (Table III). Conventional kinesin can move
microtubules in the presence of GTP (40, 41), but other kinesin-related
proteins, such as Ncd, cannot utilize GTP (44, 45). In our experimental
conditions, GTP was unable to support motility by Cin8p.
In our assay, Cin8p produced microtubule gliding rates of ~3.4
µm/min. This rate is approximately 10-fold slower than that produced
by kinesin in vitro (16) but similar to the 1.0-2.4 µm/min reported for two other BimC motors (15, 46, 47). The
distribution of microtubule velocities determined for Cin8p was broad
(Fig. 6A), ranging between 1 and 10 µm/min (although most
fell in the 1-4-µm/min range). A much narrower velocity distribution was reported for the Xenopus BimC homologue, Eg5, but this
study utilized a fragment of the motor produced in bacteria (47). The
specificity of movement in our assay to biotinated Cin8p makes it
highly unlikely that other motors were contributing to this broad range
of velocities. It is possible, therefore, that Cin8p motors with
different motile properties exist in the extracts examined. It is
tempting to speculate that the broad velocity distribution reflects
cell cycle regulation of the motile properties of Cin8p.
Comparison of Cin8p Mutants in Vivo and in Vitro--
Coupled with
the in vivo assays of spindle motors in S. cerevisiae, the rapid in vitro assay described here
allows a powerful analysis of the properties of mutant motor forms. In
this study, we examined the properties of two Cin8p mutants altered
within their motor domains. The Cin8p-3 form is altered at an arginine (changed to lysine) that is conserved among kinesin family members. The
corresponding residue in human kinesin lies in the motor domain
The second mutant, Cin8p-F467A was specifically created in loop L12,
which contains residues highly conserved among kinesin-related proteins
(42). Mutant analysis of human kinesin revealed that L12 is the major
domain for the kinesin-microtubule interaction (43). Changes of two
hydrophobic residues in the same region of human kinesin to alanine
reduced the microtubule-binding affinity (43). In our assay, the F467A
change reduced the number of microtubules bound to the Cin8p surface
10-fold (Fig. 6). Despite the pronounced effect on microtubule binding,
the gliding velocity produced by Cin8p-F467A was reduced by only 35%
(Table IV). A similar effect was also found for the human kinesin L12
mutants for which the effect on velocity was very mild as compared with
the effect on the affinity to microtubule (43). Our findings indicate
that the L12 region of Cin8p, like kinesin, is important for
microtubule binding. In vivo, cin8-F467A was
found to be defective for bipolar spindle assembly compared with either
wild-type or cin8-3 (at room temperature). Therefore,
changes in different motor domain regions can cause different
phenotypic effects. In mitosis, Cin8p acts prior to anaphase to
assemble spindles and maintain their structural integrity. During
anaphase, Cin8p is almost certainly the most important pole-separating
motor in S. cerevisiae (12). Combined with the mutant study
here, we suggest that microtubule binding ability is important for
spindle assembly, while force production may be important for anaphase
spindle elongation.
In summary, we have developed methods that allow the comparative
studies of motor proteins in vivo and in vitro.
In developing these techniques, we anticipate their applicability to
the study of other motor proteins. As was the case for Cin8p, tagging
with BCP need not interfere with the normal activity of the motor
in vivo. This allows the examination of functional motor
forms rapidly extracted from their endogenous host cell.
2 helix of the motor domain, caused a moderate defect in microtubule binding, but motility was severely affected.
cin8-F467A cells, but not cin8-3 cells, were
greatly impaired in bipolar spindle forming ability. We conclude that
microtubule binding by Cin8p is more important than motility for proper
spindle formation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-factor (Bachem) to liquid media (pH 4), for about 4 h until >85% were unbudded.
Yeast strains and plasmids
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
kip1
double mutants are
not viable, the viability of the tester strain was maintained by a
CIN8 plasmid that also carried CYH2. Despite the
presence of a recessive cyh2 cycloheximide resistance allele
in the genome, this strain is sensitive to cycloheximide due to the
dominant CYH2 allele carried on the plasmid (Fig.
1A). Transformation with a
second plasmid carrying CIN8-BCP, however, allowed cells to
live without the CIN8-CYH2 plasmid and therefore to grow on
cycloheximide-containing media. This indicated that CIN8-BCP
provides CIN8 activity. The cin8
kip1
(CIN8-BCP plasmid) cells were indistinguishable from
cin8
kip1
(CIN8 plasmid) cells for growth
rate and temperature resistance. In addition, we found that Cin8p-BCP
localized to spindle microtubules in a manner indistinguishable from
Cin8p (data not shown). We conclude that Cin8p-BCP acts similarly to
Cin8p in vivo.
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Fig. 1.
Cin8p-BCP is biotinated in yeast cells and
complements the function of wild-type Cin8p. A,
viability on medium supplemented with or without cycloheximide
(Cyh). Growth on cycloheximide medium indicates the ability
of the CIN8 form transformed, indicated on the bottom, to
rescue the viability of cin8 kip1
cells. The
lower spot in each column is a 100-fold dilution
of the upper spot. B, Western blots of
crude yeast extracts run on 7% SDS-acrylamide gel and probed with
streptavidin-conjugated alkaline phosphatase. Left, extracts
are from cells that are either deleted for CIN8 or
expressing Cin8p-BCP from its own promoter carried on a low copy
centromere-containing (CEN) or high copy 2 µm-based (2µ)
plasmid. The ~128-kDa band, absent from the cin8
strain, is consistent with the predicted size of Cin8p-BCP.
Right, comparison between levels of biotinated Cin8p under
different expression conditions. Cin8p-BCP expressed either from the
PGAL promoter or from its own promoter carried on a high copy
(2µ) plasmid. For galactose induction, cells were grown
overnight in medium containing raffinose. At t = 0, 2%
galactose was added to the cultures. Crude extracts were prepared at
the indicated time points. Relative to the level of expression provided
by the endogenous promoter/CEN plasmid construct, the endogenous
promoter/2-µm version and the galactose promoter/CEN version (induced
for 4 h) produced approximately 35- and 500-fold more Cin8p-BCP,
respectively (determined by scanning of film image of blot). The
positions of Cin8p-BCP and pyruvate carboxylase (PC) are
indicated. Strains utilized were as follows: MAY3789, -6013, and -6014 (A); MAY2063, -6013, -6015, and -6018 (B).
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Fig. 2.
Microtubule bundling by Cin8p-BCP. Crude
yeast extract from cells that overexpress Cin8p-BCP were mixed with
taxol-stabilized microtubules. Bundled (arrow) and single
microtubules (arrowheads) can be observed. The strain
utilized was MAY 6015.
Bundling of microtubules by Cin8p
cells or cells overexpressing Cin8p (without the BCP
tag). The main factors that influenced the specificity of this assay
were ionic strength and the concentration of casein, which was used to
block nonspecific interactions. We found that different extracts from
the same yeast strain sometimes required different casein
concentrations to block nonspecific microtubule binding. Therefore, for
every extract preparation we determined the casein concentration that,
in the absence of added nucleotide, would allow high microtubule
binding in the absence of a biotin block and very low binding in the
presence of a biotin block. Reducing NaCl concentrations below 150 mM also increased the nonspecific binding of microtubules.
However, NaCl concentration above 200 mM interfered with
binding of microtubules to Cin8p. The optimal conditions were achieved
with 175 mM NaCl in the buffers for both extract
preparation and assays. The number of specifically bound microtubules
was higher on modified avidin (UltraAvidinTM; see
"Materials and Methods") surfaces than on other avidin- and
streptavidin-coated surfaces.
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Fig. 3.
Microtubule binding to Cin8p-BCP on glass
surfaces. Extracts were prepared from cells that overexpressed (by
PGAL) either biotinated or untagged Cin8p or were deleted for
CIN8. The bars represent the mean ± S.E. of
microtubules bound per 400-µm2 field. For each category,
120 fields were counted (six experiments, 20 fields/category/experiment). For each experiment, one coverslip was
divided into four chambers, which each received one of the four
indicated extracts (bottom of graph). Microtubule
attachment to the glass surface was measured in the absence of added
nucleotides. During adsorption, the total extract protein concentration
in each chamber was 1 mg/ml. For blocking with biotin, the chamber
surface was saturated with biotin after the adsorption of avidin but
prior to the addition of cell extract (see "Materials and
Methods"). Strains utilized were MAY2063, -6015, and -6016.
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Fig. 4.
Cin8p-BCP-catalyzed microtubule
motility. A, time lapse sequence of a field of gliding
microtubules driven by Cin8p-BCP bound to a glass surface. The
white crosses indicate the positions of
microtubule ends at the beginning of the sequence. B,
demonstration that Cin8p exerts force toward the plus ends of
microtubules. The microtubule is marked at its minus end by the large
structure, a sea urchin axoneme fragment that was used as a seed to
grow the microtubule. The leading minus end indicates that Cin8p exerts
force toward the plus end. For both A and B, the
numbers indicate the elapsed time in minutes, and the bar
represents 1 µm. The strain utilized was MAY6015.
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Fig. 5.
Properties of Cin8p-catalyzed microtubule
motility. A, velocity distribution of moving
microtubules driven by Cin8p-BCP on glass surfaces. B,
running length of the microtubules measured in A. For these
data, a total of 85 moving microtubules were measured in four
experiments. The strain utilized was MAY6015.
cells did not show microtubule surface binding in the presence of
AMP-PNP. As expected, AMP-PNP did not support any microtubule gliding. With ADP there were fewer bound microtubule than with ATP or AMP-PNP or
in the absence of added nucleotide (Table III). This observation is
consistent with what is known for conventional kinesin for which
affinity to microtubules is strong in the presence of AMP-PNP or in the
absence of added nucleotide and is reduced in the ADP-bound state. We
also found that 1 mM GTP was unable to support
Cin8p-induced motility. GTP supports motility by kinesin at a reduced
level relative to ATP (40, 41), but tests on BimC motors have not previously been reported.
Nucleotide specificity of microtubule binding and motility
kip1
cells.
In addition, Cin8p expression from the 2-µm plasmid resembles the
cell cycle-specific fluctuations of Cin8p levels found in the wild-type
situation.4
2 helix of human kinesin (42). The second
mutant form is encoded by cin8-F467A, the change occurring
within a region corresponding to the kinesin L12 loop, important for
microtubule interaction (43). Both mutations cause
temperature-sensitive growth at 33° in the absence of the
functionally overlapping KIP1 (data not shown), but both are
clearly compromised at room temperature as well (see below). We could
detect no difference in the expression level of these two mutant forms
compared with wild-type Cin8p when produced at low copy from the
CIN8 promoter (data not shown). Therefore, the phenotypic
effects of these mutations can be attributed to the differences in
their activities. In the experiments described below, we examined
wild-type and mutant forms expressed from PGAL in cells that
were deleted for CIN8. All analyses were performed at room
temperature (~25 °C).
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Fig. 6.
Microtubule capture onto glass surfaces by
Cin8p and mutant forms. A, binding of microtubules to
glass surface in the presence of AMP-PNP. The extracts were made from
cells expressing the indicated CIN8 alleles. The
bars represent the mean ± S.E. of microtubules bound
in 40 (400-µm2) fields. The values here were corrected by
the subtraction of microtubules bound after treatment of the surfaces
with biotin (see Table III, AMP-PNP rows for examples). B,
Western blots of extracts used in A, probed with
streptavidin-conjugated alkaline phosphatase. The induction time
indicates the hours elapsed following the addition of galactose. The
strains utilized were MAY6019, -6020, and -6021.
Microtubule gliding induced by wild-type and mutant Cin8p after ATP
uncaging
-factor block
at G1, synchronized wild-type cells convert monopolar
astral arrays of microtubules into short bipolar spindles, which
subsequently elongate when cells enter anaphase. In these in
vivo experiments, we used cells deleted for KIP1
encoding the functionally overlapping BimC motor. The CIN8
allelic forms were integrated into the genome and expressed from the
native promoter (see "Materials and Methods"). Relative to
CIN8, the cin8-F467A cells were impaired in their
ability to form bipolar spindles; monopolar structures took longer to
disappear, and bipolar structures took longer to appear (Fig. 7,
A and B). The bipolar spindles formed by
cin8-F467A also were shorter than CIN8 (0.5 ± 0.1 versus 1.1 ± 0.1 µm). cin8-F467A
cells were also delayed in their entry into anaphase as evidenced by
the delayed appearance of cells with elongated spindles (>2 µm; Fig.
7C). In contrast, we could not detect any effect of
cin8-3 on spindle assembly at 25 °C, although this
mutant may exhibit a slight anaphase entry delay (Fig. 7, B
and C). Therefore, cin8-F467A, but not cin8-3, significantly reduces the ability of Cin8p to form
bipolar spindles. Further attempts to observe effects on anaphase
proficiency were inconclusive, possibly due to the difficulty in
separating the contribution of the dynein motor, which also contributes
to anaphase pole separation.
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Fig. 7.
In vivo spindle assembly
proficiency by wild-type and mutant Cin8p forms. Cells were
synchronized in G1 with -factor and released into fresh media (at
room temperature), and aliquots were fixed at the indicated times and
observed for microtubule structures by immunofluorescence microscopy.
A, percentage of total cells with monopolar spindles.
B, percentage of total cells with short (
2 µm)
spindles. C, percentage of total cells with long (>2-µm)
spindles.
, kip1
, strain MAY2180;
, kip1
cin8-F467A, strain MAY6017;
, kip1
cin8-3,
strain MAY2330.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2,
the P-loop helix (42). The role of this helix is not known. In the
conditions of our assay, Cin8p-3 was moderately reduced in its ability
to bind microtubules and greatly reduced in its ability to move
microtubules following ATP uncaging. The ability of Cin8p-3 to form
bipolar spindles in vivo (at room temperature) was not
affected, suggesting that its motility defect does not impair its
ability to assemble spindles.
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ACKNOWLEDGEMENTS |
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We thank Tami Kingsbury for providing plasmids and for sequencing the cin8-3 allele, and we thank Cindy Dougherty and Emily Hildebrandt for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM40714 (to M. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Fulbright and Rothschild postdoctoral fellowships.
To whom correspondence should be addressed: Dept. of Biology,
Mudd Hall, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD
21218. Tel.: 410-516-7299; Fax: 410-516-5213; E-mail:
hoyt{at}jhu.edu.
1 T. Kingsbury and M. A. Hoyt, unpublished results.
3 E. Hildebrandt, L. Gheber, and M. A. Hoyt, unpublished observation.
4 L. Gheber, E. Hildebrandt, and M. A. Hoyt, unpublished observations.
5 E. Hildebrandt, T. Kingsbury, and M. A. Hoyt, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
NPE-ATP, P3-(1-(2-nitrophenyl)ethyl)ester;
AMP-PNP, adenosine
5'-(,
-imino)triphosphate.
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REFERENCES |
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