CHO1, a mammalian kinesin-like protein, interacts with F-actin and is involved in the terminal phase of cytokinesis

Abstract

CHO1 is a kinesin-like protein of the mitotic kinesin-like protein (MKLP)1 subfamily present in central spindles and midbodies in mammalian cells. It is different from other subfamily members in that it contains an extra approximately 300 bp in the COOH-terminal tail. Analysis of the chicken genomic sequence showed that heterogeneity is derived from alternative splicing, and exon 18 is expressed in only the CHO1 isoform. CHO1 and its truncated isoform MKLP1 are coexpressed in a single cell. Surprisingly, the sequence encoded by exon 18 possesses a capability to interact with F-actin, suggesting that CHO1 can associate with both microtubule and actin cytoskeletons. Microinjection of exon 18-specific antibodies did not result in any inhibitory effects on karyokinesis and early stages of cytokinesis. However, almost completely separated daughter cells became reunited to form a binulceate cell, suggesting that the exon 18 protein may not have a role in the formation and ingression of the contractile ring in the cortex. Rather, it might be involved directly or indirectly in the membrane events necessary for completion of the terminal phase of cytokinesis.

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The Rockefeller University Press, 0021-9525/2002/3/783/8 $5.00
The Journal of Cell Biology, Volume 156, Number 5, March 4, 2002 783–790
http://www.jcb.org/cgi/doi/10.1083/jcb.200109090
783
Report
CHO1, a mammalian kinesin-like protein, interacts
with F-actin and is involved in the terminal phase
of cytokinesis
Ryoko Kuriyama, Charles Gustus, Yasuhiko Terada, Yumi Uetake, and Jurgita Matuliene
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
HO1 is a kinesin-like protein of the mitotic kinesin-like
protein (MKLP)1 subfamily present in central spindles
and midbodies in mammalian cells. It is different
from other subfamily members in that it contains an extra

300 bp in the COOH-terminal tail. Analysis of the chicken
genomic sequence showed that heterogeneity is derived
from alternative splicing, and exon 18 is expressed in only the
CHO1 isoform. CHO1 and its truncated isoform MKLP1 are
coexpressed in a single cell. Surprisingly, the sequence
encoded by exon 18 possesses a capability to interact
with F-actin, suggesting that CHO1 can associate with both
C
microtubule and actin cytoskeletons. Microinjection of
exon 18–specific antibodies did not result in any inhibitory
effects on karyokinesis and early stages of cytokinesis.
However, almost completely separated daughter cells
became reunited to form a binulceate cell, suggesting that
the exon 18 protein may not have a role in the formation
and ingression of the contractile ring in the cortex. Rather,
it might be involved directly or indirectly in the membrane
events necessary for completion of the terminal phase of
cytokinesis.
Introduction
To ensure high fidelity of chromosome segregation, cells
form a special device called mitotic spindles. The structural
framework of the spindle is microtubules, and their assembly
into the spindle and function in chromosome separation and
cytokinesis are largely dependent on microtubule-based
motor proteins associated with them. A number of kinesin-like
proteins have been identified and further divided into distinct
subclasses (Miki et al., 2001). One such subclass designated
as a mitotic kinesin-like protein (MKLP)*1 subfamily contains
plus-end–directed NH
2
-terminal motor proteins. Since the
first member (CHO1) was identified in CHO cells (Sellitto
and Kuriyama, 1988), other members of this subfamily have
been shown to exist in various organisms, including human
(MKLP1 [Nislow et al., 1992]),
Caenorhabditis elegans
(ZEN-4 [Raich et al., 1998]; CeMKLP1 [Powers et al.,
1998]),
Drosophila
(PAV-KLP [Adams, 1998]), zebrafish
(sequence data available from GenBank/EMBL/DDBJ under
accession no. AF139990), and sea urchin (Chui et al.,
2000). Although all of those proteins are composed of
well-conserved three subdomains (NH
2
-terminal motor
region and COOH-terminal globular tail connected with the

-helical coiled-coil central stalk), CHO1 has been noted to
be unique in a sense that it contains extra

100 amino acids
in the middle of the tail (Kuriyama et al., 1994). Since none
of the homologs have the CHO1-specific tail sequence, it is
not clear whether the proteins are heterogeneous among species
or different forms of MKLP1/CHO1 coexist in a single
cell type.
MKLP1/CHO1-related proteins show dynamic changes
in their subcellular distribution during the cell cycle. In
mammalian cells, CHO1 is present in interphase centrosomes
and nuclei and becomes associated with the mitotic spindle.
As chromosomes move toward poles, the protein shifts to
the midzone and eventually concentrates into a bright spot
in the middle of the intercellular bridge (Sellitto and
Kuriyama, 1988). Since it is a plus-end–directed motor
present in the interzonal region of the spindle, MKLP1/
CHO1 was originally thought to function in chromosome
separation and spindle elongation during karyokinesis
(Nislow et al., 1992). In fact, microinjection of CHO1
antibodies caused mitotic arrest in mammalian cells (Nislow
et al., 1990) and sea urchin embryos (Wright et al., 1993).
However, genetic analysis has suggested involvement of
zen-4
and
pavarotti
in cytokinesis rather than karyokinesis (Adams
Address correspondence to Ryoko Kuriyama, Dept. of Genetics, Cell
Biology, and Development, 6-160 Jackson Hall, 321 Church St. SE,
University of Minnesota, Minneapolis, MN 55455. Tel.: (612) 624-
0471. Fax: (612) 626-6140. E-mail: ryoko@lenti.med.umn.edu
*Abbreviations used in this paper: GFP, green fluorescent protein;
MKLP, mitotic kinesin-like protein.
Key words: actin; alternative splicing; cytokinesis; kinesin-like protein;
midbody

784 The Journal of Cell Biology
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Volume 156, Number 5, 2002
et al., 1998; Powers et al., 1998; Raich et al., 1998). Evi-
dence has also been presented that overexpression of the mu-
tant CHO1 and RNA-mediated interference specifically
blocked completion of cytokinesis in mammalian cells (Ma-
tuliene and Kuriyama, 2002). The ability of the motor pro-
teins to organize central spindles and the midbody appears
to be essential for their function in cytokinesis, which could
be achieved through their interaction with Aurora kinase
AIR-2 (Schumacher et al., 1998; Severson et al., 2001) and
RhoGAP Cyk-4 (Jantsch-Plunger et al., 2000).
To clarify the nature of molecular diversity among species
and define the role of MKLP1/CHO1 during cell division,
we examined the chicken genomic sequence. Here we report
that heterogeneity of the COOH-terminal tail is derived
from alternative splicing and that the sequence encoded by
exon 18 is expressed in only the CHO1 isoform. Exon 18
includes a polypeptide capable of interaction with F-actin in
vivo and in vitro, and microinjection of exon 18–specific an-
tibodies blocked the terminal phase of cytokinesis. A possi-
bility of CHO1 involvement in membrane events, rather
than the actin-containing contractile ring in the cell cortex,
is discussed.
Results and discussion
Coexpression of CHO1 and MKLP1 isoforms generated
by alternative splicing
Although CHO1 is a member of the MKLP1 subfamily, it is
significantly larger in size than other subfamily members.
This suggested a possibility of alternative splicing, which was
confirmed by cloning of chicken genomic sequences (Fig.
1). The chicken CHO1/MKLP1 gene spans

17.1 kb in
which 22 introns intervene between 23 exons flanked by
GT-AG consensus splice sequences (unpublished data). The
coding sequences of the motor (amino acids 1–441), stalk
(amino acids 442–654), and tail (amino acids 655–954) do-
mains are covered by exons 1–13, 13–17, and 17–23, re-
spectively. By comparing with the human sequence available
in the genome databanks, it was concluded that all exon-
intron boundaries occur at the same position between two
species (Fig. 1). Thus, the splicing pattern of CHO1/MKLP1
gene is well conserved in higher eukaryotes. The major di-
versity resides in the tail domain at the COOH terminus
(Fig. 2). Particularly prominent is the difference between
CHO1 and MKLP1, and a nearly 100 amino acid sequence
encoded by exon 18 is expressed in only CHO1 but not the
MKLP1 isoform.
Chicken cDNA encoding both CHO1- and MKLP1-type
proteins was cloned from a library (Fig. 2, GgCHO1 and
GgMKLP1), suggesting that two isoforms are likely to be ex-
pressed in a single organism. Indeed, cDNA fragments cor-
responding to each isoform were identified in EST data-
banks of not only human but also mouse. To confirm
simultaneous expression of CHO1 and MKLP1, we double
labeled CHO (Fig. 3 A) and HeLa (Fig. 3 B) cells with mono-
clonal anti-CHO1 antibody (mAb) and polyclonal anti-
body (E18) specific to exon 18. CHO1 probed by E18
was seen at the center of spindles and midbodies along with
the antigen recognized by mAb, which is reactive to the
COOH-terminal end of the stalk sequence (unpublished
data). Midbodies isolated from CHO cells are also intensely
stained with both types of antibodies (Fig. 3, C and D).
Thus, CHO1 and MKLP1 are coexpressed and tightly at-
tached to the midbody.
mAb recognizes polypeptides of 95 and 105 kD in
CHO and HeLa cells (Fig. 3 E, lanes 1 and 3). In some
occasions, the polypeptides are resolved as multiple bands,
suggesting the possibility of protein phosphorylation (un-
published data). When the same protein fractions were
probed with E18, only the band with the higher molecu-
lar mass was detected (Fig. 3 E, lanes 2 and 4). Both types
of proteins are highly enriched in isolated midbodies (Fig.
3, lanes 5 and 6). It is notable that immunostaining (Fig.
3, A and B) and immunoblotting (Fig. 3 E, lanes 2 and 4)
signals in whole cells obtained with E18 are far less in-
tense than those with mAb. This may suggest that either
CHO1 is minor or midbody staining with E18 is less ef-
fective than with mAb, or both. It was also noted that nei-
Figure 1. MKLP1/CHO1 gene organization in chicken and human. The middle column represents the full-coding chicken cDNA. Numbers
1–23 indicate the position of exons intervening between 22 introns. Although human MKLP1/CHO1 gene (23 kb) is considerably larger
than chicken (17 kb), almost all splicing sites occur at the same position between two species. A shaded column is a CHO1-specific exon
18, and dotted lines represent the gap detected in the human genomic sequence (sequence data available from EMBL/GenBank/DDBJ under
accession no. NT_010222.1; mapped to chromosome 15). Bars: (top and bottom) 5 kb; (middle) 0.5 kb.

CHO1 isoform with an actin-interacting domain |
Kuriyama et al. 785
ther PAV-KLP nor ZEN-4/CeMKLP1 includes the se-
quence corresponding to exon 18 in their genomic
sequences. However, immunostaining of
Drosophila
cul-
tured cells revealed the presence of an E18-reactive mole-
cule (

200 kD) at the center of the intercellular bridge
(unpublished data). Thus, a CHO1-related molecule with
the exon 18 sequence might colocalize with PAV-KLP at
the midzone and midbody in
Drosophila
cells.
Figure 2. Sequence alignment of the tail among MKLP1 subfamily members. In chicken and human, the COOH-terminal tail is encoded by
exons 17–23, which are differently colored. Sequences are derived from chicken (GgCHO1 and GgMKLP1; this paper), CHO cells (CgCH:
sequence data available from EMBL/GenBank/DDBJ under accession no. X83575), human EST (HsCHO1: sequence data available from EMBL/
GenBank/DDBJ under accession nos. AU123817 and BF897861), mouse EST (MmCHO1: sequence data available from EMBL/GenBank/DDBJ
under accession nos. BE333860, AA856173, and BG068324; MmMKLP1: sequence data available from EMBL/GenBank/DDBJ under accession
nos. AA798124 and AW907863), HeLa (HsMKLP1: sequence data available from EMBL/GenBank/DDBJ under accession nos. X67155 and
S46300), zebrafish (DrMKLP1: sequence data available from EMBL/GenBank/DDBJ under accession no. AF139990), sea urchin (SpMKLP1:
sequence data available from EMBL/GenBank/DDBJ under accession no. AAG18582), Drosophila (PAV-KLP: sequence data available from EMBL/
GenBank/DDBJ under accession no. AJ224882), and C. elegans (ZEN-4: sequence data available from EMBL/GenBank/DDBJ under accession nos.
AF057567 and AF057568). The E18 antibody was raised against the oligopeptide sequence encoded by exon 18 (double underlined).

786 The Journal of Cell Biology
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CHO1-specific exon 18 encodes an
actin-interacting domain
To characterize the CHO1-specific sequence, we expressed
91 amino acids encoded by exon 18 in CHO cells. The green
fluorescent protein (GFP)-tagged exogenous polypeptide is
located inside the cytoplasm in association with cytoskeletal
fibers (Fig. 4 A). Double staining with Alexa-phalloidin
(
MFs
) provided evidence that the fibers to which the exon
18 sequence attaches are actin filaments. When cells were
treated with dihydrocytochalasin B, GFP was no longer de-
tected along cytoskeletal fibers (Fig. 4 B,

DHCB
). Instead,
actin-containing dots formed by depolymerization of F-actin
become visible by GFP fluorescence. In contrast, the exon 18
polypeptide does not reveal any affinity to the microtubule
network (Fig. 4 C). This is in good agreement with our pre-
vious observation that CHO1 interacts with microtubules
through microtubule-binding sites located at the NH
2
-termi-
nal half of the protein (Matuliene and Kuriyama, 2002).
Affinity of the exon 18 sequence for F-actin was further
confirmed by in vitro cosedimentation. Fig. 4 D shows
CHO (lanes 1–4) and insect Sf9 cell lysates (lanes 5–8) con-
taining the GFP-tagged exon 18 sequence mixed with F-actin
prepared from rabbit skeletal muscles: the polypeptide
was cosedimented with F-actin (lanes 4 and 8). Lanes 9–16
demonstrate interaction of F-actin with
35
S-labeled GFP–
exon 18 synthesized in vitro: the protein was recovered in
the pellet along with F-actin (lane 14), whereas the control
35
S-luciferase remained in the supernatant (lane 16). Associ-
ation of GFP–exon 18 was also confirmed with platelet
F-actin (unpublished data). These results clearly indicate
that the sequence unique to the CHO1 isoform encodes a
polypeptide capable of interaction with F-actin. Exon 18
does not appear to contain any known consensus motifs
shared among actin-binding proteins. Thus, the question of
whether CHO1 interacts with F-actin directly or indirectly
still remains to be answered.
CHO1 is seen inside the nucleus, which is due to the pres-
ence of a nuclear localization signal at the COOH-terminal
end of the tail (unpublished data). When the full-length
CHO1 lacking nuclear localization signal was expressed in
interphase cells, the tagged molecule was located in the cyto-
plasm in association with both microtubules and F-actin
(unpublished data). However, in mitotic cells the protein
was seen predominantly in the spindle, and virtually no fluo-
rescent signal was detected in the cortex where the actin-con-
taining contractile ring is assembled. Since the exon 18 se-
quence was targeted to the cortex, especially in the vicinity of
the furrow during cytokinesis in mitotic cells (unpublished
data), the interaction of CHO1 with actin-containing struc-
tures may be controlled in a cell cycle–dependent manner.
Figure 3. Midbody staining with domain-specific antibodies. (A–D) Localization of the CHO1 isoform in CHO (A), HeLa cells (B), and isolated
CHO midbodies (C and D) probed by E18 antibodies. The same cells/structures were also stained with either mAb (A, B, and D) or anti–-tubulin
antibody (C). Bars, 10 m. (E) Immunoblot analysis of whole CHO (lanes 1 and 2), HeLa cells (lanes 3and 4), and isolated midbodies (lanes 5
and 6) stained with mAb (lanes 1, 3, and 5) and E18 (lanes 2, 4, and 6).

CHO1 isoform with an actin-interacting domain |
Kuriyama et al. 787
Cells treated with E18 antibodies failed to complete
the terminal phase of cytokinesis
By mutating the mechanochemical motor domain of
CHO1, we have shown previously that the protein is re-
quired for completion of cytoplasmic division in mamma-
lian cells (Matuliene and Kuriyama, 2002). To examine
whether inhibition of the actin-interacting domain results
in the similar phenotypes, we microinjected the affinity
purified E18 antibody into PtK
1
cells. In Fig. 5, the
metaphase cell received E18 at time zero (Fig. 5 A) under-
went normal chromosomes separation (Fig. 5 C) and cyto-
plasmic division (Fig. 5 D). Although two daughter cells
appeared to be separated completely, the cell boundary be-
came unclear (Fig. 5, E and F), and the two cells eventually
merged together by

3 h after antibody injection (Fig. 5
G). mAb staining of the fixed cell clearly indicated the for-
mation of a midbody between two separated nuclei (Fig. 5,
arrows). Nonetheless, the furrow ultimately resumed to
produce a binucleate cell (Fig. 5, DAPI), suggesting that
E18 specifically inhibits the terminal phase of cytokinesis.
The effect of E18 was specific because no major inhibition
of cell division was observed in cells treated with injection
buffer alone, commercially available nonspecific rabbit
IgG, and protein A–purified E18 preimmune antibodies
(Table I). These results contrast with those of Nislow et al.
(1990) and Wright et al. (1993) who observed mitotic ar-
rest in mAb-injected cells. The difference may be attrib-
uted to the specificity of the antibodies injected into cells
(mouse monoclonal IgM recognizing the central stalk ver-
sus rabbit E18 IgG specific to the tail). The role of
MKLP1/CHO1 in early stages of mitosis is left unsolved.
Initiation and ingression of cleavage furrows proceeded
normally in E18-injected cells, suggesting that the actin-
interacting site of CHO1 may not be involved in the for-
mation of contractile ring and the process of cell cleavage.
Rather, the protein may function to link the microtubule-
containing central spindle to the cortex/membrane at the
cell equator during the late stage of cell division. Time-
lapse analysis of dividing cells suggested that the midbody
matrix could be a structure holding microtubules and the
cell cortex/membrane together after the cessation of the
furrowing and the disassembly of the contractile ring
(Mullins and Biesele, 1977). Both the motor activity and
microtubule-bundling capacity of CHO1 are required for
completion of cytokinesis by organizing midzone microtu-
bules and the electron-dense matrix in the center of the in-
tercellular bridge (Matuliene and Kuriyama, 2002). Old
and new observations clearly indicate that complete separa-
tion of two daughter cells is achieved through stretching
and breaking the intercellular bridge at the narrowest re-
gion flanking the midbody center (Mullins and Biesele,
1977; Piel et al., 2001). Since microtubules derived from
the remnant of the central spindle are still connected to
each daughter cell, the microtubule bundle forming a core
of intercellular bridge must be under tension between two
cells. Therefore, for successful separation of daughter cells
Figure 4. Interaction of GFP–exon 18
protein with F-actin. (A–C) CHO cells
expressing the GFP-tagged exon 18
polypeptide were stained with
Alexa-conjugated phalloidin (A and B)
and -tubulin antibodies (C). In B, the
cells were treated with 5 g/ml
dihydrocytochalasin B (DHCB) for 30
min to depolymerize F-actin before
fixation. Bar, 10 m. (D) The proteins
expressed in CHO (lanes 1–4), Sf9 cells
(lanes 5–8), and reticulocyte lysates
(lanes 9–16) were mixed with () or
without () F-actin. Lanes 11, 12, 15,
and 16 include
35
S-labeled luciferase
prepared as a control protein. GFP–exon
18 bands in supernatant (S) and pellet (P)
fractions were visualized by either
immunostaining with anti-GFP antibodies
(lanes 1–8) or autoradiography (lanes
9–16). Note GFP–exon 18 prepared in
the reticulocyte lysate (lane 10) was
more easily sedimentable than that
expressed in CHO (lane 2) and Sf9 cells
(lane 6) without incubation with F-actin.

788 The Journal of Cell Biology
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Figure 5. Microinjection of E18 antibodies into PtK
1
cells. Numbers at the corner are the time after antibody injection. After fixation with
cold methanol, the cells were stained with mAb (probed by FITC-conjugated secondary antibodies, but its color was converted from green to
red), DAPI, and anti-rabbit IgG secondary antibody (E18). The coverslip was further stained with anti–-tubulin and FITC-conjugated secondary
antibodies (MTs/mAb). Arrows indicate the position of a midbody formed between two separated nuclei. Bar, 50 m.
Table I.
Effects of antibody injection into mitotic PtK
1
cells
Cells counted Normal cell division Incomplete cell division
a
Two cells appositioned
b
Arrested/no changes
c
(%)
E18
d
73 6(8) 41(56) 22(30) 4(5)
E18-pre
e
29 26(90) 0(0) 1(3) 2(7)
Control
f
41 35(85) 5(12) 0(0) 1(2)
Injection buffer
g
12 9(75) 0(0) 0(0) 3(25)
a
Although the cells underwent normal chromosome segregation and cytokinesis, regression of the cleavage furrow and/or cell fusion resulted in the forma-
tion of multinucleate cells.
b
Two almost completely separated cells were closely aligned side-by-side. A part of the cell boundary was unclear, and the two cells appeared to be par-
tially connected to each other.
c
The cells either arrested at mitosis or entered interphase without cell division.
d
5–13 mg/ml affinity purified E18 rabbit immunoglobulin molecules were prepared in injection buffer and introduced into PtK
1
cells at prophase to
anaphase. The cells were fixed at 2–6 h after antibody injection.
e
Protein A–purified preimmune E18 antibodies.
f
Two different batches of rabbit IgG fractions, which are commercially available.
g
Injection buffer alone.

CHO1 isoform with an actin-interacting domain |
Kuriyama et al. 789
the microtubule rope must be tightly attached to the mem-
brane/cortex during this tug-of-war. It might be CHO1
that is responsible for connection between the midbody
matrix and cell membrane.
Alternatively, the motor protein could be involved in
membrane events necessary for completion of cytokinesis.
It is widely believed that targeted secretion, insertion, and
fusion of membrane vesicles play a central role in progres-
sion and completion of cleavage furrows (for review see
Straight and Field, 2000). Thus, cells lacking syntaxins and
their associated proteins (Lukowitz et al., 1996; Heese et
al., 2001), a phospholipid kinase (Brill et al., 2000), dy-
namin (Gu and Verma, 1996), or Golgi-associated pro-
teins (Sisson et al., 2000), are defective in cytokinesis in a
variety of cells. Rabkinesin-6/Rab6-KIFL, a Rab6-binding
kinesin-like protein, is required for not only membrane
traffic but also cytokinesis (Hill et al., 2000). Of particular
interest is that this motor protein shares the highest degree
of sequence identity to CHO1 (Echard et al., 1998). Skop
et al. (2001) have reported recently that brefeldin, a potent
inhibitor of vesicle secretion by targeting a small GTPase-
binding protein Arf, specifically blocks the terminal phase
of cytokinesis by regressing ingressed furrows in
C. elegans
.
The phenotype detected in brefeldin-treated embryos is re-
markably similar to what we saw in E18-injected mamma-
lian cells. Importantly, CHO1/MKLP1 is capable of bind-
ing Arfs through the sequence encoded by exons 19–22,
just downstream the actin-interacting domain of exon 18
(Boman et al., 1999). Since dividing cells contain dynamic
membrane phospholipids tightly coupled with the actin
cytoskeleton (Emoto and Umeda, 2000) and Arfs are be-
lieved to be involved in both membrane traffic and actin
dynamics, it is likely that the actin- and Arf-interacting do-
mains act in concert to achieve the CHO1 function during
the late stage of cytokinesis. Further analysis of CHO1
would be of great benefit for our understanding of the
mechanism and regulation of cell division.
Materials and methods
Cloning of cDNA and genomic sequences of
chicken CHO1 and MKLP1
cDNA encoding chicken CHO1- and MKLP1-type of motor proteins were
cloned by screening of a chicken cDNA library (Stratagene) with CHO1
probes derived from CHO cells (Kuriyama et al., 1994). To extend the 5

sequence, mRNA was purified from cultured chicken lymphocytes (DT40
cells) and used for 5

-RACE with specific and degenerate primers matching
the conserved ATP-binding consensus motif (amino acids 118–124). For
cloning of genomic DNA, 10 primers (nucleotide positions 14
→
42, 3891
←
3927, 3891
→
3927, 7031
←
7073, 6294
→
6328, 9635
←
9667, 9594
→
9633, 13863
←
13902, 13851
→
13892, and 17097
←
17138) were
designed based on the nucleotide sequence of CHO1/MKLP1, and PCR
was performed with DT40 genomic templates. Five fragments in a size be-
tween 3.2 and 4.3 kb were cloned and assembled after nucleotide se-
quence analysis.
Cell culture, protein expression, and immunostaining
CHO and HeLa cells were cultured in 10% FCS containing Ham’s F-10
and DME medium, respectively. The exon 18 coding sequence (amino
acid positions 696–786) was isolated by digestion of the full-length CHO-
CHO1 with SspI and Eam1104 I and ligated into the eukaryotic expression
vector, pEGFP-C1 (CLONTECH Laboratories, Inc.). CHO cells on a cover-
slip in a 35-mm dish were transfected by addition of 0.6–2

g/ml purified
plasmid DNA and cultured overnight (Matuliene and Kuriyama, 2002). To
enrich mitotic cell populations, cells were partially synchronized by treat-
ment with thymidine followed by accumulation at M phase by addition of
nocodazole at a final concentration of 0.05

g/ml (Sellitto and Kuriyama,
1988). After washing out the drug, cells were allowed to recover for 20–50
min before fixation with cold methanol.
For immunofluorescence staining, cells were rehydrated with 0.05%
Tween-20 containing PBS and incubated with primary and secondary anti-
bodies. Primary antibodies include monoclonal anti–

-tubulin antibodies
(Sigma-Aldrich), monoclonal CHO1 (mAb [Sellitto and Kuriyama, 1988]),
and rabbit polyclonal antibodies (E18) raised against 23 amino acids in
exon 18 at amino acid positions 755–777 of chicken CHO1 (Fig. 2, double
underlined). Immunoblotting analysis was done as described before
(Kuriyama et al., 1994).
In vitro cosedimentation with F-actin
Actin was prepared from rabbit skeletal muscles (a gift from Drs. Albina
Orlova and Ewa Prochniewicz, University of Minnesota, Minneapolis,
MN) (Orlova et al., 1995), and platelet actin was purchased from Cytoskel-
eton, Inc. G-actin was polymerized and sedimented in a medium contain-
ing 5 mM Tris-HCl, pH 7.8, 0.2 mM ATP, 0.1 mM CaCl
2,
150 mM KCl,
and 1 mM MgCl
2
. For protein expression in insect cells, cDNA encoding
GFP–exon 18 was subloned into pVL1392 (PharMingen) and used for in-
fection of Sf9 cells as before (Kuriyama et al., 1994).
35
S-labeled GFP–exon
18 was synthesized using a commercially available kit (TNT Coupled
Reticulocyte Lysate System; Promega). CHO and Sf9 cells expressing exog-
enous proteins were washed once with PBS and lysed for 30–60 min at
0

C in a medium containing 10 mM Tris-HCl, pH 7.8, 0.5% Nonidet NP-
40, and protease inhibitors. Cell extracts and reticulocyte lysates recovered
after centrifugation at 200,000
g
for 30 min were mixed with F-actin and
further incubated for 30 min on ice. After layering on a 20% sucrose cush-
ion, F-actin plus associated proteins were sedimented at 100,000
g
for 30
min at 4

C.
Antibody injection
Affinity purified E18 was prepared in injection buffer (140 mM KCl, 100
mM glutamic acid, 40 mM citric acid, 1 mM MgCl
2
, 1 mM EGTA, pH 7.4)
and injected into PtK
1
cells cultured on a photoetched coverslip (Bellco).
Microinjection was performed using a Narishige microinjector attached to
a Nikon Diaphot inverted microscope, and time-lapse images were re-
corded with a Nikon Eclipse TE200 microscope using ImagePro software
packages.
This work was supported by National Institutes of Health grant GM55735
to R. Kuriyama.
Submitted: 24 September 2001
Revised: 18 January 2002
Accepted: 18 January 2002
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