Download A Novel Plant Kinesin-Related Protein Specifically

The Plant Cell, Vol. 13, 2427–2439, November 2001, www.plantcell.org © 2001 American Society of Plant Biologists
A Novel Plant Kinesin-Related Protein Specifically Associates
with the Phragmoplast Organelles
Y.-R. Julie Lee, Hoa M. Giang, and Bo Liu1
Section of Plant Biology, University of California, Davis, California 95616-8537
In higher plants, the formation of the cell plate during cytokinesis requires coordinated microtubule (MT) reorganization
and vesicle transport in the phragmoplast. MT-based kinesin motors are important players in both processes. To understand the mechanisms underlying plant cytokinesis, we have identified AtPAKRP2 (for Arabidopsis thaliana phragmoplast-associated kinesin-related protein 2). AtPAKRP2 is an ungrouped N-terminal motor kinesin. It first appeared in
a punctate pattern among interzonal MTs during late anaphase. When the phragmoplast MT array appeared in a mirror
pair, AtPAKRP2 became more concentrated near the division site, and additional signal could be detected elsewhere in
the phragmoplast. In contrast, the previously identified AtPAKRP1 protein is associated specifically with bundles of
MTs in the phragmoplast at or near their plus ends. Localization of the tobacco homolog(s) of AtPAKRP2 was altered
by treatment of brefeldin A in BY-2 cells. We discuss the possibility that AtPAKRP1 plays a role in establishing and/or
maintaining the phragmoplast MT array, and AtPAKRP2 may contribute to the transport of Golgi-derived vesicles in the
phragmoplast.
INTRODUCTION
In higher plants, cytokinesis involves the formation of the cell
plate, which depends on the cytokinetic apparatus of the
phragmoplast. The phragmoplast has a framework of microtubules (MTs) that are oriented perpendicular to the division
plane by having their plus ends at or near the division site.
Phragmoplast MTs are highly dynamic, as revealed by fluorescent analog histochemistry and the green fluorescent
protein–tagging approach (Zhang et al., 1993; Granger and
Cyr, 2000; Hasezawa et al., 2000). Derived from interzonal
MTs of the anaphase spindle, phragmoplast MTs are arranged in the pattern of a solid cylinder before the cell plate
is formed. Concomitant with the centrifugal buildup of the
cell plate, phragmoplast MTs depolymerize in the region in
which the cell plate has been formed. Remarkably, at the
same time, new MTs form at the periphery of the phragmoplast. During the whole process of phragmoplast development, MT polymerization takes place at the MT plus ends,
and new MT segments are constantly translocated away
from the division site (Vantard et al., 1990; Asada et al.,
1991). It has been shown that stabilization of MTs with taxol
prevents the centrifugal growth of the phragmoplast MT array (Yasuhara et al., 1993). The disassembly of phragmoplast MTs requires the arrival of Golgi-derived vesicles,
1 To
whom correspondence should be addressed. E-mail bliu@ucdavis.
edu; fax 530-752-5410.
Article, publication date, and citation information can be found at
www.aspb.org/cgi/doi/10.1105/tpc.010225.
because disruption of the Golgi apparatus with brefeldin A
prevents the depolymerization event in the center of the
phragmoplast (Yasuhara and Shibaoka, 2000).
During cell plate formation, Golgi-derived vesicles are
transported rapidly along the MTs toward their plus ends.
The vesicles contain xyloglucans and other components
that contribute to the cell plate (Samuels et al., 1995; Otegui
and Staehelin, 2000; Sonobe et al., 2000; Verma, 2001).
Vesicle transport along MTs depends on MT-based motor
proteins, the dyneins and the kinesins (Goldstein and Philp,
1999). Although a number of kinesin-related proteins (KRPs)
have been reported in higher plants (Reddy, 2001), the motor(s) responsible for vesicle transport in the phragmoplast
has not yet been identified.
Motor proteins play critical roles in multiple processes
during cell division (Sharp et al., 2000; Wittmann et al.,
2001). Kinesin and KRPs are members of the kinesin superfamily (Kim and Endow, 2000). In higher plants, several
KRPs have been localized to the phragmoplast (Liu and Lee,
2001). Plus end–directed KRPs in the Block-In-Mitosis C
protein (BIMC) subfamily are required for MT translocation in
the phragmoplast, likely by a sliding filament mechanism
(Asada et al., 1997; Sharp et al., 1999). One such KRP from
carrot especially concentrates in the midline of the phragmoplast MTs (Barroso et al., 2000). Minus end–directed
KRPs from the C-terminal motor KRP subfamily decorate
phragmoplast MTs as well, but their roles have not been determined (Liu and Palevitz, 1996; Liu et al., 1996; Mitsui et
al., 1996; Bowser and Reddy, 1997; Smirnova et al., 1998).
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Among them, Kinesin-like Calmodulin-Binding Protein
(KCBP) appears to be inactivated, possibly by Ca 2/calmodulin binding in the phragmoplast, although it appears to
be associated with the phragmoplast MTs (Vos et al., 2000).
In a previous study, we identified AtPAKRP1 (for Arabidopsis thaliana phragmoplast-associated kinesin-related
protein 1), an N-terminal motor KRP that does not resemble
KRPs from other organisms (Lee and Liu, 2000). It does not
associate with the preprophase band or the mitotic spindle.
During late anaphase, however, it appears along interzonal
MTs. Later, it decorates phragmoplast MTs at or near their
plus ends. AtPAKRP1 likely plays a role in establishing and/
or maintaining the phragmoplast MT array.
One of the central questions about phragmoplast operation is what the force generator(s) for unidirectional vesicle
transport is during cell plate formation. Here, we report a
phragmoplast-specific KRP, AtPAKRP2 (Arabidopsis phragmoplast-associated kinesin-related protein 2), which is named
based on its intracellular localization pattern. AtPAKRP2 has
distinct structure and localization patterns that differ from
those of AtPAKRP1. On the basis of pharmacological data,
we suggest that AtPAKRP2 is a candidate motor for transporting Golgi-derived vesicles toward the division site.
RESULTS
Isolation of AtPAKRP2 cDNA
To identify KRPs in Arabidopsis, the expressed sequence
tag clone OAO358 from green shoots (GenBank accession
number Z34049) was obtained because it overlapped with
the AtFCA1.2 locus, which was predicted to encode a KRP
(GenBank accession number Z97336). After being sequenced, however, the OAO358 clone did not show a fulllength open reading frame. To determine the 5 end of the
coding sequence, 5 rapid amplification of cDNA ends
(RACE) experiments were performed. We predicted that the
full-length open reading frame would encode a polypeptide
of 869 amino acids, which has been named AtPAKRP2
(Figure1). The deduced polypeptide has a calculated molecular mass of 97 kD and a pI of 5.8.
The deduced AtPAKRP2 sequence showed a number of
differences from the amino acid sequence of the AtFCA1.2
locus annotated by the Arabidopsis Genome Initiative, especially toward the C terminus. This difference was caused
by the discrepancy of intron/exon prediction by the Arabidopsis Genome Initiative and the cDNA sequence determined in the present study. The N-terminal part of AtPAKRP2
(amino acids 32 to 375) resembled the kinesin motor domain, although it had some unusual features (Figure 1). Typically, the motor domain of kinesin/KRPs contains an ATP
binding site, which includes a highly conserved peptide of
IF/VAYGQTGA/SGKS/T. The corresponding sequence in
AtPAKRP2, however, was IMMYGPTGAGKS. Nevertheless,
the AtPAKRP2 motor domain clearly had well-conserved
motifs, such as SSRSH, LVDMAGSE, and HVPFRDSKL,
which have been found commonly in the MT binding site.
To date, identified KRPs are grouped into nine different
subfamilies plus some ungrouped ones (Kim and Endow,
2000). To analyze the relationship between AtPAKRP2 and
other KRPs in the kinesin superfamily, phylogenetic analysis
was performed by aligning its motor sequence and the mo-
Figure 1. AtPARKP2 Is a Divergent KRP.
(A) The amino acid sequence was deduced from AtPAKRP2 cDNA clones. The N-terminal region similar to KRP motor domains is shaded. The
highly conserved peptides in the motor domain are highlighted with black background.
(B) Phylogenetic tree built from a sequence alignment of KRP motor domains.
A Novel Phragmoplast-Associated Kinesin
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tor sequences of 17 representative kinesin/KRPs, including
most if not all reported plant KRPs. First, AtPAKRP2 was
very divergent from any of these plant KRPs (Figure 1B).
Second, AtPAKRP2 did not fall into any of the existing subfamilies. Therefore, we considered AtPAKRP2 an ungrouped
or orphan KRP, like many others such as the Saccharomyces cerevisiae Smy1p protein (http://www.blocks.fhcrc.org/
~kinesin/BE6c_ungrouped.html).
Many kinesin/KRPs bear coiled-coil domains required for
oligomerization. To further analyze whether AtPAKRP2 might
oligomerize, its polypeptide sequence was analyzed for the
probability of the formation of coiled coils. Coiled coils
could be formed near the central region of the polypeptide
(amino acids 390 to 517; P 0.5) (Figure 2A). The C-terminal region after the coiled coils does not have significant
similarity to any available sequence in GenBank, nor is any
structural motif found in this so-called tail domain. A typical
tripartite structure was predicted for AtPAKRP2 (Figure 2B).
It would not be surprising if AtPAKRP2 formed a dimer in its
native form, given such a tripartite structure.
AtPAKRP2 Binds to MTs in an ATP-Dependent Manner
Because AtPAKRP2 is very divergent from other KRPs, and
the ATP binding site of AtPAKRP2 had three amino acid
substitutions compared with the majority of kinesin superfamily members, we wondered whether it could still behave
like a motor protein. Most members of the kinesin superfamily bind to MTs in an ATP-dependent manner. A glutathione S-transferase (GST) fusion protein (GST-AtPAKRP2-M)
containing the AtPAKRP2 motor domain (amino acids 1 to
435) was expressed in Escherichia coli and affinity purified
(Figure 2B). This fusion protein was subjected to MT cosedimentation experiments as described previously (Lee and
Liu, 2000). In the absence of exogenous nucleotide, GSTAtPAKRP2-M largely cosedimented with the MT pellet (Figure 2C). The presence of ATP rendered at least 50% of the
fusion protein in the supernatant instead of in the MT pellet
(Figure 2C). When ATP was replaced with the nonhydrolyzable ATP analog adenylylimidodiphosphate (AMPPNP), the
fusion protein appeared almost exclusively in the MT pellet
(Figure 2C). Apparently, the fusion protein itself did not sediment (Figure 2C). In a control experiment, the fusion tag
GST alone did not show any sign of cosedimentation with
MTs (data not shown). Therefore, we concluded that
AtPAKRP2 behaved like a typical kinesin/KRP in vitro.
Generation of Anti-AtPAKRP2 Antibodies
Antibodies against two independent regions of AtPAKRP2
were generated. Affinity-purified anti-PAKRP2-N and antiPAKRP2-T antibodies from four different animals gave
identical intracellular localization patterns in dividing Arabidopsis cells (see below). Therefore, we concluded that both
Figure 2. AtPAKRP2 Structure and Its ATP-Dependent MT Binding
Activity.
(A) Coiled-coil domains of AtPAKRP2 were predicted by the Lupus
algorithm. The x axis represents the amino acid residue number, and
the y axis represents the probability of the formation of coiled coils.
A P value 0.5 indicated that the region likely forms a coiled coil.
(B) Schemes of AtPARKP2 and its protein constructs used for MT
binding and antibody preparation. The relative positions of the motor, coiled coil, and tail are indicated.
(C) The MT binding property of the GST-AtPAKRP2-M fusion protein
was demonstrated by cosedimentation with MTs. The resulting supernatant (S) and pellet (P) were analyzed by SDS-PAGE and visualized by Coomassie blue staining. (), the GST-AtPARKP2-M fusion
protein without addition of MTs; MTs, the fusion protein incubated
with MTs alone; MTs ATP, the fusion protein incubated with MTs
and 2 M ATP; MTs AMPPNP, the fusion protein incubated with
MTs and 2 M AMPPNP.
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anti-PAKRP2-N and anti-PAKRP2-T antibodies recognized
the native AtPAKRP2 protein.
Immunoblotting experiments were performed to detect
the AtPAKRP2 polypeptide from protein extracts of etiolated
Arabidopsis seedlings and young inflorescence buds. We
could not detect the protein in extracts from etiolated seedlings. But in protein extracts of young inflorescence buds, a
single 88-kD band was detected with anti-PAKRP2-N antibodies (Figure 3A). Unfortunately, the anti-PAKRP2-T antibodies did not work in immunoblotting, possibly because of
the availability of the corresponding epitope(s) in our protein
preparations. Because of the difference in the apparent molecular mass and the calculated 97 kD, we wanted to verify
whether this 88-kD band was in fact the AtPAKRP2 protein.
We preincubated the antibodies with the AtPAKRP2-N fusion polypeptide before they were applied to the protein
blots. The preincubation prevented the 88-kD band from being revealed (Figure 3A). These results led us to believe that
our antibodies had recognized the native AtPAKRP2 protein.
AtPAKRP2 Is Concentrated in the Phragmoplast
Figure 3. Immunodetection of the AtPAKRP2 Protein in Arabidopsis.
(A) Immunoblots containing proteins extracted from inflorescence
buds were stained with affinity-purified anti-AtPAKRP2-N antibodies (lane 1) or anti-AtPAKRP2-N antibodies incubated with GSTAtPAKRP2-N proteins before immunoblot staining (lane 2). Molecular mass markers (kD) are shown at left.
(B) Intracellular localization of AtPAKRP2 in an Arabidopsis root tip
cell at cytokinesis. AtPAKRP2 is shown in green, and DNA is shown
in blue.
(C) Anti–AtPAKRP2-N staining was absent in an Arabidopsis root
tip cell at telophase when the antibodies were incubated with the
GST-AtPAKRP2-N protein before immunofluorescence staining.
(D) MTs (red) and DNA (blue) in the same cell as in (C). Bar 5 m
for (B) to (D).
To gain insights into the function of AtPAKRP2 in plant cells,
we used purified anti-AtPAKRP2-N and anti-AtPAKRP2-T
antibodies to perform immunolocalization experiments in
cells from three different species. Although both antibodies
gave identical localization signals in Arabidopsis and Brassica oleracea cells, only anti-AtPAKRP2-N antibodies worked
in tobacco BY-2 cells.
When the purified antibodies were used for immunofluorescence in Arabidopsis root tip cells, they labeled the central region of telophase cells (Figure 3B). To verify the
specificity of the purified antibodies for immunolocalization,
the anti-AtPAKRP2-N antibodies were subjected to preincubation with the AtPAKRP2-N polypeptide before they
were applied to the cells. The preincubation completely
abolished the localization signal (Figure 3C). Identical results
were obtained when the anti-AtPAKRP2-T antibodies were
used (data not shown).
Because the AtPAKRP2 protein obviously was present in
the dividing cells, we wondered whether its localization
changed dynamically during the cell division cycle by using
Arabidopsis root tip cells that were dividing actively (Figures
4 and 5). Dual localizations of AtPAKRP2 and tubulin were
performed. During interphase, AtPAKRP2 was present
throughout the cytoplasm and the nucleus, with a concentration of signals at or near the periphery of the nucleus (Figures 4A to 4D). During the progression of mitosis, no signal
was detected in the MT preprophase band and in MTs on
the nuclear envelope (data not shown). In metaphase cells,
judged by their spindle MTs, very little if any anti-AtPAKRP2
signal could be detected with MT bundles by fluorescence
microscopy (Figures 4E to 4H). The same was true for early
anaphase cells (data not shown). When sister chromatids
were separated completely during anaphase, AtPAKRP2
appeared among the interzonal MTs between segregated
chromatids (Figures 4I to 4L). Unlike clear bundles of MTs in
the spindle midzone, AtPAKRP2 appeared in a punctate
pattern among these MT bundles.
After the completion of anaphase, MTs coalesced and
were organized into an early phragmoplast array with a dark
A Novel Phragmoplast-Associated Kinesin
midline between the two mirror halves, as judged by antitubulin staining (Figure 5B). At this stage, AtPAKRP2 appeared very concentrated at the midline of the phragmoplast, and more punctate AtPAKRP2 signal could be
detected weakening away from this midline (Figures 5A to
5D). Although the phragmoplast MT array expanded toward
the cell periphery, AtPAKRP2 remained in a punctate pattern among phragmoplast MTs, with a peak of fluorescence
intensity near the midline (Figures 5E to 5H). The expansion
of AtPAKRP2 signal was concomitant with the expansion of
the MT array. MTs depolymerized from the central region of
the phragmoplast, as indicated by the anti-tubulin fluorescence signal (Figure 5J) upon the formation of daughter nuclei. At this stage, AtPAKRP2 remained distributed among
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phragmoplast MTs by being pronounced toward the midline, and the anti-AtPAKRP2 signal was more obvious where
more MTs were present toward the periphery of the phragmoplast (Figures 5I and 5L). In the area in which MTs had
been completely depolymerized, there was little if any
AtPAKRP2 signal (Figure 5I). Therefore, the results suggested that AtPAKRP2 localization correlated with phragmoplast MT organization.
Because AtPAKRP2 behaved like a typical kinesin motor
localized among phragmoplast MTs, we tested whether the
localization was dependent on MTs. The MT depolymerization agent amiprophos methyl (APM) was used to depolymerize MTs in root cells. After Arabidopsis seedlings were
treated with 100 M APM for 2 hr, no filamentous structure
Figure 4. AtPAKRP2 Was Localized among Interzonal MTs.
Triple localizations of AtPAKRP2 with anti-AtPAKRP2-N ([A], [E], and [I]), of MTs with anti–-tubulin ([B], [F], and [J]), and of DNA with DAPI
([C], [G], and [K]) are shown in Arabidopsis root tip cells at interphase ([A] to [D]), metaphase ([E] to [H]), and late anaphase ([I] to [L]). The
pseudocolored composite images ([D], [H], and [L]) show AtPAKRP2 in green, -tubulin in red, and DNA in blue. Bar in (L) 5 m for (A) to (L).
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Figure 5. Association of AtPAKRP2 with the Phragmoplast.
Triple localizations of AtPAKRP2 with anti-AtPAKRP2-N ([A], [E], and [I]), of MTs with anti--tubulin ([B], [F], and [J]), and of DNA with DAPI
([C], [G], and [K]) are shown in Arabidopsis cells during telophase/cytokinesis. Composite images ([D], [H], and [L]) are pseudocolored with
AtPAKRP2 shown in green, -tubulin shown in red, and DNA shown in blue. Bar in (L) 5 m for (A) to (L).
was detected in the root cells by anti-tubulin immunofluorescence, indicating that MTs had been depolymerized
completely (Figure 6B). In this APM-treated cell with reforming daughter nuclei indicated by 4 ,6-diamidino-2-phenylindole (DAPI) staining (Figure 6C), AtPAKRP2 no longer had
distinct localization between the nuclei; rather, it became
distributed diffusely across the cell (Figure 6A). In the control
cells treated with the identical amount of isopropanol used
to dissolve APM, intact MTs and phragmoplast-localized
AtPAKRP2 were observed (Figures 6D to 6F). Therefore, we
concluded that the localization of the AtPAKRP2 protein in
these root cells was dependent on the integrity of MTs.
Distinct Localization Patterns of AtPAKRP2
and AtPAKRP1
In a previous study, another Arabidopsis motor, AtPAKRP1,
was shown to localize in the midline of the phragmoplast
MTs (Lee and Liu, 2000). We wanted to determine whether
AtPAKRP2 colocalized with AtPAKRP1 in the phragmoplast.
Dual localizations of AtPAKRP2 and AtPAKRP1 were performed in Arabidopsis cells at different stages of cell divi-
sion (Figure 7). At an early stage of cytokinesis, as judged by
the appearance of the reforming daughter nuclei (Figure 7C),
AtPAKRP2 was detected as particles with different sizes between two daughter nuclei (Figure 7A). Compared with the
punctate localization of AtPAKRP2 in the phragmoplast,
AtPAKRP1 was much more discrete and concentrated in
the midline (Figures 7A and 7B). At a later stage of cytokinesis, the punctate localization pattern of AtPAKRP2 was still
clear in the phragmoplast (Figure 7D). AtPAKRP1, however,
remained exclusively at the equatorial plane (Figure 7E). We
realized the limitation of the resolution by immunofluorescence and did not intend to exclude the possibility of partial
colocalization of these two proteins. It was concluded that
the two proteins might have distinct functions in the phragmoplast because of the difference in localizations.
AtPAKRP2 Is Enriched in the Insoluble
Cytosolic Fraction
The punctate localization pattern of AtPAKRP2 suggested
that this motor might be associated with membranous compartment(s) such as Golgi-derived vesicles in the phrag-
A Novel Phragmoplast-Associated Kinesin
2433
moplast. To determine whether AtPAKRP2 was in fact
associated with the insoluble cytosolic fraction, protein extracts of Arabidopsis inflorescence buds were subjected to
fractionation (see Methods); the resulting supernatant was
designated the soluble cytosolic fraction, and the pellet was
designated the fraction of endomembranes and other insoluble cytosolic compartments. When equal amounts of protein from the two fractions were loaded on an SDS-PAGE gel
followed by immunoblotting with anti-AtPAKRP2-N , it appeared that AtPAKRP2 was enriched greatly in the insoluble
fraction (Figure 8A). Our results suggest that AtPAKRP2
could be a motor protein that associates with vesicles that
are enriched in such preparations. When anti-AtPAKRP1 antibodies were applied, AtPAKRP1 protein was found mainly
in the supernatant fraction, suggesting that AtPAKRP1 was
unlikely to be associated with the endomembranes (data not
shown).
AtPAKRP2 Localization Is Altered by Brefeldin A
Figure 6. Requirement of MTs for AtPAKRP2 Localization.
We further tested whether AtPAKRP2 associated with the
Golgi-derived vesicles because such vesicles were commonly found in the phragmoplast. To disrupt the distribution
of these vesicles, we used brefeldin A (BFA), which disrupts
the Golgi apparatus and inhibits cell plate formation in
BY-2 cells (Yasuhara and Shibaoka, 2000). Although BFA
could prevent MT depolymerization in the phragmoplast, it
did not change the overall organization of phragmoplast
MTs (Figure 8C) (Yasuhara and Shibaoka, 2000). Because
BFA was dissolved in methanol, control cells were treated
with an amount of methanol equal to that used to dissolve
BFA. In the phragmoplasts of control cells, the AtPAKRP2
homolog was concentrated near the equatorial plane (Figure
8E), which was similar to what was observed in cells not
treated with methanol (Figure 8H). In BFA-treated cells,
however, much reduced anti-AtPAKRP2 signal could be
detected in the same area (Figure 8B). Such a result was unlikely to be caused by an overall reduction of fluorescence
intensity because diffuse signal was present in the cytoplasm (Figure 8B). To quantify the distortion of AtPAKRP2
localization caused by BFA treatment, we measured the relative fluorescence intensities in both the BFA-treated cell and
control cells (Figures 8D, 8G, and 8J). Although a clear peak
of AtPAKRP2 signal could be detected near the equatorial
plane in the control cells, such a peak was much less obvious
in the same area of the BFA-treated cell (Figures 8D, 8G, and
8J). Moreover, the overall level of fluorescence intensity of
(A) After APM treatment, AtPAKRP2 appeared in a diffuse pattern in
the cytoplasm in an Arabidopsis root tip cell after the completion of
mitosis.
(B) MTs were depolymerized completely in this cell, as indicated by
anti–-tubulin.
(C) Daughter nuclei stained with DAPI.
(D) to (F) A control root tip cell at a similar stage as in the cell (A) to
(C), (shown by DAPI staining of the nuclei in [F]) was treated with
isopropanol alone. AtPAKRP2 localization was not disturbed (D), nor
were phragmoplast MTs (E).
Bar in (F) 5 m for (A) to (F).
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AtPAKRP2 across the cell, or the basal level, increased significantly in the BFA-treated cell (Figure 8D). We concluded that
as a result of the reduced amount of these vesicles among
phragmoplast MTs after BFA treatment, AtPAKRP2 was not
able to localize among the phragmoplast MTs properly and
was distributed randomly in the cytoplasm.
AtPAKRP2 and KNOLLE Show Distinct
Localization Patterns
The KNOLLE protein is a cytokinesis-specific syntaxin that
localizes to the developing cell plate and functions in vesicle
targeting during cell plate formation (Lauber et al., 1997).
Because no vesicle motor had been found in the phragmoplast, we tested whether AtPAKRP2 was involved in the
transport of KNOLLE-bearing vesicle(s) to the cell plate.
Dual localizations of AtPAKRP2 and KNOLLE were performed in B. oleracea because of its large cells compared
with those of Arabidopsis and because of its evolutionary
relationship with Arabidopsis. Our antibodies cross-reacted
with the B. oleracea homolog of AtPAKRP2 and gave similar
localization in B. oleracea cells and Arabidopsis cells (Figure
9A). In a cell undergoing cytokinesis, as judged by the appearance of the daughter nuclei (Figure 9C), the B. oleracea
AtPAKRP2 homolog was in a punctate pattern in the phragmoplast, being more pronounced toward the division site
(Figure 9A). In this cell, KNOLLE was in a sharp line clearly
marking the cell plate (Figure 9B). We concluded, therefore,
that the KNOLLE protein was not present in the punctate
structure(s) bearing the AtPAKRP2 homolog.
DISCUSSION
Figure 7. Distinct
AtPAKRP1.
Localization
Patterns
of
AtPAKRP2
and
Arabidopsis root tip cells at early cytokinesis ([A] to [C]) and late cytokinesis ([D] to [F]) were stained with rat anti–AtPAKRP2-T ([A] and
[D]) and rabbit anti–AtPAKRP1-C ([B] and [E]) antibodies. Daughter
nuclei are shown by DAPI staining ([C] and [F]). AtPAKRP1 appeared much more discretely at the equatorial plane than did
AtPAKRP2. Bar in (F) 5 m for (A) to (F).
We have identified AtPAKRP2 cDNA encoding a novel N-terminal motor KRP in Arabidopsis and determined the intracellular localization of this AtPAKRP2 protein. AtPAKRP2
and its homologs in tobacco and B. oleracea localize specifically to the phragmoplast during cell division. In addition to
AtPAKRP1 (Lee and Liu, 2000), AtPAKRP2 is the second
plant KRP that associates only with the phragmoplast MT
array. Unlike AtPAKRP1, which is localized exclusively at or
near the plus end of phragmoplast MT bundles, however,
AtPAKRP2 appears in a punctate pattern with more pronounced distribution toward the division site. On the basis
of biochemical and pharmacological data, AtPAKRP2
likely associates with possibly Golgi-derived vesicles in the
phragmoplast. Thus, this protein is a potential motor for vesicle transport during cell plate formation.
Although AtPAKRP2 has a tripartite structure with a
coiled-coil domain flanked by the motor and tail domains, it
does not fall into any of the established kinesin/KRP subfamilies. Phylogenetic analysis based on the alignment of
the motor domains of AtPAKRP2 and kinesin/KRPs from dif-
A Novel Phragmoplast-Associated Kinesin
2435
ferent subfamilies suggested that AtPAKRP2 is very divergent from various KRPs among eukaryotes. Therefore, it
should be considered an outgroup KRP. Such a divergence,
however, did not affect its ATP-dependent MT binding activity. There is little doubt that it behaves as a motor protein
in plant cells. AtPAKRP2 does not contain a clear consensus sequence in the neck domain that is present among
plus end–directed kinesin/KRP motors (Vale and Fletterick,
1997), nor does it have a C-terminal motor KRP-specific
neck region. Because the neck sequence of C-terminal motor KRPs has been suggested to direct KRPs to move toward the MT minus ends (Endow, 1999), we believe that
AtPAKRP2 cannot be a minus end–directed motor. Because
of the nature of the intrinsic plus end–directed activity of the
motor domain, AtPAKRP2 most likely is a plus end–directed
motor. Unfortunately, under our experimental conditions,
GST-AtPAKRP2-M failed to demonstrate motility in vitro.
This might be because the GST fusion protein was not robust enough or the conditions we tested were not favorable
for the motility assays.
The previously identified AtPAKRP1 protein also is associated exclusively with the phragmoplast (Lee and Liu,
2000). Although both AtPAKRP1 and AtPAKRP2 are N-terminal motor KRPs, they have very different structural features. AtPAKRP1 has a motor domain that is much less
Figure 8. Association of AtPAKRP2 with the Insoluble Cytosolic
Fraction and Disturbance of AtPAKRP2 Localization by BFA.
(A) Inflorescence buds of Arabidopsis were used for subcellular fractionation. Equal amounts of protein from total protein extract (lane 1),
the cytosolic fraction (lane 2), and the insoluble cytosolic fraction (lane 3)
were subjected to immunoblotting with anti–AtPAKRP2-N antibodies.
The arrow indicates a single band recognized by the anti–AtPAKRP2-N
antibodies, which is enriched in the soluble cytosolic fraction.
(B) to (D) A tobacco BY-2 cell was treated with BFA in methanol.
(E) to (G) A tobacco BY-2 cell was treated with methanol alone.
(H) to (J) A tobacco BY-2 cell was fixed and stained without any
treatment.
The cells were double stained with anti–AtPAKRP2-N ([B], [E], and
[H]) and anti–-tubulin ([C], [F], and [I]) antibodies. (D), (G), and (J)
show relative intensities of immunofluorescence of AtPAKRP2 (thick
gray lines) and tubulin (thin black lines). The x axis represents the position of the data point, which was aligned to the micrographs, and
the y axis represents the relative fluorescence intensity with the value
given arbitrarily by ImagePro software. Bar in (I) 10 m for (B) to (I).
Figure 9. Distinct Localization Patterns of AtPAKRP2 and KNOLLE
in the Phragmoplast.
Triple localizations were performed with anti–AtPAKRP2-T (A), antiKNOLLE (B), and DAPI (C) in B. oleracea cells undergoing cytokinesis as revealed by the appearance of the daughter nuclei (C). The
composite image (D) was pseudocolored, with AtPAKRP2 shown in
green, KNOLLE shown in red, and DNA shown in blue. Bar in (D) 5
m for (A) to (D).
2436
The Plant Cell
divergent from other known kinesins/KRPs than is AtPAKRP2.
AtPAKRP1 also has an extremely long nonmotor domain
and does not have a typical tripartite structure, as does
AtPAKRP2 (Lee and Liu, 2000). AtPAKRP1 has coiled coils
more scattered in the nonmotor domain. AtPAKRP2, however, has coiled coils immediately after the motor domain.
The significance of the nonmotor domains of both motors
has yet to be characterized.
Our results suggest that AtPAKRP1 and AtPAKRP2 do
not colocalize in the phragmoplast. Their distinct localizations could be distinguished from the very beginning stage
when they started to appear in the spindle midzone at late
anaphase. AtPAKRP1 clearly associated with MT bundles,
whereas AtPAKRP2 was not restricted to the MT bundles. In
later stages of phragmoplast development, AtPAKRP1 is
conspicuous in the midline of the phragmoplast, coinciding
with the plus ends of MT bundles in a narrow area, as revealed by immunofluorescence. As a result of the limitations
of our fixation and staining procedures, it is possible that we
missed some AtPAKRP1 proteins in the phragmoplast that
were not concentrated along MT bundles. But identical procedures allowed us to clearly detect AtPAKRP2 in the
phragmoplast, which was not restricted to MT bundles. It is
conceivable that phragmoplast operation requires both motors that are responsible for MT reorganization and motors
that are responsible for vesicle transport. Obviously, motors
bearing two distinct roles will have different tail domains.
Elucidation of the functions of the nonmotor domains of
both motors will shed light on the mechanisms of plant cytokinesis. For example, it would be interesting to determine
whether either tail domain could bind MTs in the absence of
the motor domain in vivo or in vitro.
Treatment with BFA suggested that AtPAKRP2 associated with Golgi vesicles that were destined to the phragmoplast, although careful examination with immunogold
labeling is needed to reveal the AtPAKRP2 localization at
the ultrastructural level. When the formation of these vesicles was hampered by BFA as it disrupted the Golgi apparatus,
fewer vesicles were expected in the phragmoplast. We suggest that binding to the vesicle might be necessary for
AtPAKRP2 to associate with the phragmoplast MTs. Therefore, when fewer vesicles were produced, AtPAKRP2 was
mislocalized. Conversely, AtPAKRP2 was required for Golgi
vesicles to travel along phragmoplast MTs because
AtPAKRP2 is a MT-based motor. It was not surprising that
AtPAKRP2 localization to the phragmoplast was dependent
on the integrity of phragmoplast MTs.
Because KNOLLE is a syntaxin-like protein that plays an
essential role in vesicle docking during cytokinesis in plant
cells (Lauber et al., 1997), it is reasonable to wonder
whether AtPAKRP2 is the motor protein that transports
KNOLLE-bearing vesicles. However, our results from double
localizations of homologs of AtPAKRP2 and KNOLLE in B.
oleracea cells suggested that they had different localization
patterns. Thus, it is possible that other motor protein(s)
might transport KNOLLE-bearing vesicles.
It is worth noting that dynamic activities of vesicles are required in animal cytokinesis as well (Straight and Field,
2000). The ras-like GTPase Rabs play critical roles in vesicle
targeting (Deacon and Gelfand, 2001). In a search for interactors with Rab6, the kinesin-like Rab6-KIFL has been identified (Echard et al., 1998). Rab6-KIFL localizes to the
spindle midzone and the midbody in mammalian cells (Hill et
al., 2000). Introduction of anti–Rab6-KIFL antibodies into dividing cells blocks cytokinesis (Hill et al., 2000). Rab6-KIFL
probably is involved in transporting Golgi-derived vesicles
during cytokinesis in animal cells. Such speculation is fostered by the evidence that Rab6-KIFL also associates with
the Golgi apparatus (Echard et al., 1998). Thus, cytokinesis
in animals requires the active transport of Golgi vesicles by
MT-based motor(s), as in plant cells.
A number of KRPs have been implicated in cytokinesis in
higher plants (Liu and Lee, 2001). The most studied ones are
members of the BIMC subfamily, which form homotetramers in their native forms (Kashina et al., 1996; Asada et al.,
1997; Barroso et al., 2000). Among them, the tobacco
TKRP125 protein and the carrot DcKRP120-2 protein are
activated to render the sliding activity of antiparallel MTs to
establish the phragmoplast MT array (Asada et al., 1997;
Barroso et al., 2000). The activity of BIMC-like KRPs likely is
balanced by C-terminal motor KRPs, most likely KatA/B/C,
which also localize to phragmoplast MTs (Liu and Palevitz,
1996; Liu et al., 1996; Mitsui et al., 1996). Moreover, downregulation of KCBP is required for the organization of
phragmoplast MTs (Vos et al., 2000). In addition to these
motors, AtPAKRP1 is believed to play a role in the maintenance of the mirror-shaped phragmoplast MT array after
the sliding activity is complete (Liu and Lee, 2001). It is
conceivable that stabilization of the phragmoplast MT array allows vesicles to be transported to the right place at
the division site, possibly by AtPAKRP2. In a simplified
view of cytokinesis, we believe there are collaborative efforts
of different motors that result in the smooth progression of
cell plate formation.
Recently, the tobacco mitogen-activated protein kinase
kinase kinase NPK1 has been implicated in a signaling
pathway that regulates cytokinesis in tobacco cells
(Machida et al., 1998). Upon overexpression of a dominant
negative construct or a kinase-negative mutant, cytokinesis can be blocked and multinucleate cells can be formed
(Nishihama et al., 2001). It appears that this kinase is required for the expansion of the phragmoplast MT array toward the cell periphery during cell plate formation
(Nishihama et al., 2001). NPK1 localizes with spindle MTs
at metaphase, at the equatorial plane during late anaphase, and in the midzone of the phragmoplast (Nishihama
et al., 2001). More interestingly, it has been mentioned that
a novel tobacco KRP, NACK1, acts as an activator of
NPK1 and localizes to the division site as well (Machida et
al., 1998; Nishihama et al., 2001). It will be interesting to
compare NACK1 and AtPAKRP1/2 once the Nack1 sequence is revealed.
A Novel Phragmoplast-Associated Kinesin
METHODS
Isolation and Characterization of AtPAKRP2 cDNAs
The Arabidopsis thaliana expressed sequence tag clone OAO358
was provided by Dr. Alain Lecharny (Laboratoire de Biologie du Developpement des Plantes, Universite de Paris Sud, France). 5 RACE
(rapid amplification of cDNA ends; Clontech, Palo Alto, CA) was applied to obtain the 5 sequence of AtPAKRP2 cDNA. Trizol (Life
Technologies, Rockville, MD) was used to isolate total RNA from inflorescence buds of Arabidopsis Columbia according to the manufacturer’s protocol. Two AtPAKRP2-specific primers were designed
for 5 RACE: one primer (5-GTGTTAAAAGAGATCTTAAAGCAGC-3)
was used for reverse transcription, and the other nested primer (5ATCTTCCCCCAACAGTTGGCACATC-3) was used for subsequent
polymerase chain reaction (PCR). DNA samples were sequenced in a
commercial laboratory (Davis Sequencing, Davis, CA). The GCG program (Genetics Computer Group, Madison, WI) was used to analyze
DNA sequences. Phylogenetic analysis of kinesin/KRP motor domains was performed with the heuristic search method of PAUP included in GCG. A maximum parsimony with random stepwise
addition was used. In addition to the AtPAKRP2 reported here, the
phylogenetic analysis included Arabidopsis AtKatA (GenBank accession number D11371), AtKatB (D21137), AtKatC (D21138), AtKatD (AF080249), AtKCBP (L40358), and AtPAKRP1 (AF193767);
Aspergillus nidulans AnBIMC (M32075); Cricetulus griseus CgMCAK
(U11790); Daucus carota DcKRP120-2 (AF283505); Drosophila melanogaster DmNcd (X52814) and DmKHC (M24441); Gallus gallus
GgChrkin (U18309); Homo sapiens HsMKLP1 (X67155); Mus musculus MmKIF1A (D29951); Nicotiana tabacum TKRP125 (D83711); Saccharomyces cerevisiae ScSmy1p (M69021); and Strongylocentrotus
purpuratus SpKRP95 (U00996). The phylogenetic tree shown in Figure 1B was one of two optimal trees and was rooted arbitrarily using
ScSmy1p as an outgroup KRP. The CoilScan program of GCG was
used to predict the coiled-coil domains of a protein.
Production of Recombinant Fusion Proteins
To construct plasmids encoding fusion proteins of glutathione
S-transferase (GST) and various regions of AtPAKRP2, new restriction sites were introduced into AtPAKRP2 cDNA by PCR. Four plasmids were made for this study: (1) pGEX-AtPAKRP2-M contained the
DNA fragment encoding amino acid residues 1 to 435, which was
amplified by PCR with the primers 5-TCTTGAATTCTGGCACCGACACCATC-3 and 5-GTGTCTCGAGAGATCTTAAAGCAGC-3; (2) pGEXAtPAKRP2-N contained the DNA fragment encoding amino acid residues 1 to 202, which was amplified with the primers 5-GCACGAATTCCGACACCATCTTCTTC-3 and 5-AGTACTCGAGTGCTTGCTCCTTTAG-3; (3) pGEX-AtPAKRP2-N contained the DNA fragment
encoding amino acid residues 1 to 127, which was amplified with the
primers 5-GCACGAATTCCGACACCATCTTCTTC-3 and 5-TAGGCTCGAGCATCATAATCGTGCA-3; and (4) pGEX-AtPAKRP2-T
contained the DNA fragment encoding amino acid residues 516 to 869,
which was amplified with the primers 5-CGCTCCATGGGAGGAAGTGTTGATG-3 and 5-AATACTCGAGACAAGACATAGCGAC-3. The
PCR fragments for the first three plasmids were digested with EcoRI and
XhoI (sites underlined in the primers) and ligated to pGEX-KG EcoRIXhoI (Guan and Dixon, 1991). The PCR fragment for the last plasmid was
digested with NcoI and XhoI and ligated to pGEX-KG NcoI-XhoI.
2437
All pGEX constructs were expressed in the E. coli strain
BL21(DE3)pLysS (Novagen, Madison, WI). GST fusion proteins were
affinity purified with glutathione–Sepharose as described by the
manufacturer (Pierce Chemical Co., Rockford, IL), except for the protein expressed by pGEX-AtPAKRP2-N. This protein was not soluble,
so it was purified from inclusion bodies with B-PER bacterial protein
extraction reagent as described by the manufacturer (Pierce).
Microtubule Sedimentation Assay
This assay was performed essentially as described previously (Lee
and Liu, 2000). In brief, purified GST-AtPAKRP2-M fusion protein (5
g) was incubated with 25 g of microtubules (MTs), 10 M taxol,
and 2 M ATP (or adenylylimidodiphosphate, AMPPNP) in BRB80
buffer (80 mM Pipes, 1 mM MgCl2, and 1 mM EGTA, pH 6.8). After incubation at room temperature, the reaction was loaded on top of a
sucrose cushion (15% sucrose and 10 M taxol in BRB80) and then
subjected to centrifugation at 25,000g. The supernatant above the
sucrose cushion and the MT pellet were analyzed by SDS-PAGE.
Proteins of the supernatant and pellet were visualized by staining the
gels with Coomassie Brilliant Blue R250 (Sigma, St. Louis, MO).
Antibody Preparation and Immunoblotting
The GST fusion proteins GST-AtPAKRP2-N and GST-AtPAKRP2-T
were used as antigens to raise polyclonal antibodies in rabbits and
rats, respectively, at commercial facilities (Strategic BioSolutions,
Romona, CA; and Antibodies, Inc., Davis, CA). Sera were passed
through a column that had the GST protein covalently linked to the
matrix (GST Orientation Kit; Pierce) to deplete antibodies against
GST. Depletion of anti-GST antibodies was confirmed by immunoblotting against the GST protein. Because the PAKRP2-N polypeptide covered approximately half of the motor domain, the anti–
PAKRP2-N sera might contain antibodies against conserved kinesin/
KRP epitopes. To eliminate such antibodies, PAKRP2-N polypeptide was used for the purpose of antibody purification because it was
upstream of the ATP binding site and few conserved residues were
found in this region. Antibodies specific to AtPAKRP2 were affinity
purified with GST-AtPAKRP2-N or GST-AtPAKRP2-T by a blot purification method (Olmsted, 1981). To test the specificity of antibodies,
control experiments were performed by incubating antibodies with
blots containing their corresponding antigens before immunostaining.
Total protein extraction from plant tissues and immunoblotting
was performed as described previously (Liu et al., 1996). To prepare
subcellular fractions, Arabidopsis inflorescence buds were ground in
the extraction buffer containing 30 mM Pipes, 10 mM EGTA, 6 mM
MgCl2, 1 mM phenylmethylsulfonyl fluoride, 2 mM DTT, and 250 mM
sucrose, pH 7.0, with a mortar and pestle. The homogenate was centrifuged at 10,000g for 10 min at 4C to remove large cellular debris.
This low-speed supernatant was centrifuged at 100,000g for 30 min
at 4C. The resulting supernatant (the cytosolic fraction) and the pellet (the insoluble cytosolic fraction) then were used for immunoblotting analysis.
Fluorescence Microscopy and Image Processing
Slides containing Arabidopsis root tip cells of 2-day-old etiolated
seedlings or tobacco BY-2 suspension cells were prepared and processed for immunofluorescence as described previously (Lee and
2438
The Plant Cell
Liu, 2000). In addition to the rabbit anti-AtPAKRP2-N and rat antiAtPAKRP2-T antibodies reported here, antibodies used in this study
included rabbit anti-AtPAKRP1-C (Lee and Liu, 2000), mouse anti-tubulin (DM1A; Sigma), fluorescein isothiocyanate–conjugated goat
anti-rabbit IgG (Sigma), Texas Red X–conjugated goat anti-rabbit IgG
(Molecular Probes, Eugene, OR), Texas Red X–conjugated goat antimouse IgG (Molecular Probes), fluorescein isothiocyanate–conjugated
goat anti-rat IgG (Sigma), and rabbit anti-KNOLLE (Rose Biotech,
Winchendon, MA). Images were acquired by conventional epifluorescence microscopy as described previously (Lee and Liu, 2000).
Bowser, J., and Reddy, A.S.N. (1997). Localization of a kinesin-like
calmodulin-binding protein in dividing cells of Arabidopsis and
tobacco. Plant J. 12, 1429–1437.
Deacon, S.W., and Gelfand, V.I. (2001). Of yeast, mice, and men:
Rab proteins and organelle transport. J. Cell Biol. 152, F21–F24.
Echard, A., Jollivet, F., Martinez, O., Lacapere, J.J., Rousselet,
A., Janoueix-Lerosey, I., and Goud, B. (1998). Interaction of a
Golgi-associated kinesin-like protein with Rab6. Science 279,
580–585.
Endow, S.A. (1999). Determinants of molecular motor directionality.
Nat. Cell Biol. 1, E163–E167.
Pharmacological Studies
To depolymerize MTs, Arabidopsis 2-day-old etiolated seedlings were
incubated in 5 mL of germination medium (half-strength Murashige
and Skoog [1962] salts [Life Technologies] and 5 mM Mes, pH 5.7)
containing 100 M APM (a gift from Dr. C. Gregg of Bayer Corp, Kansas CIty, MO) for 2 hr. The same amount of isopropanol, used to dissolve APM, was added in the control seedlings. BFA treatment was
performed in tobacco BY-2 suspension cells 4 days after subculture.
Ten-milliliter cells were treated with 20 M BFA for 2 hr. The solvent
methanol was added in the control sample. The measurement of
fluorescence intensity was performed using the Line Profile measurement function in the ImagePro software package (Media Cybernetics, Silver Spring, MD). Data then were exported to Microsoft
Excel software to generate Figures 8D, 8G, and 8J.
Accession Number
The GenBank accession number for AtPAKRP2 is AF320248.
ACKNOWLEDGMENTS
Goldstein, L.S.B., and Philp, A.V. (1999). The road less traveled:
Emerging principles of kinesin motor utilization. Annu. Rev. Cell
Dev. Biol. 15, 141–183.
Granger, C.L., and Cyr, R.J. (2000). Microtubule reorganization in
tobacco BY-2 cells stably expressing GFP-MBD. Planta 210,
502–509.
Guan, K.L., and Dixon, J.E. (1991). Eukaryotic proteins expressed
in Escherichia coli: An improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase.
Anal. Biochem. 192, 262–267.
Hasezawa, S., Ueda, K., and Kumagai, F. (2000). Time-sequence
observations of microtubule dynamics throughout mitosis in living
cell suspensions of stable transgenic Arabidopsis: Direct evidence for the origin of cortical microtubules at M/G(1) interface.
Plant Cell Physiol. 41, 244–250.
Hill, E., Clarke, N., and Barr, F.A. (2000). The Rab6-binding kinesin,
Rab6-KIFL, is required for cytokinesis. EMBO J. 19, 5711–5719.
Kashina, A.S., Baskin, R.J., Cole, D.G., Wedaman, K.P., Saxton,
W.M., and Scholey, J.M. (1996). A bipolar kinesin. Nature 379,
270–272.
Kim, A.J., and Endow, S.A. (2000). A kinesin family tree. J. Cell Sci.
113, 3681–3682.
We thank Dr. Alain Lecharny (Universite de Paris Sud) for the
OAO358 clone, Dr. Carl Gregg (Agricultural Division of Bayer Corp.)
for APM, and Mary Lai for critical comments on the manuscript. This
work was supported by a grant from the U.S. Department of Agriculture (NRI/CSREES 99-35304-8142).
Received June 4, 2001; accepted August 29, 2001.
Lauber, M.H., Waizenegger, I., Steinmann, T., Schwarz, H.,
Mayer, U., Hwang, I., Lukowitz, W., and Jurgens, G. (1997). The
Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J.
Cell Biol. 139, 1485–1493.
Lee, Y.R.J., and Liu, B. (2000). Identification of a phragmoplastassociated kinesin-related protein in higher plants. Curr. Biol. 10,
797–800.
Liu, B., and Lee, Y.R.J. (2001). Kinesin-related proteins in plant
cytokinesis. J. Plant Growth Regul. 20, 141–150.
REFERENCES
Liu, B., and Palevitz, B.A. (1996). Localization of a kinesin-like protein in generative cells of tobacco. Protoplasma 195, 78–89.
Asada, T., Sonobe, S., and Shibaoka, H. (1991). Microtubule translocation in the cytokinetic apparatus of cultured tobacco cells.
Nature 350, 238–241.
Liu, B., Cyr, R.J., and Palevitz, B.A. (1996). A kinesin-like protein,
KatAp, in the cells of Arabidopsis and other plants. Plant Cell 8,
119–132.
Asada, T., Kuriyama, R., and Shibaoka, H. (1997). TKRP125, a
kinesin-related protein involved in the centrosome-independent
organization of the cytokinetic apparatus in tobacco BY-2 cells. J.
Cell Sci. 110, 179–189.
Machida, Y., Nakashima, M., Morikiyo, K., Banno, H., Ishikawa,
M., Soyano, T., and Nishihama, R. (1998). MAPKKK-related protein kinase NPK1: Regulation of the M phase of plant cell cycle. J.
Plant Res. 111, 243–246.
Barroso, C., Chan, J., Allan, V., Doonan, J., Hussey, P., and
Lloyd, C. (2000). Two kinesin-related proteins associated with the
cold-stable cytoskeleton of carrot cells: Characterization of a
novel kinesin, DcKRP120-2. Plant J. 24, 859–868.
Mitsui, H., Hasezawa, S., Nagata, T., and Takahashi, H. (1996).
Cell cycle–dependent accumulation of a kinesin-like protein,
KatB/C, in synchronized tobacco BY-2 cells. Plant Mol. Biol. 30,
177–181.
A Novel Phragmoplast-Associated Kinesin
2439
Murashige, T., and Skoog, F. (1962). A revised medium for rapid
growth and bioassays with tobacco tissue culture. Physiol. Plant.
15, 473–497.
Intracellular distribution of subcellular organelles revealed by antibody against xyloglucan during cell cycle in tobacco BY-2 cells.
Protoplasma 213, 218–227.
Nishihama, R., Ishikawa, M., Araki, S., Soyano, T., Asada, T., and
Machida, Y. (2001). The NPK1 mitogen-activated protein kinase
kinase kinase is a regulator of cell-plate formation in plant cytokinesis. Genes Dev. 15, 352–363.
Straight, A.F., and Field, C.M. (2000). Microtubules, membranes
and cytokinesis. Curr. Biol. 10, R760–R770.
Olmsted, J.B. (1981). Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J. Biol.
Chem. 256, 11955–11957.
Vantard, M., Levilliers, N., Hill, A.M., Adoutte, A., and Lambert,
A.M. (1990). Incorporation of paramecium axonemal tubulin into
higher plant cells reveals functional sites of microtubule assembly.
Proc. Natl. Acad. Sci. USA 87, 8825–8829.
Otegui, M., and Staehelin, L.A. (2000). Syncytial-type cell plates: A
novel kind of cell plate involved in endosperm cellularization of
Arabidopsis. Plant Cell 12, 933–947.
Reddy, A.S.N. (2001). Molecular motors and their functions in
plants. Int. Rev. Cytol. 204, 97–178.
Samuels, A.L., Giddings, T.H., and Staehelin, L.A. (1995). Cytokinesis in tobacco BY-2 and root tip cells: A new model of cell plate
formation in higher plants. J. Cell Biol. 130, 1345–1357.
Sharp, D.J., McDonald, K.L., Brown, H.M., Matthies, H.J., Walczak,
C., Vale, R.D., Mitchison, T.J., and Scholey, J.M. (1999). The
bipolar kinesin, KLP61F, cross-links microtubules within interpolar
microtubule bundles of Drosophila embryonic mitotic spindles. J.
Cell Biol. 144, 125–138.
Sharp, D.J., Rogers, G.C., and Scholey, J.M. (2000). Microtubule
motors in mitosis. Nature 407, 41–47.
Smirnova, E.A., Reddy, A.S.N., Bowser, J., and Bajer, A.S. (1998).
Minus end-directed kinesin-like motor protein, KCBP, localizes to
anaphase spindle poles in Haemanthus endosperm. Cell Motil.
Cytoskeleton 41, 271–280.
Sonobe, S., Nakayama, N., Shimmen, T., and Sone, Y. (2000).
Vale, R.D., and Fletterick, R.J. (1997). The design plan of kinesin
motors. Annu. Rev. Cell Dev. Biol. 13, 745–777.
Verma, D.P.S. (2001). Cytokinesis and building of the cell plate in
plants. Annu. Rev. Plant Physiol. 52, 751–784.
Vos, J.W., Safadi, F., Reddy, A.S.N., and Hepler, P.K. (2000). The
kinesin-like calmodulin binding protein is differentially involved in
cell division. Plant Cell 12, 979–990.
Wittmann, T., Hyman, A., and Desai, A. (2001). The spindle: A
dynamic assembly of microtubules and motors. Nat. Cell Biol. 3,
E28–E34.
Yasuhara, H., and Shibaoka, H. (2000). Inhibition of cell-plate formation by brefeldin A inhibited the depolymerization of microtubules in the central region of the phragmoplast. Plant Cell Physiol.
41, 300–310.
Yasuhara, H., Sonobe, S., and Shibaoka, H. (1993). Effects of taxol
on the development of the cell plate and of the phragmoplast in
tobacco BY-2 cells. Plant Cell Physiol. 34, 21–29.
Zhang, D., Wadsworth, P., and Hepler, P.K. (1993). Dynamics of
microfilaments are similar, but distinct from, microtubules during
cytokinesis in living, dividing plant cells. Cell Motil. Cytoskeleton
24, 151–155.
A Novel Plant Kinesin-Related Protein Specifically Associates with the Phragmoplast Organelles
Y.-R. Julie Lee, Hoa M. Giang and Bo Liu
Plant Cell 2001;13;2427-2439
DOI 10.1105/tpc.010225
This information is current as of June 17, 2017
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