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Research Article
The kinesin-like protein TOP promotes Aurora
localisation and induces mitochondrial, chloroplast
and nuclear division
Yamato Yoshida1,*, Takayuki Fujiwara2, Yuuta Imoto1,3, Masaki Yoshida4, Mio Ohnuma1,5, Shunsuke Hirooka5,6,
Osami Misumi5,7, Haruko Kuroiwa1,5, Shoichi Kato8, Sachihiro Matsunaga8 and Tsuneyoshi Kuroiwa1,5,`
1
Graduate School of Science, Rikkyo University, 3-34-1 Nishiikebukuro, Toshima-ku, Tokyo 171-8501, Japan
Chromosome Dynamics Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa,
Chiba 277-8562, Japan
4
Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba,
Ibaraki 305-8572, Japan
5
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Gobancho, Chiyoda-ku, Tokyo
102-0076, Japan
6
Center for Frontier Research, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan
7
Graduate School of Medicine, Faculty of Science, Department of Biological Science and Chemistry, Yamaguchi University, 1677-1 Yoshida,
Yamaguchi, Yamaguchi 753-8512, Japan
8
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510,
Japan
2
3
Journal of Cell Science
*Present address: Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA
`
Author for correspondence ([email protected])
Accepted 18 March 2013
Journal of Cell Science 126, 2392–2400
2013. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.116798
Summary
The cell cycle usually refers to the mitotic cycle, but the cell-division cycle in the plant kingdom consists of not only nuclear but also
mitochondrial and chloroplast division cycle. However, an integrated control system that initiates division of the three organelles has not
been found. We report that a novel C-terminal kinesin-like protein, three-organelle division-inducing protein (TOP), controls nuclear,
mitochondrial and chloroplast divisions in the red alga Cyanidioschyzon merolae. A proteomics study revealed that TOP is a member of
a complex of mitochondrial-dividing (MD) and plastid-dividing (PD) machineries (MD/PD machinery complex) just prior to
constriction. After TOP localizes at the MD/PD machinery complex, mitochondrial and chloroplast divisions occur and the components
of the MD/PD machinery complexes are phosphorylated. Furthermore, we found that TOP downregulation impaired both mitochondrial
and chloroplast divisions. MD/PD machinery complexes were formed normally at each division site but they were neither
phosphorylated nor constricted in these cells. Immunofluorescence signals of Aurora kinase (AUR) were localized around the MD
machinery before constriction, whereas AUR was dispersed in the cytosol by TOP downregulation, suggesting that AUR is required for
the constriction. Taken together our results suggest that TOP induces phosphorylation of MD/PD machinery components to accomplish
mitochondrial and chloroplast divisions prior to nuclear division, by relocalization of AUR. In addition, given the presence of TOP
homologs throughout the eukaryotes, and the involvement of TOP in mitochondrial and chloroplast division may illuminate the original
function of C-terminal kinesin-like proteins.
Key words: Cell-division cycle, Cyanidioschyzon merolae, Kinesin, Mitochondrial division, Plastid division
Introduction
Almost all eukaryotic cells in the plant kingdom possess three
kinds of organelles that contain DNA and have double
membranes: one nucleus, many mitochondria and many
chloroplasts (plastids). Since mitochondria and chloroplasts
were derived from free-living a-proteobacterial and
cyanobacterial ancestors, respectively, they are never
synthesized de novo and their continuities are maintained by
division, as is the nucleus. The cell cycle, therefore, consists of
not only the mitotic cycle but also mitochondrial and chloroplast
division cycles (Suzuki et al., 1994; Imoto et al., 2010). In plant
cells, mitochondrial and plastid DNA replications take place
before nuclear DNA replication (Kobayashi et al., 2009; Imoto
et al., 2010). Recently, Kobayashi et al. have shown in
Cyanidioschyzon merolae and tobacco BY-2 cells that
mitochondrial and plastid DNA replications are signaled by the
intracellular accumulation of a tetrapyrrole intermediate,
probably Mg-ProtoIX, resulting in the activation of cyclindependent kinase A (CDKA, also known as Cdc2 in fission yeast)
and the consequent initiation of nuclear DNA replication
(Kobayashi et al., 2009). Therefore, it seems that an integrated
control system that induces division of the three types of doublemembrane organelles may be hidden in the initiation step of
mitochondrial and plastid divisions.
In the last two decades, it has been shown that mitochondrial
and chloroplast divisions occur in three steps: formation of
TOP induces organelle divisions
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mitochondrial-dividing (MD) machinery and plastid-dividing
(PD) machineries at each division site (Kuroiwa et al., 1998),
constriction of the division site, and pinching-off of the bridge of
the daughter organelle (Fig. 1A). During the formation step, MD
and PD machineries are connected with each other and form a
complex structure (MD/PD machinery complex), but this
complex separates in the constriction step (Fig. 1A) (Yoshida
et al., 2009). Both MD and PD division machineries comprise a
chimera of inner rings of bacterial-derived proteins, such as FtsZ
(Osteryoung and Nunnari, 2003; Kuroiwa et al., 2008) and
eukaryote-specific proteins, such as the MD ring, PD ring and
dynamin proteins (Bleazard et al., 1999; Miyagishima et al.,
2003; Gao et al., 2003; Nishida et al., 2003; Osteryoung and
Nunnari, 2003; Kuroiwa et al., 2008). Recent studies showed that
dynamin is localized between the PD ring filaments and is
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essential for the generation of the motive force for contraction
(Yoshida et al., 2006). In addition, the PD ring is constructed of a
bundle of glycosyltransferase protein PDR1-mediated-polyglucan
filaments. Thus, the contraction of the PD machineries is caused by
the sliding movement between dynamin and polyglucan filaments
(Yoshida et al., 2010). Similarly, it has been thought that the
contraction of the MD machineries is probably driven in the same
way as that of the PD machinery (Nishida et al., 2003; Yoshida
et al., 2009; Kuroiwa et al., 2008).
In addition, it has been reported that in C. merolae a prespindle structure, designated as the mitochondrial spindle, is
formed from each spindle pole to the division site of
mitochondria before nuclear division (supplementary material
Fig. S1, white arrowheads) (Nishida et al., 2005; Imoto et al.,
2010). Similar to the cell division spindle (Hirokawa, 1998;
Walczak and Heald, 2008), the mitochondrial spindle may be
also organized by tubulin-mediated microtubule polymers,
several types of kinesin-superfamily proteins, and many
relating factors such as mitotic kinases.
Our recent study showed that a mitotic serine/threonine kinase,
Aurora kinase (AUR), which is encoded by a single-copy gene in
the genome C. merolae, is localized not only at spindle poles
and the cell division spindle but also the mitochondrial spindle
(Kato et al., 2011). Specifically, AUR accumulates from the
mitochondrial spindle to the mitochondrial division site in the
contraction phase of mitochondrial division, indicating that it is
involved in the activation of the MD machinery. Thus, these
findings suggest the existence of an uncharacterized pathway or
factor that coordinates timing of mitochondrial and chloroplast
division and links mitochondrial- and chloroplast-division cycles
with the cell-division cycle.
C. merolae offers advantages for studying the regulation of
integrated initiation of nuclear, mitochondrial and chloroplast
divisions. The cell contains just one chloroplast, one
mitochondrion and one nucleus (Matsuzaki et al., 2004), the
division of which occurs in that order in highly synchronized
cells controlled by light/dark cycles (supplementary material Fig.
S1) (Suzuki et al., 1994). In addition, availability of the complete
genome sequence of C. merolae facilitates highly sensitive
transcriptomic and proteomic analyses (Matsuzaki et al., 2004;
Nozaki et al., 2007; Yagisawa et al., 2009; Fujiwara et al., 2009;
Yoshida et al., 2009; Yoshida et al., 2010).
In this study, we revealed that a novel kinesin-like protein, TOP,
induces divisions of the nucleus, mitochondrion and chloroplast.
TOP localized on the MD/PD machinery complex to transfer AUR
just before the contraction of the MD and PD machineries. By this
process, the mitochondrion and chloroplast were divided by the
MD and PD machineries, respectively. Nuclear division was also
accomplished by the TOP-mediated spindle. Thus, TOP regulates
division of three organelles, the nucleus, mitochondrion and
chloroplast, by relocalization of AUR protein.
Results
Fig. 1. A proteomic analysis of MD/PD machinery complexes before
constriction. (A) Schematic depiction of mitochondrial and chloroplast
division processes in C. merolae cells. Division of mitochondria and
chloroplasts is performed by the MD and PD machineries, respectively,
following the three steps; formation, constriction and pinching-off.
(B) Proteomic analysis of isolated MD/PD machinery complexes before
constriction. Identified proteins are shown in supplementary material Fig. S2;
Table S1.
Identification of a novel kinesin-like protein TOP in the MD/
PD machinery complexes
To identify proteins that regulate the integrated initiation of
nuclear, mitochondrial and chloroplast division, we performed a
proteomic analysis of the isolated MD/PD machinery complex
from cells in early S phase (Fig. 1B; supplementary material Fig.
S2; Table S1; see Materials and Methods) because division of the
organelles and organization of the mitochondrial spindle begins
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immediately after this phase. For isolation of non-constricting
MD/PD machinery complexes, synchronized cells were harvested
at the start of the second dark period (S phase) and treated with
Nonidet P-40 and n-octyl-b-D-glucopyranoside (see Materials
and Methods). After isolation of the non-constricting MD/PD
machinery complexes, proteomic analysis was performed using
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF-MS). In this fraction, known
components of the MD and PD machinery, Dynamin 1
(Dnm1), Mda1 and Dnm2 were identified. Comparing previous
results of proteomic analyses of the MD/PD machineries isolated
from cells in M phase (Yoshida et al., 2009; Yoshida et al., 2010),
we identified a novel S-phase-specific protein TOP (threeorganelle divisions inducing protein; Fig. 1B). Using the
completely sequenced genomic information of C. merolae
(http://merolae.biol.s.u-tokyo.ac.jp/), we distinguished that
TOP was one of the C-terminal motor domain-type kinesinsuperfamily proteins [C-terminal KIFs (reviewed by Hirokawa
et al., 2009)] (Fig. 2A), which had not previously been detected
in isolated constricting MD and PD machineries from cells in M
phase (Yoshida et al., 2009; Yoshida et al., 2010). By
phylogenetic analysis, we showed that a kinesin motor domain
of TOP is a classical C-terminal KIF, and TOP homologs are
widely conserved throughout eukaryotic cells, especially plants
(supplementary material Fig. S3). Some groups of C-terminal
KIFs have been reported to transport organelles (Saito et al.,
1997; Xu et al., 2002; Bananis et al., 2004; Hirokawa et al.,
2009). Also, one of the known functions of C-terminal KIFs is the
assembly of spindle poles by the cross-linking of parallel
microtubules in each half-spindle, where they focus minus ends
into spindle poles (Walczak et al., 1997; Hirokawa, 1998);
however, the involvement of TOP in mitochondrial and/or
chloroplast division has not previously been reported. We,
therefore, examined the function of TOP to reveal the regulatory
mechanism of nuclear, mitochondrial and chloroplast division.
The genes of known MD- and PD-machinery-associated
proteins (Mda1 for MD machinery and Dnm2 for PD
machinery) are selectively transcribed before organelle
division; therefore, we examined the level of TOP transcription
during the cell cycle using C. merolae microarray data (Fujiwara
et al., 2009). The level of transcription of TOP increased during S
phase and into early M phase, and this expression profile
coincided with that of other known MD- and PD-machineryassociated proteins (Fig. 2B). We then generated anti-TOP
antibodies via bacterially expressed TOP protein. Anti-TOP
antibodies detected protein of the predicted molecular mass of
TOP (,96 kDa; Fig. 2C). Also, immunoblot analyses of total
protein from synchronized cultures detected TOP during
chloroplast, mitochondrial and nuclear divisions (Fig. 2D).
Next, we examined the intracellular localization of TOP during
the cell cycle by immunofluorescence microscopy using the antiTOP antibody (Fig. 3). Although TOP was not detected during
the G1 phase, it was observed in S phase, indicating that TOP
was located near the mitochondrial division site (Fig. 3, white
arrowhead). Then, in the S–G2 phases, the fluorescence signal of
the TOP-assembled small spindle pole increased in size and split
into two spindle poles (Fig. 3, black arrowheads). During M
phase, TOP was localized on microtubules in addition to at the
two spindle poles. Therefore, we conclude that TOP is involved
in the spindle poles and the spindle microtubules for nuclear
division in M phase.
Fig. 2. mRNA and protein expression profiles of TOP. (A) Molecular
structure of the kinesin-like protein TOP. The red bar indicates the kinesin
motor domain. (B) mRNA levels of TOP (red), a-tubulin (a-Tub, blue), Mda1
(yellow), Dnm2 (green) and PDR1 (light blue) at different points of the cell
cycle, determined using microarray data. The light-dependent gene Tic22
(grey) is also shown as a control. (C) Total protein from synchronized Mphase cells was blotted with anti-TOP antibody. (D) Protein levels of TOP,
a-Tub, CENH3, Mda1 and Dnm2 at different points of the cell cycle.
Because TOP was identified in the fraction of the isolated MD/
PD machinery complex by proteomic analysis (Fig. 1B) and
appeared near the mitochondrial division site (Fig. 3, white
arrowhead), we next investigated whether TOP was involved in
mitochondrial and/or chloroplast division in addition to nuclear
division. For the purpose, we examined the effects of TOP
downregulation using antisense suppression (Fig. 4). Twentyfour hours after transformation, antisense TOP induced defects in
mitochondrial and chloroplast divisions (P,0.001; Fisher’s exact
test; Fig. 4A,B). In these cells, both MD and PD machineries
were found at the mitochondrial and chloroplast division sites,
respectively, although constriction had not commenced in either
(Fig. 4C,D). In contrast, inhibition of microtubule organization
by oryzalin treatment arrested nuclear division of the cells, but
both mitochondrial and chloroplast divisions were performed
normally (supplementary material Fig. S4) (Nishida et al., 2005).
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Fig. 3. Immunofluorescence images and models of TOP (green) and
a-tubulin (a-Tub, red) in cells. White arrowhead indicates TOP protein
near the mitochondrial division site and black arrowheads indicate the
TOP-assembled two spindle poles. Scale bar: 1 mm.
In addition, immunofluorescence microscopy showed that a
centromere marker protein, CENH3, appeared as multiple
discrete speckles in nuclei of TOP-downregulated cells arrested
in S/G2 phases (supplementary material Fig. S5). Therefore, the
obstruction of mitochondrial and chloroplast divisions by TOP
downregulation are not caused by spindle checkpoint. However,
we cannot completely exclude the possibility that the S/G2 arrest
was caused by activation of other checkpoints.
TOP binds directly to the MD machinery complex
As TOP was required for the initiation of three-organelle
divisions and seemed to be located near the mitochondrial
division site, we examined whether the timing and manner of
localization of TOP was related to the MD/PD machinery
complex, using immunofluorescence microscopy. Both MD and
PD machineries were formed prior to the expression of TOP
(Fig. 5A). TOP seemed to be located only on the MD machinery
in vivo (Fig. 5A) but could be recognized on the division site of
the isolated dividing mitochondria and chloroplasts (Fig. 5B). To
reveal whether TOP binds directly to the MD machinery
complex, we isolated the MD/PD machinery complexes from
cells in S or M phases and examined the localization of TOP by
immunofluorescence microscopy (Fig. 6A). We also measured
the circumference of isolated PD machineries to detect the stage
of division, because the circumference of the PD machinery
reduces as chloroplast division progresses (Miyagishima et al.,
Fig. 4. Downregulation of TOP. (A) Phase-contrast (PC) and
fluorescence images of antisense-TOP cells. Transiently transformed
cells express sGFP (green) in the cytosolic space. Mitochondria (Mt, red)
were immunolabeled using an anti-mitochondrial porin antibody, and
chloroplasts (Cp, red) emitted red autofluorescence. (B) In cells in which
TOP expression was downregulated by antisense suppression, the
frequency of dividing cells was reduced (P,0.001; Fisher’s exact test).
Data are from the total number of transformants examined (n) in more
than five replicates. (C) PC and immunofluorescence images of TOP,
mitochondria (Mt), MD machinery (Mda1, white arrowhead) and PD
machinery (Dnm2, black arrowhead) of cells treated with antisense TOP.
(D) Schematic model of the antisense-TOP cell division. Scale bars:
1 mm.
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Fig. 5. TOP localizes near the mitochondrial division site.
(A) Sequential immunofluorescence images and explanations of the
formation of MD machinery (Mda1, red), PD machinery (Dnm2, red),
and TOP (green, arrowhead). (B) Immunofluorescence images and a
model of TOP on an isolated dividing mitochondrion (red) and
chloroplast. Scale bars: 1 mm.
2001). The average circumference of the PD machineries
containing TOP was 2.6 mm (n525; supplementary material
Fig. S6), therefore, we concluded that TOP bound to the MD/PD
machinery complexes that were derived from cells in the early
phase of mitochondrial and chloroplast divisions. However, TOP
did not associate with small MD/PD machinery complexes
derived from cells in late stages of mitochondrial and chloroplast
division (Fig. 6A). These results were also confirmed by
immuno-electron microscopy (immuno-EM; Fig. 6B,C).
Immunogold particles indicating TOP were distributed in a
spot-like pattern on the MD machinery (Fig. 6C, inset). These
results indicate that the direct interaction between TOP and larger
MD machineries plays an important role in the initiation of
constriction of MD and PD machineries.
TOP is required for the phosphorylation of proteins in both
MD and PD machineries
Fig. 6. Direct interaction of TOP on the MD/PD machinery complex.
(A) Immunofluorescence images and diagrams of direct interaction of TOP on
isolated MD and PD machineries from early phases of division.
(B,C) Immuno-EM images of large isolated MD/PD machinery complex
(B) and large isolated MD machinery (C). Small immunogold particles
indicate Mda1 and large immunogold particles indicate TOP. A white
arrowhead indicates the MD machinery and a black arrowhead, the PD
machinery. Scale bars: 1 mm (A); 200 nm (B,C).
Reports thus far indicate that protein phosphorylation is involved
in at least the mitochondrial division. The WD40 protein Mda1
together with the MD ring assembles the outer ring of the MD
machinery, and then Mda1 is phosphorylated. In the contraction
phase of the mitochondrial division, phosphorylated Mda1
oligomers on the MD machinery are disassembled by GTP
hydrolysis of Dnm1 (Nishida et al., 2007). And finally, the
mitochondrial dynamin ring, mediated by Dnm1, severs the
bridge of the dividing mitochondrion (Nishida et al., 2003). Since
phosphorylation of Mda1 is induced with the progress of the
mitochondrial division phases, protein phosphorylation is likely
to mediate changes in the organization of the MD machinery for
constriction. Thus, it is thought that phosphorylation is one of the
most important modifications for the MD and PD machineries.
Based on these results and hypothesis, we examined whether
proteins in the isolated MD/PD machinery complexes were
phosphorylated in the early and late phases of mitochondrial and
TOP induces organelle divisions
estimated in matched peptide fragments of Dnm1, Mda1 and
Dnm2 using MALDI-TOF-MS analyses (supplementary material
Tables S2–S4). Both MD and PD machineries were highly
phosphorylated in line with the progress of mitochondrial and
chloroplast division. In addition, we showed that cells expressing
antisense TOP contained the non-constricted MD and PD
machineries at each division site (Fig. 4C), and that they
were not phosphorylated (Fig. 7E). Lastly, we used
immunofluorescence microscopy to confirm the kinases
associated with the MD/PD machinery complex. Similar to Cterminal KIFs, it is known that Aurora kinase is also involved in
spindle pole maturation because Aurora kinases were originally
identified as being required for accurate spindle pole structure
Journal of Cell Science
chloroplast division, using cytochemistry and MALDI-TOF-MS.
Large MD/PD machinery complexes, which were derived from
the cells in early phase of division, had few fluorescence signals
derived from phosphorylation, but small MD and PD
machineries, which were derived from the cells in late phase of
division, had strong fluorescence signals (Fig. 7A,B). Moreover,
differences in fluorescence signals between the control fraction
and the isolated MD/PD machinery complex fraction after
staining for phosphorylated proteins showed that many
proteins, including Dnm1, Mda1 (involved in mitochondrial
division) and Dnm2 (involved in chloroplast division) in the
isolated MD/PD machinery complex fraction, were
phosphorylated (Fig. 7C,D). Phosphorylated peptides were
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Fig. 7. Phosphorylation of MD and PD machineries during mitochondrial and chloroplast division. (A) Phase-contrast (PC) and fluorescence images of cells
stained for phosphorylated protein (PP, red). The cells were stained by Pro-Q Diamond dye. Arrowheads indicate MD machinery (white arrowhead) or PD
machinery (black arrowhead). (B) Immunofluorescence images of isolated MD machineries (Mda1, green) and PD machineries (Dnm2, green) stained for
phosphorylated protein. MD and PD machineries were isolated from cells in early M phase (upper set) or late M phase (bottom set). (C) Coomassie Brilliant Blue
(CBB)-stained and phosphoprotein-stained gels in the isolated MD/PD machinery complex (right) and control fraction (left). Each gel was stained by Pro-Q
Diamond dye before CBB staining. (D) A gel image indicates differences in fluorescence signals between the gel image of the isolated MD/PD machinery
complex fraction and the gel image of the control fraction in C. Three phosphorylated proteins in the gel were identified as Dnm1, Mda1 and Dnm2 by
immunoblotting and MALDI-TOF-MS analyses. Matched peptide sequences and estimated phosphopeptides are shown in supplementary material Tables S2–S4.
(E) Immunofluorescence images of antisense-TOP cells with staining for phosphorylated proteins. After staining phosphorylated proteins (PP, red), cells were
immunolabeled using anti-GFP antibody (GFP, green). (F) Immunoblotting and immunofluorescence images of AUR and schematic model of localization of AUR
protein (pink) during cell division. Anti-AUR antibodies detected two major polypeptides (asterisk). A higher molecular mass form of AUR is probably
phosphorylated AUR protein. (G) Phase-contrast and immunofluorescence images of AUR (green) in a TOP-downregulated cell. Transiently transformed cells
express AUR and sGFP (red) in the cytosolic space. Scale bars: 1 mm.
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(Glover, et al., 1995; Andrews et al., 2003). In addition, a recent
study showed that Aurora kinase in C. merolae (named AUR)
was involved with not only mitotic spindle formation but also
mitochondrial division (Kato et al., 2011). The level of
transcription of AUR increased in S and M phases and this
expression profile coincided with that of TOP (supplementary
material Fig. S7). Therefore, we investigated whether AUR is
involved with TOP during mitochondrial and chloroplast
divisions. Immunofluorescence microscopy identified that AUR
colocalized with TOP and was localized around the
mitochondrion before the constriction phase of mitochondrial
and chloroplast divisions (Fig. 7F, white arrowhead).
Subsequently, AUR accumulated at the mitochondrial division
site (Fig. 7F, black arrowhead). These results implied that AUR
mediates phosphorylation of the MD and PD machineries and
spindle pole maturation. Next, we investigated the localization of
AUR in the TOP-downregulated cells to examine whether AUR
is involved in the maturation of the MD and PD machineries.
AUR protein was not found to be localized around the
mitochondrial division site but scattered in the cytosolic region
in the TOP-downregulated cells (Fig. 7G). In addition, the
proteomic analysis of isolated MD/PD machinery complexes
suggested that some of the phosphorylated peptides of Dnm1,
Dnm2 and Mda1 were processed by Aurora kinase
(supplementary material Tables S2–S4). In conclusion, TOP is
a regulator of MD/PD machinery complexes for mitochondrial
and chloroplast divisions, probably by transferring of Aurora
kinase to the machinery.
Discussion
Thus far, it has been revealed that mitochondrial and chloroplast
division is performed by the MD and PD machineries which are
large protein complexes. Although the molecular mechanisms
involved are still not fully known, recent studies showed that
FtsZ and dynamin proteins can generate a force for contraction of
membranes by GTP hydrolysis (Osawa et al., 2008; Mears et al.,
2011). In particular, a series of analyses of isolated single PD and
single MD machineries showed that Dnm2 and Dnm1 are
required to generate constriction force by sliding movements of
the PD or MD ring filaments, respectively (Yoshida et al., 2006;
Yoshida et al., 2009). In addition, it was also revealed that
phosphorylation of Mda1 may be required to change the
conformation of Dnm1 in the MD machinery to constrict the
mitochondrial division site (Nishida et al., 2007). Thus, protein
modification of the components of the MD and PD machineries
are essential to accomplish mitochondrial and chloroplast
division. However, such a regulation factor for protein
modification of the MD and PD machineries has not been
identified so far. In this study, using proteomic analyses, we
identified a kinesin-like protein TOP of isolated MD/PD
machinery
complexes
before
constriction
(Fig. 1B;
supplementary material Fig. S2). Since immunofluorescence
microscopy showed that during mitochondrial and chloroplast
divisions TOP was localized on the MD/PD machinery complex
just before constriction (Figs 5, 6), it was hypothesized that TOP
is required for activation of the MD and PD machineries. Finally,
a series of analyses of the antisense suppression of TOP (Fig. 4)
and staining of phosphoproteins in the MD and PD machineries
(Fig. 7) showed that TOP is required to induce contraction of the
MD and PD machineries by phosphorylation. TOP is directly
bound to the MD machinery in late S phase, then, TOP may
induce activation of the MD/PD machinery complex through
protein phosphorylation by transferring the mitotic kinase AUR
from the cytosol to the MD/PD machinery complex
(supplementary material Fig. S8). Combined with the results of
downregulation of TOP (Fig. 4) and localization of TOP on the
MD/PD machinery complex (Fig. 5), it is thought that mitotic
kinases need to interact with a small part of the MD/PD
machinery complex which is slightly exposed in the
cytosol to activate the MD/PD machinery complex. Indeed,
immunofluorescence signals of AUR increased in this part of the
MD/PD machinery complex in line with the progresses of
mitochondrial and chloroplast division (Fig. 7F). In order for the
mitotic kinases to interact with the MD/PD machinery complex
TOP may be required (supplementary material Fig. S8). Mitotic
kinases associated with TOP can easily move to the periphery of
the MD machinery and can also move to the periphery of the PD
machinery along the MD machinery (supplementary material Fig.
S8). Although we showed that Aurora kinase may be responsible
for the MD and PD machineries, other kinases would be needed
for the regulation of the machineries. The mammalian kinase
CDK1, an orthologue of the plant CDKA, regulates
phosphorylation of mitochondrial dynamin, Drp1, to enhance
mitochondrial division during mitosis (Taguchi et al., 2007).
CDKA and CDKB are cell cycle regulators in C. merolae
(Kobayashi et al., 2011) and CDKB mRNA accumulation is
specifically detected in the mitochondrial and chloroplast
division phase of C. merolae (supplementary material Fig. S9).
Thus, CDKA and CDKB in C. merolae are candidates to
phosphorylate MD/PD machinery complexes in addition to
Aurora kinase.
Previously, it was revealed that kinesin-superfamily members
were important molecular motors that directionally transport
various cargos, including single/double-bounded membranous
organelles and large protein complexes (Hirokawa et al., 2009).
Also, it was well known that kinesin proteins that are expressed
in M phase are involved in the organization and function of the
mitotic spindle (Walczak and Heald, 2008). These mitotic
kinesins typically act in chromosome alignment and
segregation. However, the involvement of a kinesin-like protein
in mitochondrial and/or chloroplast division has never been
reported, until now. Thus, this report is the first evidence that a
member of the kinesin-superfamily proteins is involved in
mitochondrial and chloroplast division. TOP induces protein
phosphorylation of MD and PD machineries to accomplish
mitochondrial and chloroplast divisions prior to nuclear division.
And finally, nuclear division is performed by the TOP-mediated
spindle structure (Fig. 3; supplementary material Fig. S8).
Thereby, mitochondria and chloroplast division cycles are
combined with the cell division cycle.
Recently, it was revealed that the nuclear division that is
performed by TOP-mediated spindle poles (Fig. 3) is
accompanied by the inheritance of the endoplasmic reticulum
(ER) and Golgi apparatus in association with the mitotic spindle
(summarized in supplementary material Fig. S10) (Imoto et al.,
2011; Yagisawa et al., 2012a; Yagisawa et al., 2012b). Although
the interaction of plant C-terminal KIFs with organelles is still
unclear (Cai and Cresti, 2012), it was found that animal Cterminal KIFs are involved in intracellular transportation of these
single-bounded membranous organelles. KIFC3 transports the
Golgi apparatus and KIFC2 transports early endosomes as cargo
(Saito et al., 1997; Hirokawa, 1998; Xu et al., 2002; Bananis et al.,
TOP induces organelle divisions
2004; Hirokawa et al., 2009). Moreover, we showed that the
initiation of both mitochondrial and chloroplast divisions is
regulated by TOP, and recent studies revealed that the divided
mitochondria are required as the carrier of microbodies and
lysosomes, which are connected with divided mitochondria
(Fujiwara et al., 2010; Imoto et al., 2010; Imoto et al., 2011).
Thus, TOP is involved in the proliferation of all single- and
double-membrane-bound organelles (supplementary material Fig.
S10). In addition, the involvement of TOP in mitochondrial and
chloroplast division, given the presence of TOP homologs in
many members of eukaryotes (supplementary material Fig. S3),
may indicate the original function of C-terminal kinesin-like
proteins, which control proliferation of all double- and singlemembrane-bound organelles.
Materials and methods
Immunofluorescence microscopy, immunoblotting analysis and staining of
phosphorylated protein
The anti-TOP guinea pig antibody was used at a dilution of 1:1000 for
immunoblotting, or at 1:100 for immunofluorescence. Antibodies against
Dynamin 1 (Dnm1), Dynamin 2 (Dnm2), Mda1, a-tubulin, CENH3 and
mitochondrial porin were used as previously described (Nishida et al., 2003;
Miyagishima et al., 2003; Nishida et al., 2005; Maruyama et al., 2007; Nishida
et al., 2007; Fujiwara et al., 2009). Secondary antibodies used for
immunofluorescence were, Alexa Fluor 488 or Alexa Fluor 555 goat anti-guinea
pig, anti-mouse or anti-rabbit IgG, highly cross-adsorbed (Molecular Probes,
Eugene, OR). Images were captured using a BX51 fluorescence microscope
(Olympus, Tokyo, Japan), equipped with an XF37 narrow bandpass filter (Omega,
Tokyo, Japan) and a C7780-10 three charge-coupled device (CCD) camera system
(Hamamatsu Photonics, Shizuoka, Japan). Primary antibody reactions were
performed for 1 hour at 4 ˚C. Secondary antibody reactions were performed for
1 hour at 4 ˚C. For the staining of phosphorylated protein, cells or isolated MD/PD
machineries were stained with Pro-Q Diamond phosphoprotein gel stain
(Molecular Probes, Eugene, OR) for 15 minutes at 4 ˚C.
Negative staining and immunoelectron microscopy
The 10D strain of Cyanidioschyzon merolae was used (Matsuzaki et al., 2004), and
was cultured in flasks, with shaking, under continuous light (40 W/m2) at 42 ˚C.
For synchronization, the cell cultures were subcultured to ,16107 cells/ml in a
flat-bottomed flask and subjected to a 12-hour light/12-hour dark cycle at 42 ˚C
using an automatic light/dark cycle CM incubator (Fujimoto Rika, Tokyo, Japan)
(K. Suzuki et al., 1994). Synchronized cells were harvested at the start of the
second dark period (S phase) or after 2 hours of the second dark period (M phase).
Dividing chloroplasts and mitochondria and intact PD and MD machineries were
isolated as described previously (Yoshida et al., 2009).
For immunoelectron microscopy (immuno-EM), primary reactions were performed
for 1 hour at 4 ˚C with guinea pig anti-TOP or rabbit anti-Mda1, diluted 1:100 in
Can Get Signal Immunostain Solution B (TOYOBO, Osaka, Japan), and labeled
with gold particle-conjugated secondary antibody (British BioCell International,
Cardiff, UK; 15-nm for TOP or 10-nm for Mda1) at a dilution of 1/20. After MD
and PD machineries with outer membranes were incubated in organelle membrane
dissolution buffer (OMD buffer; PBS containing 100 mM n-octyl-b-Dglucopyranoside, 6 mM sodium lauryl sulphate, 20 mM urea) for 2 minutes on
ice, the lysate was negatively stained with 0.5% phosphotungstic acid (pH 7.0).
The samples were then examined with an electron microscope (model JEM-1230;
JEOL, Tokyo, Japan).
MALDI-TOF-MS analysis and MASCOT search
Plasmid construction of pCPG and pCPG-TOP-AS
Samples were analyzed by a peptide mass fingerprinting (PMF) search using a
MALDI TOF AXIMA TOF2 mass spectrometer (Shimadzu, Kyoto, Japan) in
reflectron mode. Database searches were performed using the software program
MASCOT v2.2.01 (Matrix Science, MA, USA) running on the local server
against the C. merolae genome database (including 5014 sequences) based on the
FASTA file distributed by the Cyanidioschyzon merolae Genome Project (http://
merolae.biol.s.u-tokyo.ac.jp/). The permissible value of missed cleavages was set
at one. MS tolerance values were set at 0.2–0.4 Da. Identified proteins (a
MASCOT score of more than 50) are listed in supplementary material Fig. S2;
Table S1.
PCR reactions were performed with KOD FX (TOYOBO, Osaka, Japan) using the
oligonucleotide primers, 59-cttaaccgtactgatcgtact-39 and 59-tagtctaaactgagaacagcc39 and pBSHAb-T39 as a template (Ohnuma et al., 2008), and 59-tctcagtttagactactgcactcaaagtgagtgtccg-39 and 59-atcagtacggttaagtcatgtttgacagcttatcatc-39 and
pI050P-GFP as a template (Ohnuma et al., 2009). The resultant DNA fragments
were combined and cloned to make pCPG (catalase promoter with sGFP) using the
In-FusionTM advantage PCR cloning kit (Clontech, CA, USA). The 59-flanking
region (1500 bp) and the open reading frame (ORF) of the TOP gene (2553 bp)
was amplified using the oligonucleotide primers, 59-ggcggccgctctagagaatggaaatcgcgcgcttctcc-39 and 59-tgggtaattaattaatggctcctggaaagagactcgttg-39. The pCPG
vector fragment was amplified using the oligonucleotide primers, 59-ttaattaattacccatacgatgttcctgactatgcggg-39 and 59-tctagagcggccgccaccg-39 and pCPG as a
template. The resultant DNA fragments were combined and cloned to make pCPGTOP-S using the In-FusionTM advantage PCR cloning kit (Clontech, CA, USA).
The antisense strand of the TOP ORF (2553 bp) was amplified using the
oligonucleotide primers, 59-aagtgcgcctgcgcatggctcctggaaagagactcgttg-39 and 59tgggtaattaattaaatgattcgagacagg-39. Using the oligonucleotide primers, 59-ttaattaattacccatacgatgttcctgac-39 and 59-tgcgcaggcgcacttg-39 and pCPG-TOP-S as a
template, DNA fragments were PCR amplified, then combined and cloned to
make pCPG-TOP-AS using the In-FusionTM advantage PCR cloning kit (Clontech,
CA, USA). pCPG-TOP-AS has the antisense strand of the TOP ORF instead of the
sense strand of the TOP ORF. The resultant plasmids, pCPG as a control and
pCPG-TOP-AS for antisense suppression of TOP, have a catalase promoter-sGFP
fused sequence; therefore, transformed cells could be detected by green
fluorescence under microscopic observation. Transformation of C. merolae cells
was performed as described previously (Ohnuma et al., 2009).
Cell culture and isolation of MD/PD machinery complexes
Journal of Cell Science
2399
Phylogenetic analysis
Additional amino acid sequences of C-terminal kinesin-superfamily members
were obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and were
automatically aligned using CLUSTAL X, version 2.0.9 (http://www.clustal.org/
download/current/) (Larkin et al., 2007). For phylogenetic analyses, ambiguously
aligned regions were manually arranged or deleted using BioEdit Sequence
Alignment Editor, version 4.8.10 (http://www.mbio.ncsu.edu/BioEdit/bioedit.
html), resulting in 363 amino acids (including inserted gaps) being used.
Phylogenetic tree construction and bootstrap analyses were performed using
PHYLIP, version 3.66 (http://evolution.genetics.washington.edu/phylip.html),
PROTDIST and NEIGHBOR for neighbor-joining (NJ), PROTPARS for
maximum parsimony (MP), and PROML for maximum likelihood (ML). The
JTT + C model (among-site rate variation model with four rate categories) was
selected as the probability model for NL and ML analyses. Multiple datasets for
bootstrap analyses (1000 replicates for NJ and MP, 100 replicates for ML) were
calculated using CONSENSE. No outgroup was used to root the tree.
Antibodies
To generate an anti-TOP antiserum in guinea pig, amino acids 1–431 of the
predicted 431-amino-acid sequence of the CMR497C protein was amplified by
PCR using the following primers: 59-cgggatccatgattcgagacagggttccag-39 and 59cccaagctttgcgcgaagttccatgatc-39. The resultant DNA fragment was cloned into
pQE80L (Qiagen, Hilden, Germany) following restriction digestion at BamHI and
HindIII sites. Protein expression and purification was performed as previously
described (Nishida et al., 2005). To produce a protein expression vector for
AUR, the full length of the coding sequence for AUR was amplified by PCR
with the following primer: 59-atggtaccatgcaggcgacaccaggcct-39, 59-atagaagcttctattgttccgcagcgtgca-39. The fragment was cloned into the KpnI/HindIII site of
pCold-GST (Hayashi and Kojima, 2008). The vector was transformed into
OverExpressTM C43 (DE3) (Lucigen, WI, USA). The recombinant protein GST–
AUR was purified with GSTrap HP (GE Healthcare, Buckinghamshire, UK). The
protein was injected into a rabbit and the antiserum was subjected to affinity
purification (Protein Purify Ltd, Gunma, Japan).
Acknowledgements
We thank T. Shimada for technical help with MALDI-TOF analysis.
Author Contributions
Y.Y., T.F., Y.I., M.O., S.H., O.M., H.K., S.K., S.M. and T.K. designed
the research, and Y.Y., T.F., Y.I., M.O., S.K. and S.M. carried out the
research. Y.Y., M.Y. and Y.I. performed the proteomics analyses.
Y.Y. and T.F. analyzed the C. merolae microarray data. H.K.
performed immuno-EM. Y.Y., T.F., Y.I. and M.O. performed TOP
downregulation analysis. Y.Y. and T.K. wrote the paper.
Funding
This work was supported by a Grant-in-Aid for Scientific Research
on Priority Areas (A) [grant number 22247007 to T.K.]; a Grant-inAid for Challenging Exploratory Research [grant number 22657061 to
2400
Journal of Cell Science 126 (11)
T.K.]; a Grant-in-Aid for X-Ray Free Electron Laser Priority Strategy
Program from Japan’s Ministry of Education, Culture, Sports, Science
and Technology [grant number 23370029 to S.M.]; Japan’s Ministry
of Education, Culture, Sports, Science and Technology/Japan Society
for the Promotion of Science ‘Kakenhi’ [grant number 23120518 to
S.M.]; and a Human Frontier Science Program Long Term Fellowship
[grant number LT000356/2011-L to Y.Y.].
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.116798/-/DC1
Journal of Cell Science
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