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The kinesin-like protein TOP promotes Auroralocalisation and induces mitochondrial, chloroplastand 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,`

1Graduate School of Science, Rikkyo University, 3-34-1 Nishiikebukuro, Toshima-ku, Tokyo 171-8501, Japan2Chromosome Dynamics Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan3Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa,Chiba 277-8562, Japan4Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba,Ibaraki 305-8572, Japan5Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Gobancho, Chiyoda-ku, Tokyo102-0076, Japan6Center for Frontier Research, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan7Graduate School of Medicine, Faculty of Science, Department of Biological Science and Chemistry, Yamaguchi University, 1677-1 Yoshida,Yamaguchi, Yamaguchi 753-8512, Japan8Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510,Japan

*Present address: Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA`Author for correspondence (tsune@rikkyo.ne.jp)

Accepted 18 March 2013Journal of Cell Science 126, 2392–2400� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.116798

SummaryThe cell cycle usually refers to the mitotic cycle, but the cell-division cycle in the plant kingdom consists of not only nuclear but alsomitochondrial and chloroplast division cycle. However, an integrated control system that initiates division of the three organelles has notbeen 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 ofa complex of mitochondrial-dividing (MD) and plastid-dividing (PD) machineries (MD/PD machinery complex) just prior toconstriction. 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 mitochondrialand chloroplast divisions. MD/PD machinery complexes were formed normally at each division site but they were neitherphosphorylated 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 forthe constriction. Taken together our results suggest that TOP induces phosphorylation of MD/PD machinery components to accomplishmitochondrial 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 originalfunction of C-terminal kinesin-like proteins.

Key words: Cell-division cycle, Cyanidioschyzon merolae, Kinesin, Mitochondrial division, Plastid division

IntroductionAlmost 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 cyclin-

dependent 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 double-

membrane 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

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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

essential for the generation of the motive force for contraction(Yoshida et al., 2006). In addition, the PD ring is constructed of abundle of glycosyltransferase protein PDR1-mediated-polyglucanfilaments. 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 thecontraction of the MD machineries is probably driven in the same

way as that of the PD machinery (Nishida et al., 2003; Yoshidaet al., 2009; Kuroiwa et al., 2008).

In addition, it has been reported that in C. merolae a pre-spindle structure, designated as the mitochondrial spindle, isformed from each spindle pole to the division site of

mitochondria before nuclear division (supplementary materialFig. 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 manyrelating 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 polesand 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 thecontraction phase of mitochondrial division, indicating that it isinvolved in the activation of the MD machinery. Thus, thesefindings suggest the existence of an uncharacterized pathway or

factor that coordinates timing of mitochondrial and chloroplastdivision and links mitochondrial- and chloroplast-division cycleswith the cell-division cycle.

C. merolae offers advantages for studying the regulation ofintegrated initiation of nuclear, mitochondrial and chloroplast

divisions. The cell contains just one chloroplast, onemitochondrion and one nucleus (Matsuzaki et al., 2004), thedivision 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 completegenome sequence of C. merolae facilitates highly sensitivetranscriptomic 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 thisprocess, the mitochondrion and chloroplast were divided by theMD and PD machineries, respectively. Nuclear division was alsoaccomplished by the TOP-mediated spindle. Thus, TOP regulates

division of three organelles, the nucleus, mitochondrion andchloroplast, by relocalization of AUR protein.

ResultsIdentification of a novel kinesin-like protein TOP in the MD/PD machinery complexes

To identify proteins that regulate the integrated initiation ofnuclear, mitochondrial and chloroplast division, we performed aproteomic 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 theorganelles and organization of the mitochondrial spindle begins

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.

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immediately after this phase. For isolation of non-constrictingMD/PD machinery complexes, synchronized cells were harvested

at the start of the second dark period (S phase) and treated withNonidet P-40 and n-octyl-b-D-glucopyranoside (see Materials

and Methods). After isolation of the non-constricting MD/PDmachinery complexes, proteomic analysis was performed usingmatrix-assisted laser desorption/ionization time-of-flight mass

spectrometry (MALDI-TOF-MS). In this fraction, knowncomponents of the MD and PD machinery, Dynamin 1

(Dnm1), Mda1 and Dnm2 were identified. Comparing previousresults 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 (three-organelle 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 kinesin-superfamily proteins [C-terminal KIFs (reviewed by Hirokawaet al., 2009)] (Fig. 2A), which had not previously been detected

in isolated constricting MD and PD machineries from cells in Mphase (Yoshida et al., 2009; Yoshida et al., 2010). By

phylogenetic analysis, we showed that a kinesin motor domainof 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-terminalKIFs 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 parallelmicrotubules 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/orchloroplast division has not previously been reported. We,

therefore, examined the function of TOP to reveal the regulatorymechanism of nuclear, mitochondrial and chloroplast division.

The genes of known MD- and PD-machinery-associatedproteins (Mda1 for MD machinery and Dnm2 for PDmachinery) are selectively transcribed before organelle

division; therefore, we examined the level of TOP transcriptionduring the cell cycle using C. merolae microarray data (Fujiwara

et al., 2009). The level of transcription of TOP increased during Sphase and into early M phase, and this expression profilecoincided with that of other known MD- and PD-machinery-

associated proteins (Fig. 2B). We then generated anti-TOPantibodies via bacterially expressed TOP protein. Anti-TOP

antibodies detected protein of the predicted molecular mass ofTOP (,96 kDa; Fig. 2C). Also, immunoblot analyses of total

protein from synchronized cultures detected TOP duringchloroplast, mitochondrial and nuclear divisions (Fig. 2D).Next, we examined the intracellular localization of TOP during

the cell cycle by immunofluorescence microscopy using the anti-TOP antibody (Fig. 3). Although TOP was not detected during

the G1 phase, it was observed in S phase, indicating that TOPwas located near the mitochondrial division site (Fig. 3, white

arrowhead). Then, in the S–G2 phases, the fluorescence signal ofthe TOP-assembled small spindle pole increased in size and splitinto two spindle poles (Fig. 3, black arrowheads). During M

phase, TOP was localized on microtubules in addition to at thetwo spindle poles. Therefore, we conclude that TOP is involved

in the spindle poles and the spindle microtubules for nucleardivision in M phase.

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). Twenty-

four 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).

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 M-

phase 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.

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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. 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.

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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2001). The average circumference of the PD machineriescontaining TOP was 2.6 mm (n525; supplementary material

Fig. S6), therefore, we concluded that TOP bound to the MD/PDmachinery complexes that were derived from cells in the earlyphase of mitochondrial and chloroplast divisions. However, TOP

did not associate with small MD/PD machinery complexesderived from cells in late stages of mitochondrial and chloroplastdivision (Fig. 6A). These results were also confirmed by

immuno-electron microscopy (immuno-EM; Fig. 6B,C).Immunogold particles indicating TOP were distributed in aspot-like pattern on the MD machinery (Fig. 6C, inset). Theseresults indicate that the direct interaction between TOP and larger

MD machineries plays an important role in the initiation ofconstriction of MD and PD machineries.

TOP is required for the phosphorylation of proteins in bothMD and PD machineries

Reports thus far indicate that protein phosphorylation is involved

in at least the mitochondrial division. The WD40 protein Mda1together with the MD ring assembles the outer ring of the MDmachinery, and then Mda1 is phosphorylated. In the contractionphase of the mitochondrial division, phosphorylated Mda1

oligomers on the MD machinery are disassembled by GTPhydrolysis of Dnm1 (Nishida et al., 2007). And finally, themitochondrial dynamin ring, mediated by Dnm1, severs the

bridge of the dividing mitochondrion (Nishida et al., 2003). Sincephosphorylation of Mda1 is induced with the progress of themitochondrial division phases, protein phosphorylation is likely

to mediate changes in the organization of the MD machinery forconstriction. Thus, it is thought that phosphorylation is one of themost important modifications for the MD and PD machineries.

Based on these results and hypothesis, we examined whetherproteins in the isolated MD/PD machinery complexes werephosphorylated in the early and late phases of mitochondrial and

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.

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).

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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

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 C-

terminal 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

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 recentstudy showed that Aurora kinase in C. merolae (named AUR)

was involved with not only mitotic spindle formation but alsomitochondrial division (Kato et al., 2011). The level oftranscription of AUR increased in S and M phases and thisexpression profile coincided with that of TOP (supplementary

material Fig. S7). Therefore, we investigated whether AUR isinvolved with TOP during mitochondrial and chloroplastdivisions. Immunofluorescence microscopy identified that AUR

colocalized with TOP and was localized around themitochondrion before the constriction phase of mitochondrialand chloroplast divisions (Fig. 7F, white arrowhead).

Subsequently, AUR accumulated at the mitochondrial divisionsite (Fig. 7F, black arrowhead). These results implied that AURmediates phosphorylation of the MD and PD machineries andspindle pole maturation. Next, we investigated the localization of

AUR in the TOP-downregulated cells to examine whether AURis 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 regionin the TOP-downregulated cells (Fig. 7G). In addition, theproteomic 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 mitochondrialand chloroplast divisions, probably by transferring of Aurorakinase to the machinery.

DiscussionThus far, it has been revealed that mitochondrial and chloroplastdivision is performed by the MD and PD machineries which are

large protein complexes. Although the molecular mechanismsinvolved are still not fully known, recent studies showed thatFtsZ 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 andsingle MD machineries showed that Dnm2 and Dnm1 arerequired 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 thatphosphorylation of Mda1 may be required to change the

conformation of Dnm1 in the MD machinery to constrict themitochondrial division site (Nishida et al., 2007). Thus, proteinmodification of the components of the MD and PD machineries

are essential to accomplish mitochondrial and chloroplastdivision. However, such a regulation factor for proteinmodification of the MD and PD machineries has not beenidentified so far. In this study, using proteomic analyses, we

identified a kinesin-like protein TOP of isolated MD/PDmachinery complexes before constriction (Fig. 1B;supplementary material Fig. S2). Since immunofluorescence

microscopy showed that during mitochondrial and chloroplastdivisions TOP was localized on the MD/PD machinery complexjust 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 theMD and PD machineries by phosphorylation. TOP is directlybound to the MD machinery in late S phase, then, TOP may

induce activation of the MD/PD machinery complex throughprotein phosphorylation by transferring the mitotic kinase AUR

from the cytosol to the MD/PD machinery complex(supplementary material Fig. S8). Combined with the results ofdownregulation of TOP (Fig. 4) and localization of TOP on theMD/PD machinery complex (Fig. 5), it is thought that mitotic

kinases need to interact with a small part of the MD/PDmachinery complex which is slightly exposed in thecytosol to activate the MD/PD machinery complex. Indeed,

immunofluorescence signals of AUR increased in this part of theMD/PD machinery complex in line with the progresses ofmitochondrial and chloroplast division (Fig. 7F). In order for the

mitotic kinases to interact with the MD/PD machinery complexTOP may be required (supplementary material Fig. S8). Mitotickinases associated with TOP can easily move to the periphery ofthe 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 responsiblefor the MD and PD machineries, other kinases would be needed

for the regulation of the machineries. The mammalian kinaseCDK1, an orthologue of the plant CDKA, regulatesphosphorylation 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 chloroplastdivision phase of C. merolae (supplementary material Fig. S9).Thus, CDKA and CDKB in C. merolae are candidates tophosphorylate MD/PD machinery complexes in addition to

Aurora kinase.

Previously, it was revealed that kinesin-superfamily memberswere important molecular motors that directionally transport

various cargos, including single/double-bounded membranousorganelles 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 themitotic spindle (Walczak and Heald, 2008). These mitotickinesins typically act in chromosome alignment andsegregation. However, the involvement of a kinesin-like protein

in mitochondrial and/or chloroplast division has never beenreported, until now. Thus, this report is the first evidence that amember of the kinesin-superfamily proteins is involved in

mitochondrial and chloroplast division. TOP induces proteinphosphorylation of MD and PD machineries to accomplishmitochondrial and chloroplast divisions prior to nuclear division.

And finally, nuclear division is performed by the TOP-mediatedspindle 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 isperformed by TOP-mediated spindle poles (Fig. 3) isaccompanied 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 stillunclear (Cai and Cresti, 2012), it was found that animal C-terminal KIFs are involved in intracellular transportation of these

single-bounded membranous organelles. KIFC3 transports theGolgi apparatus and KIFC2 transports early endosomes as cargo(Saito et al., 1997; Hirokawa, 1998; Xu et al., 2002; Bananis et al.,

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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 single-

membrane-bound organelles.

Materials and methodsCell culture and isolation of MD/PD machinery complexes

The 10D strain of Cyanidioschyzon merolae was used (Matsuzaki et al., 2004), andwas cultured in flasks, with shaking, under continuous light (40 W/m2) at 42 C̊.

For synchronization, the cell cultures were subcultured to ,16107 cells/ml in aflat-bottomed flask and subjected to a 12-hour light/12-hour dark cycle at 42 C̊using 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 wereisolated as described previously (Yoshida et al., 2009).

MALDI-TOF-MS analysis and MASCOT search

Samples were analyzed by a peptide mass fingerprinting (PMF) search using a

MALDI TOF AXIMA TOF2 mass spectrometer (Shimadzu, Kyoto, Japan) inreflectron mode. Database searches were performed using the software programMASCOT v2.2.01 (Matrix Science, MA, USA) running on the local server

against the C. merolae genome database (including 5014 sequences) based on theFASTA 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 (aMASCOT score of more than 50) are listed in supplementary material Fig. S2;Table S1.

Phylogenetic analysis

Additional amino acid sequences of C-terminal kinesin-superfamily members

were obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and wereautomatically 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 SequenceAlignment 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 formaximum parsimony (MP), and PROML for maximum likelihood (ML). The

JTT + C model (among-site rate variation model with four rate categories) wasselected as the probability model for NL and ML analyses. Multiple datasets forbootstrap 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 thepredicted 431-amino-acid sequence of the CMR497C protein was amplified byPCR using the following primers: 59-cgggatccatgattcgagacagggttccag-39 and 59-

cccaagctttgcgcgaagttccatgatc-39. The resultant DNA fragment was cloned intopQE80L (Qiagen, Hilden, Germany) following restriction digestion at BamHI andHindIII sites. Protein expression and purification was performed as previously

described (Nishida et al., 2005). To produce a protein expression vector forAUR, the full length of the coding sequence for AUR was amplified by PCRwith the following primer: 59-atggtaccatgcaggcgacaccaggcct-39, 59-atagaagcttc-tattgttccgcagcgtgca-39. The fragment was cloned into the KpnI/HindIII site of

pCold-GST (Hayashi and Kojima, 2008). The vector was transformed intoOverExpressTM 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 affinitypurification (Protein Purify Ltd, Gunma, Japan).

Immunofluorescence microscopy, immunoblotting analysis and staining ofphosphorylated protein

The anti-TOP guinea pig antibody was used at a dilution of 1:1000 forimmunoblotting, or at 1:100 for immunofluorescence. Antibodies againstDynamin 1 (Dnm1), Dynamin 2 (Dnm2), Mda1, a-tubulin, CENH3 andmitochondrial porin were used as previously described (Nishida et al., 2003;Miyagishima et al., 2003; Nishida et al., 2005; Maruyama et al., 2007; Nishidaet al., 2007; Fujiwara et al., 2009). Secondary antibodies used forimmunofluorescence were, Alexa Fluor 488 or Alexa Fluor 555 goat anti-guineapig, 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 wereperformed for 1 hour at 4 C̊. Secondary antibody reactions were performed for1 hour at 4 C̊. For the staining of phosphorylated protein, cells or isolated MD/PDmachineries were stained with Pro-Q Diamond phosphoprotein gel stain(Molecular Probes, Eugene, OR) for 15 minutes at 4 C̊.

Negative staining and immunoelectron microscopy

For immunoelectron microscopy (immuno-EM), primary reactions were performedfor 1 hour at 4 C̊ with guinea pig anti-TOP or rabbit anti-Mda1, diluted 1:100 inCan Get Signal Immunostain Solution B (TOYOBO, Osaka, Japan), and labeledwith 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 MDand PD machineries with outer membranes were incubated in organelle membranedissolution buffer (OMD buffer; PBS containing 100 mM n-octyl-b-D-glucopyranoside, 6 mM sodium lauryl sulphate, 20 mM urea) for 2 minutes onice, 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).

Plasmid construction of pCPG and pCPG-TOP-AS

PCR reactions were performed with KOD FX (TOYOBO, Osaka, Japan) using theoligonucleotide primers, 59-cttaaccgtactgatcgtact-39 and 59-tagtctaaactgagaacagcc-39 and pBSHAb-T39 as a template (Ohnuma et al., 2008), and 59-tctcagtttagac-tactgcactcaaagtgagtgtccg-39 and 59-atcagtacggttaagtcatgtttgacagcttatcatc-39 andpI050P-GFP as a template (Ohnuma et al., 2009). The resultant DNA fragmentswere combined and cloned to make pCPG (catalase promoter with sGFP) using theIn-FusionTM advantage PCR cloning kit (Clontech, CA, USA). The 59-flankingregion (1500 bp) and the open reading frame (ORF) of the TOP gene (2553 bp)was amplified using the oligonucleotide primers, 59-ggcggccgctctagagaatggaaa-tcgcgcgcttctcc-39 and 59-tgggtaattaattaatggctcctggaaagagactcgttg-39. The pCPGvector fragment was amplified using the oligonucleotide primers, 59-ttaattaatta-cccatacgatgttcctgactatgcggg-39 and 59-tctagagcggccgccaccg-39 and pCPG as atemplate. The resultant DNA fragments were combined and cloned to make pCPG-TOP-S using the In-FusionTM advantage PCR cloning kit (Clontech, CA, USA).The antisense strand of the TOP ORF (2553 bp) was amplified using theoligonucleotide primers, 59-aagtgcgcctgcgcatggctcctggaaagagactcgttg-39 and 59-tgggtaattaattaaatgattcgagacagg-39. Using the oligonucleotide primers, 59-ttaattaa-ttacccatacgatgttcctgac-39 and 59-tgcgcaggcgcacttg-39 and pCPG-TOP-S as atemplate, DNA fragments were PCR amplified, then combined and cloned tomake 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 thesense strand of the TOP ORF. The resultant plasmids, pCPG as a control andpCPG-TOP-AS for antisense suppression of TOP, have a catalase promoter-sGFPfused sequence; therefore, transformed cells could be detected by greenfluorescence under microscopic observation. Transformation of C. merolae cellswas performed as described previously (Ohnuma et al., 2009).

AcknowledgementsWe thank T. Shimada for technical help with MALDI-TOF analysis.

Author ContributionsY.Y., T.F., Y.I., M.O., S.H., O.M., H.K., S.K., S.M. and T.K. designedthe research, and Y.Y., T.F., Y.I., M.O., S.K. and S.M. carried out theresearch. 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 TOPdownregulation analysis. Y.Y. and T.K. wrote the paper.

FundingThis work was supported by a Grant-in-Aid for Scientific Researchon Priority Areas (A) [grant number 22247007 to T.K.]; a Grant-in-Aid for Challenging Exploratory Research [grant number 22657061 to

TOP induces organelle divisions 2399

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T.K.]; a Grant-in-Aid for X-Ray Free Electron Laser Priority StrategyProgram from Japan’s Ministry of Education, Culture, Sports, Scienceand Technology [grant number 23370029 to S.M.]; Japan’s Ministryof Education, Culture, Sports, Science and Technology/Japan Societyfor the Promotion of Science ‘Kakenhi’ [grant number 23120518 toS.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

ReferencesAndrews, P. D., Knatko, E., Moore, W. J. and Swedlow, J. R. (2003). Mitotic

mechanics: the auroras come into view. Curr. Opin. Cell Biol. 15, 672-683.Bananis, E., Nath, S., Gordon, K., Satir, P., Stockert, R. J., Murray, J. W. and

Wolkoff, A. W. (2004). Microtubule-dependent movement of late endocytic vesiclesin vitro: requirements for Dynein and Kinesin. Mol. Biol. Cell 15, 3688-3697.

Bleazard, W., McCaffery, J. M., King, E. J., Bale, S., Mozdy, A., Tieu, Q., Nunnari,

J. and Shaw, J. M. (1999). The dynamin-related GTPase Dnm1 regulatesmitochondrial fission in yeast. Nat. Cell Biol. 1, 298-304.

Cai, G. and Cresti, M. (2012). Are kinesins required for organelle trafficking in plantcells? Front. Plant Sci. 3, 170.

Fujiwara, T., Misumi, O., Tashiro, K., Yoshida, Y., Nishida, K., Yagisawa, F.,

Imamura, S., Yoshida, M., Mori, T., Tanaka, K. et al. (2009). Periodic geneexpression patterns during the highly synchronized cell nucleus and organelle divisioncycles in the unicellular red alga Cyanidioschyzon merolae. DNA Res. 16, 59-72.

Fujiwara, T., Kuroiwa, H., Yagisawa, F., Ohnuma, M., Yoshida, Y., Yoshida, M.,

Nishida, K., Misumi, O., Watanabe, S., Tanaka, K. et al. (2010). The coiled-coilprotein VIG1 is essential for tethering vacuoles to mitochondria during vacuoleinheritance of Cyanidioschyzon merolae. Plant Cell 22, 772-781.

Gao, H., Kadirjan-Kalbach, D., Froehlich, J. E. and Osteryoung, K. W. (2003).ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast divisionmachinery. Proc. Natl. Acad. Sci. USA 100, 4328-4333.

Glover, D. M., Leibowitz, M. H., McLean, D. A. and Parry, H. (1995). Mutations inaurora prevent centrosome separation leading to the formation of monopolar spindles.Cell 81, 95-105.

Hayashi, K. and Kojima, C. (2008). pCold-GST vector: a novel cold-shock vectorcontaining GST tag for soluble protein production. Protein Expr. Purif. 62, 120-127.

Hirokawa, N. (1998). Kinesin and dynein superfamily proteins and the mechanism oforganelle transport. Science 279, 519-526.

Hirokawa, N., Noda, Y., Tanaka, Y. and Niwa, S. (2009). Kinesin superfamily motorproteins and intracellular transport. Nat. Rev. Mol. Cell Biol. 10, 682-696.

Imoto, Y., Fujiwara, T., Yoshida, Y., Kuroiwa, H., Maruyama, S. and Kuroiwa,T. (2010). Division of cell nuclei, mitochondria, plastids, and microbodies mediatedby mitotic spindle poles in the primitive red alga Cyanidioschyzon merolae.Protoplasma 241, 63-74.

Imoto, Y., Yoshida, Y., Yagisawa, F., Kuroiwa, H. and Kuroiwa, T. (2011). The cellcycle, including the mitotic cycle and organelle division cycles, as revealed bycytological observations. J. Electron Microsc. (Tokyo) 60 Suppl. 1, S117-S136.

Kato, S., Imoto, Y., Ohnuma, M., Matsunaga, T. M., Kuroiwa, H., Kawano, S.,Tsuneyoshi, K. and Matsunaga, S. (2011). Aurora Kinase of the red algaCyanidioschyzon merolae is related to both mitochondrial division and mitoticspindle formation. Cytologia (Tokyo) 76, 455–462.

Kobayashi, Y., Kanesaki, Y., Tanaka, A., Kuroiwa, H., Kuroiwa, T. and Tanaka,

K. (2009). Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclearDNA replication in plant cells. Proc. Natl. Acad. Sci. USA 106, 803-807.

Kobayashi, Y., Imamura, S., Hanaoka, M. and Tanaka, K. (2011). A tetrapyrrole-regulated ubiquitin ligase controls algal nuclear DNA replication. Nat. Cell Biol. 13,483-487.

Kuroiwa, T. (1998). The primitive red algae Cyanidium caldarium and Cyanidioschyzon

merolae as model system for investigating the dividing apparatus of mitochondria andplastids. Bioessays 20, 344-354.

Kuroiwa, T., Kuroiwa, H., Sakai, A., Takahashi, H., Toda, K. and Itoh, R. (1998).The division apparatus of plastids and mitochondria. Int. Rev. Cytol. 181, 1-41.

Kuroiwa, T., Misumi, O., Nishida, K., Yagisawa, F., Yoshida, Y., Fujiwara, T. andKuroiwa, H. (2008). Vesicle, mitochondrial, and plastid division machineries withemphasis on dynamin and electron-dense rings. Int. Rev. Cell Mol. Biol. 271, 97-152.

Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A.,

McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R. et al. (2007).Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948.

Maruyama, S., Kuroiwa, H., Miyagishima, S. Y., Tanaka, K. and Kuroiwa,

T. (2007). Centromere dynamics in the primitive red alga Cyanidioschyzon merolae.Plant J. 49, 1122-1129.

Matsuzaki, M., Misumi, O., Shin-I, T., Maruyama, S., Takahara, M., Miyagishima,S. Y., Mori, T., Nishida, K., Yagisawa, F., Nishida, K. et al. (2004). Genome

sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature

428, 653-657.

Mears, J. A., Lackner, L. L., Fang, S., Ingerman, E., Nunnari, J. and Hinshaw, J. E.

(2011). Conformational changes in Dnm1 support a contractile mechanism for

mitochondrial fission. Nat. Struct. Mol. Biol. 18, 20-26.

Miyagishima, S., Takahara, M., Mori, T., Kuroiwa, H., Higashiyama, T. and

Kuroiwa, T. (2001). Plastid division is driven by a complex mechanism that involves

differential transition of the bacterial and eukaryotic division rings. Plant Cell 13,

2257-2268.

Miyagishima, S., Nishida, K., Mori, T., Matsuzaki, M., Higashiyama, T., Kuroiwa,

H. and Kuroiwa, T. (2003). A plant-specific dynamin-related protein forms a ring at

the chloroplast division site. Plant Cell 15, 655-665.

Nishida, K., Takahara, M., Miyagishima, S. Y., Kuroiwa, H., Matsuzaki, M. and

Kuroiwa, T. (2003). Dynamic recruitment of dynamin for final mitochondrial

severance in a primitive red alga. Proc. Natl. Acad. Sci. USA 100, 2146-2151.

Nishida, K., Yagisawa, F., Kuroiwa, H., Nagata, T. and Kuroiwa, T. (2005). Cell

cycle-regulated, microtubule-independent organelle division in Cyanidioschyzon

merolae. Mol. Biol. Cell 16, 2493-2502.

Nishida, K., Yagisawa, F., Kuroiwa, H., Yoshida, Y. and Kuroiwa, T. (2007). WD40

protein Mda1 is purified with Dnm1 and forms a dividing ring for mitochondria

before Dnm1 in Cyanidioschyzon merolae. Proc. Natl. Acad. Sci. USA 104, 4736-

4741.

Nozaki, H., Takano, H., Misumi, O., Terasawa, K., Matsuzaki, M., Maruyama, S.,

Nishida, K., Yagisawa, F., Yoshida, Y., Fujiwara, T. et al. (2007). A 100%-

complete sequence reveals unusually simple genomic features in the hot-spring red

alga Cyanidioschyzon merolae. BMC Biol. 5, 28.

Ohnuma, M., Yokoyama, T., Inouye, T., Sekine, Y. and Tanaka, K. (2008).

Polyethylene glycol (PEG)-mediated transient gene expression in a red alga,

Cyanidioschyzon merolae 10D. Plant Cell Physiol. 49, 117-120.

Ohnuma, M., Misumi, O., Fujiwara, T., Watanabe, S., Tanaka, K. and Kuroiwa,

T. (2009). Transient gene suppression in a red alga, Cyanidioschyzon merolae 10D.

Protoplasma 236, 107-112.

Osawa, M., Anderson, D. E. and Erickson, H. P. (2008). Reconstitution of contractile

FtsZ rings in liposomes. Science 320, 792-794.

Osteryoung, K. W. and Nunnari, J. (2003). The division of endosymbiotic organelles.

Science 302, 1698-1704.

Saito, N., Okada, Y., Noda, Y., Kinoshita, Y., Kondo, S. and Hirokawa, N. (1997).

KIFC2 is a novel neuron-specific C-terminal type kinesin superfamily motor for

dendritic transport of multivesicular body-like organelles. Neuron 18, 425-438.

Suzuki, K., Ehara, T., Osafune, T., Kuroiwa, H., Kawano, S. and Kuroiwa,

T. (1994). Behavior of mitochondria, chloroplasts and their nuclei during the mitotic

cycle in the ultramicroalga Cyanidioschyzon merolae. Eur. J. Cell Biol. 63, 280-288.

Taguchi, N., Ishihara, N., Jofuku, A., Oka, T. and Mihara, K. (2007). Mitotic

phosphorylation of dynamin-related GTPase Drp1 participates in mitochondrial

fission. J. Biol. Chem. 282, 11521-11529.

Walczak, C. E. and Heald, R. (2008). Mechanisms of mitotic spindle assembly and

function. Int. Rev. Cytol. 265, 111-158.

Walczak, C. E., Verma, S. and Mitchison, T. J. (1997). XCTK2: a kinesin-related

protein that promotes mitotic spindle assembly in Xenopus laevis egg extracts. J. Cell

Biol. 136, 859-870.

Xu, Y., Takeda, S., Nakata, T., Noda, Y., Tanaka, Y. and Hirokawa, N. (2002). Role

of KIFC3 motor protein in Golgi positioning and integration. J. Cell Biol. 158, 293-

303.

Xue, Y., Ren, J., Gao, X., Jin, C., Wen, L. and Yao, X. (2008). GPS 2.0, a tool to

predict kinase-specific phosphorylation sites in hierarchy. Mol. Cell. Proteomics 7,

1598-1608.

Yagisawa, F., Nishida, K., Yoshida, M., Ohnuma, M., Shimada, T., Fujiwara, T.,

Yoshida, Y., Misumi, O., Kuroiwa, H. and Kuroiwa, T. (2009). Identification of

novel proteins in isolated polyphosphate vacuoles in the primitive red alga

Cyanidioschyzon merolae. Plant J. 60, 882-893.

Yagisawa, F., Fujiwara, T., Kuroiwa, H., Nishida, K., Imoto, Y. and Kuroiwa,

T. (2012a). Mitotic inheritance of endoplasmic reticulum in the primitive red alga

Cyanidioschyzon merolae. Protoplasma 249, 1129-1135.

Yagisawa, F., Fujiwara, T., Ohnuma, M., Kuroiwa, H., Nishida, K., Imoto, Y.,

Yoshida, Y. and Kuroiwa, T. (2012b). Golgi inheritance in the primitive red alga,

Cyanidioschyzon merolae. Protoplasma (in press).

Yoshida, Y., Kuroiwa, H., Misumi, O., Nishida, K., Yagisawa, F., Fujiwara, T.,

Nanamiya, H., Kawamura, F. and Kuroiwa, T. (2006). Isolated chloroplast

division machinery can actively constrict after stretching. Science 313, 1435-1438.

Yoshida, Y., Kuroiwa, H., Hirooka, S., Fujiwara, T., Ohnuma, M., Yoshida, M.,

Misumi, O., Kawano, S. and Kuroiwa, T. (2009). The bacterial ZapA-like protein

ZED is required for mitochondrial division. Curr. Biol. 19, 1491-1497.

Yoshida, Y., Kuroiwa, H., Misumi, O., Yoshida, M., Ohnuma, M., Fujiwara, T.,

Yagisawa, F., Hirooka, S., Imoto, Y., Matsushita, K. et al. (2010). Chloroplasts

divide by contraction of a bundle of nanofilaments consisting of polyglucan. Science

329, 949-953.

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