Aurora Kinase of the Red Alga Cyanidioschyzon Merolae Is Related to Both Mitochondrial Division and Mitotic Spindle Formation

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Aurora Kinase of the Red Alga Cyanidioschyzon merolae is Related to Both Mitochondrial Division and Mitotic Spindle Formation

Shoichi Kato1, Yuuta Imoto2,3, Mio Ohnuma3, Tomoko M. Matsunaga1, Haruko Kuroiwa3, Shigeyuki Kawano2, Tsuneyoshi Kuroiwa3 and Sachihiro Matsunaga1,*

1 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278–8510, Japan 2 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277–8562, Japan 3 Laboratory of Cell Biology, Department of Life Science, College of Science, Rikkyo University, 3–34–1 Nishi-ikebukuro, Toshima, Tokyo 171–8501, Japan

Received September 28, 2011; accepted October 26, 2011

Summary In eukaryotic cells, the Aurora kinase family contributes to the successful progression of mitosis by regul ating centrosome maturation, spindle formation, microtubule attachment to kineto chores, and cytokinesis. A primitive red alga, Cyanidioschyzon merolae, contains a single mitochondrion, which divides just before nuclear division. In this study, we identiﬁed a single ortho- logue of Aurora kinase in C. merolae, which was designated CmAUR. Analysis of the dynamics during mitosis of CmAUR fused with the green ﬂuorescent protein showed CmAUR is localized to the mitotic spindle and polar microtubules along the mitochondrion. Interestingly, CmAUR signals were also detected in the mitochondrial division site. These results indicate Aurora kinase in C. merolae is related to mitochondrial division in addition to mitotic spindle formation.

Key words Aurora kinase, Mitochondrial division, Spindle, Cyanidioschyzon merolae.

As cell cycle regulators, mitotic kinases promote correct mitotic progression by phosphorylation of each substrate. Aurora kinase, a mitotic serine/threonine kinase, is highly conserved in eukary otic organisms (Cheeseman et al. 2002, Andrews et al. 2003, Carmena and Earnshaw 2003). Animal paralogues of Aurora kinase are mainly classiﬁed into 2 groups, namely Aurora A and B (Glover et al. 1995, Roghi et al. 1998, Schumacher et al. 1998, Mesilaty-Gross et al. 1999, Adams et al. 2000). Aurora A, which is localized to the spindle during mitosis (Kimura et al. 1997), functions in centrosome maturation, maintenance, duplication, and spindle assembly (Giet et al. 1999, Berdinik and Knoblich 2002). Aurora B is localized to the centromere in prometaphase and metaphase, and regulates accurate binding of the kinetochore to microtubules. In anaphase, Aurora B is transferred to the spindle midzone and regulates cytokinesis (Andrews et al. 2003, Carmena and Earnshaw 2003). Three Aurora kinase paralogues are known in Arabidopsis thaliana, namely AtAUR1, AtAUR2 and AtAUR3. AtAUR1/AtAUR2 and AtAUR3 are regarded as functional homo logues of Aurora A and Aurora B, respectively, although AtAUR1 is also related to cytoplas- mic division (Demidov et al. 2005, Kawabe et al. 2005, Kurihara et al. 2006, Kurihara et al. 2007). Microtubules are involved in mitochondrial division or distribution in addition to construction of the spindle for nuclear division (Yaffe et al. 2003, Nishida et al. 2005, Imoto et al. 2010). Thus it is possible that Aurora kinase, which is responsible for microtubule dynamics, directly regulates

* Corresponding author, e-mail: [email protected] 456 S. Kato et al. Cytologia 76(4) mitochondrial division. Therefore, we embarked on the elucidation of the relationship between Aurora kinase and mitochondrial division. However, plant and animal cells contain many mitochondria which divide and fuse randomly; therefore, it has been difﬁcult to elucidate the division. To avoid this problem, we used the primitive red alga Cyanidioschzon merolae, which possesses a minimum set of organelles, e.g., a single mitochondrion and a single chloroplast in interphase. Such a simple organelle set has provided novel insights into organelle dynamics (Kuroiwa et al. 2006, Yoshida et al. 2010, Imoto et al. 2011). Additionally, the complete genome sequence C. merolae was decoded (Matsuzaki et al. 2004, Nozaki et al. 2007). When C. merolae undergoes cell division, the mitochondrion bends at the dividing plane and divides just before nuclear division. Around the bending site, the mitochondrial division ring (MD ring) is assembled (Kuroiwa et al. 1998, Miyagishima et al. 2003, Kuroiwa et al. 2008). The MD ring mediates mitochondrial ﬁssion (Nishida et al. 2003), and then polar microtubules distribute mitochondria to the daughter cells (Imoto et al. 2010). In this study, we visualized and analyzed Aurora kinase of C. merolae, and observed the relationship between the Aurora kinase and polar microtubules or mitochondrion.

Materials and methods

Cell culture Cyanidioschyzon merolae strain 10D was used in this study. Cells were cultured in MA2 me- dium (Ohnuma et al. 2008) in a glass vessel under continuous white light (100 μmol photon m-2 s-1) at 40°C.

Search for Aurora kinase candidate gene Aurora kinase candidate genes from the C. merolae genome that showed high similarity to previously known Aurora kinases were searched against the complete C. merolae genome sequence (Matsuzaki et al. 2004) by means of a BLAST search. Amino acid sequences of the Aurora kinases were aligned using ClustalW (Thompson et al. 1994). A phylogenetic tree was constructed by the neighbor-joining method with a Poisson correction distance model using MEGA2 software (Kumar et al. 2000). Bootstrap probabilities for each node were calculated with 1000 replicates.

Plasmid construction To create the fusion protein pCmAUR-sGFP, a 2787 bp fragment of CmAUR, which extended from the 5′ flanking region to the end of the open reading frame (ORF), was generated by fusing two individual fragments of the sequence. The 5′-flanking region and 5′ portion of the CmAUR ORF (1738 bp) was amplified with the primer set CmAUR1F; 5′-AGGAAGCTTCCTT- GGGATTTTTCTGGACT-3′ and CmAUR1R; 5′-CTCGGAAGCATAGATGACAGC-3′ (HindIII site underlined). This polymerase chain reaction (PCR) was carried out for 35 cycles (95°C for 20 s, 55°C for 20 s, and 72°C for 1.5 min) with ExTaq® (TaKaRa Bio, Otsu, Japan). Similarly, the 3′ portion of the CmAUR ORF (1266 bp) was amplified with the primer set CmAUR2F; 5′-CAAA- GGCATCACCCGAGAAA-3′ and CmAUR2R; 5′-CAAGCCATGGCTTGTTCCGCAGCGTGC- ATTC-3′ (NcoI site underlined). This PCR was carried out for 35 cycles (95°C for 20 s,5° C5 for 20 s, and 72°C for 1.5 min) with ExTaq® (TaKaRa Bio, Otsu, Japan). These fragments were designed to have overlapping complementary regions. PCR was performed with these fragments for 10 cycles (98°C for 10 s and 72°C for 1 min) to generate a fused DNA fragment (2787 bp). This fragment was amplified with the primer set CmAUR1F and CmAUR2R. This PCR was carried out for 20 cycles (98°C for 6 s, 60°C for 10 s, and 72°C for 1.5 min) with PrimeSTAR® HS DNA Polymerase (TaKaRa Bio, Otsu, Japan). The amplified fragment was digested with HindIII and NcoI, and cloned into the HindIII/NcoI site of pTH2PL (Ohnuma et al. 2009). The created plasmid, pCmAUR-sGFP has a 1408-bp of promoter region of CmAUR and a 1359-bp CmAUR ORF fused 2011 Aurora Kinase is Related to Mitochondrial Division 457 to sGFP.

Transformation Using 12 μg of pCmAUR-sGFP, the transformation of C. merolae cells was performed according to the methods of Ohnuma et al. (2008). After culture for 24 h, the cells were collected for immunoﬂuorescence microscopy.

Immunofluorescence microscopy Cell ﬁxation and immunoﬂuorescence microscopy were performed as described previously (Nishida et al. 2004). Primary and secondary antibodies were used at the following concentrations: 1 : 400 for rabbit anti-GFP antibody (MBL, Nagoya, Japan) (costaining with α-tubulin), 1 : 100 for rabbit anti-GFP antibody (costaining with mitochondrial Porin and Mda1), 1 : 1000 for Alexa-488 goat antirabbit antibody, 1 : 600 for mouse anti-α-tubulin antiserum, 1 : 1000 for Alexa-555 goat anti mouse antibody, 1 : 200 for guinea pig anti-mitochondrial Porin antiserum (a mitochondrial marker protein; Fujiwara et al. 2009), 1 : 1000 for Alexa-555 goat antiguinea pig antibody, 1 : 100 for rabbit anti-Mda1 (Nishida et al. 2007), and 1 : 1000 for Alexa-555 goat antirabbit antibody. Staining with 4′,6-diamidino-2-phenylindole (DAPI) was performed as described previously (Yagisawa et al. 2007). Images were captured as described previously (Nishida et al. 2005).

Results and discussion

We identified a single-copy gene homologous to Aurora kinase in the C. merolae genome data base (Matsuzaki et al. 2004), which was designated CmAUR. The kinase domain showed high similarity to those of homologues in other eukaryotic organisms. Moreover, both the active center and the phosphorylation sites in the Aurora kinases were conserved (Fig. 1). Therefore, CmAUR is expected to show kinase activity. According to the neighbor-joining analysis, CmAUR belonged to the clade of plant Aurora kinases (Fig. 2). Ipl1, an Aurora kinase of an unicellular budding yeast, does not have similarity with CmAUR, which suggested evolutionary diversification of Aurora kinases occurred independently in each kingdom of fungi, plants and animals. To visualize CmAUR dynamics, we used GFP system as described by Ohnuma et al. (2009). sGFP was fused to the C-terminal of CmAUR which is driven by CmAUR promoter. The plasmid was transformed into C. merolae. After cell fixation, immunostaining was performed using antibodies against GFP (Fig. 3 Ag–Al) and α-tubulin (Fig. 3 Am–Ar). DNA was stained by DAPI (Fig. 3 Aa–Af). The mitotic phase of cells in the fluorescent and phase contrast images were distin- guished by localization of α-tubulin or shape of nuclei as previously described (Imoto 2010). In the G2 phase, CmAUR was localized to the microtubule-organizing center and the plus end of polar microtubules elongating along the mitochondrion (Fig. 3 Ag, Am, As, Ba). At prophase, when polar microtubules were organized along the mitochondrion indicating the mitochondrial division period as shown in previous study (Imoto et al. 2010), intense GFP signal was observed from the central micro tubules between the 2 poles, in addition to microtubules (Fig. 3 Ah, An, At, Bb). At prometaphase and metaphase, the polar microtubules on the mitochondrion disappeared and a mitotic spindle was formed. CmAUR was localized to the spindle poles, mitotic spindle, and region between divided organelles (Fig. 3 Ai, Aj, Ao, Ap, Au, Av, Bc, Bd). After nuclear division, CmAUR was detected in the spindle midzone (Fig. 3 Ae, Ak, Aq, Aw, Be). When mitosis terminates, CmAUR looked to exist around mitochondria (Fig. 3 Al, Ar, Ax, Bf). CmAUR was localized to a number of positions at each phase during mitosis. Localization to the mitotic spindle and the spindle pole is a representative characteristic of Aurora A (Kimura et al. 1997, Giet et al. 1999, Berdinik and Knoblich 2002). CmAUR might have similar functions to Aurora A, which induces spindle pole maturation and spindle assembly. In contrast, localization to 458 S. Kato et al. Cytologia 76(4)

Fig. 1. Alignment of amino acid sequences of C. merolae Aurora kinase CmAUR (accession no. CMQ044C), 2 human Aurora kinases comprising Aurora A and Aurora B (O14965 and Q96GD4, respectively), 3 Arabidopsis thaliana Aurora kinases AtAUR1, AtAUR2, AtAUR3 (AB196733, AB196734 and AB196735, respectively), and a budding yeast Ipl1 (AAA20496). Phosphorylation sites of Protein Kinase A, the active centers and the kinase domains are shown in a gray shaded box, an open box, and dotted line boxes, respectively. Asterisks indicate the presence of identical residues in all sequences. 2011 Aurora Kinase is Related to Mitochondrial Division 459

Fig. 2. Phylogenetic relationships among Aurora kinases. A Neighbor–Joining tree constructed from amino acid sequences of the kinase domains only is shown. Bootstrap probabilities from 1000 replications are provided for each node. Respective accession numbers for proteins included in the analysis are: Aurora kinase A and B from Homo sapiens (O14965 and Q96GD4), Mus musculus (O70126 and P97477), Xenopus laevis (S52243 and AAG10787), Caenorhabditis elegans (NP_505119 and NP_491714), and Drosophila melanogaster (CAA58469 and AAD34349); Aurora C from Homo sapiens (BAA76292) and Mus musculus (NP_065597); and proteins from Saccharomyces cerevisiae (AAA20496), Schizosaccharomyces pombe (O59790), Arabidopsis thaliana (AB196733, AB196734 and AB196735), Oryza sativa (OsAUR1, AB193736; and OsAUR2, AB196737), and Cyanidio schyzonmerolae (CMQ044C). the spindle midzone indicates CmAUR also shows characteristics of Aurora B, which is responsible for cytokinesis (Andrews et al. 2003, Carmena and Earnshaw 2003). These characteristics are similar to those of Arabidopsis thaliana Aurora kinase AtAUR1, which is localized to the spindle and phragmoplast (Kawabe et al. 2005). CmAUR is a single homologue of Aurora kinase, suggesting that CmAUR would have the function of an evolutionarily primitive Aurora kinase before duplication such as Aurora kinase A and B (Fig. 2). Surprisingly, the dynamics of CmAUR were consistent with those reported for Aurora kinases of other organisms, but were also localized to polar microtubules on the mitochondrion and the center region of the mitochondrion. According to previous studies, the central position was considered to be a mitochondrial division site (Kuroiwa et al. 1998, Nishida et al. 2003). The immunostaining of GFP and mitochondrial porin, which is localized to mitochondria (Fujiwara et al. 2009), revealed CmAUR-sGFP is localized to polar microtubules on mitochondria and the mitochondrial bending site in the dividing plane (Fig. 4 a–c). The MD ring is formed at the dividing plane and the mitochondrion divides at this site (Kuroiwa et al. 1998, Nishida et al. 2003, Yoshida et al. 2009). These observations suggest that CmAUR is localized to MD ring (Fig. 3 At, Au, Bb, Bc, 4a–c). To conﬁrm whether CmAUR is localized to the MD ring, we performed immunostaining of GFP and Mda1, which are components of the MD ring (Nishida et al. 2007). CmAUR localization to the MD 460 S. Kato et al. Cytologia 76(4)

Fig. 3. Dynamics of CmAUR immunolocalization in C. merolae. (A) Fluorescent and phase contrast images of mitotic cells. (Aa–Af) Blue ﬂuorescence of DNA after staining with DAPI and phase contrast images. (Ag–Al) Images of ﬂuorescence signal of CmAUR-sGFP in green and (Am–Ar) α-tubulin in red after immunostaining. (As–Ax) Merged images of (Aa–Ar). (B) Schematic images of C. merolae and localization of CmAUR in the cell division show localization of nuclear (n), mitochondrion (mt), plastid (p) and CmAUR-sGFP (CmAUR). G2, Pro, Prometa, Meta, Ana and Tero represent cells at G2 phase, prophase, prometaphase, metaphase, anaphase and telophase, respectively. A scale bar represents 2 μm.

Fig. 4. Relationship between CmAUR and mitochondrion. Images of the fluorescence signal of CmAUR- sGFP in green (a, d), mitochondrial porin in red (b), and Mda1 in red (e) after immunostaining. The 2 fluorescence images were merged with the corresponding phase-contrast autofluorescence image (c, f). A schematic image of C. merolae in a moment of mitochondrial division shows localization of nuclear (n), mitochondrion (mt), plastid (p) and CmAUR-sGFP (CmAUR) (g). A scale bar represents 2 μm. 2011 Aurora Kinase is Related to Mitochondrial Division 461 ring was observed (Fig. 4 d–g). The mitochondrial polar microtubules are required for segregation of mitochondria (Imoto et al. 2010). Taken together, these results indicate CmAUR could be related to formation of polar microtubules on the mitochondrion and mitochondrial division directly. Therefore, CmAUR might regulate mitochondrial division in addition to nuclear division and cytokinesis.

Acknowledgements

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant nos. 23370029, 23120518 and 23012027) to S. M. and by a SENTAN Grant from the Japan Science and Technology Agency to S. M.

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