Evaluation of the Aurora kinase ortholog CmAUR

Although some previous studies have focused on CmAUR localization in C. merolae23,24, little is known about its role as an Aurora kinase, such as in mitotic progression through histone H3 Ser10 phosphorylation in mitosis25. Thus, we considered that it was important to test the Aurora kinase function of CmAUR. First, we conducted immunostaining to determine the cellular localization of CmAUR. Previously, we reported a CmAUR antibody and confirmed its specificity24. The immunostaining showed that CmAUR localized to the mitotic spindle and mitochondrion (Fig. 1a, Supplementary Fig. 1). To investigate its function in cell cycle progression, we transiently expressed a kinase-dead version of CmAUR (CmAURK208R) in C. merolae. This mutant of CmAUR was designed by reference to previous reports of kinase-dead human Aurora A26 and B27, and we confirmed that recombinant CmAURK208R protein had no autophosphorylation activity (Fig. 1b). Expression of CmAURK208R in C. merolae delayed M phase, and this was most likely caused by a dominant-negative effect, analogous to a previous study in human27 (Fig. 1c). These results suggested that CmAUR has an important role in M phase in C. merolae, like its orthologs in other eukaryotes. We also tested whether CmAUR has capability as a kinase of histone H3 Ser10. Similar to other species, C. merolae showed phosphorylation of histone H3 Ser10 during M phase, as reported previously28 (Fig. 1d, Supplementary Fig. 2). Recombinant glutathione-S-transferase (GST)-CmAUR strongly phosphorylated H3 Ser10 in vitro (Fig. 1e). Although a weak histone H3 phospho Ser10 band was detected in the GST only-treated sample, the intensity was not different from the band in the non-GST-treated sample (Fig. 1e). Therefore, we consider that H3 Ser10 phosphorylation by GST-CmAUR depends on CmAUR. For further confirmation of whether CmAUR works as an Aurora kinase, we treated GST-CmAUR in vitro with hesperadin, an Aurora kinase inhibitor. Phosphorylation of H3 Ser10 by GST-CmAUR was inhibited by hesperadin (Fig. 1f). In summary, these results suggest that CmAUR functions as an Aurora kinase.

Fig. 1: The Cyanidioschyzon merolae Aurora kinase CmAUR has conserved properties of Aurora kinase orthologs. a Localization of CmAUR by immunofluorescence analysis. CmAUR was stained with CmAUR antibody. Mitochondria were stained by Ef-Tu antiserum. DNA was stained with DAPI. White arrowheads indicate signals localized to the spindle or spindle pole. Black arrowheads indicate plastid autofluorescence. Arrows indicate plastid DNA. The Pearson correlation coefficient (PCC) in each cell was calculated using areas without plastids. We observed 24 cells and present representative images in this figure. b Autophosphorylation assay of wild-type and mutant CmAUR. ATP-γS was used as a substrate for autophosphorylation, and the phosphorylation was detected by western blotting using thiophosphate ester antibody. c Mitotic inhibition in a dominant-negative mutant of CmAUR. CmAURK208R is a kinase-dead mutant; GFP was used as a negative control. Relevant genes were transiently overexpressed in C. merolae and mitotic cells among transfectants were counted. The error bars indicate standard error of the mean. d Immunofluorescence image of histone H3 Ser10 phosphorylation in mitosis. More than 18 cells were observed. e In vitro phosphorylation assay of histone H3 Ser10 with recombinant CmAUR. Glutathione-S-transferase (GST) was used as a negative control. Distilled water was used as a blank sample. f In vitro phosphorylation assay of histone H3 Ser10 treated with hesperadin. The concentration of hesperadin was 10 µm. The concentration of dimethyl sulfoxide in the final solution was 0.1% (v/v). Bars: 1 µm a, 2 μm c. Full size image

Relationship between mitochondrial division and CmAUR

To investigate the molecular processes regulated by CmAUR, we conducted immunostaining analyses of autophosphorylated CmAUR. Previous studies suggested that Aurora kinase activates itself through autophosphorylation29,30. Because the self-phosphorylation site of Aurora kinase is well conserved among Aurora kinase orthologs, a commercial human phospho-Aurora (phAUR) antibody was able to detect self-phosphorylated CmAUR (Supplementary Fig. 3a–d). By using the phAUR antibody, we demonstrated that activated CmAUR partially localized to the mitochondrion (Fig. 2a, Supplementary Fig. 4). Co-immunostaining using phAUR and an antibody against the mitochondrial division ring component Mda1 revealed that phAUR was localized to the mitochondrial division ring (Fig. 2b). To investigate whether the localization changed depending on cell cycle progression, we analyzed phAUR and Mda1 in both G2 and M phase. The intensity of the phAUR signals on the mitochondrial division ring increased as mitochondrial division progressed (Student’s t test, p < 0.01) but not Mda1 signals (Fig. 2b, c). Because Mda1 is a protein that accumulates on the mitochondrial division ring, this result suggests that the phAUR signals increased independently of the accumulation of other mitochondrial division-related proteins in M phase such as Mda1. After mitochondrial division, the phAUR signal was observed on structures other than the mitochondrion (Fig. 2a, white arrowheads). Considering the morphological features and a previous report on C. merolae mitosis31, we deduced that this structure was the mitotic spindle. However, localization on speckles, which are clearly different from the spindle, was also observed. This suggests that CmAUR does not co-localize exclusively with mitochondria, as already reported in human Aurora A25. We conclude that CmAUR activity is involved in both mitochondrial division and mitotic spindle formation.

Fig. 2: CmAUR involvement in mitochondrial division in mitosis. a Localization of activated CmAUR. Activated CmAUR was visualized using antibody to phosphorylated Aurora kinase. Mt indicates mitochondria, which were stained by Ef-Tu antiserum. Arrows indicate speckles localized to the mitochondrial division site. White arrowheads indicate signals that seemed to be localized to the spindle or spindle pole. Asterisks indicate speckles, which are not predicted to be localized to a specific organelle. Black arrowheads indicate plastid autofluorescence signals. Inter, interphase; Pt/Mt division, plastid/mitochondrial dividing phase; PC, phase contrast image. The PCC of each cell was calculated using areas without plastids. More than 30 cells were observed. b Colocalization of activated CmAUR and mitochondrial ring protein Mda1. More than 30 cells were observed. c Intensity of activated CmAUR speckles localized to the mitochondrial division ring. The relative intensity of human phospho-Aurora antibody (phAUR) and Mda1 in each cell is indicated on the right and left, respectively. **p = 0.0045 (two-sided Student’s t test). d Scheme of mitochondrial division in C. merolae. e, f Effect of kinase-dead mutant e and overexpression f of CmAUR on mitochondrial division in C. merolae. GFP was used as a negative control. GFP, CmAURK208R-GFP, and CmAUR-GFP were transiently overexpressed in C. merolae. According to the classification in d, transfectants were counted. g Co-staining of CmAUR and with ProQ Diamond. CmAUR was stained with α-CmAUR antibody. The two images are of representative M-phase cells. The red autofluorescence signal indicates plastids. h Colocalization analysis of ProQ Diamond and CmAUR. Co-stained M-phase cells were analyzed. Each point indicates the PCC of individual C. merolae cells. The area in each cell without plastids was used for analysis. n = 135. Bars: 1 μm (a, b, left), 250 nm (b, right), 2 µm g. Full size image

To confirm the role of CmAUR in mitochondrial division during mitosis, we overexpressed both wild-type and kinase-dead type CmAUR. Then we measured the frequency of each phase of the transformants (interphase, pre-mitochondrial division phase, and post-mitochondrial division phase) in the mitochondrial replication process (Fig. 2d). The transformants overexpressing both CmAURK208R and wild-type CmAUR were arrested at the pre- and post-mitochondrial division phases (Fig. 2e, f; Supplementary Tables 1, 2). Because the proportion of wild-type and CmAURK208R overexpressing cells in each phase did not seem to be different, it is not clear whether the kinase activity of CmAUR had an impact on the mitochondrial division. However, these data indicate that CmAUR kinase activity and/or another CmAUR function can regulate both nuclear and mitochondrial division. Division of the mitochondrion occurs before nuclear division in C. merolae31. Thus, the inhibition of mitochondrial division was most likely not caused by nuclear division.

To predict the localization of substrates phosphorylated by CmAUR in vivo, we conducted co-staining for CmAUR and with ProQ diamond. ProQ diamond binds total phosphorylated proteins and we previously demonstrated that ProQ diamond staining is effective in C. merolae24. In co-stained M-phase C. merolae cells, CmAUR signals were partially co-localized with ProQ signals (Fig. 2g, h). Because CmAUR was localized to the mitochondrion (Fig. 1a, Supplementary Fig. 1), this result implies that CmAUR phosphorylates substrates in mitochondria.

Identification of the substrates of CmAUR

Because localization analysis of phAUR suggested a relationship between mitochondrial division and CmAUR, we predicted that CmAUR is involved in mitochondrial division through phosphorylation of mitochondrial division ring components. To verify this presumption, we performed in vitro kinase assays using recombinant CmDnm1, Mda1, and TOP, which are known to be involved in mitochondrial division in C. merolae11,24,32. CmDnm1 is a highly conserved protein among eukaryotes and an orthologue of human Drp1 (DNM1L), which forms a ring around mitochondria and induces their division in eukaryotes such as mammalian, nematode, and yeast cells33,34,35. In these assays, CmAUR phosphorylated CmDnm1, Mda1, and TOP (Fig. 3a–c). These results suggested that CmAUR regulates mitochondrial division through phosphorylation of mitochondrial division ring components in C. merolae.

Fig. 3: CmAUR phosphorylated mitochondrial division-related proteins in vitro. a–c In vitro kinase assays of recombinant CmAUR with recombinant mitochondrial division ring proteins. CBB staining indicates Coomassie Brilliant Blue staining. Full size image

Determination of phosphorylation sites of CmDnm1 by CmAUR

On the basis of our findings that CmAUR is involved in mitochondrial division and in the phosphorylation of proteins related to mitochondrial division, we hypothesized that CmAUR regulates mitochondrial division via direct phosphorylation of mitochondrial division ring proteins. We predicted that phosphorylation of CmDnm1 by CmAUR would be important for mitochondrial division because CmDnm1, as a component of the mitochondrial division ring, has a crucial role in mitochondrial division in C. merolae4,11. To further analyze phosphoregulation of CmDnm1 by CmAUR, in vitro phosphorylation sites in CmDnm1 were identified by mass spectrometry (Supplementary Fig. 5). Taking evolutionary conservation among eukaryotes into consideration, nine residues were determined as candidate sites for phosphorylation. Through in vitro kinase assays of point mutants of these nine residues, four phosphorylation sites (T139, S570, S726, S732) were confirmed to be phosphorylated by CmAUR (Fig. 4a). In addition, to confirm each single phosphorylation site, we quantified the western blot signal reduction of phosphorylation site variants. As a result, signal densities of T139A and S726A variants were reproducibly reduced compared with wild-type CmDnm1 signals (Fig. 4b).

Fig. 4: Effects of amino-acid substitutions of phosphorylation sites of Dnm1 by Aurora kinase in Dnm1 on mitochondrial division. a In vitro kinase assay of recombinant CmAUR and site-directed Dnm1 mutants and those with mutations of 4, 7, 8, and 9 residues (see Supplementary Table 3). b Quantification of in vitro kinase assays of CmDnm1 single amino-acid variants. Signals from different membranes were normalized against the luminosity of wild-type CmDnm1. The relative value of wild-type CmDnm1 was 1. n = 3 for T139A, n = 4 for S726A. The error bars indicate standard error of the mean. c Frequency of transformants expressing CmDnm1 variants. Alanine and phosphomimetic glutamic acid mutants of CmDnm1T139 and CmDnm1S726 were overexpressed in C. merolae cells. According to classification Fig. 2d, transfectants were counted. Transfectants with an aberrant number of plastids (three or more) were not counted. d Normal phenotype of dividing mitochondrion in cell overexpressing wild-type CmDnm1. e Phenotype of single mitochondrion without division in cell overexpressing CmDnm1S570E. f Phenotype of multiple chloroplasts in CmDnm1T139A overexpression mutant. g Frequency of cells with multiple chloroplasts among Dnm1-mutant transfectants. Total countable transfectants were counted. h In vitro kinase assay of human Aurora kinases with recombinant GST-human Drp1. Bars: 1 μm. Full size image

To reveal the function of these phosphorylation sites in mitochondrial division, we produced Dnm1 variants in which phosphorylation sites T139, S570, S726, and S732 were substituted to nonphosphorylatable alanine or phosphomimetic glutamic acid and overexpressed them in C. merolae. The effects of these variants were quantified by determining the frequency of each mitochondrial division phase in each transformant (Fig. 2d). The population in each mitochondrial division phase was not affected by overexpression of CmDnm1S570A/E, CmDnm1S726A, or CmDnm1S732A/E, but the number of cells in pre-mitochondrial division phase was increased by overexpression of CmDnm1T139A/E and CmDnm1S726E (Fig. 4c; Supplementary Table 4). This result suggests that the expression of CmDnm1T139A/E and CmDnm1S726E causes suppression of mitochondrial division. Previously, it was reported that mitochondrial division is stalled by overexpression of dominant-negative Drp1 mutants (phosphodeficient or mimetic substitution within the GTPase domain)33. We observed that overexpression of CmDnm1T139A/E and CmDnm1S726E caused mitochondrial division arrest. These results suggest that T139A/E and S726E function, respectively, in a dominant-negative mode. Interestingly, overexpression of CmDnm1 with T139 variations caused abnormal phenotypes such as mitochondrial distribution on one side of the cell (Fig. 4c–e; Supplementary Table 4) and multiple chloroplasts (Fig. 4f, g; Supplementary Table 5), as described previously31. Because aberrant phenotypes were not frequent among the transformants of CmDnm1S726E, we predict that phosphoregulation of T139 has a more important role in mitochondrial division than does phosphoregulation of S726.

To investigate whether the phosphorylation of T139 regulates the localization of CmDnm1, we constructed stably transformed lines that expressed superfolder GFP (sfGFP)-CmDnm1, sfGFP-CmDnm1T139A, and sfGFP-CmDnm1T139E under the control of a heat-inducible promoter16. The heat-inducible expression enables CmDnm1 variant localization to be observed without the effect of a transfection reagent containing polyethylene glycol. After heat induction, we immunostained cells using an Ef-Tu36 antibody as a mitochondrial marker and a GFP antibody. We found no differences in the intracellular localization of each CmDnm1 variant (Supplementary Fig. 6). This result indicates that phosphorylation of T139 may regulate CmDnm1 independently from the intracellular localization control.

Finally, we examined if recombinant human Aurora kinase could directly phosphorylate recombinant Drp1, which is a CmDnm1 orthologue found in human. Aurora A and Aurora B could phosphorylate Drp1 in vitro (Fig. 4h). This result suggests that the direct phosphoregulation of CmDnm1 orthologs by Aurora kinase is conserved between C. merolae and human.