Limits...
Identification and characterization of plant Haspin kinase as a histone H3 threonine kinase.

Kurihara D, Matsunaga S, Omura T, Higashiyama T, Fukui K - BMC Plant Biol. (2011)

Bottom Line: Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana.Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT

Background: Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear.

Results: Here, we identified a Haspin kinase, designated AtHaspin, in Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during prometaphase and metaphase, and around the cell plate during cytokinesis. This localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11 of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was expressed in root meristems, shoot meristems, floral meristems, and throughout the whole embryo at stages of high cell division. Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.

Conclusions: Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. The expression and dominant-negative analysis showed that AtHaspin may have a role in mitotic cell division during plant growth. Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

Show MeSH
Phosphorylation of histone H3 at Thr3 and Thr11 in vivo. (A, B) Phosphorylation of histone H3 at Thr3 and Thr11 during cell cycle. BY-2 cells immunostained using anti-H3T3ph (A), anti-H3T11ph (B), or anti-H3S28ph antibodies. DNA was stained with DAPI. Merged images of DNA (blue), H3S10ph (red) and H3S28ph (green) are shown in color. Scale bars: 10 μm. (C) After 48-h induction with 10 μM 17-β-estradiol, BY-2 cells inducibly expressing AtHaspin-tdTomato were fixed with 4% (w/v) paraformaldehyde for 20 min. DNA was stained with DAPI. Merged images of DNA (blue) and AtHaspin-tdTomato (red) are shown in color. Scale bars: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113928&req=5

Figure 4: Phosphorylation of histone H3 at Thr3 and Thr11 in vivo. (A, B) Phosphorylation of histone H3 at Thr3 and Thr11 during cell cycle. BY-2 cells immunostained using anti-H3T3ph (A), anti-H3T11ph (B), or anti-H3S28ph antibodies. DNA was stained with DAPI. Merged images of DNA (blue), H3S10ph (red) and H3S28ph (green) are shown in color. Scale bars: 10 μm. (C) After 48-h induction with 10 μM 17-β-estradiol, BY-2 cells inducibly expressing AtHaspin-tdTomato were fixed with 4% (w/v) paraformaldehyde for 20 min. DNA was stained with DAPI. Merged images of DNA (blue) and AtHaspin-tdTomato (red) are shown in color. Scale bars: 10 μm.

Mentions: To reveal whether AtHaspin phosphorylates histone H3 at Thr3 and Thr11 in vivo, we performed indirect immunofluorescence using anti-H3T3ph, H3T11ph, and H3S28ph antibodies in BY-2 cells (Figures 4A and 4B). During interphase, no signals of H3T3ph, H3T11ph, and H3S28ph were observed. The H3T3ph signal was first detected on chromosomes in early prophase before NEBD, whereas the H3T11ph signal was first detected on chromosomes in late prophase after NEBD. During late prophase, H3T3ph signals were observed along the chromosome, while H3S28ph signals were observed at pericentromeric regions. During prometaphase, the H3T3ph signals were stronger at pericentromeric regions than on the chromosome arms. Signals of H3T3ph increased at the pericentromeric region until late metaphase. In contrast, phosphorylated H3T11 was entirely localized on the chromosome from prometaphase to anaphase. Signals of H3T3ph disappeared after chromosome segregation during anaphase, while H3T11ph signals were still localized on the chromosome. A moderate-strength AtHaspin-tdTomato signal was observed on the chromosome during prometaphase and metaphase (Figures 3A and 3B, arrowhead). This localization of AtHaspin-tdTomato overlapped with those of phosphorylated H3T3 and H3T11 from after NEBD until anaphase in BY-2 cells (Figures 4A and 4B).


Identification and characterization of plant Haspin kinase as a histone H3 threonine kinase.

Kurihara D, Matsunaga S, Omura T, Higashiyama T, Fukui K - BMC Plant Biol. (2011)

Phosphorylation of histone H3 at Thr3 and Thr11 in vivo. (A, B) Phosphorylation of histone H3 at Thr3 and Thr11 during cell cycle. BY-2 cells immunostained using anti-H3T3ph (A), anti-H3T11ph (B), or anti-H3S28ph antibodies. DNA was stained with DAPI. Merged images of DNA (blue), H3S10ph (red) and H3S28ph (green) are shown in color. Scale bars: 10 μm. (C) After 48-h induction with 10 μM 17-β-estradiol, BY-2 cells inducibly expressing AtHaspin-tdTomato were fixed with 4% (w/v) paraformaldehyde for 20 min. DNA was stained with DAPI. Merged images of DNA (blue) and AtHaspin-tdTomato (red) are shown in color. Scale bars: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113928&req=5

Figure 4: Phosphorylation of histone H3 at Thr3 and Thr11 in vivo. (A, B) Phosphorylation of histone H3 at Thr3 and Thr11 during cell cycle. BY-2 cells immunostained using anti-H3T3ph (A), anti-H3T11ph (B), or anti-H3S28ph antibodies. DNA was stained with DAPI. Merged images of DNA (blue), H3S10ph (red) and H3S28ph (green) are shown in color. Scale bars: 10 μm. (C) After 48-h induction with 10 μM 17-β-estradiol, BY-2 cells inducibly expressing AtHaspin-tdTomato were fixed with 4% (w/v) paraformaldehyde for 20 min. DNA was stained with DAPI. Merged images of DNA (blue) and AtHaspin-tdTomato (red) are shown in color. Scale bars: 10 μm.
Mentions: To reveal whether AtHaspin phosphorylates histone H3 at Thr3 and Thr11 in vivo, we performed indirect immunofluorescence using anti-H3T3ph, H3T11ph, and H3S28ph antibodies in BY-2 cells (Figures 4A and 4B). During interphase, no signals of H3T3ph, H3T11ph, and H3S28ph were observed. The H3T3ph signal was first detected on chromosomes in early prophase before NEBD, whereas the H3T11ph signal was first detected on chromosomes in late prophase after NEBD. During late prophase, H3T3ph signals were observed along the chromosome, while H3S28ph signals were observed at pericentromeric regions. During prometaphase, the H3T3ph signals were stronger at pericentromeric regions than on the chromosome arms. Signals of H3T3ph increased at the pericentromeric region until late metaphase. In contrast, phosphorylated H3T11 was entirely localized on the chromosome from prometaphase to anaphase. Signals of H3T3ph disappeared after chromosome segregation during anaphase, while H3T11ph signals were still localized on the chromosome. A moderate-strength AtHaspin-tdTomato signal was observed on the chromosome during prometaphase and metaphase (Figures 3A and 3B, arrowhead). This localization of AtHaspin-tdTomato overlapped with those of phosphorylated H3T3 and H3T11 from after NEBD until anaphase in BY-2 cells (Figures 4A and 4B).

Bottom Line: Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana.Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT

Background: Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear.

Results: Here, we identified a Haspin kinase, designated AtHaspin, in Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during prometaphase and metaphase, and around the cell plate during cytokinesis. This localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11 of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was expressed in root meristems, shoot meristems, floral meristems, and throughout the whole embryo at stages of high cell division. Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.

Conclusions: Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. The expression and dominant-negative analysis showed that AtHaspin may have a role in mitotic cell division during plant growth. Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

Show MeSH