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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.

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Subcellular localization of AtHaspin in living tobacco BY-2 cells. (A) DNA staining with Hoechst 33342 (top row), GFP fluorescence (middle row), and merged images (bottom row) showing DNA (blue) and GFP (green). Magenta arrowhead indicates fluorescent signal on chromosomes. (B) Live cell imaging was carried out in BY-2 cells expressing GFP-α-tubulin after more than 48-h induction of AtHaspin-tdTomato with 10 μM 17-β-estradiol. Merged images show AtHaspin-tdTomato (magenta) and GFP-α-tubulin (green). Magenta arrowhead indicates fluorescent signal on chromosomes. Numbers indicate time of observation (h: min) in additional file 1. (C) Kymographs representing fluorescence on yellow lines in left column. Arrows indicate PPB. Letters indicate mitotic stages as shown in (B). Scale bars: 10 μm (left), 10 min (bottom).
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Figure 3: Subcellular localization of AtHaspin in living tobacco BY-2 cells. (A) DNA staining with Hoechst 33342 (top row), GFP fluorescence (middle row), and merged images (bottom row) showing DNA (blue) and GFP (green). Magenta arrowhead indicates fluorescent signal on chromosomes. (B) Live cell imaging was carried out in BY-2 cells expressing GFP-α-tubulin after more than 48-h induction of AtHaspin-tdTomato with 10 μM 17-β-estradiol. Merged images show AtHaspin-tdTomato (magenta) and GFP-α-tubulin (green). Magenta arrowhead indicates fluorescent signal on chromosomes. Numbers indicate time of observation (h: min) in additional file 1. (C) Kymographs representing fluorescence on yellow lines in left column. Arrows indicate PPB. Letters indicate mitotic stages as shown in (B). Scale bars: 10 μm (left), 10 min (bottom).

Mentions: To analyze the subcellular localization of AtHaspin during cell division, we transformed Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2) cultured cells with GFP-fused AtHaspin and observed tobacco BY-2 cells stably expressing AtHaspin-GFP with DNA stained by Hoechst 33342 (Figure 3A). During interphase, AtHaspin was mainly localized in the cytoplasm and at the nuclear periphery. After nuclear envelope breakdown (NEBD), AtHaspin invaded the nuclear region. During metaphase, fluorescent signals of AtHaspin-GFP were also observed on the chromosome (Figure 3A, arrowhead). After metaphase, AtHaspin-GFP was localized with the phragmoplast from its initial formation at the center of the equatorial plane to its expansion towards the cell periphery as the cell cycle progressed.


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)

Subcellular localization of AtHaspin in living tobacco BY-2 cells. (A) DNA staining with Hoechst 33342 (top row), GFP fluorescence (middle row), and merged images (bottom row) showing DNA (blue) and GFP (green). Magenta arrowhead indicates fluorescent signal on chromosomes. (B) Live cell imaging was carried out in BY-2 cells expressing GFP-α-tubulin after more than 48-h induction of AtHaspin-tdTomato with 10 μM 17-β-estradiol. Merged images show AtHaspin-tdTomato (magenta) and GFP-α-tubulin (green). Magenta arrowhead indicates fluorescent signal on chromosomes. Numbers indicate time of observation (h: min) in additional file 1. (C) Kymographs representing fluorescence on yellow lines in left column. Arrows indicate PPB. Letters indicate mitotic stages as shown in (B). Scale bars: 10 μm (left), 10 min (bottom).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Subcellular localization of AtHaspin in living tobacco BY-2 cells. (A) DNA staining with Hoechst 33342 (top row), GFP fluorescence (middle row), and merged images (bottom row) showing DNA (blue) and GFP (green). Magenta arrowhead indicates fluorescent signal on chromosomes. (B) Live cell imaging was carried out in BY-2 cells expressing GFP-α-tubulin after more than 48-h induction of AtHaspin-tdTomato with 10 μM 17-β-estradiol. Merged images show AtHaspin-tdTomato (magenta) and GFP-α-tubulin (green). Magenta arrowhead indicates fluorescent signal on chromosomes. Numbers indicate time of observation (h: min) in additional file 1. (C) Kymographs representing fluorescence on yellow lines in left column. Arrows indicate PPB. Letters indicate mitotic stages as shown in (B). Scale bars: 10 μm (left), 10 min (bottom).
Mentions: To analyze the subcellular localization of AtHaspin during cell division, we transformed Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2) cultured cells with GFP-fused AtHaspin and observed tobacco BY-2 cells stably expressing AtHaspin-GFP with DNA stained by Hoechst 33342 (Figure 3A). During interphase, AtHaspin was mainly localized in the cytoplasm and at the nuclear periphery. After nuclear envelope breakdown (NEBD), AtHaspin invaded the nuclear region. During metaphase, fluorescent signals of AtHaspin-GFP were also observed on the chromosome (Figure 3A, arrowhead). After metaphase, AtHaspin-GFP was localized with the phragmoplast from its initial formation at the center of the equatorial plane to its expansion towards the cell periphery as the cell cycle progressed.

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.

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