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Figure 1. Multiple alignment of Haspin kinases in the kinase domain. (A) Kinase domains from Anopheles gambiae (EAA05110), Aquilegia caerulea (AcoGoldSmith_v1.025146m), Arabidopsis rylata (XP_002889750), Arabidopsis thaliana (NP_172416), Aspergillus fumigatus (XP_751829), Aspergillus nidulans (XP_659658), Brachypodium distachyon (Bradi1g20070.1), Caenorhabditis elegans F22H10.5 (NP_510696), C. elegans C01H8.9 (NP_492043), C. elegans Y18H1A.10 (NP_490768), Carica papaya (evm.model.supercontig_48.218), Chlamydomonas reinhardtii (XP_001699957), Chlorella variabilis (EFN57276), Cucumis sativus (Cucsa.050880.1), Drosophila melanogaster (P83103), Encephalitozoon cuniculi (NP_597598), Gallus gallus (XP_425408), Glycine max 03g38780 (Glyma03g38780.1), G. max 19g41380 (Glyma19g41380.1), Homo sapiens (AAH47457), Manihot esculenta (cassava4.1_028012m), Medicago truncatula (AC235094_20.1), M. truncatula (Medtr7g135040.1), Mimulus guttatus (mgv1a027116m), Micromonas pusilla (XP_003057374), Micromonas RCC299 (XP_002502153), Mus musculus (NP_034483), Physcomitrella patens (XP_001777245), Populus trichocarpa (XP_002329997), Prunus persica (ppa015455m), Oryza sativa (BAC16406), Ostreococcus lucimarinus (XP_001417826), Ostreococcus tauri (XP_003079484), Ricinus communis (XP_002512572), Saccharomyces cerevisiae Ybl009wp (NP_009544), S. cerevisiae ALK-1 (CAA61012), Schizosaccharomyces pombe (CAB16874), Selaginella moellendorffii (XP_002986955), Setaria italica (SiPROV006697m), Tetraodon nigroviridis (CAF92724), Vitis vinfera (XP_002276683), Volvox carteri (XP_002952488), Xenopus laevis (TC388096), and Zea mays (NP_001149827). Accession numbers from the DNA Data Bank of Japan (DDBJ) or transcript names from the genome database (Phytozome v6.0) are given in parentheses. (B) Amino acid structure of Haspin proteins from A. thaliana, H. sapiens, and S. pombe. Black boxes show NLSs (nuclear localization signals) predicted by the PSORT algorithm (http://psort.nibb.ac.jp/form.html). Gray box indicates kinase domain. (C) Multiple alignment of kinase domain of AtHaspin, human Haspin, and fission yeast Haspin. Missing residues are shown as dashes, identical amino acids are shaded in gray, and residues of ATP/Mg2+ ion-binding sites are shown in magenta. Important residues for histone H3 phosphorylation in catalytic cleft are shown in green.
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Figure 2. GST-AtHaspin phosphorylates histone H3 at Thr3 and Thr11 in vitro. (A) GST-AtHaspin and GST-AtHaspin-KD were incubated with or without ATP, and phosphorylated proteins were stained with ProQ Diamond Phosphoprotein stain. (B) GST-AtHaspin and GST-AtAUR3 were incubated with GST-H3 tail (left and right lanes). Negative control: GST-H3 tail only (middle lane). Phosphorylated GST-H3 tail was immunostained using anti-H3T3ph, H3T11ph, H3S10ph, and H3S28ph antibodies. (C) GST-AtHaspin and GST-AtHaspin-KD were incubated with GST-H3 tails or mutants as substrates. Phosphorylated GST-H3 tails were immunostained with anti-H3T3ph and anti-H3T11ph antibodies.
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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).
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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.
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Figure 5. Expression patterns of AtHaspin-GFP in Arabidopsis. (A) Expressions of AtHaspin, AtAURs, and AtCycB1;3 during mitotic cell cycle in synchronized Arabidopsis cultured cells. Expression data were obtained from publicly available microarray data [30]. Figure shows expressions of genes after removal of the DNA synthesis inhibitor, aphidicolin. (B) Total RNA was extracted from 3-day-old seedlings (3), roots (R), young leaves (YL), leaves (L), stems (S), flower buds (FB), flowers (F), siliques (Si), and genomic DNA (G). Expression was monitored by RT-PCR. Number of PCR cycles is shown in parentheses after gene names. GAPDH was used as an internal control. (C-N) Expression of AtHaspin-GFP in root tip (C), lateral root (D), shoot meristem and leaf primordia (E-H), leaf primordia and first true leaves (F, G), leaf primordia and second true leaves (H), inflorescence meristem and floral meristem in cauline leaves (I), floral meristem (J), ovules in closed flowers (K), one-cell stage embryo (L), four-cell stage embryo (M), heart stage embryo (N), and torpedo stage embryo (O). Scale bars: 100 μm (C, D, F, H, I, J), 50 μm (E), 30 μm (G, K), 10 μm (L, M), and 20 μm (N, O).
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Figure 6. Root growth defects in plants overexpressing AtHaspin-KD-Venus. (A) At 11 days after imbibition, root growth was decreased in AtHaspin-KD-Venus overexpression plants compared with roots of Col. and AtHaspin-Venus overexpression plants. Scale bar: 10 μm. (B, C) Root length of transformants with inducible AtHaspin-Venus and AtHaspin-KD-Venus with (B) or without induction (C). Mean values ± standard error are shown (B; n ≥ 15 plants, C; n = 10 plants).
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Figure 7. Overexpression of AtHaspin-KD-Venus decreased the size of the root meristem. (A, C) At 6 days after imbibition, DNA was stained with DAPI in transformants with inducible AtHaspin-Venus and AtHaspin-KD-Venus with (A) or without induction (C). Arrowheads indicate position of first endocycles in the epidermis. Position of first endoduplicated cells was estimated as described in materials and methods. Scale bar: 50 μm. (B, D) Distance of first endoduplicated cells from QC in epidermis and cell cortex in transformants with inducible AtHaspin-Venus and AtHaspin-KD-Venus with (B) or without induction (D). Mean values ± standard error are shown (n = 5 plants). One-way ANOVA with Bonferroni post-hoc test showed a significant difference between AtHaspin-KD-Venus overexpression plants and Col. (p < 0.001). (E) AtHaspin-GFP was expressed in root tips, except for QC and columella. Cell walls were stained with PI. Merged images show AtHaspin-GFP (green) and PI (magenta). Scale bar: 20 μm.
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