Introduction [1] [2] [3] [4] Drosophila [5] Drosophila Materials and methods Molecular and immunological techniques [6,7] [5] [8] [9] Immunofluorescence microscopy [10,11] [10] [12] [13] Cultured cells were examined using a Plan-Apochromat objective lense (100×, 1.4NA; Zeiss) attached to an Axioplan2 (Zeiss). Images were captured by a CCD camera (Orca; Hamamatsu) using OpenLab2 (Improvision). Larval central nervous systems were taken using a Plan-Apochromat lense (63×, 1.4NA; Zeiss) attached to an Axiovert 200 M (Zeiss) with a confocal scan head (LSM510meta; Zeiss). Confocal images were presented as a maximum intensity projection of the Z-stacks. All digital images were imported to Photoshop (Adobe) and adjusted for brightness and contrast. Phosphatase treatment p For phosphatase treatment of fixed cells for immunofluorescence with the anti-dH2A-pT119 antibody, cells were fixed with 4% paraformaldehyde in PBS followed by incubation with lambda phosphatase for 1 h at 37 °C. Cells were then washed and immunostained as described above. Microscope images with the same exposure settings were taken of immunostained cells with and without phosphatase treatment. Average pixel intensity of dH2A-pT119 staining on the DNA was measured in interphase and mitotic cells (16 cells in 2 separate experiments). Fly stocks [14] nhk-1 nhk-1 E107 [15] Results H2A T119 phosphorylation is specific to centromeres in mitosis Drosophila [5] Fig. 1 Fig. 1 [8,16] Figs. 1 Fig. 1 Supplementary Fig. 1 Supplementary Fig. 1 [5,17] [18] Supplementary Fig. 2A Centromeric H2A T119 phosphorylation depends on Aurora B kinase [5] NHK-1 [17] Supplementary Figs. 3 and 5 NHK-1 nhk1 E107 [15] Supplementary Fig. 2B [19] Fig. 2 Supplementary Fig. 5 [19] Supplementary Fig. 4 Polo kinase down-regulates the H2A phosphorylation on chromosome arms [20] Fig. 2 Fig. 2 Polo suppresses phosphorylation by the H2A kinase NHK-1 Fig. 2 Fig. 3 Supplementary Fig. 5 These epistasis studies suggested that Polo functions upstream of NHK-1 to suppress H2A T119 phosphorylation, but is independent of Aurora B. Cyclin B degradation triggers a loss of H2A phosphorylation at initiation of anaphase [21] [22,23,13] Figs. 4 Discussion Drosophila Fig. 4 Currently we do not know what the function of this H2A phosphorylation is in cells. In higher eukaryotes which have many copies of histone genes, the function of histone modifications has been studied only indirectly by down-regulating responsible modifying enzymes. Unfortunately this approach is not suitable for kinases as they are likely to have multiple substrates (for example, Cdc2 and Aurora B mediating H1 and H3 phosphorylation). [19,20] Appendix A Supplementary data Supplementary Fig. 1  Supplementary Fig. 2  Supplementary Fig. 3  Supplementary Fig. 4  Supplementary Fig. 5  Appendix A Supplementary data doi:10.1016/j.yexcr.2007.04.038