Megakaryocytes express functional Aurora-B kinase in endomitosis by Amy E. Geddis, and Kenneth Kaushansky Blood Volume 104(4):1017-1024 August 15, 2004 ©2004 by American Society of Hematology

Aurora-B kinase is expressed in EnM MKs Aurora-B kinase is expressed in EnM MKs. (A) RT-PCR assay for Aurora-B kinase using RNA isolated from polyploid murine MKs and unsynchronized BaF3/Mpl cells. Aurora-B kinase is expressed in EnM MKs. (A) RT-PCR assay for Aurora-B kinase using RNA isolated from polyploid murine MKs and unsynchronized BaF3/Mpl cells. Two independent primer pairs were designed to yield products of about 300 base pair (bp). (B) Western blot comparing Aurora-B kinase expression in polyploid murine MKs and diploid cells. Gradient-purified MKs compared to unsynchronized BaF3/Mpl cells. Equivalent amounts of total cell protein were loaded in each lane. Aurora-B kinase is 41 kDa. (C) Kinase assay comparing Aurora-B kinase activity in polyploid murine MKs and diploid cells. Aurora-B kinase was immunoprecipitated from mature MK or BaF3/Mpl lysates, and kinase activity was assayed using purified histone H3 as a substrate. Phosphorylation was detected by blotting with phospho-specific histone H3 antibody. Blots were reprobed with antibody to histone H3. Lanes 1 and 2 contain no substrate, lane 3 contains purified histone H3 alone, lane 4 is empty, and lanes 5 and 6 show the kinase assay of MK and BaF3/Mpl immunoprecipitates against the purified histone H3 (19 kDa). Amy E. Geddis, and Kenneth Kaushansky Blood 2004;104:1017-1024 ©2004 by American Society of Hematology

Diploid controls for Aurora-B kinase localization in mitosis. Diploid controls for Aurora-B kinase localization in mitosis. (A) Diploid CD34+ cells showing expected pattern of Aurora-B kinase localizing to centromeres in prophase through metaphase, then transferring to the midzone cortex and the condensing midbody in anaphase. Note chromosomal organization defining the different stages with condensation beginning at prophase, chromosomal alignment occurring at metaphase, sister chromatid separation in early anaphase with spindle elongation, and the beginning of chromosome decondensation in late anaphase and telophase. (B) Comparison of a diploid metaphase (bipolar spindle, single metaphase plate) with a polyploid metaphase (more than 2 spindle poles and more than a single metaphase plate). Amy E. Geddis, and Kenneth Kaushansky Blood 2004;104:1017-1024 ©2004 by American Society of Hematology

Functional Aurora-B kinase is expressed in EnM MKs Functional Aurora-B kinase is expressed in EnM MKs. (A) Deconvolution images of a series of murine polyploid MKs showing localization of Aurora-B kinase (green) to centromeres. Functional Aurora-B kinase is expressed in EnM MKs. (A) Deconvolution images of a series of murine polyploid MKs showing localization of Aurora-B kinase (green) to centromeres. DNA is blue. Cells shown are in prometaphase with chromosomes not yet completely aligned, metaphase with chromosomal alignment evident to several plates, and anaphase. Note centromere localization of Aurora-B kinase in up through metaphase with indistinct localization in anaphase. (B) Histone H3 is phosphorylated on Ser10 in EnM murine MKs. Phospho-histone H3 is shown in green and tubulin in red; left image shows a cell with chromosomal alignment suggesting metaphase, whereas the very large cell on the right appears to be in early anaphase with chromosomal separation. (C) Localization of Aurora-B kinase (green) to centromeres does not require intact kinase activity. Purified mouse MKs were incubated for 2 hours with 5 μM ZM447439 prior to fixation and immunostaining for Aurora-B kinase (green) and phospho-histone H3 (red). Phosphorylation of histone H3 is absent due to inhibition of aurora kinase. Amy E. Geddis, and Kenneth Kaushansky Blood 2004;104:1017-1024 ©2004 by American Society of Hematology

The chromosomal passengers, INCENP and survivin, are present at centromeres in EnM MKs. (A) RT-PCR showing expression of INCENP and survivin in polyploid murine MKs. RNA was isolated from gradient-purified MKs or BaF3/Mpl cells as a diploid control. The chromosomal passengers, INCENP and survivin, are present at centromeres in EnM MKs. (A) RT-PCR showing expression of INCENP and survivin in polyploid murine MKs. RNA was isolated from gradient-purified MKs or BaF3/Mpl cells as a diploid control. Lanes 1, MKs; lanes 2, BaF3/Mpl; lanes 3, negative control. (B) Aurora-B kinase is present and functional in human MKs. Aurora-B kinase is shown in green and phospho-histone H3 is shown in red. Note the localization to midzone structures in the two rightmost images shown in anaphase. (C-D) INCENP and survivin (green) are present and properly localized in human MKs. Tubulin is shown in red, and DNA is blue. Note the localization to centromeres in the leftmost 2 images of metaphase cells, then midzone structures in the next 2 images shown in anaphase. Amy E. Geddis, and Kenneth Kaushansky Blood 2004;104:1017-1024 ©2004 by American Society of Hematology

Phosphorylation of histone H3 is similar in EnM and mitosis and is ablated by an inhibitor of aurora kinase. Phosphorylation of histone H3 is similar in EnM and mitosis and is ablated by an inhibitor of aurora kinase. (A) EnM murine MK and mitotic cell adjacent to each other allowing direct comparison of phosphorylation of histone H3. Phospho-histone H3 is shown in green; tubulin is stained in red. (B) Murine MKs were purified and incubated for 2 hours with either vehicle (DMSO) or 5 μM ZM447439 prior to fixation and immunostaining for phospho-histone H3 (green). (C) Spindle structure in MKs treated with an aurora inhibitor is not the same as that in MKs treated with an Eg5 inhibitor. Murine MKs were incubated for 2 hours with either 5 μM ZM447439 or 100 μM monastrol prior to fixation and immunostaining for tubulin and phospho-histone H3. Amy E. Geddis, and Kenneth Kaushansky Blood 2004;104:1017-1024 ©2004 by American Society of Hematology

Aurora-related proteins are correctly localized in EnM MKs Aurora-related proteins are correctly localized in EnM MKs. (A) Murine MKs were fixed and stained for CENP-E (green) and α-tubulin (red). Aurora-related proteins are correctly localized in EnM MKs. (A) Murine MKs were fixed and stained for CENP-E (green) and α-tubulin (red). DNA is shown in blue. Note that CENP-E is localized to kinetochores and bi-orientation of chromosomes is visible at top of the spherical arrangement of spindle poles. (B) Murine MKs were fixed and stained for aurora-A kinase (green) and α-tubulin (red). DNA is shown in blue. Note localization of aurora-A kinase to spindle poles. Image is shown as a projection to display multiple spindle poles in the same view. Amy E. Geddis, and Kenneth Kaushansky Blood 2004;104:1017-1024 ©2004 by American Society of Hematology