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Activation of the endomitotic spindle assembly checkpoint and thrombocytopenia in Plk1-deficient mice

Marianna Trakala, David Partida, María Salazar-Roa, María Maroto, Paulina Wachowicz, Guillermo de Cárcer and Marcos Malumbres

Data supplements

Article Figures & Data

Figures

  • Figure 1

    Defective megakaryocyte maturation in the absence of Plk1. (A) Bone marrow sections from 8-week-old mice were stained with hematoxylin and eosin (H&E) or antibodies against Plk1 or the VWF. Scale bars, 100 μm (insets, 20 μm). (B) The quantification of VWF area per cell is shown in the histogram. Horizontal bars indicate the average from >500 cells per condition from at least 3 different animals. ***P < .001; Student t test. (C) Phosphorylation of pH3 in Plk1-deficient or control megakaryocytes. Scale bars, 100 μm (insets, 20 μm). The quantification of pH3-positive cells is shown in the right histogram. Data are mean ± standard deviation (SD; n = 3 mice per genotype). ***P < .001; Student t test. (D) Representative images of mitotic cells in Plk1-deficient or control mice after staining with H&E or immunodetection of pH3. Scale bars, 20 μm. (E) Analysis of nuclear complexity in Plk1-null and control megakaryocytes (MKs). Scale bars, 20 μm. In these analyses, at least 250 cells from ≥3 different animals per genotype were used. Student t test; ***P < .001.

  • Figure 2

    Defective megakaryocyte ploidy and circulating platelets in Plk1(Δ/Δ) mice. (A) Analysis of DNA content in gated CD41+ cells from the bone marrow of 8-week-old Plk1(Δ/Δ) or control mice. The plot is representative of 3 different animals analyzed per genotype. (B) DNA content in CD41+ cells from the bone marrow of mice with the indicated genotypes. Data are mean ± SD (n = 4 mice per genotype). *P < 0.05; **P < .01; ***P < .001; Student t test. (C) Number of circulating platelets in Plk1(Δ/Δ) or control mice. As a comparison, the average count is ∼100 × 109 platelets/L in Cdc20-deficient mice.12 ***P < .001; Student t test.

  • Figure 3

    Defective mitotic exit in Plk1-deficient megakaryocytes. (A) Representative mitotic progression and exit in wild-type megakaryocytes. Cells were transduced with lentiviral vectors expressing GFP-histone H2B (green) and mCherry-Geminin (red). Mitotic entry (time 0) can be monitored by chromosome condensation and pancellular staining of geminin. Mitotic exit (1 hour) is monitored by chromosome decondensation, reformation of nuclear lobules, and geminin degradation. (B) Two independent examples of defective mitotic progression in Plk1-deficient megakaryocytes. Mitotic entry (chromosome condensation and pancellular staining of geminin) is shown at time 0 (third frame in the top cell and second frame in the bottom cell). Geminin is slowly degraded 27 to 32 hours after mitotic entry, resulting in cell death (top cell) or mitotic exit (bottom) after approximately 37 hours. (C) Representation of cell fate in wild-type and Plk1(Δ/Δ) megakaryocytes. Every row represents a single cell. Geminin high phases are shown in red, whereas geminin low phases are in green. Endomitosis is indicated by a purple box and cell death is indicated by a red circle. Mitotic entry (condensation of chromatin and pancellular localization of geminin) is normalized as time 0. (D) Duration of mitosis (DOM; in hours) in Plk1-deficient or control megakaryocytes. Cdc20-deficient megakaryocytes12 are used as a control. ***P < .001; Student t test.

  • Figure 4

    Lack of Plk1 results in defective centrosome maturation and spindle formation in mitotic megakaryocytes. (A) Reduced polyploidization in Lin bone marrow cells 3 days after stimulation with TPO. Data are mean ± SD (n = 4 mice per genotype). *P <0.05; **P < .01; ***P < .001; Student t test. (B) Representative images and quantification of mitotic cells (arrowheads) in these cultures 3 days after stimulation with TPO. Scale bars, 20 μm (top) and 50 μm (bottom). Data are mean ± SD. At least 250 cells per genotype were scored. ***P < .001; Student t test. (C) Immunodetection of Plk1 (red) in Plk1(lox/lox) and Plk1(Δ/Δ) stimulated in vitro with TPO. DAPI (blue) was used to stain DNA. Scale bars, 10 μm. (D) Quantification of ring structures in the mitotic cells in these cultures. Data are mean ± SD. n = 50 (control) or 250 (Plk1- or Cdc20-null) mitotic cells per condition. ***P < .001; Student t test. (E) Immunodetection of α-tubulin (green) and pericentrin (red) in Plk1-null or control cells treated with the indicated compounds. DAPI (blue) was used to stain DNA. Scale bars, 10 μm. (F) Quantification of pericentrin median fluorescence intensity (MFI) in these cultures. Horizontal bars indicate averages. ns, not significant; ***P < .001; Student t test.

  • Figure 5

    Accumulation of SAC proteins in the kinetochores of Plk1-deficient megakaryocytes. (A) Immunodetection of Bub1 (green) and centromeres (ACAs, magenta) in Plk1(Δ/Δ) cells or control cells treated with Taxol or the Plk1 inhibitor BI2536. Cdc20-deficient cells were used as a control of SAC-independent mitotic arrest. DAPI (blue) was used to stain DNA. (B) Immunodetection of Mad2 (red) and centromeres (ACAs, magenta) in Plk1(Δ/Δ) cells or control cells treated with Taxol or the Plk1 inhibitor BI2536. Cdc20-deficient cells were used as a control of SAC-independent mitotic arrest. DAPI (blue) was used to stain DNA. Scale bars, 10 μm (insets, 2 μm).

  • Figure 6

    Lack of Plk1 results in a SAC-dependent mitotic arrest. (A) Representative images of Plk1(Δ/Δ) megakaryocytes in the absence or presence of the SAC inhibitor reversine. Spindles are shown by α-tubulin staining (green), whereas pH3 is shown in magenta. DAPI (blue) was used to stain DNA. Scale bars, 50 μm (or 20 μm in the 2 right columns). (B) Representative images of the indicated cultures in the absence or presence of reversine. Spindles are shown by α-tubulin staining (green), whereas pH3 is shown in magenta and DAPI (blue) was used to stain DNA. Scale bars, 50 μm (or 20 μm in the bottom row). (A-B) Arrows indicate curved nuclei around a single tubulin aster. (C) Mitotic index, as determined by detection of pH3, in Plk1- or Cdc20-deficient cells or control cells treated with Taxol or BI2536 in the absence or presence of the Mps1 inhibitor reversine. Data are mean ± SD. n = 50 cells per condition. **P < .01; ***P < .001; Student t test.