Fig 1: Centrosome clustering is essential for proplatelet formation and is enhanced during emergency thrombopoiesis.(A) Proplatelet-forming FLMKs were spun down onto coverslips and stained for EB3 and pericentrin. Scale bar, 30 μm. Arrowheads highlight clustered centrosomes. (B and C) Centrosome clustering was quantified in dimethyl sulfoxide (DMSO)–or Gris-treated FLMKs transduced with MSCV–β1-tubulin–dendra2. Images were acquired every hour for 24 hours. n = 3. At least 200 cells were analyzed per mouse. Data are presented as means ± SD. Scale bars, 10 μm. (D and E) Proplatelet formation of FLMKs treated with the interphase declustering drug Gris (D) or the CPAP inhibitor CCB02 (E) was assessed using the IncuCyte imaging system and a customized analysis pipeline (87). n = 3; four technical replicates. Data are presented as means ± SD. (F) BM MKs were treated with DMSO or 50 μM Gris and allowed to form proplatelets on a CD31-coated surface. MKs were stained for α-tubulin and F-actin. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 50 μm. (G and H) Proplatelet tip number and tip diameter were analyzed using ImageJ software. n = 2. Three representative cells were analyzed per mouse. Data are presented as means ± SD. Unpaired, two-tailed Student’s t test. **P < 0.01; ****P < 0.0001. FSC-A, forward scatter area. (I and J) Representative flow plot (I) and ploidy distribution (J) of DMSO- and Gris-treated, in vitro differentiated BM MKs were assessed using flow cytometry. Data are presented as means ± SD. Two-way ANOVA with Sidak correction for multiple comparisons. **P < 0.01; ****P < 0.0001. (K and L) CD42d MFI (K) and percentage of CD41/CD42d+ cells in DMSO- and Gris-treated cultures (L) were analyzed by flow cytometry. n = 4. Data are presented as means ± SD. Unpaired, two-tailed Student’s t test. **P < 0.01. (M to O) Representative images (M) and quantification of MKs containing clustered centrosomes (N) and MK numbers (O) in mice in situ upon platelet depletion. Samples were taken 96 hours after administration of an anti-GPIbα antibody. Scale bars, 50 μm. n = 10. Data are presented as means ± SD. Unpaired, two-tailed Student’s t test. *P < 0.05; ****P < 0.0001.