Relationship with the endomitotic process. (A-C) The number of invaginating pre-DMS correlates with the number of nuclear lobes. (A) Confocal images show that the tubular membrane connections (green) are situated between the nuclear lobes (blue). Note that the number of cell surface connections (arrowheads) increased with the number of nuclear lobes (stars). Bars, 10 µm. (B) Quantification of the number of invaginating pre-DMS and nuclear lobes using Z-stack analysis on whole cells. Note that the number of nuclear lobes are not equal to the number of chromosome copies. The Z-step size was 0.25 µm. Overall, a total of 80 MKs (39 × 2 lobes; 14 × 3 lobes; 12 × 4 lobes, and 10 × >6 lobes) were included in our analysis, and the means ± standard error of the mean are presented. (C) Quantification of the DNA staining as a function of the different developmental stages of the DMS (pre-, intermediate, and late DMS). The Z-step size was 0.25 µm, and the Amira Software was used to calculate the DNA content in whole cells. (D) Immunostaining for the centrosome marker γ tubulin reveals the presence of a single fluorescent spot with an increased fluorescent size (arrowhead in upper left panel) compared with γ tubulin staining in late stage MK cells (arrowheads in lower right panel), suggesting multiple centrosomes assembly. The images are maximum projections of whole cells. Bars: upper panels, 10 µm; lower panels, 1 µm. (E) TEM image showing the presence of four centrioles (arrowheads) in proximity to the pre-DMS. Bar, 500 nm. (F) Image of an MK undergoing its first anaphase. Immunostaining for PRC-1, a marker of the midzone. PRC-1 is localized between the condensed chromosomes (arrow). Note the distinct GPIbβ-positive cell surface pool (arrowhead). Bar, 10 µm. (G) Identification of a pre-DMS (arrowhead) in MKs undergoing the first anaphase. Cells were permeabilized with 0.5% Triton X-100, stained with β-tubulin and DAPI to characterize the mitotic spindle and the condensed chromosomes, respectively. Bar, 10 µm.