Tmod3^−/− MKs show abnormal ultrastructure with incomplete DMS formation, abnormal organelle distribution, and defects in F-actin. (A) Representative TEM images of Tmod3^+/+ and Tmod3^−/− MKs at lower (upper panels) and higher (lower panels) magnification. All Tmod3^+/+ MKs showed DMS tubules surrounding platelet territories with abundant electron-dense α-granules (left). In contrast, about half of Tmod3^−/− MKs contained large clumps of vesicles (middle), whereas others appeared with some DMS tubules surrounding platelet territories, which were often larger and with fewer electron-dense granules (right). Bar, 5 µm (upper panels) and 1 µm (lower panels). (B-C) Immunofluorescence staining of GPIbα, F-actin, mitochondria (TOM20), and nuclei (Hoechst) in isolated Tmod3^+/− and Tmod3^−/− fetal liver MKs. Grayscale images are shown for each stain, and the merge shows GPIbα (green), mitochondria (red), and nuclei (blue). In Tmod3^+/− MKs, GPIbα and F-actin are associated with DMS membranes surrounding platelet territories that contain mitochondria. Tmod3^+/− MK morphology is indistinguishable from Tmod3^+/+ (data not shown). In Tmod3^−/− MKs, GPIbα-labeled DMS membranes form fewer territories, which are deficient in mitochondria, F-actin accumulates in abnormal foci (B, red arrows), and mitochondria aggregate in the cytoplasm and around the nucleus (C, white arrowheads). Images are single optical sections selected from Z stacks acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a ×100 oil-immersion objective (N.A. 1.4) at zoom 2 (B) or zoom 5 (C). Asterisk in (C), nucleus. Bars, 8 μm (B); 2 µm (C).