Tmod3 regulates F-actin polymerization and organization in MKs. (A) Tmod3 is associated with F-actin bundles that assemble in MKs spread on collagen. Immunofluorescence staining of F-actin (red) and Tmod3 (green) in WT MKs spread on collagen for 30 minutes. F-actin polymerizes into bundles and foci on the ventral surface of the MKs. Boxed regions shown at high magnification (lower panels) reveal Tmod3 puncta associated with the F-actin bundles (yellow arrrowheads). Bars, 10 μm. (B-C) Immunofluorescence staining of F-actin (red) and NMIIA (green) in Tmod3^+/+ and Tmod3^−/− MKs spread on collagen. In WT MKs, F-actin assembles into parallel bundles, as well as intensely stained foci on the ventral surface, termed podosomes.¹⁷  NMIIA is associated with the F-actin bundles (but not the podosomes) in a striated pattern in WT MKs, whereas both F-actin and NMIIA form amorphous aggregates near the cell periphery in Tmod3^−/− MKs. Boxed regions shown at higher magnification (lower sets of panels in [B-C]). Scale bars, 10 µm. Images are single optical sections from the ventral surface of MKs, acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a ×63 oil-immersion objective (N.A. 1.4) at zoom 1 (top panels) and zoom 3 (boxed regions).