Figure 7
Figure 7. Tmod3−/− proplatelets have defects in F-actin and TM4 organization, and circulating Tmod3−/− platelets fail to organize an F-actin ring after spreading on collagen. (A) Immunofluorescence staining of F-actin (red) and α-tubulin (green) in proplatelet-forming Tmod3+/+ (top) and Tmod3−/− (bottom) fetal liver MKs. Bar, 20 µm. (B) Relative level of F-actin vs α-tubulin per proplatelet bud, calculated from fluorescence intensity ratios as in Figure 5. Tmod3+/+: 0.82 ± 0.44 (n = 51); Tmod3−/−: 1.72 ± 1.0 (n = 45). ***P < .001. (C) F-actin/tubulin intensity per proplatelet bud with respect to area. (D) High magnification images and representative line scans of α-tubulin (green) and F-actin (red) fluorescence in Tmod3+/+ and Tmod3−/− proplatelets. Bars, 5 µm. (E) F-actin cytoplasm vs cortex intensity per proplatelet bud. Tmod3+/+: 0.55 ± 0.17 (n = 50); Tmod3−/−: 1.47 ± 0.74 (n = 42). ***P < .001. (F) F-actin cytoplasm vs cortex intensity per proplatelet bud with respect to area. (G) High magnification images and representative line scans of TM4 (green), α-tubulin (blue), and F-actin (red) fluorescence in Tmod3+/+ and Tmod3−/− proplatelets. Bars, 5 µm. (H) TM4 cytoplasm vs cortex intensity per proplatelet bud. Tmod3+/+: 0.75 ± 0.17 (n = 60); Tmod3−/−: 1.17 ± 0.24 (n = 60). (I) TM4 cytoplasm vs cortex intensity per proplatelet bud with respect to area. (J) Rhodamine-phalloidin staining for F-actin in Tmod3+/+ (left) and Tmod3−/− (right) embryonic platelets spread on collagen for 30 minutes. Bar, 2 µm. Images are single optical sections acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a 100× oil-immersion objective (N.A. 1.4) at zoom 1 (A) or zoom 3 (D,G,J). Fluorescence intensities of α-tubulin and F-actin in proplatelets (B-C) were determined using ImageJ software, with areas of proplatetets determined from α-tubulin staining as described in Figure 5. Ratios of cytoplasm to cortex staining intensities for F-actin and TM4 (E-F,H-I) were determined from intensities within small ∼0.5 μm diameter circles placed over the edge or middle of the proplatelet, using ImageJ software. Line scans of TM4 and F-actin signals across proplatelets were performed also with ImageJ software (D,G). Blue dotted lines indicate maximum x- and y-coordinate values of Tmod3+/+ data.

Tmod3−/− proplatelets have defects in F-actin and TM4 organization, and circulating Tmod3−/− platelets fail to organize an F-actin ring after spreading on collagen. (A) Immunofluorescence staining of F-actin (red) and α-tubulin (green) in proplatelet-forming Tmod3+/+ (top) and Tmod3−/− (bottom) fetal liver MKs. Bar, 20 µm. (B) Relative level of F-actin vs α-tubulin per proplatelet bud, calculated from fluorescence intensity ratios as in Figure 5. Tmod3+/+: 0.82 ± 0.44 (n = 51); Tmod3−/−: 1.72 ± 1.0 (n = 45). ***P < .001. (C) F-actin/tubulin intensity per proplatelet bud with respect to area. (D) High magnification images and representative line scans of α-tubulin (green) and F-actin (red) fluorescence in Tmod3+/+ and Tmod3−/− proplatelets. Bars, 5 µm. (E) F-actin cytoplasm vs cortex intensity per proplatelet bud. Tmod3+/+: 0.55 ± 0.17 (n = 50); Tmod3−/−: 1.47 ± 0.74 (n = 42). ***P < .001. (F) F-actin cytoplasm vs cortex intensity per proplatelet bud with respect to area. (G) High magnification images and representative line scans of TM4 (green), α-tubulin (blue), and F-actin (red) fluorescence in Tmod3+/+ and Tmod3−/− proplatelets. Bars, 5 µm. (H) TM4 cytoplasm vs cortex intensity per proplatelet bud. Tmod3+/+: 0.75 ± 0.17 (n = 60); Tmod3−/−: 1.17 ± 0.24 (n = 60). (I) TM4 cytoplasm vs cortex intensity per proplatelet bud with respect to area. (J) Rhodamine-phalloidin staining for F-actin in Tmod3+/+ (left) and Tmod3−/− (right) embryonic platelets spread on collagen for 30 minutes. Bar, 2 µm. Images are single optical sections acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a 100× oil-immersion objective (N.A. 1.4) at zoom 1 (A) or zoom 3 (D,G,J). Fluorescence intensities of α-tubulin and F-actin in proplatelets (B-C) were determined using ImageJ software, with areas of proplatetets determined from α-tubulin staining as described in Figure 5. Ratios of cytoplasm to cortex staining intensities for F-actin and TM4 (E-F,H-I) were determined from intensities within small ∼0.5 μm diameter circles placed over the edge or middle of the proplatelet, using ImageJ software. Line scans of TM4 and F-actin signals across proplatelets were performed also with ImageJ software (D,G). Blue dotted lines indicate maximum x- and y-coordinate values of Tmod3+/+ data.

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