Figure 5.
Figure 5. CI-induced podosome assembly in primary murine MKs. (A) CI-induced podosome assembly in 129Sv-derived MKs: the Lin- bone marrow cells harvested from 129Sv mice were grown for 72 hours in the presence of TPO, seeded on coverslips coated with CI for 30 minutes (i) or 2 hours (ii-iv), processed for staining with rhodamine-phalloidin (red) and antivinculin (green) to label F-actin and vinculin, respectively, and analyzed by confocal microscopy. Labeling with anti-VWF (blue pseudocolor) shows that the observed cells are, indeed, MKs. Podosomes are readily discernible 30 minutes following adhesion to CI (i), and their assembly is optimal within 2 hours (ii,iv). Optical sections from the bottom (ii: section = 0.70 μm) to the top of the cell (iii: section = 1.40 μm) illustrate ventral localization of punctate F-actin structures. Vinculin rings were noticeable mainly at the cell margin (iv). Bar, 10 μm. (B) Adhesion to the high-affinity substrates of GPVI or α2β1 integrin CVX (i-ii) or GFOGER peptide (iii-iv), respectively, induced similarly the formation of actin-rich podosome structures with vinculin rings in 129Sv-derived MKs at the ventral side of the cells mainly at the cell periphery. Bar, 10 μm. (C) CI induced podosome assembly in C57Bl/6-derived similarly to 129Sv-derived MKs: vinculin (green) delineates the core of F-actin structures mainly in the cell margin (i). Bar, 10 μm. The inset is a phase contrast of a 5-fold magnification of the area marked by a rectangle. Dark dots corresponding to actin punctate structures are located in the leading edges and the tips of lamellae. (ii) High-power view of actin-rich podosome-like structures in the same area (section = 0.35 μm). Actin dots are circled, individually numbered (for example: 1 to 10) and measured using CLSM5 Zeiss Browse Image software (diameter of circles: no. 1 = 0.44 μm; no. 2 = 0.45 μm; no. 3 = 0.54 μm; no.4 = 0.41; no. 5 = 0.45; no. 6 = 0.55; no. 7 = 0.55; no. 8 = 0.44; no. 9 = 0.55; and no. 10 = 0.41 μm). Bar, 2 μm. (iii-iv) Podosome assembly induced by adhesion to CVX (iii) or GFOGER (iv) substrates is illustrated. The magnified region confined in a white square (0.35 μm, bottom panel) illustrates the ringlike shape of vinculin lining actin dots mainly at the cell periphery. Bar, 2 μm. (D) CI-induced podosome assembly is similarly induced by GPVI and α2β1 integrin in primary murine MKs: quantification of the experiments illustrated in panels A-C. Note that only a negligible proportion of MKs show podosome structures on PLL substrate. At least 300 MKs showing podosome-like structures were counted in each experiment. Bars represent the mean ± standard deviation of 3 independent experiments. The results are not significantly different between C57Bl/6 and 129Sv mice in all tested conditions.

CI-induced podosome assembly in primary murine MKs. (A) CI-induced podosome assembly in 129Sv-derived MKs: the Lin- bone marrow cells harvested from 129Sv mice were grown for 72 hours in the presence of TPO, seeded on coverslips coated with CI for 30 minutes (i) or 2 hours (ii-iv), processed for staining with rhodamine-phalloidin (red) and antivinculin (green) to label F-actin and vinculin, respectively, and analyzed by confocal microscopy. Labeling with anti-VWF (blue pseudocolor) shows that the observed cells are, indeed, MKs. Podosomes are readily discernible 30 minutes following adhesion to CI (i), and their assembly is optimal within 2 hours (ii,iv). Optical sections from the bottom (ii: section = 0.70 μm) to the top of the cell (iii: section = 1.40 μm) illustrate ventral localization of punctate F-actin structures. Vinculin rings were noticeable mainly at the cell margin (iv). Bar, 10 μm. (B) Adhesion to the high-affinity substrates of GPVI or α2β1 integrin CVX (i-ii) or GFOGER peptide (iii-iv), respectively, induced similarly the formation of actin-rich podosome structures with vinculin rings in 129Sv-derived MKs at the ventral side of the cells mainly at the cell periphery. Bar, 10 μm. (C) CI induced podosome assembly in C57Bl/6-derived similarly to 129Sv-derived MKs: vinculin (green) delineates the core of F-actin structures mainly in the cell margin (i). Bar, 10 μm. The inset is a phase contrast of a 5-fold magnification of the area marked by a rectangle. Dark dots corresponding to actin punctate structures are located in the leading edges and the tips of lamellae. (ii) High-power view of actin-rich podosome-like structures in the same area (section = 0.35 μm). Actin dots are circled, individually numbered (for example: 1 to 10) and measured using CLSM5 Zeiss Browse Image software (diameter of circles: no. 1 = 0.44 μm; no. 2 = 0.45 μm; no. 3 = 0.54 μm; no.4 = 0.41; no. 5 = 0.45; no. 6 = 0.55; no. 7 = 0.55; no. 8 = 0.44; no. 9 = 0.55; and no. 10 = 0.41 μm). Bar, 2 μm. (iii-iv) Podosome assembly induced by adhesion to CVX (iii) or GFOGER (iv) substrates is illustrated. The magnified region confined in a white square (0.35 μm, bottom panel) illustrates the ringlike shape of vinculin lining actin dots mainly at the cell periphery. Bar, 2 μm. (D) CI-induced podosome assembly is similarly induced by GPVI and α2β1 integrin in primary murine MKs: quantification of the experiments illustrated in panels A-C. Note that only a negligible proportion of MKs show podosome structures on PLL substrate. At least 300 MKs showing podosome-like structures were counted in each experiment. Bars represent the mean ± standard deviation of 3 independent experiments. The results are not significantly different between C57Bl/6 and 129Sv mice in all tested conditions.

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