Figure 5
Figure 5. Treatment with t-PA/Plgn causes marked changes in the actin cytoskeleton, an increase in focal adhesion size, and generation of contractile forces in primary mouse astrocytes. (A) Representative immunofluorescence images of primary mouse astrocytes showing changes in F-actin and focal adhesion after treatment with t-PA (1μM) or its vehicle. Marked increase in stress fiber formation (left images, green) and vinculin size (right magnified images, red, indicated by arrows) are demonstrated after treatment with t-PA. Nuclei appear in blue. Scale bars represent 20 μm. (B) Representative immunofluorescence images of primary mouse astrocytes demonstrating changes in focal adhesion 24 hours after treatment with t-PA (50nM) + Plgn (20nM). Marked increase in vinculin (white, arrows) dimension is observed. Nuclei appear in red. Scale bars represent 20 μm. (C) Phase-contrast images of primary mouse astrocytes treated with vehicle, t-PA (50nM) + Plgn (20nM), or blebbistatin (a selective myosin ATPase inhibitor; 10μM) either alone or in combination for 24 hours. The full blockade of t-PA/Plgn–mediated morphology changes by blebbistatin indicates that contractile forces are generated in astrocytes by these proteases. Scale bar represents 40 μm. (D) Representative immunofluorescence images of serum-starved primary mouse astrocytes (48 hours) subjected to treatment with vehicle, t-PA (50nM) + Plgn (20nM) or t-PA alone (500nM) for 4.5 hours. Immunocytochemical analysis shows an increase in pMLC staining intensity and distribution (white) in astrocytes after stimulation with t-PA and Plgn or with high levels of t-PA alone. Cell nuclei are counterstained red. Scale bar represents 40 μm. (E) Western blot analysis confirming a concentration-dependent increase in pMLC as well as in pERK and pAkt levels in primary mouse astrocytes after t-PA/Plgn treatment. Astrocytes were serum-starved for 4 hours and then treated for 2 hours with t-PA (50nM) in the presence of either 20 or 100nM Plgn. A representative blot is shown on the right, and quantitation of fold changes in phospho-proteins after treatment with t-PA 50nM + Plgn (100nM) is presented on the left (n = 4-5). (F) Representative Western blot analysis demonstrating a strong attenuation of t-PA (10nM) + Plgn (50nM)–mediated increase in pMLC, pERK, and pAkt levels by coaddition of ct-PA (200nM) and TXA (20mM). Stimulation was performed as described in panel E.

Treatment with t-PA/Plgn causes marked changes in the actin cytoskeleton, an increase in focal adhesion size, and generation of contractile forces in primary mouse astrocytes. (A) Representative immunofluorescence images of primary mouse astrocytes showing changes in F-actin and focal adhesion after treatment with t-PA (1μM) or its vehicle. Marked increase in stress fiber formation (left images, green) and vinculin size (right magnified images, red, indicated by arrows) are demonstrated after treatment with t-PA. Nuclei appear in blue. Scale bars represent 20 μm. (B) Representative immunofluorescence images of primary mouse astrocytes demonstrating changes in focal adhesion 24 hours after treatment with t-PA (50nM) + Plgn (20nM). Marked increase in vinculin (white, arrows) dimension is observed. Nuclei appear in red. Scale bars represent 20 μm. (C) Phase-contrast images of primary mouse astrocytes treated with vehicle, t-PA (50nM) + Plgn (20nM), or blebbistatin (a selective myosin ATPase inhibitor; 10μM) either alone or in combination for 24 hours. The full blockade of t-PA/Plgn–mediated morphology changes by blebbistatin indicates that contractile forces are generated in astrocytes by these proteases. Scale bar represents 40 μm. (D) Representative immunofluorescence images of serum-starved primary mouse astrocytes (48 hours) subjected to treatment with vehicle, t-PA (50nM) + Plgn (20nM) or t-PA alone (500nM) for 4.5 hours. Immunocytochemical analysis shows an increase in pMLC staining intensity and distribution (white) in astrocytes after stimulation with t-PA and Plgn or with high levels of t-PA alone. Cell nuclei are counterstained red. Scale bar represents 40 μm. (E) Western blot analysis confirming a concentration-dependent increase in pMLC as well as in pERK and pAkt levels in primary mouse astrocytes after t-PA/Plgn treatment. Astrocytes were serum-starved for 4 hours and then treated for 2 hours with t-PA (50nM) in the presence of either 20 or 100nM Plgn. A representative blot is shown on the right, and quantitation of fold changes in phospho-proteins after treatment with t-PA 50nM + Plgn (100nM) is presented on the left (n = 4-5). (F) Representative Western blot analysis demonstrating a strong attenuation of t-PA (10nM) + Plgn (50nM)–mediated increase in pMLC, pERK, and pAkt levels by coaddition of ct-PA (200nM) and TXA (20mM). Stimulation was performed as described in panel E.

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