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A. Gomez–Cabrero, E. Abad, J. Pales, A. Gual, X. Gasull, M. Morales; Effects of PTD4–Profilin I on Actin Cytoskeleton of Trabecular Meshwork Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1852.
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Fusion proteins with a protein transduction domain (PTD) are able to cross biological membranes, and they have been used successfully in a wide variety of cells. Profilin I is a nucleotid exchange protein that binds monomeric actin and regulates actin polimerization. Our previous studies have shown that PTD4–Profilin I increases aqueous humor (AH) outflow in perfused anterior segments of bovine eyes. Here, we further investigated its effects on actin cytoskeleton and cell shape on bovine trabecular meshwork (BTM) cells in culture.
Effects of purified PTD4–Profilin I protein, jasplakinolide, phalloidin–oleate, PAO and ML–7 were studied by immunocytochemistry in BTM cells from primary cultures.
In serum starved BTM cells, incubation with PTD4–Profilin I (10 µM) for 30 min or 2 h showed an increase on both lamellipodia number per cell and number of cells with lamellipodia. General actin filament stabilizing drugs such as jasplakinolide (2 µM, 30 min) and phalloidin–oleate (100 µM, 30 min) inhibited lamellipodia formation induced by PTD4–Profilin I. Since profilin I is regulated by PIP2 binding, decrease on PIP2 formation by PAO (PI(4)K inhibitor; 5 µM, 30 min) clearly inhibited lamellipodia formation, even in presence of PTD4–Profilin I. Using ML–7, a myosin light chain kinase inhibitor (50 µM, 30 min) that alters, not only the ability of the cell to keep the actin cytoskeleton tensioned for cell shape, but also lamellipodia formation, we observed that lamellipodia formation was inhibited too.
The present study indicates that Profilin I participates in polymerization of pre–existing actin filaments and nucleation of new ones in lamellipodia. Our data suggest that polymerization induced by PTD4–Profilin I could cause changes in trabecular meshwork cell shape or contractility that would explain the aqueous humor outflow increase previously reported.
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