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S. Chifflet, V. Nin, V. Correa, J.A. Hernandez; Effect of Plasma Membrane Hyperpolarization on the Actin Cytoskeleton and on the Adherens Junctions Stability in Cultured Bovine Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4509.
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Purpose: In previous work we showed that the depolarization of the plasma membrane potential (PMP) of several cultured epithelia exhibiting well–developed adherens junctions (AJ) provokes a gradual loss of the peripheral actin fibers. In culture conditions, confluent bovine corneal endothelial cells (BCEC) predominantly organize their actin cytoskeleton in the form of circumferential bundles along their lateral membranes. Nevertheless, these actin bundles are not as tightly packed as in vivo. We study here whether the PMP hyperpolarization determines modifications in the organization of the actin cytoskeleton and the AJ stability of cultured BCEC. Methods: Confluent BCEC monolayers were incubated at room temperature in normal or hyperpolarizing media (i.e., with valinomycin or with sodium replaced by choline) for up to 30 minutes. Membrane potential was monitored by fluorescence microscopy using oxonol V. Fluorescent probes were employed for F–actin (FITC–phalloidin), vinculin and cadherin (IIF). The quantitative aspects of the changes in actin distribution were assessed by image analysis. The stability of the AJ was evaluated by studying the modifications induced by calcium removal on the organization of the cortical actin cytoskeleton, cadherin and vinculin, under normal and hyperpolarizing conditions. Results: Incubation of BCEC in PMP–hyperpolarizing media determines an increase in the tightness of the peripheral actin and a decrease in the amount of focal contacts. The effect of calcium removal on the AJ is significantly delayed in the presence of hyperpolarizing solutions. Conclusions: These results suggest that PMP hyperpolarization of cultured BCEC monolayers increases the degree of actin compactness along the lateral membrane and, possibly by this means, contributes to determine a greater structural stability of the AJ. This work further contributes to the idea that the PMP may participate in signaling processes in epithelia.
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