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C. Ramachandran, S. P. Srinivas; Actin Cytoskeleton Regulates Both Formation and Disassembly of Adherens and Tight Junctions in the Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1457.
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To determine the role of actin cytoskeleton, in the formation and disassembly of adherens (AJs) and tight junctions (TJs).
Experiments were conducted with cultured bovine and feshly peeled rabbit corneal endothelium. Disassembly and reformation of AJs and TJs were induced by exposure to Ca2+-free Ringers (containing 2 mM EGTA) and subsequent add-back of Ca2+, respectively. Changes in trans-endothelial electrical resistance (TER; a measure of barrier integrity) were assessed from electrical cell-substrate impedance which was measured in real-time. Associated changes in junctional components and phosphorylation of MLC (myosin light chain) were followed by immunolocalization. RhoA activation was assessed by Western blot analysis.
Exposure to Ca2+ free medium led to RhoA mediated MLC phosphorylation, a precipitous drop in TER, formation of contractile F-actin ring, and redistribution of cadherins and ZO-1. While ZO-1 was found co-localized with the contractile F-actin ring, cadherins were internalized. These effects were reversed upon Ca2+ add-back in about 3 hrs. Pre-treatment with Y-27632 or blebbistatin (inhibitors of actomyosin contraction) reduced the rate of decline in TER, opposed the formation of contractile F-actin ring and blocked the redistribution of cadherins and ZO-1 upon Ca2+ depletion. Both the drugs reduced the rate of recovery in TER, and opposed the relocalization of cadherins and ZO-1 upon Ca2+ add-back. Cytochalasin D, which inhibits actin polymerization, also reduced the rate of recovery upon Ca2+ add-back.
Although enhanced actomyosin contractility breaks down the barrier integrity, it is essential for the reestablishment of AJs and TJs.
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