Abstract
Purpose: :
To determine the role of actin cytoskeleton, in the formation and disassembly of adherens (AJs) and tight junctions (TJs).
Methods: :
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.
Results: :
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.
Conclusions: :
Although enhanced actomyosin contractility breaks down the barrier integrity, it is essential for the reestablishment of AJs and TJs.
Keywords: cytoskeleton • cell adhesions/cell junctions • cornea: basic science