Disassembly and Formation of Apical Junctions in the Corneal Endothelium:A Study Based on Ca2+ Switch Protocol

Abstract

Purpose: : To determine the role of actomyosin contraction in disassembly and formation of adherens junctions (AJs) and tight junctions (TJs) in corneal endothelium.

Methods: : Experiments were conducted with cultured bovine corneal endothelial cells. Disassembly of AJs, and hence TJs, was induced by exposure to Ca²⁺-free medium containing 2 mM EGTA. Subsequently, cells were exposed to Ca²⁺-rich medium (2 mM Ca²⁺) to follow the dynamics of AJs and TJs reformation. Actomyosin contraction was prevented by inhibiting Rho kinase (Y-27632; 5 µM) or Myosin II ATPase (blebbistatin; 5 µM). Trans-endothelial electrical resistance (TER) was measured in real-time (1 Hz) to follow the status of the tight junction assembly. Changes in peri-junctional actomyosin ring (PAMR) and localization of ZO-1 and E-cadherin, during Ca²⁺ depletion and add back, were followed by immunocytochemistry. RhoA activation was assessed by a pull-down assay using Rhotekin as bait.

Results: : Exposure to Ca²⁺-free medium led to activation of RhoA, concomitant with a precipitous fall (< 30 sec) in TER. It also resulted in compaction of PAMR, dispersion of ZO-1, and internalization of E-cadherin. Repletion of Ca²⁺ led to a recovery of TER and reversal of the effects on junction proteins within 3 hrs. Pre-exposure to Y-27632 and blebbistatin opposed the decline in TER and also opposed dispersion of ZO-1 and contraction of PAMR produced by Ca²⁺depletion. In addition, the same agents also reduced the rate of recovery in TER upon Ca²⁺ add-back.

Conclusions: : Since cadherin-dependent AJs require external Ca²⁺, the reduction in the rate of decline in TER upon Ca²⁺removal in the presence of blebbistatin and Y-27632 indicates that disassembly of TJs is enhanced by actomyosin contraction. Significant inhibition in the rate of increase in TER following Ca²⁺ add-back by blebbistain and Y-27632 suggests that a basal actomyosin contraction is necessary for reformation of AJs and TJs.

Keywords: cell adhesions/cell junctions • cytoskeleton • cornea: endothelium