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C. Ramachandran, S. P. Srinivas; Disassembly and Formation of Apical Junctions in the Corneal Endothelium:A Study Based on Ca2+ Switch Protocol. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3164.
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To determine the role of actomyosin contraction in disassembly and formation of adherens junctions (AJs) and tight junctions (TJs) in corneal endothelium.
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.
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.
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.
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