May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Disassembly and Formation of Apical Junctions in the Corneal Endothelium:A Study Based on Ca2+ Switch Protocol
Author Affiliations & Notes
  • C. Ramachandran
    School of Optometry, Indiana University, Bloomington, Indiana
  • S. P. Srinivas
    School of Optometry, Indiana University, Bloomington, Indiana
  • Footnotes
    Commercial Relationships  C. Ramachandran, None; S.P. Srinivas, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3164. doi:https://doi.org/
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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. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 
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