April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Actin Cytoskeleton Regulates Both Formation and Disassembly of Adherens and Tight Junctions in the Corneal Endothelium
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  NIH R21-EY019119 (SPS), FRSP Award from the Office of Vice President for Research, Indiana University (SPS)
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 1457. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      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.

      Download citation file:


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

      ×
  • Supplements
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 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×