Formation and Disassembly of Adherens and Tight Junctions in the Corneal Endothelium: Regulation by Actomyosin Contraction

Abstract

Purpose. To determine the role of actin cytoskeleton in the disassembly and reformation of adherens junctions (AJs) and tight junctions (TJs) in bovine corneal endothelial monolayers. Methods. Disassembly and reformation of AJs and TJs were induced by extracellular Ca(2+) depletion and subsequent add-back of Ca(2+), respectively. Resultant changes in the transendothelial electrical resistance (TER), an indicator of integrity of TJs, were measured based on electrical cell-substrate impedance. Phosphorylated myosin light chain (ppMLC), a biochemical measure of actomyosin contraction, and activation of its upstream regulatory molecule RhoA-GTP were assessed by Western blot analysis. Results. Extracellular Ca(2+) depletion led to activation of RhoA, increase in ppMLC, decrease in TER, contraction of the perijunctional actomyosin ring (PAMR), and redistribution of zonula occludens-1 (ZO-1) and cadherins. These effects were reversed on Ca(2+) add-back. Pretreatment with Y-27632 and blebbistatin (as inhibitors of actomyosin contraction) reduced the rate of decline in TER, opposed the contraction of the PAMR, and blocked the redistribution of ZO-1 and cadherins. Both drugs reduced the recovery in TER and opposed the normal redistribution of ZO-1 and cadherins on Ca(2+) add-back. Cytochalasin D, which led to dissolution of the PAMR, also reduced the recovery of TER on Ca(2+) add-back. Conclusions. The (Ca(2+) depletion)-induced disassembly of AJs accelerates the breakdown of TJs through a concomitant increase in the actomyosin contraction of the PAMR. However, these data on reassembly show that a contractile tone of the PAMR is essential for assembly of the apical junctional complex.

rhAPC reduces the endothelial cell permeability via a decrease of contractile tensions induced by endothelial cells

“…Our results show that the contractile tension of endothelial cells was attenuated with Y-27632 application. Unlike calyculin and Y-27632, cytoclasin inhibits actin polymerization, thereby attenuating contraction (39). Cytoclasin D was also shown in this study to reduce the contractile tension of endothelial cells (Fig.…”

Microtubule stabilization opposes the (TNF-α)-induced loss in the barrier integrity of corneal endothelium

“…As a distinct pool of F-actin at the AJC, the PAMR is structurally and functionally coupled to TJs and AJs [8]. Upon excessive actomyosin contraction of the PAMR, the centripetal force generated around AJC reduces cell-cell tethering and this opposes the stable interaction of trans-membrane domains of the TJs and AJs [8,30]. In light of these observations, we examined whether the influence of cAMP-elevating agents also manifested at the level of the cytoskeleton.…”

The Hippo pathway: regulators and regulations

“…In keratinocytes and mammary epithelial cells, calcium depletion led to disruption of cell junctions and YAP/TAZ nuclear enrichment (Varelas et al 2010;Schlegelmilch et al 2011). Calcium-chelating agents may regulate intracellular actin dynamics, as disruption of cell junctions by calcium depletion resulted in an increase of actomyosin contraction in bovine corneal endothelial cells (Ramachandran and Srinivas 2010). However, it is also possible that the effect of actin rearrangements on the Hippo pathway is mediated by other cellular processes; for instance, the actin cytoskeleton is required for the establishment of cell polarity (Li and Gundersen 2008).…”