Formation and disassembly of adherens and tight junctions in the corneal endothelium: regulation by actomyosin contraction.
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.
Ramachandran
Charanya
C
School of Optometry, Indiana University, Bloomington, Indiana, USA.
Srinivas
Sangly P
SP
D000199:Actins / Q000502:physiology*; D000205:Actomyosin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D022005:Adherens Junctions / Q000378:metabolism*; D000577:Amides / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D018929:Cell Culture Techniques; D017097:Electric Impedance; D004728:Endothelium, Corneal / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011725:Pyridines / Q000494:pharmacology; D019108:Tight Junctions / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000199:Actins; D000577:Amides; D015820:Cadherins; D006576:Heterocyclic Compounds, 4 or More Rings; D008565:Membrane Proteins; D018994:Myosin Light Chains; D010750:Phosphoproteins; D011725:Pyridines; D062826:Zonula Occludens-1 Protein; C108830:Y 27632; C472645:blebbistatin; D000205:Actomyosin; D020742:rhoA GTP-Binding Protein; D002118:Calcium