Restricted accessResearch articleFirst published online 2006-12
Non-Muscle Myosin-II-B Filament Regulation of Paracellular Resistance in Cervical Epithelial Cells Is Associated With Modulation of the Cortical Acto-Myosin
Objective:To understand myosin regulation of epithelial permeability.
Methods:This was an experimental study, using human cervical epithelial cells CaSki. End points were paracellular permeability (determined in terms of transepithelial electrical resistance); non-muscle myosin-II-B (NMM-II-B) cellular localization; NMM-II-B phosphorylation status; NMM-II-B-actin interaction (determined in vitro by the immunoprecipitation-immunoreactivity method); and NMM-II-B filamentation (determined in vitro using purified NMM-II-B filaments in terms of filaments disassembly/assembly ratios.
Results:Treatment of cells with the Rho-associated kinase (ROCK) inhibitor Y-27632 or with the phosphatase inhibitor okadaic acid decreased the resistance of the lateral intercellular space (RLIS), and increased phosphorylation of NMM-II-B on threonine and serine residues. Y-27632 induced disorganization of the cortical acto-myosin and decreased co-immunoprecipitation of actin with NMM-II-B. Homodimerization assays using NMM-II-B filaments from cells treated with Y-27632 or okadaic acid revealed decreased filamentation compared to control cells. However, okadaic acid blocked Y-27632 decreased filamentation. Treatment with DRB, a casein kinase-II (CK2) inhibitor, induced opposing effects to those of Y-27632 and okadaic acid. Treatment with 5,6-dichloro-1-β-(D)-ribofuranosylbenzimidazole (DRB) did not involve modulation of actin depolymerization, suggesting that NMM-II-B regulation of the RLIS was independent of actin polymerization status. Exposure of NMM-II-B filaments to CK2 increased filamentation, regardless of prior treatments in vivo with Y-27632, okadaic acid, or DRB.
Conclusions:The results suggest that NMM-II-B filaments are in steady-state equilibrium of phosphorylation-dephosphorylation mediated by CK2 and by ROCK-regulated myosin heavy chain phosphatase, respectively. Increased phosphorylation would tend to inhibit assembly of NMM-II-B filaments and lead to decreased actin-myosin interaction, which would tend to decrease the RLIS and increase the paracellular permeability.
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