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MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function
Dan Yu, Amanda M. Marchiando, Christopher R. Weber, David R. Raleigh, Yingmin Wang, Le Shen, Jerrold R. Turner and W. James Nelson
Proceedings of the National Academy of Sciences of the United States of America
Vol. 107, No. 18 (May 4, 2010), pp. 8237-8241
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/25665522
Page Count: 5
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The perijunctional actomyosin ring contributes to myosin light chain kinase (MLCK)-dependent tight junction regulation. However, the specific protein interactions involved in this process are unknown. To test the hypothesis that molecular remodeling contributes to barrier regulation, tight junction protein dynamic behavior was assessed by fluorescence recovery after photobleaching (FRAP). MLCK inhibition increased barrier function and stabilized ZO-1 at the tight junction but did not affect claudin-1, occludin, or actin exchange in vitro. Pharmacologic MLCK inhibition also blocked in vivo ZO-1 exchange in wild-type, but not long MLCK -/- , mice. Conversely, ZO-1 exchange was accelerated in transgenic mice expressing constitutively active MLCK. In vitro, ZO-1 lacking the actin binding region (ABR) was not stabilized by MLCK inhibition, either in the presence or absence of endogenous ZO-1. Moreover, the free ABR interfered with full-length ZO-1 exchange and reduced basal barrier function. The free ABR also prevented increases in barrier function following MLCK inhibition in a manner that required endogenous ZO-1 expression. In silico modeling of the FRAP data suggests that tight junction-associated ZO-1 exists in three pools, two of which exchange with cytosolic ZO-1. Transport of the ABR-anchored exchangeable pool is regulated by MLCK. These data demonstrate a critical role for the ZO-1 ABR in barrier function and suggest that MLCK-dependent ZO-1 exchange is essential to this mechanism of barrier regulation.
Proceedings of the National Academy of Sciences of the United States of America © 2010 National Academy of Sciences