Modulation of tight junction protein molecular remodeling: A potential mechanism of barrier regulation

Tight junctions (TJs) create a regulated paracellular barrier to the movement of water, solutes and immune cells between both epithelial and endothelial cells. The signaling events that underlie barrier dysregulation in disease are beginning to be understood. However, the molecular structural events that regulate function remain incompletely defined, largely due to our limited understanding of the molecular details of TJ organization and regulation. Recent studies have shown TJ undergoes molecular remodeling at steady state and lead to the hypothesis that molecular remodeling contributes to barrier regulation.;To test this hypothesis, I investigated the role of protein remodeling in myosin light chain kinase (MLCK)-dependent tight junction regulation. Dynamic behavior of tight junction proteins was assessed by fluorescence recovery after photobleaching (FRAP). MLCK inhibition in vitro increased barrier function and immobilized ZO-1 at the tight junction but did not affect claudin-1, occludin, or actin exchange. 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, the exchange of ZO-1 lacking the actin binding region (ABR) was not affected 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 intracellular ZO-1. Exchange of one pool is regulated by MLCK, whereas the ABR stabilizes ZO-1 at the tight junction. These data demonstrate an important role of the ZO-1 ABR and suggest that MLCK-dependent ZO-1 exchange is an essential component of this mechanism of barrier regulation.;Charge selectivity of the TJ barrier function is defined by claudins. Previous studies have examining claudin-1 by FRAP show that it slowly exchanges at the tight junction. In contrast, we found that claudin-4 is highly mobile at the tight junction. Analysis of claudin-1/4 tail and claudin-4/1tail chimeric constructs suggest that diffusion of claudin within TJ is influenced by the C- terminal tails. To better define this, we expressed claudin-2 with C-terminal tails of claudin1-8 and expressed these in MDCK cells lacking endogenous claudin-2. Chimeras containing tails of claudin1, 2, 5 and 8 were stable at the TJ, decreased TER and increased Na+ conductance. In contrast, chimeras containing claudin3, 4, 6, 7 tails were highly mobile and had a limited impact on TER and Na+ conductance. These data demonstrate that the highly divergent C-terminal tail defines FRAP behavior and suggest that stabilization within the tight junction affects pore formation.