MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function

Abstract

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.

Fig. 1.

MLCK inhibition uniquely stabilizes ZO-1 at the tight junction in vitro. (A) Caco-2 monolayers stably expressing fluorescent fusion constructs were studied by FRAP 15 min after addition of the specific MLCK inhibitor PIK (250 μM). (Scale bar, 3 μm.) (B) Mean recovery curves, n = 4 per condition. § P < 0.001 vs. ZO-1 without PIK. (C) TER and FRAP of ZO-1 and actin were assessed 15 min after addition of 0.5 μM latrunculin A (latA); 1 μM jasplakinolide (jas); 10 μM blebbistatin (blebb); 250 μM PIK; 10 μM ML-7; or 40 ng/mL toxin B, n = 4 per condition.

Fig. 2.

MLCK regulates ZO-1 exchange in vivo. (A) mRFP1-ZO-1 FRAP was assessed in wild-type (WT), long MLCK^−/−, and CA-MLCK transgenic mice. PIK (250 μM) was added 15 min before analysis. (Scale bar, 3 μm.) (B) Mobile fraction of mRFP1-ZO-1, n = 5 per condition. § P < 0.001 vs. corresponding mucosa after PIK treatment.

Fig. 3.

The ABR is required for ZO-1 stabilization after MLCK inhibition. (A) EGFP-ZO-1^ΔABR FRAP was assessed after addition of drugs, as in Fig. 1, and mobile fraction determined, n = 4 per condition. § P < 0.001 vs. untreated (control) monolayers. ‡ P < 0.001 vs. PIK-treated monolayers. (B) Mobile fraction of EGFP–ZO-1 or EGFP-ZO-1^ΔABR expressed in ZO-1 knockdown (kd) monolayers, n = 3 per condition. † P < 0.05 vs. control ZO-1 expressing monolayers. § P < 0.001 vs. corresponding PIK-treated monolayers.

Fig. 4.

ABR expression reduces barrier function and prevents TER increases after MLCK inhibition. (A) Mobile fraction of mRFP1–ZO-1 FRAP in monolayers also expressing EGFP-ZO-1 or EGFP-ABR, as indicated, without or with 250 μM PIK, n = 3. § P < 0.001 vs. monolayers without PIK. ‡ P < 0.01 vs. full-length EGFP-ZO-1-expressing monolayers. (B) PIK induces a dose-dependent increase in TER of control (ct) monolayers but not ZO-1 knockdown (kd) or EGFP-ABR-expressing monolayers, n = 3 per condition. ‡ P < 0.01 vs. all other conditions.

Fig. 5.

In silico models of ZO-1 exchange. (A) ZO-1 was distributed in one cytosolic and three tight junction-associated pools. (B) Representative data showing in vitro ZO-1 FRAP (circles) in control monolayers and in silico models of total recovery (black line) as the sum of slow and fast components of recovery.