The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state

Abstract

The tight junction defines epithelial organization. Structurally, the tight junction is comprised of transmembrane and membrane-associated proteins that are thought to assemble into stable complexes to determine function. In this study, we measure tight junction protein dynamics in live confluent Madin-Darby canine kidney monolayers using fluorescence recovery after photobleaching and related methods. Mathematical modeling shows that the majority of claudin-1 (76 +/- 5%) is stably localized at the tight junction. In contrast, the majority of occludin (71 +/- 3%) diffuses rapidly within the tight junction with a diffusion constant of 0.011 microm(2)s(-1). Zonula occludens-1 molecules are also highly dynamic in this region, but, rather than diffusing within the plane of the membrane, 69 +/- 5% exchange between membrane and intracellular pools in an energy-dependent manner. These data demonstrate that the tight junction undergoes constant remodeling and suggest that this dynamic behavior may contribute to tight junction assembly and regulation.

Figure 1.

Individual tight junction proteins display distinct FRAP behaviors in polarized epithelia. (A) EGFP-occludin, –claudin-1, –ZO-1, and –β-actin were studied by FRAP. High magnification images of tight junction segments before and at the indicated time points after photobleaching are shown in the left panels. Corresponding kymographs are shown at the right. (B) Quantitative analysis of FRAP from experiments similar to those shown in B (n = 7, 6, 8, and 6 for occludin, claudin-1, ZO-1, and β-actin, respectively). (C) The mobile fraction and t_1/2 of recovery for each protein were calculated from the recovery curves in B. Error bars represent SEM. Bars, 2 μm.

Figure 2.

Occludin and ZO-1 FRAP are differentially dependent on membrane composition and metabolic energy. (A) FRAP experiments were performed on monolayers of EGFP-occludin– and EGFP–ZO-1–expressing cells incubated at 37 (control), 18, or 14°C, with MBCD, or after ATP depletion. Representative kymographs are shown. (B and C) The t_1/2 and mobile fraction were calculated from FRAP experiments (n ≥ 5 at each temperature). TER was measured in parallel. (D–F) TER and mobile fraction were determined in monolayers incubated with MBCD or after ATP depletion. n ≥ 5 for each treatment. Error bars represent SEM. Bar, 2 μm.

Figure 3.

Occludin diffuses within tight junction. (A) EGFP-occludin–expressing cells within confluent monolayers were studied by FRAP after photobleaching elongated tight junction regions. Representative images before and at the indicated time points after photobleaching and the corresponding kymograph are shown. (B) The effect of continuous photobleaching of EGFP-occludin within a region of the tight junction is shown in representative images at the indicated times and in the corresponding kymograph. (A and B) Quantitative analysis of the individual sites indicated by the colored arrows is shown at the right. (C) Fluorescence of PA-GFP was activated in the white areas shown in the image collected during activation. Images collected at subsequent times show diffusion of activated PA-GFP–occludin to adjacent regions. The corresponding kymograph and quantitative analysis of the indicated activation region and adjacent region are shown. (D) The effect of continuous intracellular photobleaching of an EGFP-occludin–expressing cell within a confluent monolayer is shown. The kymographs and quantitative analyses show FLIP analysis of tight junction–associated EGFP-occludin within the indicated regions of photobleached and adjacent control cells. Bars: (A and B) 5 μm; (C and D) 10 μm.

Figure 4.

Tight junction–associated ZO-1 exchanges with an intracellular pool. (A) EGFP–ZO-1–expressing cells within confluent monolayers were studied by FRAP after photobleaching elongated tight junction regions. Representative images before and at the indicated times after photobleaching and the corresponding kymograph are shown. (B) The effect of continuous photobleaching of EGFP–ZO-1 within a region of the tight junction is shown in representative images at the indicated times and in the corresponding kymograph. (A and B) Quantitative analysis of the individual sites indicated by the colored arrows is shown at the right. (C) The effect of continuous intracellular photobleaching of an EGFP–ZO-1–expressing cell within a confluent monolayer is shown. The kymograph and quantitative analysis show tight junction–associated EGFP–ZO-1 fluorescence within the indicated tight junction region of a photobleached cell. Bars: (A and B) 5 μm; (C) 10 μm.

Figure 5.

Limited claudin-1 exchange occurs by diffusion within the tight junction. (A) EGFP–claudin-1–expressing cells within confluent monolayers were studied by continuous photobleaching of a region of the tight junction. Representative images at indicated times and the corresponding kymograph show the effect on tight junction fluorescence. Quantitative analysis of the individual sites indicated by the colored arrows is shown at the right. (B) The effect of continuous intracellular photobleaching of an EGFP–claudin-1–expressing cell within a confluent monolayer is shown. The kymograph and quantitative analysis shows tight junction–associated EGFP–claudin-1 fluorescence within the indicated region of a photobleached cell before and at intervals after photobleaching. (C) High magnification images and corresponding kymograph of EGFP–claudin-1^ΔYV are shown. The mobile fraction and t_1/2 of wild-type EGFP–claudin-1 and EGFP–claudin-1^ΔYV are shown in the graph at the right. Bars: (A) 5 μm; (B) 10 μm; (C) 2 μm.

Figure 6.

In silico simulations accurately model tight junction protein dynamic behavior. Computer models were established to predict tight junction protein dynamics (pink and cyan lines in A–L) and were compared with experimental data (red and blue symbols in A–L) for occludin (A–D), claudin-1 (E–H), and ZO-1 (I–L). Small area FRAP (A, E, and I), large area FRAP (B, F, and J), tight junction FLIP (C, G, and K), and intracellular FLIP experiments (D, H, and L) are compared. The models at the left show the assumptions required for the simulation to fit the experimental data. Specific values are given in Table II.