Dynamic behavior of paired claudin strands within apposing plasma membranes

Abstract

The tight junction (TJ) strand is a linear proteinaceous polymer spanning plasma membranes, and each TJ strand associates laterally with another TJ strand in the apposing membranes of adjacent cells to form "paired" TJ strands. Claudins have been identified as the major constituents of TJ strands, and when exogenously expressed in L fibroblasts, they polymerize into paired strands, which are morphologically similar to paired TJ strands in epithelia. Here, we show that a fusion protein of GFP with claudin-1 can also form similar paired strands in L fibroblasts, allowing us to directly observe individual paired claudin strands in live cells in real time. These paired strands showed more dynamic behavior than expected; they were occasionally broken and annealed, and dynamically associated with each other in both an end-to-side and side-to-side manner. Through this behavior of individual paired claudin strands, the network of strands was reorganized dynamically. Furthermore, fluorescence recovery after photobleaching analyses revealed that claudin molecules were not mobile within paired strands. Although these observations are not necessarily representative of TJ strands per se in epithelial cells, they provide important information on the structural and kinetic properties of TJ strands in situ with significant implications for barrier function of TJs.

Figure 1

Live observation of individual paired claudin strands formed in cultured L fibroblasts. (A) Schematic drawing of the observation system. L cell transfectants expressing GFP-claudin-1 (C1GL cells) were used, and in these cells GFP-claudin-1 was polymerized into paired strands. When the cell–cell contact plane was oriented obliquely or perpendicularly to the observation axis, GFP-claudin-1 was clearly visualized to be concentrated as strand-like structures (Lower). This image was taken by conventional fluorescence microscopy. (B) Immuno-replica electron microscopy. Freeze-fracture replicas were obtained from C1GL cells and immunolabeled with anti-GFP monoclonal antibody (10-nm gold particles). GFP was detected exclusively on intramembranous particle strands. Bars = 4 μm (A) and 300 nm (B).

Figure 2

Coculture of C1GL cells with L transfectants expressing FLAG-claudin-1 (C1FL cells). Cocultured cellular sheets were fixed, permeabilized, and stained with anti-FLAG mAb in red. (A) GFP signals. Two types of GFP-positive networks, bright (arrows) and dim (arrowheads), were clearly distinguished in terms of their green fluorescence intensity. (B) FLAG staining. The GFP-bright networks lacked the FLAG signal, whereas the GFP-dim one was always positive for the FLAG signal, indicating that these two types of networks were formed at C1GL/C1GL and C1GL/C1FL cell contacts, respectively. (C) Merged image. Note that, in the GFP-dim networks, the pattern of GFP-positive strands coincided precisely with that of FLAG-positive strands (Inset). Bar = 5 μm.

Figure 3

Time-lapse images of the dynamic behavior of paired claudin strands. Elapsed time is indicated at the top (in min:s). (A) Breaking and annealing. Two strands were annealed to generate a single strand (arrowheads), and another single strand was broken into two shorter strands (arrows). (B) End-to-side association. Three strands formed two T-shaped junctions by end-to-side association (arrowheads) (see Movies 1–4). Bars = 1 μm.

Figure 4

Time-lapse images of the dynamic reorganization of networks of paired claudin strands. Elapsed time is indicated at the top (in min:s). In the first frame of time-lapse series, an arbitrary continuous membrane domain delineated by strands was colored red. When this domain became continuous to an adjacent domain during time-lapse observation, this adjacent domain was also colored red. The network was reorganized continuously and dynamically, while retaining the structural integrity of the network as a whole (see Movie 5). Bar = 2.5 μm.

Figure 5

Time-lapse images of fluorescence recovery after photobleaching of reconstituted strands. Elapsed time is indicated at the top (in min:s). The bleached zone is outlined by a white box. (A) A band of strand 0.9 μm in width was photobleached, and no recovery was detected. The length of the nonbleached strand segment did not appear to change. (B) A narrow (0.15-μm-wide) band of a strand was bleached. The bleached mark (arrows) remained detectable, and its relative position within the strand did not change for 13 min after photobleaching. The positions of the bleached point (arrows) relative to both ends (arrowheads and double arrowheads) were measured and summarized schematically in Right (see Movies 6 and 7). Bars = 0.5 μm (A) and 1 μm (B).