Establishment of tight junctions between epithelial cells ( electron microscopy / electrophysiology / intercellular contacts / membranes / wound healing )

Epithelia serve as barriers to the diffusion of solutes between body compartments, and must do so despite the frequent loss of cells. When single cells are experimentally removed from the Necturus gallbladder epithelium, contiguous cells migrate to fill the defect within 30 min. Electrophysiological measurements show that the local electrical resistance across the epithelium in the region of a wound returns to normal in the same period of time; electron microscopy demonstrates that tight junctions are formed concurrently. Physiologically functional and morphologically recognizable tight junctions can thus be established within 30 min, demonstrating a mechanism for the rapid restoration of epithelial integrity after cell loss. A distinctive structural feature of most vertebrate epithelia is the tight junction, or zonula occludens, which joins every cell with its neighbors. Each such junction is generally located near the cells' apices, and consists of one to several circumferential bands of close contacts between the apposed plasma membranes, manifested as membrane fusions in conventional electron microscopy (1, 2) and as intramembrane strands in freeze-fracture preparations (3-5). The close membrane contacts in effect form a series of gaskets surrounding each cell, and thereby constitute the principal barrier to the movement of solutes across an epithelium by diffusion between cells (6). Since the regulation of fluid composition within body compartments is one of the primary functions of epithelia, the integrity of tight junctions is essential to normal epithelial function, and must be maintained despite the frequent loss of cells through ageing and trauma (7). The present report documents the rapid formation of tight junctions between newly apposed epithelial cells. Gallbladders were dissected from freshly killed and pithed mudpuppies (Necturus maculosus) and cut into pieces roughly 5 mm square. Each piece was mounted on a glass slide, with the epithelial surface uppermost, secured at its corners with metal spring clips, and bathed in modified L-15 tissue culture solution (8). Preparations were viewed and photographed through Nomarski differential interference contrast optics with a modified Zeiss WL microscope equipped with a X40, water-immersion lens. The formation of tight junctions was initiated by bringing epithelial cells into contact with each other through the removal of one to several intervening cells. A selected cell (Fig. 4) was damaged by traumatic impalement or pricking with a fine glass microelectrode. After minimal damage, a cell generally rounded up and left the epithelium within 20 min. The process could be accelerated by pulling the damaged cell from the epithelium with the microelectrode. In either case, the loss of a cell (or group of cells) reliably pro* Present address: Division of Biology, California Institute of Technology, Pasadena, Calif. 91125. 2711 voked a stereotyped response of the adjacent cells (Figs. 4-6). Within 5 min, cells began to extend processes and to move into the lacuna (Fig. 5). The defect was usually grossly closed in 15 min, and often undetectable after 30 min (Fig. *6). To obtain a physiological measure of the reconstitution of tight junctions between newly apposed cells, electrical current pulses were passed across the epithelium while a pair of microelectrodes connected to a differential amplifier monitored the resultant potentials just above the epithelial surface (9, 10). Because more ionic current flows across an epithelium at regions of lower transepithelial resistance in such a recording configuration, greater potentials are recorded above sites of epithelial leakage. Immediately after the removal of a cell, a high leakage current was demonstrable at the site of the lesion (Figs. 1 and 2). This subsided gradually as contiguous cells advanced into the defect left by the missing cell, and reached control values by 30 min after damaging (Fig. 3). High-resolution experiments, which detected the normal slight leakage of current through tight junctions (9, 10), confirmed that transepithelial resistance at the site of removal of a cell reached control values after about 30 min. To corroborate the physiological findings, the sites of cell removal were examined by electron microscopy. Epithelia were fixed without damaging and at various stages of recovery from lesions by immersion for 50-90 min in a fixative consisting of 150 mM glutaraldehyde, 800 mM sodium cacodylate, and 5 mM calcium chloride at pH 7.3. After fixation, specimens were washed in cacodylate buffer, postfixed in osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in ethanol, and embedded in Epon. Thin sections stained with uranyl acetate and lead citrate were examined with a Philips EM 200 or EM 301 microscope. Necturus gallbladder cells are normally joined together by typical tight junctions which appear in transmission electron micrographs as 5-10 points of membrane fusion (Fig. 8), and in freeze-fracture micrographs as a comparable number of anastomosing strands (Fig. 9). After a lesion, the surviving cells extended numerous microspike processes which met at the defect's center in roughly 15 min. After 30 min of recovery, the cells were closely apposed to one another (Fig. 12), and their apical membranes showed multiple points of fusion characteristic of tight junctions (Figs. 10 and 11). These fusions, which were not always in their normal, ordered positions at cellular apices and which lacked the usual neat spacing of unperturbed tight junctions, nevertheless extended through multiple serial sections, and thus constituted at least segments of, if not complete, tight junctions. If the recording scans or microscopical sectioning had been misoriented, normal intercellular contacts (rather than nascent ones) would have been the objects of study. To exProc. Nat. Acad. Sci. USA 72 (1975)