Further Observations on the Fine Structure of Freeze-Cleaved Tight Junctions

Abstract

The fine structure of freeze-cleaved tight junctions has been examined in high-resolution replicas of rat small intestine. By carefully comparing the changes in the surface topography of the cleavage faces of specimens, which, before freezing, were either prefixed with glutaraldehyde and then infiltrated with glycerol, infiltrated with glycerol alone, or infiltrated with glycerol and then fixed with glutaraldehyde, a more precise definition of the tight-junction architecture at the supramolecular level has become possible. The results of this analysis suggest that the bilayer membranes contributing to a tight junction are held together along inter-connected lines of attachment that are arranged in the form of a continuous band-like mesh-work. Each line consists of 2 parallel rows, one in each membrane, of closely spaced adhesion particles. The sensitivity of these particles to glutaraldehyde and their cleaving behaviour under different experimental conditions indicate that they represent globular proteins which bridge the width of the adjoining membranes and are linked together in the plane of the inter-cellular space. Thus, the morphology of a tight-junction seal resembles a modified zipper with the locking units making head to head contact. Similarly, the presence of many open-ended sealing elements between crypt cells has been interpreted as suggesting that the formation of tight-junction seals could resemble a ‘zippering-up’ process. At the juncture of 3 cells the tight-junction meshwork is both modified and extended basally to produce a characteristic pattern. In the central area of such a triple junction, 3 parallel and very closely spaced vertical seals have been resolved, each joining a pair of adjacent plasma membranes. Small, regularly spaced, cross-bridging elements interlink pairs of central seals within the plane of each membrane. At different levels each individual central seal may further give rise to horizontal sealing elements which connect in a ladder-like fashion either to the major network or to vertical elements positioned at greater distances from the central axis. Evidence is presented suggesting that fragments of tight junctions can be internalized and broken down in lysosome-like vesicles.