A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

Abstract

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as ‘markers’ for the sequential events of synthesis and assembly of zonulae occludentes: (1) development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, (2) appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, (3) appearance of 13–15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and (4) alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: (1) reshaping the cell surface to align future junction-containing regions with those of adjacent cells, (2) delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and (3) aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements. To test the functional significance of these morphological features of junction-forming cells and to validate the interpretation that they are reliable indicators of the stages of tight junction genesis, primary cultures of bovine brain capillary endothelium were treated with 25 μg/ml of Cytochalasin-D or 0.25 mg/ml of n-ethylmaleimide (Sigma Chemical Co.) in order to prevent cytoskeletal mediation of surface contouring (step 1) or to inhibit vesicle fusion with the plasmalemma (step 2) and thereby prevent junction formation as a consequence of failure of the vesicle fusions to insert tight junctional intramembrane particles into the plasma membrane. Examination of platinum replicas of freeze-fractured control and treated endothelial monolayer cultures confirmed the absence of surface contours in Cytochalasin-D-treated cells, which exhibited no zonulae occludentes, and also clearly showed that n-ethylmaleimide-treated cells, which lacked tight junctions, did not have the rich endowment of vesicle fusion sites (and IMPs) which were conspicuous in control cells. Demonstration of the failure of MDCK cells to form tight junctions when cultured in the presence of 5–10 μg/ml of cycloheximide (Griepp et al., 1983) lends further support for the schemata proposed above. Advantages of this model include: (1) all stages of de novo tight junction formation are present in each monolayer culture, and (2) cultures possess vast areas of tight junction-containing membrane which are easily sampled by freeze-fracture. This model will provide the basis for future attempts to identify the signals that regulate tight junction formation, and will facilitate studies to characterize the protein(s) of the endothelial tight junctions, the messages (m-RNA) that code for them, and ultimately, the genes bearing their blueprint.