The preimplantation mammalian embryo: characterization of intercellular junctions and their appearance during development.
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Inhibition of intercellular communication has been hypothesized to play a role in tumor promotion. The compound 2,2',4,4',5,5'-hexabromobiphenyl (245-HBB) is a tumor promoter in vivo and blocks intercellular communication in vitro. The scrape-loading/dye-transfer (SL/DT) assay was used to assess this in vitro effect at varying concentrations of 245-HBB. The SL/DT technique is based on the intracellular loading of a fluorescent dye, lucifer yellow (LY), and monitoring its transfer into adjacent cells via patent gap junctions. Confluent WB-F344 (rat epithelial) cells were exposed to various noncytolethal concentrations of 245-HBB. Transfer of LY was then quantified with anchored cell analysis/sorting (ACAS 470, Meridian Instruments, Okemos, Mich.). The results indicate an inverse correlation between the degree of fluorescence in secondary LY-recipient cells and the treatment concentration. The coupling of these two new methods of cellular biology provided rapid quantitative analysis of dye transfer in measuring the concentration/response of modulation of gap-junctional permeability in cultured cells.
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Junctions between the cells in the reticular lamina of the organ of Corti were examined in thin sections and after freeze-fracturing to find a structural basis for the large ionic differences between the endolymph and perilymph. The apices of the cells in the reticular lamina are joined by a band of tight junctions spaced at 140 A intervals. Beneath this apical band the organization of the tight junctions depends on whether they join a supporting cell and a hair cell, or two supporting cells. At hair cell junctions with supporting cells, there is an extensive labyrinth of tight junctions enclosing lengthy, tortuous passages whose walls are composed of either multiple parallel or single junctions. At appositions between two supportinc cells, maculae or fasciae occludentes lie immediately beneath the apical bands of closely spaced tight junctions, near the top of the zonulae adherentia which are characteristic of appositions between supporting cells. The complexes of tight junctions, or zonulae occludentes, between extralaminar supporting cells differ from those in the reticular lamina. The extralaminar cells are joined by a band of four to seven branching, anastomotic tight junctions. Thus, these junctions are like zonulae occludentes in other tissues. The novel organizations of the tight junctions in the reticular lamina, different from those between the extralaminar supporting cells, suggests a special role for these junctions in the reticular lamina. Two sizes of gap junctions link, and presumably couple, supporting cells in the reticular lamina.
The ultrastructure of gap and tight junctions and the cell-to-cell transfer of small molecules were studied in primary cultures and freshly isolated sheets of endothelial cells from calf aortae and umbilical veins. In thin sections and in freeze-fracture replicas, the gap and tight junctions in the freshly isolated cells from both sources appeared similar to those found in the intimal endothelium. Most of the interfaces in replicas had complex arrays of multiple gap junctions either intercalated within tight junction networks or interconnected by linear particle strands. The particle density in the center of most gap junctions was noticeably reduced. In confluent monolayers, after 3-5 days in culture, gap and tight junctions were present, although reduced in complexity and apparent extent. Despite the relative simplicity of the junctions, the cell-to-cell transfer of potential changes, dye (Lucifer Yellow CH), and nucleotides was readily detectable in cultures of both endothelial cell types. The extent and rapidity of dye transfer in culture was only slightly less than that in sheets of freshly isolated cells, perhaps reflecting a reduced gap junctional area combined with an increase in cell size in vitro.
Two bronchiolo-alveolar carcinomas of Clara cell type have been studied by thin section and freeze-fracture techniques. Thin sections of the tumour cells showed junctional complexes (tight junctions and intermediate junctions) at the luminal aspect of the lateral cell membranes and desmosomes below them. Freeze-fracture replicas showed well-developed tight junctions with strands arranged in parallel or polygonal patterns. The junctions were continuous 'zonula occludens' type, usually three or more strands deep, with focal proliferation in some areas. The granules in the tumour cells tended to be larger than those of normal human Clara cells, although they were morphologically similar. There was no evidence of secretion of granules from the tumour cells. The relationships between tumour differentiation and growth pattern, and junctional development are discussed.
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Freeze-etch preparations of mesothelial cells taken from the peritoneum of mouse reveal the presence of vesicles invaginating the apical and the basal cell surfaces. These vesicles are scarcely seen within the cytoplasm. Long tortuous tubular profiles extend for considerable distance within the cytoplasm and are frequently associated with the vesicles. The possible nature and role of the vesicles and the tubules in transport phenomena across the mesothelial barrier, are discussed in relation to the pore theory advanced by physiologists and the "stomata" concept observed by early German and contemporary anatomists. "Occludens" junctions of the leaky type are seen though their macular or zonular nature is yet to be established.