Tight junctions are one mode of cell-cell adhesion in epithelial and endothelial cellular sheets. They act as a primary barrier to the diffusion of solutes through the intercellular space, create a boundary between the apical and the basolateral plasma membrane domains, and recruit various cytoskeletal as well as signalling molecules at their cytoplasmic surface. New insights into the molecular architecture of tight junctions allow us to now discuss the structure and functions of this unique cell-cell adhesion apparatus in molecular terms.

Multifunctional strands in tight junctions.

Polarized epithelial cells play fundamental roles in the ontogeny and function of a variety of tissues and organs in mammals. The morphogenesis of a sheet of polarized epithelial cells (the trophectoderm) is the first overt sign of cellular differentiation in early embryonic development. In the adult, polarized epithelial cells line all body cavities and occur in tissues that carry out specialized vectorial transport functions of absorption and secretion. The generation of this phenotype is a multistage process requiring extracellular cues and the reorganization of proteins in the cytoplasm and on the plasma membrane; once established, the phenotype is maintained by the segregation and retention of specific proteins and lipids in distinct apical and basal-lateral plasma membrane domains.

https://science.sciencemag.org/content/sci/245/4919/718.full.pdf

Morphogenesis of the polarized epithelial cell phenotype

The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions

Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm.

https://rupress.org/jcb/article-pdf/120/3/695/1065059/695.pdf

Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells.

Considerable evidence suggests that Ca2+ modulates endothelial cell metabolic and morphologic responses to mediators of inflammation. We have used the fluorescent Ca2+ indicator, quin2, to monitor endothelial cell cytosolic free Ca2+, [Ca2+]i, in cultured human umbilical vein endothelial cells. Histamine stimulated an increase in [Ca2+]i from a resting level of 111 +/- 4 nM (mean +/- SEM, n = 10) to micromolar levels; maximal and half-maximal responses were elicited by 10(-4) M and 5 X 10(-6) M histamine, respectively. The rise in [Ca2+]i occurred with no detectable latency, attained peak values 15-30 s after addition of stimulus, and decayed to a sustained elevation of [Ca2+]i two- to threefold resting. H1 receptor specificity was demonstrated for the histamine-stimulated changes in [Ca2+]i. Experiments in Ca2+-free medium and in the presence of pyrilamine or the Ca2+ entry blockers Co2+ or Mn2+, indicated that Ca2+ mobilization from intracellular pools accounts for the initial rise, whereas influx of extracellular Ca2+ and continued H1 receptor occupancy are required for sustained elevation of [Ca2+]i. Ionomycin-sensitive intracellular Ca2+ stores were completely depleted by 4 min of exposure to 5 X 10(-6) M histamine. Verapamil or depolarization of endothelial cells in 120 mM K+ did not alter resting or histamine-stimulated [Ca2+]i, suggesting that histamine-elicited changes are not mediated by Ca2+ influx through voltage-gated channels. Endothelial cells grown on polycarbonate filters restricted the diffusion of a trypan blue-albumin complex; histamine (through an H1-selective effect) promoted trypan blue-albumin diffusion with a concentration dependency similar to that for the histamine-elicited rise in [Ca2+]i. Exposure of endothelial cells to histamine (10(-5) M) or ionomycin (10(-7) M) was associated with a decline in endothelial F-actin (relative F-actin content, 0.76 +/- 0.07 vs. 1.00 +/- 0.05; histamine vs. control, P less than 0.05; relative F-actin content, 0.72 +/- 0.06 vs. 1.00 +/- 0.05; ionomycin vs. control, P less than 0.01). The data support a role for cytosolic calcium in the regulation of endothelial shape change and vessel wall permeability in response to histamine.

/pdf/histamine-type-i-receptor-occupancy-increases-endothelial-53cmegrk6d.pdf

Histamine type I receptor occupancy increases endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers.

Single mouse intestinal epithelial cells (IEC) may be isolated by the use of a combination of methods used for the isolation of IEC from other species. Isolated cells remain viable for several hours. The membrane integral enzymes alkaline phosphatase and leucine aminopeptidase of isolated IEC are localized to the brush borders of IEC in tissue and in most newly isolated IEC. With time, both enzymes are found distributed over the entire cell surface. Redistribution appears to occur by diffusion in the plane of the membrane. It is slowed, but not blocked, if cells are maintained at 0 degrees C instead of at 37 degrees C, and it is not blocked by fixation in 0.5-3% paraformaldehyde. Drugs that alter cell membrane potential or that affect cell levels of ATP enhance the rate of redistribution of the enzymes.

https://rupress.org/jcb/article-pdf/86/3/849/1074485/849.pdf

Seth Schulman

Papers

Redistribution of membrane proteins in isolated mouse intestinal epithelial cells.