The function of occludin (Occ) in the tight junction is undefined. To gain insight into its role in epithelial cell biology, occludin levels in Madin-Darby canine kidney II cells were suppressed by stably expressing short interfering RNA. Suppression of occludin was associated with a decrease in claudins-1 and -7 and an increase in claudins-3 and -4. Claudin-2 levels were unaffected. The tight junction "fence" function was not impaired in suppressed Occ (Occ-) clones, as determined by BODIPY-sphingomyelin diffusion in the membrane. The most striking changes were those related to control of the cytoskeleton and the "gate" function of tight junctions. A reduced ability of Occ- clones to extrude apoptotic cells from the monolayers suggested that neighbors of apoptotic cells either failed to sense their presence or were unable to coordinate cytoskeletal activity necessary for their extrusion. To further test the extent to which actin cytoskeletal activity depends on the presence of occludin, Occ- and Occ+ monolayers were depleted of cholesterol. Previous studies showed that cholesterol depletion is associated with reorganization of the actin cytoskeleton and a fall in transepithelial electrical resistance. In contrast to control Occ (Occ+) cells, transepithelial electrical resistance did not fall significantly in cholesterol-depleted Occ- monolayers and they failed to generate Rho-GTP, one of the signaling molecules involved in regulating the actin cytoskeleton. While steady-state transepithelial electrical resistance was similar in all clones, tight junction permeability to mono- and divalent inorganic cations was increased in Occ- monolayers. In addition, there was a disproportionately large increase in permeability to monovalent organic cations, up to 6.96 A in diameter. Chloride permeability was unaffected and there was little change in mannitol flux. The data suggest that occludin transduces external (apoptotic cells) and intramembrane (rapid cholesterol depletion) signals via a Rho signaling pathway that, in turn, elicits reorganization of the actin cytoskeleton. Impaired signaling in the absence of occludin may also alter the dynamic behavior of tight junction strands, as reflected by an increase in permeability to large organic cations; the permeability of ion pores formed of claudins, however, is less affected.

No abstract

Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol (CH) depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with detergent resistant membranes (DRMs) into two groups. Group A proteins (occludin, claudin-2 and -3) were detected in large, intermediate and small aggregates in both detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH altered the distribution of group A and B proteins among the three size categories in a detergent-specific manner. In lysates produced with octyl glucoside, a detergent that selectively extracts proteins from DRMs, group A proteins were undetectable in large aggregates and CH depletion did not alter the distribution of either group A or B proteins in intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in intimate enough contact with CH to be cross-linked to [ 3 H]-photo-cholesterol. However, antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein. Unlike claudins, occludin's presence in TJs and DRMs did not require palmitoylation. Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergent-related differences in the densities of TJ-bearing DRMs. There was little or no change in those densities after CH depletion. Removing CH from the plasma membrane increased tyrosine and threonine phosphorylation of occludin, and transepithelial electrical resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER fell below control values. We conclude that the association of integral TJ proteins with DRMS, pelleted at low speeds, is partially CH dependent. However, the buoyant density of TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH.

Transepithelial electrical resistance (TER), a measure of tight junction (TJ) barrier function, develops more rapidly and reaches higher values after preincubation of MDCK cells for 24 h with 2 microM Lovastatin (lova), an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase. While this effect was attributed to a 30% fall in cholesterol (CH), possible effects of lova on the supply of prenyl group precursors could not be excluded. In the current study, strategies were devised to examine effects on TER of agents that simultaneously lower CH and increase the flux of intermediates through the CH biosynthetic pathway. Zaragozic acid, 20 microM, an inhibitor of squalene synthase known to increase the synthesis of isoprenoids and levels of prenylated proteins, lowered cell CH by 30% after 24 h, while accelerating development of TER in the same manner as lova. TER was also enhanced, despite a 23% increase in the rate of [3H]acetate incorporation into CH, when total CH was reduced by 45% during a 2-h incubation with 2 mM methyl beta-cyclodextrin (MBCD), an agent that stimulates CH efflux from cells. The fact that the rate of TER development was diminished when cell CH content was elevated by incubation with a complex of CH and MBCD is further evidence that this sterol modulates development of the epithelial barrier. Cell associated CH derived from the complex was similar to endogenous CH with respect to its accessibility to cholesterol oxidase. Lova's effect on TER was diminished when 5 micrograms/mL of CH was added to the medium during the last 11 h of incubation with lova.

Gene drives hold promise for use in controlling insect vectors of diseases, agricultural pests, and for conservation of ecosystems against invasive species. At the same time, this technology comes with potential risks that include unknown downstream effects on entire ecosystems as well as the accidental or nefarious spread of organisms that carry the gene drive machinery. A code of ethics can be a useful tool for all parties involved in the development and regulation of gene drives and can be used to help ensure that a balanced analysis of risks, benefits, and values is taken into consideration in the interest of society and humanity. We have developed a code of ethics for gene drive research with the hope that this code will encourage the development of an international framework that includes ethical guidance of gene drive research and is incorporated into scientific practice by gaining broad agreement and adherence.

That changes in membrane lipid composition alter the barrier function of tight junctions illustrates the importance of the interactions between tetraspan integral tight junction proteins and lipids of the plasma membrane. Application of methyl-β-cyclodextrin to both apical and basolateral surfaces of MDCK cell monolayers for 2 hours, results in an ~80% decrease in cell cholesterol, a fall in transepithelial electrical resistance and a 30% reduction in cell content of occludin, with a smaller reduction in levels of claudins -2, -3 and -7. There were negligible changes in levels of actin, and the two non-tight junction membrane proteins GP-135 and caveolin-1. While in untreated control cells breakdown of occludin, and probably other tight junction proteins, is mediated by intracellular proteolysis, our current data suggest an alternative pathway whereby in a cholesterol-depleted membrane, levels of tight junction proteins are decreased via direct release into the intercellular space as components of membrane-bounded particles. Occludin, along with two of its degradation products and several claudins, increases in the basolateral medium after incubation with methyl-β-cyclodextrin for 30 minutes In contrast caveolin-1 is detected only in the apical medium after adding methyl-β-cyclodextrin. Release of occludin and its proteolytic fragments continues even after removal of methyl-β-cyclodextrin. Sedimentation and ultrastructural studies indicate that the extracellular tight junction proteins are associated with the membrane bounded particles that accumulate between adjacent cells. Disruption of the actin filament network by cytochalasin D, did not diminish methyl-β-cyclodextrin-induced release of tight junction proteins into the medium, suggesting that the mechanism underlying their formation is not actin-dependent. The 41 and 48 kDa C-terminal occludin fragments formed during cholesterol depletion result from the action of a GM6001-sensitive metalloproteinase(s) at some point in the path leading to release of the membrane particles.

Occludin and 18 distinct members of the claudin family are tetra-span transmembrane proteins that are localized in cell-specific tight junctions (TJs). A previous study showed that expression of chick occludin in Madin-Darby canine kidney (MDCK) cells raised transepithelial electrical resistance (TER) and, paradoxically, increased mannitol flux. In the present study, we employed epitope tagged canine occludin expression, under the control of the tetracycline repressible transactivator, to determine the extent to which the unexpected parallel increase in TER and mannitol flux was related to a structural mismatch between avian and canine occludins, which are only 50% identical. To determine whether the paradoxical changes in permeability was specific to occludin, we assessed the effect of over-expressing epitope tagged murine claudin-1. Our data revealed that over-expression of either of the epitope tagged mammalian tight junction proteins increased TER, mannitol and FITC-dextran flux. We observed a 2- and up to 5.6-fold over-expression of occludin-VSV-G and claudin-1-myc, respectively, with no change in ZO-1, endogenous occludin or claudin-1 expression. Confocal microscopy revealed that occludin-VSV-G, claudin-1-myc and ZO-1 co-localized at the TJ. In addition, claudin-1-myc formed aberrant strands along the lateral cell surface without an underlying ZO-1 scaffold. In fracture labeled replicas these strands consisted of claudin-1-myc with little accompanying occludin. These observations suggest that in epithelial cells claudin-1 can assemble into TJ strands without the participation of either ZO-1 or occludin. The proximity of the myc tag to the COOH-terminal YV sequence of claudin-1 appeared to interfere with its interaction with ZO-1, since over-expression of non-tagged claudin-1 increased TER but had a minimal effect on solute flux and no aberrant strands formed. From our data we conclude that differences in structure between avian and mammalian occludin do not account for the observed paradoxical increase in mannitol flux. Levels of ZO-1 remained unchanged despite substantial increases in induced TJ integral protein expression, suggesting that an imbalance between levels of ZO-1 and occludin or claudin-1 leads to altered regulation of pores through which non-charged solute flux occurs. We suggest that ion and solute flux are differentially regulated at the TJ.

Phospholemman (PLM) is a 72-amino acid transmembrane protein thought to function in Na,K-ATPase regulation or assembly, similar to other members of the FXYD family of proteins. Unique to PLM among these regulatory proteins are sites for C-terminal phosphorylation by PKA and PKC, although a role for phosphorylation in PLM function remains unclear. To study PLM phosphorylation, we used PLM phosphopeptides to generate antibodies to specifically detect phosphorylated PLM. Peptide affinity chromatography isolated two populations of antibodies: one reacting with standard PLM, a collection of closely-spaced 15-kDa protein bands by SDS-PAGE. About 20% of PLM antibodies reacted specifically with a single distinct form of PLM. Levels of this second immunological form (PLM-b) were increased with overexpression of PLM cDNA, and also reacted with a monoclonal antibody against the PLM N-terminus. In complete contrast to standard PLM, however, PLM-b was quantitatively insoluble in nonionic detergents and was released from tight binding by colchicine. Antibodies to PLM-b were present in two different antisera raised to the phosphorylated C-terminal peptide (residues 57-70), but not in antiserum raised to the non-phosphorylated C-terminal peptide. Despite an apparent relationship between PLM-b and phosphorylated PLM, PLM-b levels were not affected by treatment of heart cells with isoproterenol. PLM-b appears to represent a cytoskeleton-attached detergent-insoluble form of PLM with distinctive C-terminal immunoreactivity that might have implications for PLM structure and function.