Biomedical subjects

James Melvin Anderson

Publications and source records attributed to James Melvin Anderson.

6 recordsLinked to original sources

PubMed2004

Setting up a selective barrier at the apical junction complex.

Across the animal kingdom the apical junction complex of epithelial cells creates both a permeability barrier and cell polarity. Although based on overlapping and evolutionarily conserved proteins, the cell-cell contacts of nematodes, flies and mammals appear to differ in morphology and functional organization. Emerging evidence shows that the selective pore-like properties of vertebrate and invertebrate barriers are created by the claudin family. Similarly, assembly of the barriers requires a conserved set of polarity-generating protein complexes, particularly the PAR protein complexes.

Adherens Junctions↗

PubMed2004

The molecular physiology of tight junction pores.

Tight junctions form selective barriers that regulate paracellular transport across epithelia. A large family of tetraspanning cell-cell adhesion proteins called claudins create the barrier and regulate electrical resistance, size, and ionic charge selectivity. Study of inherited human claudin diseases and the outcome of the genetic manupulation of claudins in mice, Drosophila, and Caenorhabditis elegans are furthering our understanding of paracellular physiology.

Amino Acid Sequence↗

PubMed2003

Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture.

Tight junctions (TJs) regulate paracellular permeability across epithelia and vary widely in their transepithelial electrical resistance (TER) and charge selectivity. The claudin family of transmembrane proteins influences these properties. We previously reported that claudin-4 increased TER approximately 300% when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells and decreased the paracellular permeability for Na(+) more than Cl(-) (Van Itallie C, Rahner C, and Anderson JM. J Clin Invest 107: 1319-1327, 2001). In comparison, we report here that expression of claudin-2 increases TER by only approximately 20% and does not change the ionic selectivity of MDCK II cells from their cation-selective background. To test whether the extracellular domains of claudins-4 and -2 determine their unique paracellular properties, we determined the effects of interchanging these domains between claudins-4 and -2. Inducible expression of wild-type claudins and extracellular domain chimeras increased both the number and depth of fibrils, but the characteristic fibril morphologies of claudin-4 or -2 were not altered by switching extracellular domains. Like claudin-4, chimeras expressing the first or both extracellular domains of claudin-4 on claudin-2 increased TER severalfold and profoundly decreased the permeability of Na(+) relative to Cl(-). In contrast, chimeras expressing the first or both extracellular domains of claudin-2 on claudin-4 increased the TER by only approximately 60 and approximately 40%, respectively, and only modestly altered charge selectivity. These results support a model in which the claudins create paracellular channels and the first extracellular domain is sufficient to determine both paracellular charge selectivity and TER.

Amino Acid Sequence↗

PubMed2003

Expression, solubilization, and biochemical characterization of the tight junction transmembrane protein claudin-4.

The tight junction tetraspan protein claudin-4 creates a charge-selective pore in the paracellular pathway across epithelia. The structure of the pore is unknown, but is presumed to result from transcellular adhesive contacts between claudin's extracellular loops. Here we report the expression of claudin-4 by baculovirus infection of Sf9 cells and describe the biochemical analysis suggesting it has a hexameric quaternary configuration. We show the detergent perfluoro-octanoic acid is able to maintain oligomeric claudin species. Sucrose velocity centrifugation and laser light scattering are also used to investigate the oligomeric state of claudin-4. In contrast to proteins of similar topology, such as gap junction family connexins, the oligomeric state of claudins appears more dynamic. These data suggest the structural organization of claudins in tight junction pores is unique.

Animals↗

PubMed2002

Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1.

Zonula occludens (ZO)-1 is a member of the MAGUK (membrane-associated guanylate kinase homologs) family of membrane-associated signaling molecules that binds directly to both cytosolic and transmembrane components of the tight junction and is believed to organize these proteins within the apical junctional complex. It also binds directly to F-actin, although the functional relevance of this interaction is unknown. To address this issue, we have used VSVG-tagged transgenes to dissect ZO-1 and have identified a 220 amino acid region of ZO-1 that is necessary for its association with F-actin in MDCK cell pull-down assays. A GST fusion expressing this region can bind directly to F-actin in vitro, whereas a GFP fusion expressing this domain decorates actin stress fibers when expressed in MDCK cells. These results indicate that this actin-binding region (ABR) is both necessary and sufficient for binding to F-actin in vitro and in vivo. VSVG-tagged transgenes that lack the ABR still accumulate at both early and late cell-cell contacts in MDCK cells, suggesting that the ABR is not required for tight junction localization. However, accumulation of constructs lacking the ABR is markedly reduced at tight junctions in confluent cells, suggesting that the ABR does play an important role in the localization of ZO-1 at junctions. Furthermore, the ABR is required for localization to a novel actin-rich pool of ZO-1 that accumulates in puncta at the free edge of cells before initiation of cell-cell contact. We conclude that direct interactions between ZO-1 and F-actin play a role in several different steps of junction assembly.

Actins↗

PubMed2002

Claudins create charge-selective channels in the paracellular pathway between epithelial cells.

Epithelia separate tissue spaces by regulating the passage of ions, solutes, and water through both the transcellular and paracellular pathways. Paracellular permeability is defined by intercellular tight junctions, which vary widely among tissues with respect to solute flux, electrical resistance, and ionic charge selectivity. To test the hypothesis that members of the claudin family of tight junction proteins create charge selectivity, we assessed the effect of reversing the charge of selected extracellular amino acids in two claudins using site-directed mutagenesis. Claudins were expressed in cultured Madin-Darby canine kidney cell monolayers under an inducible promoter, and clones were compared with and without induction for transmonolayer electrical resistance and dilution potentials. Expression and localization of claudins were determined by immunoblotting, immunofluorescence microscopy, and freeze-fracture electron microscopy. We observed that substituting a negative for a positive charge at position 65 in the first extracellular domain of claudin-4 increased paracellular Na+ permeability. Conversely, substituting positive for negative charges at three positions in the first extracellular domain of claudin-15, singly and in combination, reversed paracellular charge selectivity from a preference for Na+ to Cl-. These results support a model where claudins create charge-selective channels in the paracellular space.

Amino Acid Sequence↗