Role for Actin Organization vs. Tight Junction Membrane Protein Cross-Linking in Regulation of Renal Paracellular Permeability to Macromolecules

Background: The Leak Pathway mediates macromolecule paracellular permeability across the tight junctions of epithelia. Changes in Leak Pathway permeability have been associated with many renal and other epithelial diseases states. The basic cellular processes and regulation of the Leak Pathway are poorly understood. The cytoplasmic ZO-1 protein provides a scaffold for crosslinking tight junction membrane proteins and for linking these proteins to the actin cytoskeleton. It also shuttles TOCA-1 protein, which facilitates formation of branching actin networks, to the tight junction. In MDCK II cells, knockdown of ZO-1 (ZO-1 KD) and knockout of TOCA-1 (TOCA-1 KO) increase Leak Pathway permeability. Methods: The flux rates of a size panel of fluorescein-dextran molecules (FD) were determined across monolayers of wild type MDCK II cells, ZO-1 KD cells, and TOCA-1 KO cells. Results: We hypothesized that depletion of these two proteins would similarly affect the Leak Pathway properties, opening number and opening size. Both TOCA-1 KO and wild type MDCK II cells exhibited similar proportional decreases in flux rate with increasing FD molecular size. ZO-1 KD cells exhibited a diminishing difference from wild type cells with increasing FD size. Plotting apparent permeability (Papp) versus solute Stokes radius on semilog plots demonstrated that the rate of decrease in Papp as a function of FD size was similar for wild type MDCK II and TOCA-1 KO cell monolayers. In contrast, ZO-1 KD cells exhibited a more rapid decline in Papp as solute size increased such that a large FD molecular size exhibited a similar Papp in wild type and ZO-1 KD MDCK II cells. Conclusion: Based on theoretical considerations, opening number should not affect Papp as a function of solute size. Opening size, however, should affect Papp as a function of solute size. Our results support the conclusion that depletion of TOCA-1 increases opening number without significantly affecting opening size. In contrast, ZO-1 depletion increases opening number and decreases opening size. These results suggest that crosslinking of tight junction membrane proteins, a property of ZO-1 but not TOCA-1, may be important for determining the size of the Leak Pathway openings, whereas, interaction of the tight junction with actin structures may affect preferentially opening number.