ZO‐2 Protein But Not ZO‐1 Protein Limits Occludin Lateral Mobility in MDCK Type II Renal Epithelial Cells

The tight junction‐associated Zonula Occludens proteins, ZO‐1 and ZO‐2, possess binding sites for multiple tight junction membrane proteins, including occludin, multiple tight junction‐associated proteins, and F‐actin filaments. It is believed that these ZO proteins function as scaffolding proteins that crosslink tight junction membrane and associated proteins to the actin cytoskeleton to form a supramolecular complex. Fluorescence Recovery After Photobleaching (FRAP) studies reveal the existence of subpopulations of tight junction membrane proteins with limited lateral mobility (immobile fractions). It is hypothesized that the immobile proteins represent those tight junction membrane proteins tethered to the actin cytoskeleton via scaffolding proteins, including ZO‐1 and ZO‐2. To examine the roles of ZO‐1 and ZO‐2 in limiting the lateral mobility of the tight junction membrane protein occludin, we compared the FRAP behavior of GFP‐occludin constructs with and without the Ca‐terminal cytoplasmic domain or the ZO binding region contained in the C‐terminal cytoplasmic region. Wild type MDCK Type II renal epithelial cells were transduced with a viral vector containing full‐length GFP‐occludin, C terminal‐deletion GFP‐occludin, or ZO binding region‐deletion GFP‐occludin. In cells expressing the full‐length GFP‐occludin construct, approximately half (44.5 + 18.8%, n=31) of the GFP‐occludin was not mobile in the plane of the plasma membrane (immobile fraction). Deletion of virtually the entire C‐terminal region of GFP‐occludin decreased the immobile fraction (30.3 + 11.8%, n=23; p=0.0024 versus full‐length GFP‐occludin). Deletion of the ZO binding region produced a more dramatic decrease in the occludin immobile fraction (immobile fraction = 21.0 + 9.4%, n=26; p=3.4085 X 10‐7 versus full‐length GFP‐occludin). These results support the hypothesis that binding of occludin to ZO proteins limits occludin lateral mobility. To investigate whether this reflects binding to ZO‐1, ZO‐2, or both proteins, we examined the effect of knockdown of either ZO‐1 protein content or ZO‐2 protein content in MDCK II cells on the FRAP behavior of full‐length GFP‐occludin. Full‐length GFP‐occludin expressed in ZO‐1 knockdown MDCK Type II cells exhibited an immobile fraction similar to the value obtained in wild type MDCK Type II cells (immobile fraction = 44.4 + 14.2%, n=28; p=0.9711). In contrast, full‐length GFP‐occludin expressed in ZO‐2 knockdown MDCK Type II cells exhibited a decreased immobile fraction (25.9 + 11.2%, n=27; p=3.4713 X 10‐5 versus wild type MDCK II cells) similar to the immobile fraction observed in wild type MDCK Type II cells expressing ZO binding region‐deletion GFP‐occludin. These results indicate that about half of the immobile occludin protein is tethered to ZO‐2 protein in MDCK Type II renal epithelial cells. The other half of the immobile occludin fraction is tethered via a non‐ZO protein mechanism. ZO‐1 protein does not appear to tether any of the immobile occludin fraction in these cells. These results point to an unexpected complexity to the organization of the tight junction supramolecular complex.