Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Primer sequences used for construction of ZO-1 transgenes. Details of the cloning strategy are elaborated in the Supplementary Methods. Restriction sites used for subcloning of each amplicon are indicated in the right column.
• Supplemental Figure 2 - Alignment of the U6 motifs in ZO-1, ZO-2, ZO-3 and PYD. Amino acid sequences of the human polypeptides were aligned using the ClustalW WWW Service at the European Bioinformatics Institute (http://www.ebi.ac.uk/clustalw). Amino acid identities between two or more polypeptides are highlighted in red. The amino acid residues aligned are indicated in parenthesis.
• Supplemental Figure 3 - The polarized distribution of E cadherin and pg135 are unaltered in MDCK cells expressing the ΔU6 Transgene. MDCK tet-off cells were plated in filter inserts in the absence of doxycycline and grown for 10 days before they were fixed and stained for c-myc, the lateral membrane protein E cadherin (E cad.) and the apical marker gp135. Despite the presence of numerous ectopic strands, the distribution of E cadherin and gp135 are restricted to the lateral and apical membranes, respectively. This is evident in both enface maximum density projections (top panels) and z-axis scans (bottom panels). Furthermore, there is no apparent incorporation of E cadherin into the ectopic fibrils formed by ΔU6.
• Supplemental Figure 4 - Cingulin is recruited into ectopic fibrils in cells expressing the ΔU6 Transgene. MDCK cells stably expressing the ΔU6 Transgene were plated on glass coverslips, incubated for 3 days and subsequently fixed and stained with antibodies against the myc transgene (ΔU6) and cingulin. The recruitment of cingulin into the ectopic fibrils of MDCK cells expressing ΔU6 is in marked contrast to the lack of incorporation of cingulin into the fibrils produced in cells that express ZNG (Figure 9G, H), which lacks the C-terminal domain. These results suggest that it is the C-terminal domain that recruits cingulin to tight junction fibrils.
• Supplemental Figure 5a - Transepithelial Electrical Resistance is Unaltered in Cell Lines Expressing the ΔU6 Transgenes. A. Steady state TER was examined in three different clonal lines expressing full length (ZO1 FL) or ΔU6 transgenes grown for 14 days in the presence or absence of doxycycline. TER is unaffected by transgene expression. Untransfected MDCK tet-off cells and cell lines transfected with empty vector (pTRE empty) are included as controls.
• Supplemental Figure 5b - Transepithelial Electrical Resistance is Unaltered in Cell Lines Expressing the ΔU6 Transgenes. B. Calcium-switch assay; Clonal lines expressing ZO1 FL (clone A1), ΔU6 (clone O2) pTRE-empty (clone B3) were plated and grown in the presence or absence of DOX for 3 days prior to calcium removal. TER recovery was measured at the indicated time-points over 24 hours following re-addition of calcium to depleted monolayers. Although the profile of TER recovery can differ from clone to clone, it is unaffected by induction of any of the transgenes. This was true of all the clones in Figure 3A.
• Supplemental Data