The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis

Teoh, Kim-Tat; Siu, Yu-Lam; Chan, Wing-Lim; Schlüter, Marc A.; Liu, Chia-Jen; Peiris, J. S. Malik; Bruzzone, Roberto; Margolis, Benjamin; Nal, Béatrice

We then decided to analyze the morphology and polarity of MDCKII monolayers at 2 h post-calcium switch, when the control and the HA-E (ΔPBM) expressing cell lines had reached their maximal TER values (Figure 7). Monolayers of MDCKII eGFP-PALS1, eGFP-PALS1, HA-E (wt), and eGFP-PALS1, HA-E (ΔPBM) were fixed, permeabilized, and stained with appropriate antibodies to study the subcellular distribution of GP135 (apical marker), E-cadherin (AJ protein), ZO-1 (TJ marker), and HA-E wt and truncated proteins. Relative localization of eGFP-PALS1 was also analyzed. Confocal microscopy and Z-sectioning allowed three-dimensional analysis of samples and monitoring of TJ formation and polarity establishment. At 2 h post-calcium switch, MDCKII eGFP-PALS1 cells had formed a regular monolayer of cubical cells (Figure 7A, panels a and b). In these cells, eGFP-PALS1 was present at cell–cell contacts in apical regions where it colocalized with ZO-1. Analysis of XZ and YZ dimensions showed that both proteins were present at TJ (Figure 7A, panel b, black arrowheads). The GP135 marker was found on apical surface of the cells (Figure 7A, panel a), whereas the E-cadherin protein was present at cell–cell junctions on lateral membranes, underneath eGFP-PALS1 and ZO-1, delineating AJ (Figure 7A, panels a and b). We concluded that at t = 2 h post-calcium switch, MDCKII eGFP-PALS1 cells had correctly formed TJ and were polarized.