👤 Verena Höffken

In epithelia, apicobasal cell polarization is closely linked to cell-cell contact formation, both controlled by the conserved Crumbs (CRB) complex, which includes the transmembrane protein Crumbs (CRB3a) and adapter proteins PALS1, PATJ, and LIN7c. In MDCK II cells, a model for cell polarization, depletion of PALS1 - which binds to all CRB components - leads to defective cell polarization and improper distribution of tight junction proteins, resulting in severe epithelial barrier defects in 3D cyst models. This study investigated whether this phenotype is associated with transcriptional changes by analyzing wildtype (WT) and PALS1 knockout (KO) MDCK II cell lines grown under non-confluent conditions and in 3D cyst cultures. Our results indicate that the transition from non-confluent cells to 3D cysts involves numerous differentially expressed genes (DEGs) in both WT and KO cells. Importantly, the analyses revealed significant overlaps between WT and KO cells in their maturation processes, suggesting that most identified DEGs are linked to differentiation from non-confluent to polarized MDCK cells and likely not a result of PALS1 deficiency. Gene Ontology (GO) enrichment and over-representation analyses using REACTOME and KEGG databases confirmed these similarities. In contrast, the direct comparison of WT and KO cells at the two stages showed fewer DEGs and overlaps in associated biological processes and signaling pathways. DEGs associated with the 3D stage, in which the phenotype manifests, contain DEGs and pathways that were predominantly linked to cell cycle linked processes, centromere assembly, or DNA replication. Furthermore, the transcription of genes encoding key junction proteins, additional polarity proteins, and cell-substrate interaction proteins is less affected by the loss of PALS1, indicating that PALS1 influences the transcriptional profiles in epithelial cells as a modulating factor. Show less

The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier. Show less