IIIG9 and PP1α form a protein complex in the adherens junctions of polarized ependymal and MDCK cells

IIIG9 was originally identified as a novel regulatory subunit of protein phosphatase 1 (PP1, PPP1R32) in ependymal cells, where it localizes to cilia and adherens junctions. However, it remains unclear whether IIIG9 forms complexes with PP1α, β, or γ to regulate ciliary function and/or the stability of adherens junctions in polarized cells. MDCK and ependymal cells, both polarized epithelial cell types, provide valuable models for investigating the molecular mechanisms underlying epithelial polarization and neoplastic events. We characterized the expression of PP1 subunits in ependymal cells using RT-qPCR in combination with laser microdissection (LMD) and in situ hybridization. We analyzed the colocalization of catalytic PP1 isoforms and IIIG9 by confocal microscopy, both in situ and in vitro. Similar analyses were performed in monolayers and cysts of MDCK cells. To assess the interaction between IIIG9 and PP1α, we used fluorescence resonance energy transfer (FRET) analysis in HEK cells and proximity ligation assays (PLA) in adult ependymal cells and MDCK cells. PP1α is the predominant PP1 isoform expressed in ependymal cells lining the ventricular wall of the adult brain, similar to IIIG9. Additionally, IIIG9 mRNA was detected in ependymal cilia, hippocampal neurons, and the cerebral cortex. Both proteins were found in the cytoplasm, at adherens junctions (positive for E-cadherin), and in cilia (positive for acetylated α-tubulin) throughout the entire ventricular system. In polarized MDCK cell cultures (monolayers and cysts), IIIG9 and PP1α colocalized with adherens junction proteins, including β-catenin and Par3. FRET analysis revealed a high-efficiency interaction between IIIG9 and PP1α, both in the cytoplasm and in puncta near the plasma membrane in HEK cells. Additionally, PLA detected positive interactions between IIIG9 and PP1α, as well as between IIIG9 and E-cadherin, in adult brain ependymal cells. Our data confirm the interaction between IIIG9 and PP1α in ependymal and MDCK cells. This interaction likely plays a key role in mediating the subcellular functions of PP1 at adherens junctions and cilia in polarized cells, thereby contributing to the regulation of cell polarity.