Correct cyst morphogenesis of epithelial cells requires apical–basal polarization, which is partly regulated by mitotic spindle orientation, a process dependent on the heterotrimeric G protein subunit Gαi and its binding protein LGN. Here, we show that in three-dimensional culture of mammalian epithelial Madin–Darby canine kidney (MDCK) cells, the Gαi-activating protein Ric-8A is crucial for orientation of the mitotic spindle and formation of normal cysts that comprise a single layer of polarized cells with their apical surfaces lining an inner lumen. Consistent with the involvement of LGN, cystogenesis can be well organized by ADP-ribosylated Gαi, retaining the ability to interact with LGN, but not by the interaction-defective mutant protein Gαi2 (N150I). In monolayer culture of MDCK cells, functional tight junction (TJ) assembly, a process associated with epithelial cell polarization, is significantly delayed in Ric-8A-depleted cells as well as in Gαi-depleted cells in a mitosis-independent manner. Ric-8A knockdown results in a delayed cortical delivery of Gαi and the apical membrane protein gp135, and an increased formation of intercellular lumens surrounded by membranes rich in Gαi3 and gp135. TJ development also involves LGN and its related protein AGS3. Thus, Ric-8A regulates mammalian epithelial cell polarity for TJ assembly and cystogenesis probably in concert with Gαi and LGN/AGS3.
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