Tight junctions (TJs) establish the epithelial barrier and are thought to form a membrane fence to regulate epithelial polarity, although the roles of TJs in epithelial polarity remain controversial. Claudins constitute TJ strands in conjunction with the cytoplasmic scaffolds ZO-1 and ZO-2 and play pivotal roles in epithelial barrier formation. However, how claudins and other TJ membrane proteins cooperate to organize TJs remains unclear. Here, we systematically knocked out TJ components by genome editing and show that while ZO-1/ZO-2–deficient cells lacked TJ structures and epithelial barriers, claudin-deficient cells lacked TJ strands and an electrolyte permeability barrier but formed membrane appositions and a macromolecule permeability barrier. Moreover, epithelial polarity was disorganized in ZO-1/ZO-2–deficient cells, but not in claudin-deficient cells. Simultaneous deletion of claudins and a TJ membrane protein JAM-A resulted in a loss of membrane appositions and a macromolecule permeability barrier and in sporadic epithelial polarity defects. These results demonstrate that claudins and JAM-A coordinately regulate TJ formation and epithelial polarity.

Directional cell movements during morphogenesis require the coordinated interplay between membrane receptors and the actin cytoskeleton. The WAVE regulatory complex (WRC) is a conserved actin regulator. Here, we found that the atypical cadherin Fat2 recruits the WRC to basal membranes of tricellular contacts where a new type of planar-polarized whip-like actin protrusion is formed. Loss of either Fat2 function or its interaction with the WRC disrupts tricellular protrusions and results in the formation of nonpolarized filopodia. We provide further evidence for a molecular network in which the receptor tyrosine phosphatase Dlar interacts with the WRC to couple the extracellular matrix, the membrane, and the actin cytoskeleton during egg elongation. Our data uncover a mechanism by which polarity information can be transduced from a membrane receptor to a key actin regulator to control collective follicle cell migration during egg elongation. 4D-live imaging of rotating MCF10A mammary acini further suggests an evolutionary conserved mechanism driving rotational motions in epithelial morphogenesis.

The PAR-3–atypical protein kinase C (aPKC)–PAR-6 complex has been implicated in the development of apicobasal polarity and the formation of tight junctions (TJs) in vertebrate epithelial cells. It is recruited by junctional adhesion molecule A (JAM-A) to primordial junctions where aPKC is activated by Rho family small guanosine triphosphatases. In this paper, we show that aPKC can interact directly with JAM-A in a PAR-3–independent manner. Upon recruitment to primordial junctions, aPKC phosphorylates JAM-A at S285 to promote the maturation of immature cell–cell contacts. In fully polarized cells, S285-phosphorylated JAM-A is localized exclusively at the TJs, and S285 phosphorylation of JAM-A is required for the development of a functional epithelial barrier. Protein phosphatase 2A dephosphorylates JAM-A at S285, suggesting that it antagonizes the activity of aPKC. Expression of nonphosphorylatable JAM-A/S285A interferes with single lumen specification during cyst development in three-dimensional culture. Our data suggest that aPKC phosphorylates JAM-A at S285 to regulate cell–cell contact maturation, TJ formation, and single lumen specification.