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Summary: Ben Margolis studies the role of protein interaction domains and signal transduction pathways in epithelial cell polarity.

Our laboratory focuses on mechanisms underlying protein targeting and cell polarity. The cell polarity process—where discrete membrane surfaces in a single cell contain different proteins—operates in a large number of cells within the body, including neurons and epithelia. In neurons, membrane surfaces within the dendrite spines receive neuronal signals, whereas membranes that compose the axon are involved in sending neuronal signals. To accomplish these tasks, neurons are polarized: different proteins are localized to the axon and the dendrite.

Similarly, epithelial cells contain two membrane surfaces with different protein compositions, the apical and basolateral membranes. The apical surface of epithelial cells faces the lumen of organs; the basolateral surface contacts the basement membrane and other cells. Between the apical and basolateral surfaces sits a seal called the tight junction. In the intestine, nutrients are absorbed into the cell via the apical surface and then transported into the bloodstream via the basolateral surface. The tight junction prevents nutrients in the intestine from directly entering the bloodstream so that reabsorption can be controlled by transport through the epithelial cell. To accomplish this vectorial transport, the apical and basolateral surfaces must have different protein components. Our laboratory focuses on the control mechanisms within the epithelial cell that lead to this differential protein composition.

	PALS1 is essential for epithelial cell polarity... PALS1 and zona occludens-1 in the kidney...

Many proteins involved in polarity determination are scaffold proteins with multiple protein-protein interaction domains. Several of these scaffolds contain PDZ domains that can bind to the carboxyl-terminal tails of cell surface proteins directing their localization in cells. Our studies began with the analysis of the mammalian PDZ domain protein, LIN-7. L in-7 was first identified by Stuart Kim and his colleagues (Stanford University) as a gene that affects the trafficking of the C aenorhabditis elegans epidermal growth factor receptor. In mammalian cells, we have identified a family of LIN-7–binding proteins that we call proteins associated with LIN seven (PALS). PALS1 is an essential polarity protein localized to the tight junction in mammalian epithelial cells. We have found that the PDZ domain of PALS1 interacts with the carboxyl terminus of Crumbs3, a small transmembrane protein of the apical surface. PALS1 has two L27 domains, a new domain we identified that allows PALS proteins to interact with LIN-7. The L27C domain of PALS1 binds mammalian LIN-7, while the L27N domain binds a new protein we have named PATJ. In addition to an amino-terminal L27 domain that binds the L27N domain of PALS1, PATJ has 10 PDZ domains. PATJ is also a tight-junction protein and is responsible for targeting PALS1 to this junction. Together Crumbs, PALS1, and PATJ form a complex that connects apical membrane proteins with the tight junction. (This work was supported in part by a grant from the National Institutes of Health).

We hypothesize that the Crumbs-PALS1-PATJ complex marks a crucial spatial cue that can control the subsequent polarization of mammalian epithelial cells. We have found that overexpression of the Crumbs3 protein disrupts the localization of this complex, leading to defects in cell polarity and tight junction formation. Similarly, we have used small interfering RNA to deplete epithelial cells of PALS1 and PATJ and found significant defects in cell polarization and tight-junction formation. These results provide convincing evidence that this complex is important in epithelial cell polarity and agree with studies that have examined highly related proteins found in Drosophila epithelia.

We have gone further to explore the mechanism whereby the Crumbs-PALS1-PATJ complex controls polarity. We have found that PALS1 can interact with an important polarity complex found at tight junctions. This complex, containing the proteins Par3, Par6, and atypical protein kinase C (aPKC), also plays an important role in many polarity decisions in lower organisms such as Drosophila and C. elegans . We demonstrated that PALS1 can interact directly with Par6 and that disruption in PALS1 localization affects the targeting of the Par3-Par6-aPKC complex to the tight junction. Similarly, it can be shown that Crumbs3 can recruit the Par3-Par6-aPKC complex to membrane surfaces when the proteins are transfected into a heterologous system. We hypothesize that once aPKC is targeted to the tight junction, it phosphorylates proteins that can regulate trafficking of proteins to the different membrane surfaces.

In more recent studies we have examined the role of the Crumbs-PALS1-PATJ complex in the formation of epithelial cilia, highly polarized microtubular structures that arise from the apical surface of epithelia. Ciliary defects have recently been implicated in polycystic kidney disease, a common cause of kidney failure. We have found that Crumbs3 is highly concentrated in cilia and that knockdown of Crumbs3 prevents cilia formation. Other polarity proteins—Par3, Par6, and aPKC—are also concentrated in cilia. Furthermore, inhibition of aPKC inhibited cilia formation. Finally, we demonstrated that Par3 interacts with a major microtubular motor of the cilia, kinesin II. These results suggest the multifunctional nature of polarity-signaling complexes and their likely control of microtubular networks.

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