1. How is the polarization of epithelial cells determined? Epithelial cells are highly polarized, with an apical surface facing the lumen of the cavity and a basolateral surface facing other cells and extracellular matrix. Epithelial cells have a conserved set of protein complexes, such as the Par3/Par6 complex, which are needed to become polarized. Upstream of these complexes, we have discovered a new pathway that determines the orientation of polarization, that is which way the cell points. Alterations in this pathway can cause cells to reverse polarity, so that the apical surface now points away from the central lumen. We have found that this pathway starts with assembly of the laminin-rich extracellular matrix around the cells, followed by integrin signaling at the basolateral surface. The signal for orientation of polarity must then move across the cells to cause the assembly of the apical surface; we are studying the molecular mechanism of this movement.

2. Once the initial orientation of polarity is determined, how does the cell execute this program to become fully polarized? The apical and basolateral surfaces of the cell have completely different protein and lipid compositions, and so the cell has mechanisms to specifically sort these components to one surface or the other. The lipids phosphatidyl inositol 4,5-P2 (PIP2) and 3,4,5-P3 (PIP3) are crucial determinants of the identities and formation of the apical and basolateral surfaces, respectively. Normally PIP3 is found only at the basolateral surface. Ectopic localization of PIP3 to the apical surface can transform this apical surface into basolateral surface, by causing the relocalization of normally basolateral proteins to the apical surface, as well as removal of apical proteins from this transformed surface. Inhibition of generation of endogenous PIP3 reduces the size of the basolateral surface. The major human pathogen, Pseudomonas aeruginosa , hijacks this pathway by causing the accumulation of PIP3 and basolateral proteins at the apical surface, thereby promoting bacterial internalization and disease. Conversely, PIP2 is enriched at the apical surface. The lipid phosphatase, PTEN, is needed for the segregation of PIP2 and PIP3. Exogenous PIP2 added to the basolateral surface causes apical markers to relocalize to the basolateral surface. The PIP2 recruits Annexin 2 to the apical surface, which in turn recruits the GTP-bound form of Cdc42 to the apical surface. PTEN, PIP2, Annexin 2 and Cdc42 are all needed to form normal apical surfaces and lumens.

3. The basic building block of most epithelial organs are tubes, lined by epithelial cells. How do epithelial cells organize themselves into tubes? We have found that when epithelia cells remodel to form tubes, they start by partially dedifferentiating, losing their epithelial polarity and taking on many of the properties of migrating fibroblasts. The transcription factor Slug is needed for cells to survive during this process. (This differentiation and migration is a good in vitro model for cancer metastasis as well as various fibrotic diseases.) The cells then migrate to positions where they will form the new tube. The cells then re-differentiate, reacquiring epithelial polarity and creating new lumens between the cells. Tubulogenesis can thus be conceptualized as occurring in two stages, dedifferentiation and re-differentiation.