Control of epithelial cell polarity
Loss of epithelial cell polarity is associated with the overwhelming majority of human cancers. This has prompted intense research on mechanisms for epithelial polarity. Over the last decades, we have gained insight into how epithelial cell establish polarized surface domains and carry out vectorial protein transport, how cell-cell adhesion is mediated and how simple epithelia maintain a monolayer organization. This groundwork has set the stage for a new challenge: to identify and characterize the signaling mechanisms that integrate these individual aspects of epithelial polarity and architecture during morphogenesis and in pathology. The recent acknowledgement that basic mechanisms for the establishment of cell polarity are conserved between epithelial and non-epithelial systems allows us to take advantage of work in invertebrates and yeast where genetic approaches have identified and delineated signaling mechanisms for various aspects of polarity. My lab is interested in characterizing such novel polarity signaling cascades for epithelial polarity by means of cell biological approaches in established model cell lines for non-stratified epithelia of the kidney and liver.
Most of our current effort is focused on Par1, a serine/threonine kinase that was identified as polarity determinant in the one-cell embryo of C. elegans. We have determined that dysfunction of Par1 in the kidney epithelial cell line MDCK inhibits various aspects of epithelial polarization, notably the establishment of an apical surface domain and protein trafficking to the apical surface, the development of a columnar cell shape and the organization of an epithelial-specific microtubule array. We are now working to identify the Par1 substrates that regulate these individual morphogenetic processes. We anticipate that this will give us mechanistic insight into how these different aspects of polarization are linked. It is known, for instance, that protein trafficking to the apical domain is microtubule-dependent. Thus, we expect that the Par1 substrate(s) involved in epithelial microtubule-organization also regulate apical protein targeting and the formation of the apical cell surface.
As few Par1 substrates are known so far, we are employing both, a candidate approach and an unbiased proteome-wide screen. Putative Par1 targets are tested for their Par1-dependent phosphorylation in vitro and in vivo. Subsequently, we are characterizing their cell biological roles in MDCK cultures.
Our repertoire of approaches include the analysis of microtubule-dynamics and of apical surface formation in intact cells by time lapse imaging of GFP and RFP-tagged proteins, immunofluorescence and confocal laser microscopy to study cell morphology and sub-cellular protein distribution, biochemical assays that measure the kinetics of different protein trafficking steps and biochemical assays that measure protein-protein interactions and enzyme activities in cell lysates.
Cohen D, Tian Y, Müsch A. Par1b Promotes Hepatic-type Lumen Polarity in MDCK Cells via Myosin II and E-Cadherin-dependent Signaling. 2007 Mol Biol Cell. 18(6):2203-15
Elbert M, Cohen D, Müsch A. PAR1b promotes cell-cell adhesion and inhibits dishevelled-mediated transformation of Madin-Darby canine kidney cells. 2006 Mol Biol Cell. 17(8):3345-55.
Cohen D, Rodriguez-Boulan E, Müsch A. Par-1 promotes a hepatic mode of apical protein trafficking in MDCK cells. 2004 Proc Natl Acad Sci U S A. 101(38):13792-7.
Cohen D, Brennwald PJ, Rodriguez-Boulan E, Müsch A. Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton. 2004 J Cell Biol. 164(5):717-27.
Cohen D, Müsch A. Apical surface formation in MDCK cells: regulation by the serine/threonine kinase EMK1. 2003 Methods. 30(3):269-76.