We are using cell biological approaches to understand signaling mechanisms that govern the establishment and maintenance of epithelial cell polarity and their relevance for morphogenesis and cell transformation.
1. Identification of Par1b signaling pathways in epithelial cells - The role of Par1b in H. pylori pathology
Par1 isoforms are serine/threonine kinases that have emerged as "core determinants" of cell polarity in different contexts, including in mammalian epithelial cells. In collaboration with the microbiologist M. Stein (Univ Edmonton, Alberta) we found that Par1b is inactivated by the cytotoxic protein CagA of Helicobacter pylori. H. pylori is a human adapted bacterium that colonizes the gastric mucosa of an estimated half of the worlds’ population and destroys the epithelial lining of the stomach in a fraction of infected carriers, resulting in gastritis and gastric ulcers. H. pylori induced gastric atrophy can progress to gastric cancer, and CagA has been classified as a bacterial oncogene by the WHO. Our goal is to elucidate CagA signaling mechanisms that are mediated by Par1b inhibition. In addition to the effects on epithelial morphology and cell-cell junctional integrity we are studying the contribution of Par1b-inhibition to the accumulation of DNA doublestrand breaks in the gastric epithelium upon H. pylori infection, which has been linked to the transformation risk. To identify relevant Par1b- substrates we have conducted an unbiased screen that yielded more than 70 putative novel Par1b substrates and their Par1b phosphorylation sites in polarized epithelial cells. We are elucidating their roles in H. pylori infected gastric epithelial monolayers derived from organoids, which we establish from human tissue that is leftover from bariatric surgery.
2. Mechanisms for the establishment of distinct epithelial phenotypes in tubule-forming epithelia and hepatocytes
Kidney epithelia and hepatocytes embody the two major vertebrate epithelial phenotypes. Kidney epithelial cells are monopolar; each cell in the kidney tubules has a single apical surface lying opposite a single basolateral surface, separated from each other by tight junctions. The apical surfaces collectively form the luminal aspect of the tubular monolayer, whereas the basal surfaces lie on a basal lamina. Hepatocytes are instead multipolar; they organize in one- or two-cell thick plates, with each cell having multiple luminal domains that form a branched canalicular network and multiple basal surfaces with no underlying basal lamina; this organization favors extensive bidirectional exchange of macromolecules with the blood, central to hepatocyte function. Developmentally, multipolar hepatocytes arise from monopolar intermediates, but the mechanisms directing this phenotypic conversion remain unknown. Using gain- and loss- of function studies in kidney- (MDCK) and hepatocyte-derived (WIFB and HepG2) cell cultures, we discovered a key role for the serine/threonine kinase Par1b in this conversion. We further discerned extracellular matrix (ECM) signaling dependent regulation of RhoA/Rho Kinase activity as central for Par1b signaling in this process. We are currently exploiting the results from our unbiased epithelial Par1b substrate screen to characterize the details of the Par1b-ECM-RhoA signaling cascade(s) that mediate monopolar->hepatocyte polarity conversion.
We are also elucidating the mechanisms responsible for the dramatically different trafficking phenotypes of kidney and hepatocytic epithelial cells. Whereas kidney cells target proteins directly to the apical and basolateral membranes from the Golgi apparatus, hepatocytes target proteins initially to the basolateral membrane and then relocate apical membrane proteins to their final destination by transcytosis. We are utilizing fast live-cell imaging and manipulations in intact cells and developed a fluorescence-based cell fractionation approach to determine how and where apical and basolateral proteins become segregated during their trafficking itineraries in the direct and transcytotic apical targeting mode, respectively.
Cohen, D., Rodriguez-Boulan, E. and Müsch, A. (2004). "Par-1 promotes a hepatic mode of apical protein trafficking in MDCK cells". PNAS 101, 13792-97.
Cohen, D., Brennwald, P.J., Rodriguez-Boulan, E. and Müsch, A. (2004)."Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton". Journal Cell Biology, 164, 717-727.
Rodriguez-Boulan, E., Kreitzer G and Müsch, A. (2005) "Protein Sorting and Post-Golgi Trafficking in Epithelia."Nature Reviews Molecular Cell Biology, 6, 233-47.
Elbert, M., Cohen D.and Müsch, A, (2006): " Par1b promotes cell-cell adhesion and inhibits Dishevelled-mediated transformation of MDCK cells", Mol.Biol.Cell, 17, 3345-55.
Cohen D., Tian Y and Müsch, A (2007): "Par1b promotes hepatic-type lumen polarity in MDCK cells via myosin II and E-cadherin dependent signaling", Mol. Biol. Cell 18, 2203-15.
Zaher Zeaiter, David Cohen, Anne Müsch, Fabio Bagnoli, Antonello Covacci, and Markus Stein (2008) "Analysis of detergent resistant membranes of Helicobacter pylori infected gastric adenocarcinoma cells reveals a role for MARK2/Par1b in CagA-mediated disruption of cellular polarity", J Cellular Microbiology, 10, 781-94.
Cohen D., Fernandez D., Lazaro-Dieguez, F. and Anne Müsch,. “The serine/threonine kinase Par1b regulates epithelial lumen polarity via IRSp53-mediated cell-ECM signaling”, (2011) J Cell Biol. 192, 525-540.
Treyer A., Müsch, A. “Hepatiocyte Polarity” , (2013) Comprehensive Physiology, 3, 243-87.
Ispolatov I, Müsch, A. " A model for the self-organization of vesicular flux and protein distributions in the Golgi apparatus", (2013), PLOS Computational Biology, Jul;9(7):e1003125. doi: 10.1371/journal.pcbi.1003125
Lazaro-Dieguez, F. Cohen D., Fernandez D., Louis Hodgson, S. van IJzendoorn and Müsch, A. “Par1b links lumen polarity with LGN/NuMA positioning for distinct epithelial cell division phenotypes”, (2013) J Cell Biol. 203(2): 251-64
Slim CL, Lázaro-Diéguez F, Bijlard M, Toussaint MJ, de Bruin A, Du Q, Müsch A, van Ijzendoorn SC. Par1b induces asymmetric inheritance of plasma membrane domains via LGN-dependent mitotic spindle orientation in proliferating hepatocytes. PLoS Biol. 2013 Dec;11(12):e1001739.
Nachbar J, Lázaro-Diéguez F, Prekeris R, Cohen D, Müsch A. KIFC3 promotes mitotic progression and integrity of the central spindle in cytokinesis. Cell Cycle. 2014 13(3): 426-33
Müsch, A. “The unique polarity phenotype of hepatocytes” Exp. Cell Research 2014. 328(2):276-83.
Mishra JP, Cohen D, Zamperone A, Nesic D, Muesch A, Stein M. Cell Microbiol. 2015 Jun 3. doi: 10.1111/cmi.12464. [Epub ahead of print]: CagA of Helicobacter pylori interacts with and inhibits the serine-threonine kinase PRK2.
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Albert Einstein College of Medicine
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