Identification of Novel Regulators of Epithelial Polarity and Cell Migration
Abstract
The ability of cells to form separate apical and basolateral domains of their plasma membranes is an important aspect of epithelial integrity, and loss of epithelial polarity results in the acquisition of mesenchymal characteristics which contribute to tumorigenesis. Epithelial-to-mesenchymal transition (EMT), which occurs during certain stages of development, frequently occurs during tumour development in epithelia. When epithelial cells become mesenchymal as a result of EMT, they acquire the ability to migrate. In this context it is important to identify regulators of epithelial polarity and cell migration in order to prevent tumorigenesis. We have focused on the role of the structurally related pleckstrin homology domain leucine-rich repeat protein phosphatases (PHLPP1 and PHLPP2) in epithelial polarity. These proteins regulate the intracellular trafficking of the adherens junction protein E-cadherin, thereby controlling its recruitment to cell-cell contacts as well as its lateral targeting in a three-dimensional environment (paper I). Interestingly, while both proteins regulate this process, only PHLPP1 depletion resulted in increased adenocarcinoma cell migration. Cell migration is an important aspect of embryonic development, wound healing and immune responses. It also plays a role in pathological processes that include cancer, vascular disease, osteoporosis, chronic inflammatory diseases and mental retardation. This therefore explains the interest in understanding the mechanisms that regulate the cell's ability to move. Intracellular transport of a variety of cell-surface receptors is known to be important for cell migration, and special focus has been given to the integrins. Integrins are extracellular matrix receptors that undergo endocytosis and recycling, and this transport is thought to contribute to the ability of cells to detach and re-attach. Suprisingly, we observed that in addition to be recycled, integrins were also degraded in lysosomes, and that this requires their prior ubiquitination (paper II). Only fibronectin-bound integrins were ubiquitinated and sorted into multivesicular endosomes (MVEs), and this step required the endosomal complex required for transport (ESCRT) machinery. We propose that this trafficking route exists in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form non-productive adhesion sites. Upon ESCRT depletion, integrins accumulate intracellularly together with the nonreceptor tyrosine kinase Src at enlarged early endosomes. This results in the inability of Src to regulate myosin light chain kinase, MLCK, localized at the cell periphery, which thereby cannot phosphorylate the myosin regulatory light chain MRLC. These signaling pathways govern Golgi orientation, focal adhesion turnover and cell spreading, as well as cell migration (paper III). Cell migration is also regulated by phosphoinositides as has been demonstrated for PtdIns(4,5)P2 and PtdIns(3,4,5)P3. We identified a role for a recently discovered phosphoinositide, PtdIns5P, in the regulation of fibroblast migration, which is synthesized by the kinases VPS34 and PIKfyve and the phosphatase MTMR3. Importantly, we observed that cells with reduced PtdIns5P levels were unable to orient their Golgi apparatus or their actin cytoskeleton during directed cell migration, suggesting that PtdIns5P may regulate proteins involved in actin-myosin contractility. These findings contribute to our understanding of the regulation of epithelial polarity as well as fibroblast cell migration by the identification of novel players that control important players in these biological processes. By identifying novel regulators of epithelial polarity and cell migration, our studies have provided a framework for further studies of EMT and tumorigenesis
Series
Doktoravhandlinger ved NTNU, 1503-8181; 2012:281Dissertations at the Faculty of Medicine, 0805-7680; 573