Cell polarity and tight junctions (TJs) are necessary for intact epithelia. Loss of tight junction integrity is consistent with a display of mesenchymal phenotype which is characteristic of metastatic progression. Occludin, the first identified transmembrane protein identified to localize at tight junctions, has been implicated in regulating tight junction function and cellular polarity. The N-terminal region of occludin has been shown to be an important factor in tight junction maintenance but the mechanism is not yet known.Extensive studies by other researchers have been performed to examine factors that drive EMT, while little is known about their functional reversibility in MET. A model system of EMT ↔ MET was previously established using Pa4 cells, an immortalized epithelial cell line derived from rat parotid gland. Stable introduction of an oncogenic form of the kinase Raf1 into Pa4 was used to derive a mesenchymal phenotype (Pa4Raf1) that demonstrates anchorage-independent growth that coincides with a loss of functional TJs including down-regulation of occludin. The N-terminal region of occludin (66 amino acids) was cloned and used as a bait to pull down protein binding partners in Pa4 and Pa4Raf1 cell lysates. This region of occludin was coupled to a hexa-histidine sequence (His-tag). The resulting protein was expressed in E. coli, purified using magnetic nickel beads, and used as a bait to pull down potential binding partners for this region of occludin present in two cell lines: Pa4 and Pa4-Raf1. Importantly, N-terminal region of occludin has a series of serine and tyrosine residues that are predicted to be phosphorylated by a wide range of kinases. In this regard, Ser8, Ser45, Tyr12, Tyr22, Tyr29 and the poly-proline region (Pro-Leu-Ser-Pro-Pro-Pro-Tyr-Arg-Pro) of the N-terminal bait peptide were mutated in order to further elucidate the functions of these residues. Serine was mutated to aspartic acid, tyrosine to glutamic acid, and proline to alanine. The binding partners were also knocked down to identify their effects on occludin colocalization.Many of the potential binding partners that were identified are involved in apoptosis, metastasis and/or cellular oxidation. This research seeks for ways by which manipulating these identified binding partners will transform a cell from a mesenchymal phenotype to an epithelial phenotype.
|Date of Award||13 Jun 2016|
|Supervisor||Randy Mrsny (Supervisor) & Matthew Lloyd (Supervisor)|