Terry, S.J.; (2011) Identification of regulators and effectors of RhoGTPase signalling in corneal epithelial cells. Doctoral thesis , UCL (University College London).

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Abstract

Epithelial cells adhere to each other and are connected via a series of junctions. Tight junctions (TJs) are a specific type of junction consisting of heteromeric protein complexes that are linked to the actin cytoskeleton and are important in regulating paracellular permeability and cell polarity. RhoGTPases are small molecular switch proteins that are important regulators of the cytoskeleton and modulators of gene expression. RhoGTPases have thus been identified as being major signalling components associated with TJs. However little is known about how RhoGTPases are regulated to control junction formation and gene expression in corneal epithelial cells. I used a siRNA screening approach combined with functional assays to identify components of RhoGTPase signalling that affect the assembly of junctions and gene expression in Human corneal epithelial cells (HCE). I identified and validated several candidates that regulate junction assembly. One of these candidates was p114RhoGEF, a novel TJ localised guanine nucleotide exchange factor (GEF) important for the assembly of functional TJs. p114RhoGEF is a widely expressed and I discovered its depletion effects junction formation and morphogenesis in three dimensional culture systems in different epithelial cell types. p114RhoGEF is required for activation of RhoA at cell-cell junctions and junctional actinomyosin activity, p114RhoGEF is present in a complex containing Myosin II-A, the RhoA effector Rock II and the junctional adaptor protein Cingulin; indicating p114RhoGEF is a component of a junction associated RhoA-signalling module. p114RhoGEF, thus regulates spatial activation of RhoA at cell-cell junctions and organisation of the junctional cytoskeleton. p114RhoGEF may also have a role in cell migration, as depletion in HCE cells, caused cells to migrate at a slower rate during wound healing assays. I have also started to explore the function of a putative p114RhoGEF ortholog, cg10188 in Drosophila melanogaster. Preliminary experiments have identified cg10188 to be important in larval development.

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