DDX3X, a helicase, can interact directly with mRNA and translation initiation factors, regulating the selective translation of mRNAs that contain a structured 5′ untranslated region (5’UTR). This activity modulates the expression of mRNAs controlling cell cycle progression and mRNAs regulating actin dynamics, contributing to cell adhesion and motility. Previously, we have shown that ribosomes and translation initiation factors localise to the leading edge of migrating fibroblasts in loci enriched with actively translating ribosomes, thereby promoting steady-state levels of ArpC2 and Rac1 proteins at the leading edge of cells during spreading. As DDX3X can regulate Rac1 levels, cell motility and metastasis, we have examined DDX3X protein interactions and localisation using a number of complementary approaches. We now show that DDX3X can physically interact and co-localise with PABP1 and caprin-1 at the leading edge of spreading cells. Furthermore, as depletion of DDX3X leads to decreased cell motility, this provides a functional link between DDX3X, caprin-1 and initiation factors at the leading edge of migrating cells to promote cell migration and spreading.

This thesis undertakes to elucidate the roles of the proteins Fragile-X Mental Retardation Protein (FMRP), Cytoplasmic FMRP Interacting Protein (CYFIP1) and the helicase DDX3X, within mesenchymal cells.

Total Internal Reflection Microscopy (TIRFM) was used to look for spatiotemporal colocalisation of FMRP and CYFIP1, as prior work suggests that they function in concert, acting as translational repressors within neuronal cells.

Biochemical methods provided evidence of a Ribonucleoprotein complex consisting of four participants – mRNA, eIF4E, CYFIP1 and FMRP. Using super-resolution microscopy techniques, the interaction of FMRP and CYFIP were established at the leading edge of spreading fibroblasts with a resolution of 50nm.

DXX3X has a known structural helicase domain and evidence suggests that it may be involved in the up-regulation of mRNA translation. Within this thesis, biochemical methods are complemented with standard immunocytochemistry (ICC) to elucidate the role and subcellular location of DDX3X. Using ICC and post-acquisition imaging techniques such as deconvolution, colocalisation of DDX3X with actin stress fibres suggests that DDX3X may be transported intracellularly via actin filaments. Using in situ Proximity Ligation Assay (PLA), a bespoke analysis program ‘PLAY’ provides evidence to suggest that DDX3X interacts with the eukaryotic initiation factor eIF4E, whilst travelling along filamentous actin structures to the cell periphery. The interaction between DDX3X and eIF4E at the leading edge of cells appears to correlate with an upregulation in local metabolism and morphology change, suggesting that DDX3X may be involved in the transport and localised translation of proteins needed for cell spreading and motility.