University of Sussex
Cooper, Simon J..pdf (19.11 MB)

Investigating the role of FMRP, CYFIP1 and DDX3X in the processes of mRNA localisation and translation within mesenchymal cells

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posted on 2023-06-09, 05:11 authored by Simon J Cooper
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.


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University of Sussex

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