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Title: Deciphering the in vivo role of the Drosophila RNA-binding protein Imp in cell motility
Author: Pritchard, Clare
ISNI:       0000 0004 5371 0284
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2016
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Cultured motile cells show a highly enriched belt of actin at the tip of their lamellipodial protrusions, termed the leading edge. It is at this leading edge that researchers have uncovered pools of highly localized mRNAs encoding actin and actin-regulatory proteins, including β-actin, Profilin, Cofilin and all seven subunits of the Arp2/3 complex, whose local translation is then required for proper cell migration. The localization and local translation of these mRNAs are regulated by RNA-binding proteins, including Imp, which localizes β-actin mRNA at the leading edge. However, published studies have so far failed to determine if Imp is required to localize β-actin mRNA and/or other mRNAs at the leading edge of migratory cells in vivo. Our examination of Drosophila embryonic macrophage migration in vivo revealed that actin is not enriched at the leading edge, compared with cultured macrophages, demonstrating that a single cell population employs different mechanisms of cytoskeletal arrangement when migrating in vivo, compared with an ex vivo migration along a 2D substrate. It is therefore not surprising that we did not observe Imp at the leading edge of macrophages in vivo. However, overexpression of Imp reduced both the velocity and directionality of macrophages and inhibited cell-to-cell contact inhibition, suggesting a defect in microtubule dynamics, although we have yet to establish a mechanism for this. We show that Imp binds the 3’UTR of β-actin, profilin, and β-integrin mRNAs and reveal three sites of primary sequence that are required for Imp binding to β-actin mRNA. Our results suggest that, in contrast to cultured migratory cells, β-actin mRNA is unlikely to be localized to, and locally translated, at the leading edge of macrophages in vivo. However, cytoplasmic mRNA regulation is likely to play some kind of role in cell migration, as revealed by overexpression of Imp, which impairs macrophage motility. This thesis highlights a crucial requirement for studies to determine the mechanisms of cytoplasmic mRNA regulation in motile cells in vivo, which appear to be distinct from those employed in some cultured cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH301 Biology