Investigating GSK3 and Lpd regulation of actin-based protrusions during mouse neural crest migration

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Neural crest cells are uniquely migratory cells that delaminate from the neural plate border during embryogenesis, contributing to diverse tissues including the craniofacial skeleton and peripheral nervous system. While mammalian neural crest cells are known to migrate efficiently in culture, the relative contribution of actin-based protrusions, such as lamellipodia, to the migration of these cells in vitro and in vivo are poorly understood. This is largely owing to the relative inaccessibility of this cell population during in utero development, making analysis of actin dynamics very challenging. The actin regulator Lamellipodin (Lpd) has been shown to promote lamellipodia formation by balancing actin branching (Scar/WAVE, Arp2/3) versus actin elongation (Ena/VASP) at the leading edge of various cell lines. However, a role for Lpd in regulating actin-based protrusions in mammalian neural crest cells has not been explored. Previous work has shown that the serine/threonine kinase, glycogen synthase kinase-3 (GSK3) is required for lamellipodia formation in mouse neural crest cells. Additionally, inhibition of GSK3 mislocalises Lpd away from the cell leading edge. I therefore hypothesised that GSK3 may act through Lpd to regulate actin-based protrusions in mouse neural crest cells. We have recently established a robust, reproducible explant culture system allowing the systematic assessment of genetically-modified mouse neural crest cells as they undergo EMT, delaminate and migrate away from the neural plate. Here, using neural crest-specific knockouts and explant cultures, I have shown that Lpd is required for lamellipodia formation and migration of mouse cranial neural crest cells ex vivo. Using mass spectrometry and coimmunoprecipitation assays, I then identified Lpd as a novel substrate of GSK3. GSK3 phosphorylation promoted Lpd interactions with the Scar/WAVE complex. Similarly, inhibition of GSK3 activity led to a failure of the Scar/WAVE complex component, Abi1, and Ena/VASP proteins, VASP and Mena, to localise at the lamellipodial edge. Consistent with this, GSK3 negatively regulates Lpd interaction with Ena/VASP proteins, VASP and Mena. Finally, GSK3-Lpd also appears to function by increasing the turnover of nascent adhesions while inhibiting the maturation of mature focal adhesions underneath the lamellipodium. My results define a novel role for GSK3 phosphorylation in controlling Lpd-dependent actin dynamics, as well as a novel requirement for GSK3-Lpd signalling in mouse neural crest development. Altogether, my work provides an improved understanding of cytoskeletal regulation in the neural crest, which will have general implications for neurocristopathies and neural crest-associated cancers.
Date of Award1 Mar 2023
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorMatthias Krause (Supervisor) & Karen Liu (Supervisor)

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