Casamento, Alessandra;
(2024)
Investigating the regulation of Fast Endophilin-Mediated Endocytosis.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
While the best characterised endocytic pathway is Clathrin-mediated endocytosis (CME), several Clathrin-independent endocytic routes exist, including Fast Endophilin-Mediated Endocytosis (FEME). FEME is prominent at the leading edge of migrating cells, following the activation of cargo receptors upon stimulation by their cognate ligands. It specifically controls Beta 1 Adrenergic Receptor (β1-AR) uptake and signalling and, additionally, regulates the internalization of other G Protein-Coupled Receptors (GPCRs), Receptor Tyrosine Kinases, Cytokine Receptors as well as Axon Guidance Receptors. Within 10 seconds, such cargoes are captured and internalised by locally pre-enriched Endophilin. The mechanism by which FEME is regulated has not been fully elucidated. While FEME is not constitutively active but triggered by activation of discrete receptors, high levels of Growth Factors in the culture media elicit spontaneous FEME in mammalian cells. Growth Factor deprivation, instead, suppresses the pathway. Therefore, a role for Growth Factor-activated protein kinases in FEME regulation was predicted. Using chemical and genetic inhibition, it was found that Cdk5 and GSK3 inhibit FEME. In this thesis, the mechanism by which the two aforementioned kinases act on FEME was further outlined by employing an array of experimental techniques, including pull-down assays, microscopy, and proteomics. Specifically, it was shown that concurrent inhibition of GSK3 and its priming kinase Cdk5 promoted the interaction of Endophilin with Dynamin and Dynein, two FEME players involved in carrier budding. Furthermore, this research demonstrated that Endophilin binds to and recruits active GSK3, thereby locally inhibiting FEME. Finally, this study laid a foundation for understanding the role of the Endophilin-related protein Bin1 in FEME, revealing that Bin1 interacts with the FEME components Lamellipodin and Dynein.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Investigating the regulation of Fast Endophilin-Mediated Endocytosis |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10191151 |
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