Identifying proteins that preferentially bind ATP- over ADP-F-actin

Claydon, Sophie (2024) Identifying proteins that preferentially bind ATP- over ADP-F-actin. PhD thesis, University of Glasgow.

Abstract

The cytoskeleton is a dynamic network of proteins that grants cells internal organisation, structure and mobility. This gives cells the ability to participate in all manner of physiological processes, such as immune response, growth and development. However, the same genes and proteins that control these processes can also be altered and participate in pathological events such as cancer and metastasis. The actin cytoskeleton is responsible for the formation of cellular protrusions that allow cells to migrate. The process that gives rise to these protrusions is the polymerisation of actin into its filamentous form, Factin. This is a highly dynamic process that involves regulation by many other proteins and some degree of self-regulation. Actin monomers incorporated into the polarised filament are ATP bound. Upon polymerisation, actin intrinsic hydrolytic activity gives rise to differing states of nucleotide bound conformations throughout the actin filament. Certain proteins are known to bind the aged, ADP-bound portion of F-actin, and contribute to negative regulation for example by promoting depolymerisation. Similarly, we hypothesise that some proteins would preferentially bind the ATP-bound portion of F-actin and be involved in positive regulation of polymerisation.

To investigate this, we optimised a methodology for creating F-actin filaments that mimic the ATP-bound portion using non-hydrolysable analogues of ATP, and constructed affinity chromatography columns that were used to isolate specific interactors. Subsequent mass spectrometry revealed a pool of proteins that preferentially bind ATP-bound over ADP-bound F-actin. One of the identified hits, Eps8, was employed to explore the mechanism of ATP-F-actin binding. Combining molecular biology cloning techniques and super resolution microscopy, we identified Eps8’s SH3 domain as the main regulator of this interaction. Many of the proteins identified as preferentially binding ATP-F-actin contain SH3 domains, suggesting a more generalised role of this domain in the binding to ATP-actin, either directly or indirectly. SH3 domains are known to interact with proline rich sequences. Recent work from our lab has shown the importance of the polyproline domains of members of the WAVE regulatory complex on the formation of actin protrusions. Based on these combined data, we hypothesise that the SH3/polyproline domain interaction could constitute an axis for the recruitment of different proteins involved in actin polymerisation to the site of ATP-F-actin, generating a positive feedback loop of polymerisation. More work, for example using purified proteins and binding assays, is needed to determine the exact method of ATP-F-actin binding, the role it has in positive regulation of polymerisation and its biological relevance.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Insall, Professor Robert
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84477
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 23 Jul 2024 15:25
• Last Modified: 23 Jul 2024 15:25
• Thesis DOI: 10.5525/gla.thesis.84477
• URI: https://theses.gla.ac.uk/id/eprint/84477

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