Adhesion complexes (ACs) are large macromolecular complexes of integrins and associated proteins that connect the actin cytoskeleton to the extracellular matrix. In migrating cells, ACs are highly dynamic -- forming and maturing at the cell front and disassembling at the cell rear. The turnover of ACs enables and localises the necessary traction forces required for cell migration. There is evidence for the spatiotemporal recruitment of specific proteins during AC maturation or disassembly; however, a holistic understanding of the compositional changes to ACs during these states is lacking. To this end, we sought to characterise the dynamic changes that occur at ACs during turnover using a mass spectrometry (MS)-based proteomics approach. A major challenge in studying AC turnover is the desynchronised nature of AC formation, maturation and disassembly within a population of cells. Therefore a nocodazole-washout assay was used to synchronise microtubule-induced AC maturation and disassembly. To study the dynamics of AC turnover by MS, an AC isolation method was optimised for use with the nocodazole-washout assay. Subsequently, the maturation of ACs by the loss of microtubules was studied by MS-based proteomics, and it was found that this resulted in the overall accumulation of adhesion proteins, and also the conversion of fibrillar adhesions to focal adhesions. Studying the dynamic process of AC disassembly requires a sensitive MS quantification method; as such, label-free quantitative methods were compared, and it was found that LC-MS peak ion intensity quantification performed better than spectral counting. Using optimised methodologies for isolation of ACs and MS quantification, the dynamics of AC disassembly was analysed over the course of the nocodazole-washout assay. It was found that in general, microtubules were enriched around ACs, whereas many structural AC proteins decreased over time. In summary, we have optimised methods for the study of ACs by MS-based proteomics, and applied these methods to the study of AC turnover.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:603114 |
| Date | January 2013 |
| Creators | Ng, Daniel |
| Contributors | Humphries, Martin; Ballestrem, Christoph |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-dynamics-of-adhesion-complex-turnover-by-mass-spectrometry-based-proteomics(4e6d3051-c007-4715-a290-9acfd45d38a7).html |
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