George, Adams
(2022)
Stem cell adhesome: Deciphering the role of
distinct integrin-signalling networks in
regulating stem cell behaviour.
Doctor of Philosophy thesis, University of Liverpool.
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Abstract
Pluripotent stem cells are characterized by their capacity for self-renewal and differentiation, playing a critical role in the maintenance and regeneration of tissues. The composition, organization, and topology of the extracellular matrix (ECM) within the tissue microenvironment, collectively known as the niche regulates stem cell fate. Integrins are heterodimeric receptors that interact with extracellular matrix proteins, forming a mechanical link between the ECM and the actin cytoskeleton. The dimensionality, chemistry, topography, and elasticity of the microenvironment can be tuned to modulate gene expression and stem cell fate. These force-induced changes in gene expression and stem cell fate are mediated by dynamic regulation of signalling, adaptor and cytoskeletal proteins, nuclear morphology and chromatin organization in response to mechanical stress. This study aimed to characterise the integrin heterodimers and integrin-dependent signalling networks recruited on the ECM ligands vitronectin and fibronectin, and two self-assembling nano engineered polymers that serve as stem cell substrates that promote self-renewal, ZTFN and ZT910 Immunofluorescence imaging demonstrated that iPSCs plated on vitronectin fibronectin, ZTFN and ZT910 recruited αVβ5, α5β1, αVβ1 and α5β1 respectively to integrin-associated adhesion complexes. The distinct integrin heterodimers recruited on vitronectin, fibronectin, ZTFN and ZT910 were confirmed using adhesion complex enrichment and integrin inhibition assays. Traction force microscopy of iPSCs plated on hydrogels of different rigidities (5kPa to 70-100 kPa) demonstrated that vitronectin fibronectin, ZTFN and ZT910 induced differential force transmission on soft and stiff, but not intermediate rigidity matrices. Suggesting that the different integrin heterodimers possess distinct biophysical properties that are tuneable in a rigidity-dependent manner. To determine how the mechanical properties of different integrin heterodimers influence stem cell behaviour, nuclear morphology, chromatin organisation and YAP localisation, iPSCs were plated on substrate-coated hydrogels of different rigidities. Under these conditions, iPSCs demonstrated no difference in nuclear morphology or YAP localisation but exhibited significant changes in substrate and rigidity dependent chromatin organisation suggesting that distinct biophysical properties integrin dependent gene regulation. Adhesion complex enrichment was employed to isolate and characterise for the first-time integrin adhesion complexes recruited by iPSC on vitronectin, fibronectin, ZTFN and ZT910. The αVβ5-VIT, α5β1-FN, αVβ1-ZTFN and α5β1-ZT910 adhesomes exhibited broad convergence when comparing the α5-mediated adhesomes (fibronectin and ZT910) and αV mediated adhesomes (vitronectin and ZTFN). Bioinformatic analysis of α5β1-IACs and αVβ5- IACs recruited on fibronectin and vitronectin respectively revealed enrichment of gene ontology terms containing Focal Adhesion and Metabolism proteins. Integrative analysis revealed that SLC3A2 may play a potential role in regulating both integrin mechanosignalling and metabolism. Immunofluorescence imaging demonstrated that SLC3A2 localises to α5- mediated adhesions, but not αV-mediated adhesions, suggesting that SLC3A2 may modulate mechanosignalling and metabolism in an α5β1-heterodimer specific manner. Traction force microscopy revealed that SLC3A2 knockdown did not affect force transmission of iPSCs plated on vitronectin or fibronectin. However, treatment of iPSCs with SLC3A2 siRNA alone or in combination SLC3A2 light chain inhibitor LAT1 significantly reduced overall intracellular ATP 3 levels, demonstrating a putative impact on cellular energetics and metabolism. Analysis of the SLC3A2-LAT1 cryo-EM structure enabled the identification of SLC3A2-LAT1 regulatory domains and constructs were generated to target different structural domains of the SLC3A2- LAT1 complex. These constructs will allow dissection of the relative contribution of SLC3A2- integrin-binding to SLC3A2-LAT1-mediated amino acid transport, in force transduction, metabolic reprogramming and transcriptional regulation. Together, these advances contribute to our understanding of the mechanisms by which integrin-ECM engagement regulates global cellular processes in stem cells but will likely also have relevance in somatic cell biology and disease.
| Item Type: | Thesis (Doctor of Philosophy) |
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| Divisions: | Faculty of Health and Life Sciences |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 02 Sep 2022 10:39 |
| Last Modified: | 18 Jan 2023 20:53 |
| DOI: | 10.17638/03160821 |
| Supervisors: |
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| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3160821 |
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