Systems-level analyses of the adhesion nexus

Abstract

Cell adhesion to the extracellular matrix is mediated by the integrin family of adhesion receptors. Integrin receptor engagement initiates the formation of multimolecular protein complexes, termed integrin adhesion complexes (IACs), at the cell membrane. IACs are complex signalling hubs that are enriched in tyrosine-based phosphorylation events and form a mechanochemical connection between integrin receptors and the actin cytoskeleton. Dysregulation of individual IAC components has been reported to influence a wide range of biological processes that contribute to disease. Literature-curated and proteomic analyses of IACs have revealed an unanticipated molecular complexity of IACs in a variety of experimental contexts; however, a global consensus view of the composition of IACs, and a description of how the complex network of interactions in IACs influences global cell function, is currently lacking.Here, multiple existing and new proteomic datasets detailing the protein composition of IACs were analysed to identify a systems-level description of IACs and to enable interrogation of IAC structure, topology and dynamics. Quantitative IAC proteomes derived from multiple cell types were integrated to generate a 2,412-protein 'meta-adhesome' database of proteins enriched to fibronectin-induced IACs. To investigate the putative functional adhesion landscape in an objective manner, the meta-adhesome was analysed using a combination of hierarchical clustering, gene ontology and interaction network analyses. An emergent property of the meta-adhesome was the definition of a consensus adhesome: 60 proteins commonly identified from IAC datasets that likely represent an IAC protein core composition. The consensus adhesome highlights how integrins connect to actin via multiple pathways and consists of both canonical and underappreciated IAC components.To investigate the robustness of the IAC network, the effects of pharmacological perturbation of the key IAC kinases FAK and Src on IACs were examined. FAK activity was inhibited with the small molecule inhibitor AZ13256675, and mass spectrometry-based protein quantification revealed that IAC protein composition was unaffected upon FAK inhibition. Moreover, IAC composition was also insensitive to Src inhibition using AZD0530 and to simultaneous FAK and Src inhibition. In contrast, phosphorylation of IAC components, cell migration and cell proliferation were reduced upon FAK and/or Src inhibition. These data suggest that IAC protein composition is robust to perturbation of key kinases, while flux of signals propagated through IACs via phosphorylation is kinase dependent.To examine IAC dynamics, the composition of IACs during IAC assembly and IAC disassembly were examined in the context of the meta-adhesome and consensus adhesome using IAC proteomic datasets. These analyses revealed the temporal dynamics of specific functional protein modules at IACs and detailed the compositional dynamics of the core cell adhesion machinery. In summary, these studies describe both a systems-level and a reductionist view of the IAC proteome, investigate the effects of kinase inhibition on IAC composition and chart IAC dynamics during their assembly and disassembly. These data demonstrate the usefulness of the meta-adhesome and consensus adhesome for future analyses of IAC proteomes.

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Humphries (Supervisor) & Pedro Pedrosa Mendes (Supervisor)