Insulin controls whole body glucose homeostasis and is the most potent anabolic hormone known, driving glucose uptake into insulin-sensitive cells (primarily adipocytes, muscle cells and kidney podocytes). Cellular insulin resistance is the hallmark of type 2 diabetes and metabolic syndrome, which have a rapidly increasing worldwide incidence in association with increasing obesity. The kidney is a disease target in diabetes, and diabetic nephropathy is the commonest cause of kidney failure. The main mechanism of insulin resistance is free fatty acid-induced perturbation of insulin receptor signalling networks. Normally, insulin receptors (IR) phosphorylate insulin receptor substrate (IRS) proteins that are linked to the activation of two main signalling pathways, the PI3K/Akt pathway and the Ras/MAPK pathway. A number of studies have described alterations in insulin signalling pathways as a consequence of insulin resistance; however, the molecular basis for insulin resistance is complex and remains poorly understood.

Insulin resistance is effectively a defect in signal transduction and we hypothesise that an integrative approach, as opposed to the reductionist approaches used to date, will offer new mechanistic insights. The student will therefore adopt a holistic, proteomic approach and aim to identify the perturbations in signalling that occur under conditions of insulin resistance. Building on a methodology and workflow established in MH’s laboratory for integrins [Humphries, JD et al. (2009) Science Sig. 2: ra51], IR-associated protein complexes will be isolated and purified from human podocyte cell lines. Insulin resistance will be generated in wild-type cells by addition of the free fatty acid palmitate, and tested by radio-labelled glucose uptake assays. IR-associated complexes will be isolated by binding anti-IR antibody-coated microbeads to cells, stabilisation with a membrane-permeable cross-linker, detergent lysis, sonication and elution. The peptide and phosphopeptide composition of these complexes will be determined by mass spectrometry, and then alterations in complex composition will be quantified in the presence or absence of palmitate and insulin. Bioinformatic interrogation of the data will take the form of ontological surveys (to identify protein classes that change), hierarchical clustering based on spectral count (to identify proteins with similar quantitative changes), and protein-protein interaction network analyses (to identify potential connections between isolated proteins). This process will identify candidates for follow-up in (initially) cell biological and (eventually) whole animal studies.

The student will be co-supervised by Martin Humphries and Rachel Lennon, who share a lab. This multidisciplinary environment will provide technical training in niche [mass spectrometry/bioinformatics (MH)] and core [biochemistry/molecular biology/cell biology (MH/RL)] areas, and biomedical training in diabetes, renal physiology and pathology (RL).

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• Humphries, JD et al. (2009) Science Sig. 2: ra51.
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Why adhesion? Cell surface adhesion receptors play key roles in the control of cell movement, survival, division and differentiation, and they underpin a metazoan (multicellular) existence. By studying how adhesion receptors signal, it will be possible to understand how cell fate is controlled.

What don’t we understand? Following binding to extracellular matrix ligands, adhesion receptors trigger the assembly of multi-protein complexes on the cytoplasmic face of the plasma membrane. These complexes contain signalling molecules and cytoskeletal proteins, and their role is to control the location of signalling pathways and provide a physical link to the contractile actomyosin polymer network. The identity of the molecules that transduce signals, the mechanisms of complex assembly and disassembly, the stoichiometry of complex components, the key control points, and the extent of variation between complexes in different cells are not known.

How to get answers to these questions? Recently, we have developed the first methodology for employing mass spectrometric analyses of isolated complexes to define the adhesion receptor-associated proteome (ref. 1). This technique allows an unbiased approach to studying adhesion signalling. This project will use this method to identify candidate proteins that mediate signalling. Candidates will then be validated by a combination of biochemical (e.g. using IP-blotting and phosphorylation analysis) and/or cell biological approaches (e.g. using fluorescence imaging).

How does adhesion control diverse cell behaviours? The bioinformatic analyses that we have carried out of adhesion complexes so far suggest that they not only contain factors that link receptors to the cytoskeleton, but that they also contain factors involved in regulating protein synthesis and controlling cell division. It will be interesting to identify the mechanisms that link adhesion to these global cell functions.

Associated skills: Mass spectrometry, RNA interference, DNA manipulation, PCR, Mammalian cell culture, Transfection/Immunoprecipitation, Western blotting, Immunofluorescence microscopy, Bioinformatics.

• Humphries, J.D., Byron, A., Bass, M.D., Craig, S.E., Pinney, J.W., Knight, D. and Humphries, M.J. (2009) Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6. Science Sig. 2: ra51
• Askari, J.A., Buckley, P.A., Mould, A.P. and Humphries, M.J. (2009) Linking integrin conformation to function. J. Cell Sci. 122: 165-170
• Askari, J.A., Tynan, C.J., Webb, S.E.D., Martin-Fernandez, M.L., Ballestrem, C. and Humphries, M.J. (2010) Focal adhesions are sites of integrin extension. J. Cell Biol. 188: 891-903
• Morgan, M.R., Humphries, M.J. and Bass, M.D. (2007) Synergistic control of cell adhesion by integrins and syndecans. Nature Rev. Mol. Cell Biol. 8: 957-969