Professor Steve Winder
Professor of Molecular Cell Biology
Room: B2 06 Florey building
Brief career history
My group is using molecular and cellular approaches to understand the biology of the adhesion receptor dystroglycan. We are focussed on modulating dystroglycan signalling as a therapeutic route to treat Duchenne muscular dystrophy. We are also investigating the functions of dystroglycan in organising and stabilising the nuclear lamina, in cancer and in muscular dystrophies.
Regulation of dystroglycan function in muscular dystrophy and cancer
The laminin binding protein dystroglycan plays multiple roles in cell adhesion, signalling and membrane cytoskeleton stability. Perturbation of dystroglycan function underlies several muscular dystrophies and is also a secondary consequence of adenocarcinoma progression. Changes to the post-translational modification of dystroglycan are crucial in directing the associations, cellular localisation and ultimately degradation of dystroglycan. Our aim is to elucidate the mechanisms and consequences of these post-translational modifications in order to better understand dystroglycan function and to identify potential therapeutic targets for the treatment of muscular dystrophy or cancer.
We employ in vitro, in/ex vivo fish and mouse genetic models with clinically relevant archival tissue samples or immortalised cell lines. Dystroglycan function is dissected through the use of molecular cell biology approaches, and potential therapeutic targets are assessed in vitro and in vivo. Recently we have developed a novel therapeutic approach for the treatment of Duchenne muscular dystrophy using inhibitors of tyrosine phosphorylation and proteasomal degradation. Through the use of zebrafish screening and phenotypic analysis in mdx mice and human DMD myoblasts we are in the process of validating the potential for repurposed drugs as a precursor to initiating clinical trials. Physiological analysis is carried out I collaboration with Nic Wells at the RVC London.
In vitro models of prostate cancer have revealed a role for the post-translational proteolytic processing and nuclear targeting of dystroglycan. Current efforts are centred around characterising a role as part of the LINC complex in the inner nuclear membrane. These studies form part of an ongoing collaboration with Bulmaro Cisneros, CINVESTAV Mexico City.
Undergraduate and postgraduate taught modules
Level 4/Maters (MSc)
Dystroglycan function in mammalian auditory hair cells and neurons - Action on Hearing Loss Studentship
3 year fully funded PhD studentship (Uk and EU only)
Supervisors: Professor Steve Winder and Professor Walter Marcotti, Department of Biomedical Science
This PhD offers an exciting opportunity for a talented student to work on a multidisciplinary project in the areas of auditory physiology and molecular cell biology investigating the role of dystroglycan in mammalian hearing.
This proposal focuses on the function of a transmembrane protein called b-dystroglycan, which forms the core of dystroglycan complexes that enable cells to establish mechanical connections and communication pathways between their internal structures and the extracellular environment.
The inner ear interprets mechanical vibrations induced by sound. This requires the precise transduction of incoming auditory stimuli into an electrical signal that can be perceived by the brain. This sophisticated biological process occurs at the sensory hair cells. The hair cell mechanotransducer apparatus projects into a unique extracellular fluid compartment that is rich in potassium ions and that provides the ‘battery’ for the inner ear. Furthermore, hair cells transfer electrical information rapidly to their sensory nerves via specialised ‘ribbon’ synapses. All of these processes are expected to involve dystroglycan complexes, although published evidence suggests that b-dystroglycan is not present in the sensory organ. This is puzzling because there is clear evidence for its partner, a-dystroglycan, which is encoded by the same gene. We also know that defects in a number of other proteins linked to dystroglycan complexes are associated with hearing loss. One of these proteins is dystrophin, mutations of which is normally linked to muscular dystrophy.
Recently, we found that tyrosine phosphorylation of b-dystroglycan occurs at unusually high levels in the ear and that this explains why the protein was not previously detected. It changes the binding properties to other proteins, which is of general biological interest because dystroglycans are ubiquitously expressed yet they serve a wide range of different functions. In muscle, dystrophin binds to dystroglycan complexes and dissipates contraction forces to the extracellular connective tissue, thus protecting muscle membrane integrity. Mutations in dystrophin weaken these connections so that muscle cells become more easily damaged and degenerate, leading to muscular dystrophy. The strength of the attachment can be altered through the modification to b-dystroglycan that we observe in the ear and the mechanism has been proposed as a potential therapy to stabilise the muscle. Similar modifications to b-dystroglycan act as switches to alter association with other proteins in non-muscle cells, including nerves, and they are important for the organisation of signalling molecules at synaptic junctions and of molecules that regulate the flow of water and inorganic ions across fluid compartments. Finally, longer term changes in the function of dystroglycan complexes have been associated with aging. Thus phosphorylation of b-dystroglycan in the ear is likely to be critical for auditory function and for greater understanding of noise-induced and age-related hearing loss.
Outline of the research methods
Animal models: We have made a knock-in mouse (Dag1Y890F) with a phenylalanine substitution that prevents tyrosine phosphorylation of β-dystroglycan . This will allow us to focus on the specific function of this modification in the ear. We will characterise the model, using the structural, physiological and biochemical techniques listed below. To identify potential hearing loss linked to the inability to phosphorylate Y890 in b-dystroglycan we will perform in vivo electrophysiological recording (auditory brainstem responses, ABRs) from normal and Dag1Y890F mice at 1, 4, 8 and 12 months old at frequencies of 4, 8, 16 and 32kHz.
Cell lines and antibodies: We have a conditionally immortal, mouse otic neuronal cell line, US/VOT-N33  that expresses β-dystroglycan and eps8, which has been assayed both by oligonucleotide gene expression arrays and by immunolabeling. The cells also react strongly with antibodies to pY890 b-dystroglycan. Eps8 is known to be expressed in neurons and it is associated with synaptic plasticity and the morphology of dendritic spines . We have the hybridoma cell line that produces antibodies to non-phosphorylated β-dystroglycan (Mandag2) and our own polyclonal antibody to pY890 b-dystroglycan (Ab1709) with which we will be able to estimate relative expression of the two forms in tissue sections, organotypic whole-mounts of the organ of Corti and in western blots and immunoprecipitations. We will use antibodies to ribeye (ribbon synapse), eps8 and eps8l2 (hair bundles and neurons), and Kir4.1 and aqp4 (fluid regulation) to study the distribution of these proteins in Dag1Y890F mice.
Single cell electrophysiology - Patch clamp recording will carried out on hair cells as described previously . We will use acutely dissected tissue to record mechanoelectrical transducer current from hair cells in response to displacements of hair bundles with a fluid jet and to assess basolateral membrane properties such as ion currents and vesicle release at the ribbon synapses.
Microscopy – Confocal fluorescence microscopy will be used to localise proteins in cells, cryostat sections and organotypic cultures. Scanning and transmission electron microscopy will be used to study the hair bundles and the localisation and structure of ribbon synapses.
Biochemistry – We will validate the Eps8 SH3 domain interaction with β-dystroglycan using peptide SPOT arrays to define the precise binding site on dystroglycan and using reciprocal fusion protein pull down assays from otic epithelial cell lysates. The interaction will be further validated by immunoprecipitation, immunofluorescence localisation and proximity ligation in otic epithelial cells in vitro. We have used similar approaches to validate SH3 domain interactions between β-dystroglycan and other cytoskeletal proteins [5, 6].
Informal enquiries about this project welcome.
Targeting dystroglycan to the nucleus in muscular dystrophy and cancer
This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.
Open to self funded students as well as possible BMS funded.
Dystroglycan is an essential cell adhesion receptor required for early embryonic development. Genetic loss of function gives rise to severe muscular dystrophies with neuronal involvement. Post-translational loss of function also occurs in Duchenne muscular dystrophy and in some cancers. This includes phosphorylation, proteolysis and ubiquitination. Moreover some proteolytic fragments of dystroglycan are targeted to the nucleus where they have effects on transcription. As part of our analysis of dystroglycan post-translational modifications, we identified a lipid modification - palmitoylation of a conserved cysteine residue that could act to anchor bioactive dystroglycan fragments to the membrane, both at the cell surface and in the nucleus.
To examine the function of dystroglycan palmitoylation in cellular targeting and in cellular phenotypes associated with muscular dystrophy and cancer.
PCR-based site-directed mutagenesis, and cloning of dystroglycan mutants. Overexpression of dystroglycan mutants in tissue culture cells. Analysis of subcellular distribution of dystroglycan by quantitative immunofluorescence microscopy and cell fractionation. In vitro assays of cell invasion and metastatic growth.
For further information about these projects and how to apply, please see our PhD Opportunities page.
- Lipscomb L, Piggott RW, Emmerson T & Winder SJ (2016) Dasatinib as a treatment for Duchenne muscular dystrophy. Human Molecular Genetics, 25(2), 266-274. View this article in WRRO
- Mathew G, Mitchell A, Down JM, Jacobs LA, Hamdy FC, Eaton C, Rosario DJ, Cross SS & Winder SJ (2013) Nuclear targeting of dystroglycan promotes the expression of androgen regulated transcription factors in prostate cancer.. Sci Rep, 3, 2792. View this article in WRRO
- Martínez-Vieyra IA, Vásquez-Limeta A, González-Ramírez R, Morales-Lázaro SL, Mondragón M, Mondragón R, Ortega A, Winder SJ & Cisneros B (2013) A role for β-dystroglycan in the organization and structure of the nucleus in myoblasts. Biochimica et Biophysica Acta - Molecular Cell Research, 1833(3), 698-711.
- Miller G, Moore CJ, Terry R, La riviere T, Mitchell A, Piggott R, Dear TN, Wells DJ & Winder SJ (2012) Preventing phosphorylation of dystroglycan ameliorates the dystrophic phenotype in mdx mouse. Human Molecular Genetics, 21(20), 4508-4520.