2018 PhD opportunities
MRC funded 3.5 year industrial PhD studying new type of membrane injury causing muscle weakness (Deadline 20th of January 2018)
Supervisory team: Isuru Jayasinghe (Leeds), Tim Etheridge (Exeter), Catherine Arden (Newcastle) & Badrilla Ltd (Leeds)
Exercise, in both the young and old, causes microscopic injuries to skeletal muscle cells. Such micro-injuries often manifest themselves as muscle weakness. In a recent ground-breaking study, we discovered that these micro-injuries can acutely re-shape the plasma membrane locally into fluid-filled pockets called ‘vacuoles’. Vacuoles withdraw the muscle’s calcium, required for its contraction, thereby paralysing the muscle fibres. Whilst this is likely to be an adaptation of the muscles to limit further injury during exercise, it also manifests in muscle weakness long after the conclusion of exercise. Such weakness correlates with the persistence of vacuoles for several days following exercise. One of the fundamental features of ageing is the higher propensity for exercise-induced muscle weakness. For the individual, this limits mobility and capacity to gain the benefits of regular exercise (e.g. risk management of cardiovascular diseases and diabetes). Strikingly, vacuoles appear to persist in the muscles of elderly subjects for up to a few weeks. Membrane injury-sensing proteins, membrane re-shaping proteins and proteins mediating autophagy (which facilitate the removal and break down of vacuoles) are thought to be crucial to observing vacuoles following exercise. Some of these proteins are also prime suspects for the apparent inability to turn over vacuoles in the muscles of elderly people due to changes in their expression levels. However, the exact molecular mechanisms of vacuole formation and turnover are still unknown. This is because vacuoles are often small membrane structures. Their compact nature means that such proteins interacting with vacuoles are located in a space that is so compact that most microscopes available to the modern biologist are unable to visualise their precise interactions. It this PhD project, you will study the interaction mechanism of membrane-injury and autophagy proteins with vacuoles formed during intense exercise in human subjects. With the utility of the newest version of the Nobel Prize winning super-resolution microscopy technology – DNA-PAINT – you will visualise how these proteins interact, form and repair vacuoles at an exquisite level of detail. To achieve this, you will work with co-supervisor Dr Tim Etheridge (University of Exeter, expert in molecular and metabolic muscular adaptation to exercise) to: a) access and analyse existing human muscle samples from exercised young and older humans and, b) perform new human clinical exercise trials to obtain muscle biopsies from young and older volunteers before and after exercise at specific time points. They will initially be screened with in vitro biochemistry and bioinformatics for proteins which are different between young and aged muscle. With the guidance of primary supervisor Dr Isuru Jayasinghe (University of Leeds, expert in calcium signalling in muscle) and co-supervisor Dr Catherine Arden (Newcastle University, expert in autophagy), you will immuno-label membrane injury sensing and autophagy proteins in histological sections of the biopsies. You will apply Dr Jayasinghe’s DNA-PAINT protocols to then determine the nanometre-scale locations of these proteins in relation to vacuoles. DNA-PAINT images will reveal the positions of each protein type at a precision of ~ 5-10 nm, allowing you to ‘count’ them and determine the ratios at which they interact using an image-analysis algorithm called qPAINT. To facilitate this quantification, you will work with industrial supervisor Prof John Colyer (Badrilla Ltd, specialising in quantitative technologies for antibody assays) to develop a peptide-based antibody calibration structure which will be embedded within the histology samples during DNA-PAINT imaging. The image-based measurements on membrane injury-sensing and autophagy proteins will provide novel and important insight of the protein interactions which orchestrate the formation and repair of vacuoles in muscle. Comparison of these mechanisms will crucially shed light on the predisposition of the elderly to experience prolonged muscle weakness following exercise. Your findings here will spark new avenues of targeted treatments and rehabilitation strategies which can improve the quality of life of Britain’s ageing population.
Supervisory team: Prof Nikita Gamper & Isuru Jayasinghe
Peripheral sensory neurons collect and transduce to CNS wealth of somatosensory information, including pain. Intracellular Ca2+ plays a paramount role in mediating sensory mechanisms in these neurons. Thus, Ca2+ release from intracellular stores underlies responses of pain-sensing neurons to inflammation. An important organelle for maintenance of cellular Ca2+ homeostasis is endoplasmic reticulum (ER). It serves as a Ca2+ reservoir and releases Ca2+ in response to action of inflammatory mediators. Upon release, ER is then refilled by store-operated Ca2+ entry (SOCE) from the extracellular space. Recent work in the host laboratory identified multiprotein signaling complexes assembled at junctions between the ER and plasma membrane (PM) in the peripheral pain-sensing neurons. Furthermore, our unpublished data suggest that scaffolding protein junctophilin-4 (JPH4) plays a key role in such complexes. We will aim to build upon this foundation to elucidate roles of junctophilins in Ca2+ signaling in sensory neurons. To answer our research questions we will employ cell biology and electrophysiology methods with novel imaging approaches (including super-resolution) and in vivo behavioral tests. Mechanistic understanding of localized intracellular signaling in sensory neurons is necessary to understand principles of somatic sensations; moreover, such understanding will pave a way for future treatments of inflammatory pain.
Supervisory team: Lynn McKeown, Robin Bonn & Isuru Jayasinghe
Statins, in addition to their ability to lower cholesterol levels, have beneficial effects on the vasculature by preventing the inappropriate release of pro inflammatory factors from endothelial cells. How they do this is not fully understood, however, it is known that statins also inhibit a biochemical pathway that is necessary for the addition of lipid anchors (prenylation) to Rab GTPases preventing them from being inserted into their target membranes. Rab GTPases regulate all aspects of intracellular trafficking including exocytosis and several
Rabs have been shown to play roles in the secretion from endothelial cells. The identification of a Rab GTPase whose lipid modulation mimics statin therapy would provide a specific target for developing novel therapeutics for cardiovascular diseases.
This project will address the effects of statins on endothelial cell secretion by using state of the art Super-resolution microscopy in combination with chemical, molecular and biochemistry technologies.